A liquid phase preparation, separation and purification method for high-purity deoxynucleoside triphosphate monomers

Abstract

This application relates to a method for preparing deoxynucleoside triphosphate monomers, specifically disclosing a method for preparing high-purity deoxynucleoside triphosphate monomers using high-pressure preparative liquid chromatography. In this method, mobile phase A uses TEAA or TEAB as an ion-pairing reagent while simultaneously adding an alkali metal salt, wherein the alkali metal salt is one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate, and the concentration of the alkali metal salt is 0.2-0.4%; mobile phase B is one or more selected from methanol, acetonitrile, ethanol, or tetrahydrofuran; and the preparative liquid chromatography column is a reverse-phase column.

Description

Technical Field

[0001] This application relates to the field of compound purification technology, specifically designing a method for preparing deoxynucleoside triphosphate monomers using high-pressure liquid chromatography. Background Technology

[0002] Deoxynucleoside triphosphates (dNTPs) include deoxyadenosine triphosphate (dNTPs), deoxycytidine triphosphate (dNTPs), deoxyguanosine triphosphate (dNTPs), and deoxythymidine triphosphate (dNTPs). In biochemistry and molecular biology, dNTPs have various applications, such as in PCR polymerase chain reaction (PCR) and in vitro transcription (IVT), both of which require high-purity dNTP monomers as raw materials. With the increasing demand for high-fidelity sequences, the market demand for these four high-purity dNTPs will increase significantly.

[0003] In existing technologies, the biosynthesis reaction solution for deoxynucleoside monophosphate (dNMP) to deoxynucleoside triphosphate (dNTP) is either separated by sedimentation to obtain the generated dNTPs as barium salts, followed by ion exchange resin purification; or the dNTP biosynthesis reaction solution is directly eluted with an ion exchange resin-packed column, followed by high-performance liquid chromatography (HPLC) purification. In summary, high-performance preparative liquid chromatography (HPLC) is primarily used for the separation and purification of high-purity (greater than 95%) dNTPs.

[0004] In existing technologies, the preparation of dNTPs using high-pressure preparative liquid phase generally employs ion-pairing reagents as the mobile phase, with the separation mode primarily being reversed phase, or uses salt solutions as the mobile phase, with the separation mode primarily being anion exchange. Both methods suffer from poor separation effect, slow separation speed, and excessive consumption of mobile solvents, failing to meet the needs of large-scale industrial production. Summary of the Invention

[0005] The alkali metal salts mentioned in this application refer to sodium or potassium salts.

[0006] In this application, the mobile phase B used to prepare the liquid phase is also referred to as the eluent.

[0007] In this application, the sample loading amount can be calculated based on the crude product concentration and the injection volume.

[0008] To address the aforementioned technical problems, this application provides a method for purifying and preparing high-purity deoxynucleoside triphosphate monomers using high-pressure preparative liquid phase purification. Specifically, this method can be summarized as follows:

[0009] Mobile phase A uses TEAA or TEAB as an ion-pairing reagent and adds an alkali metal salt, wherein the alkali metal salt is one or more of an alkali metal salt or a potassium salt, and the concentration of the alkali metal salt is 0.2-0.4%; mobile phase B is one or more of methanol, ethanol, acetonitrile or tetrahydrofuran; the preparative liquid column is a reverse-phase chromatography column.

[0010] By adopting the above technical solution, using TEAA / TEAB as ion-pairing reagents in mobile phase A can effectively increase the retention time of the target compound. The addition of alkali metal salts further increases the ion exchange mode and enhances the separation degree between the target compound and nearby impurities (from 0 to 1.7). Without the addition of alkali metal salts, the purity of the obtained dGTP is only about 95%, an increase of only 2% compared to before loading. However, with the addition of alkali metal salts, the purity of dGTP prepared with alkali metal salts, represented by sodium chloride, can reach 98.5%, while the purity of dGTP prepared with sodium (potassium) carbonate or sodium (potassium) bicarbonate is higher than 99%. Simultaneously, the introduction of alkali metal salts also increases the solubility of dNTP series molecules in water, thereby increasing the loading amount, i.e., increasing the concentration of the crude product. This is because when the crude product concentration reaches 45 g / L, the purity is still greater than 99%. All the above data are statistically significant.

[0011] Mobile phase B uses one or more of methanol, acetonitrile, ethanol or tetrahydrofuran as the eluent; preferably, one or more of methanol, acetonitrile or ethanol is used as the eluent, and most preferably, methanol is used as the eluent.

[0012] By adopting the above-mentioned preferred technical solutions, the solubility of alkali metal salts can be effectively increased, and the compatibility of the method can be enhanced. Ultimately, under these separation conditions, the separation degree and loading amount of dNTP are significantly improved. For example, compared with ethanol, when methanol is used as mobile phase B and the crude product purity is 75%, the dGTP prepared is still higher than 99%, while ethanol is not higher than 99%. Or, for example, when the crude product concentration is as high as 45 g / L, when methanol is used as mobile phase B, the dGTP prepared is still higher than 99%, while if acetonitrile or ethanol is used, the yield or purity will be lower than that of methanol. Therefore, the above-mentioned optimized technical solution solves the problem that the main peak and adjacent impurities (adjacent impurities refer to the preceding and following impurities) cannot be effectively separated when using a single TEAA / TEAB ion-pair mobile phase. Analysis of the above results suggests that methanol as the optimal solution may not only be related to the polarity of mobile phase B. While it's reasonable to prioritize methanol over ethanol or tetrahydrofuran when using a reverse-phase column as the preparative column, methanol's polarity is weaker than acetonitrile's. Experimental results show that methanol significantly improves the solubility of alkali metal salts compared to acetonitrile, increasing the method's compatibility. Ultimately, this leads to a significant improvement in the resolution and loading capacity of dNTPs under these separation conditions. Using acetonitrile as mobile phase B shifts the eluent time of dGTP and its impurities forward and causes severe tailing. To obtain dGTP with a purity of over 99%, some yield loss is required. When preparing dGTP of the same concentration and purity, the yield of mobile phase B using acetonitrile is 3% lower than that of methanol, which is statistically significant. This means that the elution effect of methanol as an eluent is unexpectedly better than that of acetonitrile.

[0013] The mobile phase used in this application for high-pressure preparation of liquid phase is based on the commonly used TEAA / TEAB mobile phase, with the addition of alkali metal salts to increase the ion concentration in the mobile phase; the alkali metal salts are sodium chloride, sodium (potassium) carbonate, sodium (potassium) bicarbonate, potassium chloride, sodium (potassium) nitrate, sodium (potassium) bromide, sodium (potassium) sulfate, or sodium (potassium) bisulfate, etc.; preferably, the alkali metal salts are carbonate or bicarbonate alkali metal salts, such as sodium (potassium) carbonate and sodium (potassium) bicarbonate.

[0014] By adopting the above technical solutions, alkali metal salts can increase the separation degree between the target compound and nearby impurities. Simultaneously, the introduction of alkali metal salts also increases the solubility of dNTP series molecules in water, thereby significantly improving the sample loading capacity of high-pressure preparative liquid chromatography (HPLC), which is beneficial for large-scale dNTP preparation and more suitable for industrial production. Furthermore, sodium chloride (potassium), sodium phosphate (potassium), sodium monohydrogen phosphate (potassium), sodium dihydrogen phosphate (potassium), sodium nitrate (potassium), sodium bromide (potassium), sodium sulfate (potassium), or sodium bisulfate (potassium) exhibit unexpectedly weaker separation degrees than sodium carbonate (potassium) or sodium bicarbonate (potassium) due to the difficulty in removing their anions. In the experiment of screening the types of salts in mobile phase A, the purity of dGTP represented by sodium chloride was no more than 99%. However, when sodium carbonate (potassium) or sodium bicarbonate (potassium) was added to mobile phase A, and their concentrations were between 0.2% and 0.4%, the purity of the prepared dGTP was all above 99%. That is, the purity of dGTP prepared by adding sodium carbonate (potassium) or sodium bicarbonate (potassium) to mobile phase A was 1 percentage point higher than that prepared by other alkali metal salts, and this was statistically significant. Attached Figure Description

[0015] Figure 1 To prepare crude dGTP before separation, the four compound peaks were identified in order of elution time as impurity dGDP, dGTP pre-impurity, dGTP, and dGTP post-impurity.

[0016] Figure 2 The mobile phase A is salt-free.

[0017] Figure 3 0.2% sodium chloride was added to mobile phase A;

[0018] Figure 4 0.2% sodium bicarbonate was added to mobile phase A;

[0019] Figure 5 Adding 0.2% sodium bicarbonate or potassium bicarbonate to mobile phase A yielded similar results to adding 0.4% or 0.2% sodium bicarbonate. The purity of the collected fraction was analyzed by HPLC chromatogram, and the purity was not less than 99.5%.

[0020] Figure 6 Adding 0.2% sodium bicarbonate or potassium bicarbonate to mobile phase A yielded similar results to adding 0.4% or 0.2% sodium bicarbonate. The MS spectrum of the dGTP fraction was collected.

[0021] Figure 7 0.4% sodium chloride was added to mobile phase A;

[0022] Figure 8 Add 0.4% potassium chloride to mobile phase A, and mix with the adsorbent... Figure 7 similar;

[0023] Figure 9 Add 0.2% potassium bicarbonate to mobile phase A, and... Figure 4 similar;

[0024] Figure 10 The preparative liquid phase diagram shows that by adding 0.2% potassium bicarbonate or sodium bicarbonate to mobile phase A, the concentration of crude dGTP was increased from 35 g / L to 45 g / L. Figure 4 similar;

[0025] Figure 11 The HPLC chromatogram shows the purity of the main peak fraction, obtained by adding 0.2% potassium bicarbonate or 0.2% sodium bicarbonate to mobile phase A, which increased the crude dGTP concentration from 35 g / L to 45 g / L.

[0026] Figure 12 (See figure below) 0.6% sodium carbonate (potassium) or sodium bicarbonate (potassium) is added to the mobile phase A;

[0027] Figure 13 Acetonitrile was used as the eluent to prepare the liquid mobile phase B;

[0028] Figure 14 Ethanol was used as the eluent to prepare the liquid mobile phase B. Detailed Implementation

[0029] To clearly and concisely illustrate the implementation scheme of this application, one or two embodiments with similar results are used as representatives, for example:

[0030] In the embodiments of this application, the separation effect of sodium chloride is similar to that of potassium chloride, sodium nitrate (potassium), sodium bromide (potassium), sodium sulfate (potassium), sodium bisulfate (potassium), sodium phosphate (potassium), sodium dihydrogen phosphate (potassium), and sodium monohydrogen phosphate (potassium). That is, no matter how other parameters such as pH and salt concentration are adjusted, only the diphosphate impurity dGTP can be effectively separated from the main peak. The impurity peak before P cannot be separated from the dGTP main peak, and the impurity after dGTP can be separated from the main peak, but the peak shape is poor. For example, mobile phase B is one or more of methanol, ethanol, acetonitrile, or tetrahydrofuran. Ethyl acetate, a commonly used organic solvent, is used as a representative mobile phase that interferes with the absorption peak of dGTP. That is, ethyl acetate has a strong absorption peak at 254 nm, which is not conducive to observation. Therefore, it is not used as the eluent in this application. Tetrahydrofuran represents a mobile phase with strong elution ability and is more volatile, which poses a production risk. Therefore, it is not preferred.

[0031] The deoxyguanosine triphosphate mentioned in this application is abbreviated as dGTP.

[0032] The dGDP described in this application is an alkali metal salt of 2'-deoxyguanosine-5'-bisphosphate, which is an impurity of dGTP;

[0033] The dGTP precursor described in this application is a single dGTP isomer. The elution time in the HPLC chromatogram is located between dGDP and dGTP. Because the elution time is before dGTP, this application names it as the dGTP precursor.

[0034] The dGTP impurity described in this application is a dGTP dehydration impurity. The peak time in the HPLC chromatogram is after dGTP, so this application names it as dGTP impurity.

[0035] In this application, the pre- and post-impurities of DATP, DTTP, DCTP, and DUTP are the same as those of DGTP and are processed in the same way. Therefore, this application only uses DGTP as an example for explanation.

[0036] Each embodiment or comparative example in this application has at least three parallel samples, meaning that each data point is statistically significant.

[0037] The duration of the method described in this application embodiment is the total retention time after each injection of the prepared liquid phase.

[0038] The types of impurities in the crude dGTP used in this application are listed below. Figure 1 As shown.

[0039] The liquid chromatography conditions for dGTP purity testing in this application are shown in the table below:

[0040]

[0041]

[0042] Example 1: Effect of the type of salt used in mobile phase A on resolution

[0043] The preparation conditions for Example 1 were as follows:

[0044] The mobile phase was phase A: ion pairs were prepared using 100 mM ± 5 mM triethylamine carbonate TEAB aqueous solution. The effect of the type of salt used in mobile phase A on the resolution was investigated. The pH was adjusted to 7.0–7.5 with carbon dioxide or sodium hydroxide.

[0045] Phase B: Methanol, 0–20 min, methanol gradient increased from 0% to 30% and maintained at 30% for 10 min, the methanol used was industrial grade, the purified water was grade III water, and the other additives were all analytical grade.

[0046] The mobile phase flow rate was 15 mL / min;

[0047] The equipment uses a dynamic axial compression column DAC-50, and the packing material is C18-100-8.

[0048] The detection wavelength is 254nm;

[0049] The crude dGTP concentration was 35 g / L, and the HPLC purity of the crude product in this experiment was 93%.

[0050] The types of salts and their amounts are shown in Table 1. Sodium chloride represents potassium chloride, sodium phosphate, potassium phosphate, sodium monohydrogen phosphate (potassium), sodium dihydrogen phosphate (potassium), sodium nitrate (potassium), sodium bromide (potassium), sodium sulfate (potassium), sodium bisulfate (potassium), etc.

[0051] The experimental results are shown in Table 1.

[0052]

[0053] Example 1.1 Preparative liquid phase diagram of mobile phase A without the addition of alkali metal salts is shown below. Figure 2 As shown, the results indicate that if no alkali metal salt is added to the mobile phase A, the purity of the prepared dGTP is only about 95%, which is only about 2% higher than the initial purity. This is because if no alkali metal salt is added to the mobile phase A, only the diphosphate impurity dGDP is effectively separated from the main peak of dGTP, but the front and back impurity peaks cannot be separated from the main peak of dGTP.

[0054] Example 1.2 When the alkali metal salts in mobile phase A are sodium chloride and potassium chloride, sodium (potassium) phosphate, sodium (potassium) dihydrogen phosphate, sodium (potassium) monohydrogen phosphate, sodium (potassium) nitrate, sodium (potassium) bromide, sodium (potassium) sulfate, etc., the prepared liquid phase diagram is as follows: Figure 3 As shown, because their separation effects are similar, they can only effectively separate the diphosphate impurity dGDP from the main peak, while the pre-impurity peak cannot be separated from the dGT main peak. Although the post-impurity dGTP is separated from the main peak, the peak shape is poor. Furthermore, when their concentration is 0.2%, the purity of the prepared dGTP does not exceed 98%, which is nearly 5% higher than the initial purity.

[0055] In Examples 1.3 and 1.4, when 0.2% of an alkali metal salt, either sodium carbonate (potassium) or sodium bicarbonate (potassium), is added to mobile phase A, the pH is adjusted using carbon dioxide or sodium hydroxide. Their separation effects are comparable, and the prepared liquid phase diagrams are shown below. Figure 4 or Figure 9 As shown, the purity of the dGTP main peak fraction obtained by HPLC purity analysis was above 99% (e.g., Figure 5 As shown in the figure, the purity was increased by 6% or more compared to the initial purity.

[0056] Example 2: Effect of salt concentration in mobile phase A on resolution

[0057] The preparation conditions for Example 2 were as follows:

[0058] The mobile phase was phase A: ion pairs were prepared using 100 mM ± 5 mM triethylamine carbonate TEAB aqueous solution. The effect of the concentration of the salt used in mobile phase A on the resolution was investigated. The pH was adjusted to 7.0–7.5 with carbon dioxide or sodium hydroxide.

[0059] Phase B: Methanol, 0–20 min, methanol gradient increased from 0% to 30% and maintained at 30% for 10 min, the methanol used was industrial grade, the purified water was grade III water, and the other additives were all analytical grade.

[0060] The mobile phase flow rate was 15 mL / min;

[0061] The equipment uses a dynamic axial compression column DAC-50, and the packing material is C18-100-8.

[0062] The detection wavelength is 254nm;

[0063] The crude dGTP concentration was 35 g / L, and the HPLC purity of the crude product in this experiment was 93%.

[0064] The types and concentrations of salts are shown in Table 2. Sodium chloride represents potassium chloride, sodium phosphate, potassium phosphate, sodium monohydrogen phosphate (potassium), sodium dihydrogen phosphate (potassium), sodium nitrate (potassium), sodium bromide (potassium), sodium sulfate (potassium), etc.; sodium bicarbonate represents sodium carbonate (potassium) or potassium bicarbonate.

[0065] The experimental results are shown in Table 2.

[0066]

[0067] Preparation liquid phase diagrams of Examples 2.1 and 2.3 and attached Figure 3 Similarly; Example 2.2 uses sodium chloride as the preparation liquid phase diagram is attached. Figure 7 As shown, the liquid phase diagrams obtained by replacing sodium chloride with potassium chloride, sodium phosphate, potassium phosphate, sodium monohydrogen phosphate (potassium), sodium dihydrogen phosphate (potassium), sodium nitrate (potassium), sodium bromide (potassium), sodium sulfate (potassium), and sodium bisulfate (potassium) are attached. Figure 8 As shown, compare the attached Figure 7 and attached Figure 8 The peak shapes and separation effects are very similar, and the integral data do not show statistically significant differences.

[0068] Examples 2.1-2.3 illustrate that when an alkali metal salt, represented by sodium chloride, is added to mobile phase A at a concentration ranging from 0.1% to 0.4%, the purity of the prepared dGTP increases with increasing concentration. However, when the concentration reaches 0.4%, the purity of dGTP no longer increases because there is no significant difference between the purity at 0.6% and 0.4%, with a maximum purity of approximately 98.5%. The liquid phase diagrams for Examples 2.4 and 2.6 are attached. Figure 12As shown.

[0069] Examples 2.4-2.6 illustrate that when sodium (potassium) carbonate or sodium (potassium) bicarbonate is added to mobile phase A at concentrations ranging from 0.1% to 0.4%, there is no significant difference between sodium and potassium, because the purity of the prepared dGTP increases with increasing concentration (e.g., ...). Figure 5 The purity of dGTP prepared when the concentration of alkali metal salt is in the range of 0.2% to 0.4% is above 99%, while the purity of dGTP prepared when the salt concentration is too high (e.g., 0.6%) or too low (0.1%) is below 99% (see attached). Figure 12 As shown in the figure, the main problem is that the salt concentration is too low or too high, resulting in severe tailing of the main peak and poor separation of both the preceding and following impurities. Compared with the preceding impurities, the separation of the following impurities is even worse. That is, the purity of dGTP prepared by adding sodium carbonate (potassium) or sodium bicarbonate (potassium) to the mobile phase A at a concentration in the range of 0.2% to 0.4% is 1 percentage point higher than that of dGTP prepared by other alkali metal salts, and this is statistically significant.

[0070] Compared with sodium carbonate (potassium) or sodium bicarbonate (potassium) in the alkali metal salts represented by sodium chloride in Tables 1 and 2, the purity of the prepared dGTP and the degree of separation between dGTP and its impurities are both lower.

[0071] Example 3: Effect of Ion Pair Species on Separation Efficiency and Recovery Rate

[0072] Unlike Example 1, the ion pairs in mobile phase A were 100 mM ± 5 mM TEAA. The experimental results are shown in Table 3.

[0073]

[0074] Comparing the experimental results in Tables 1 and 3, it can be concluded that there is no significant difference between ion pairs TEAA and TEAB in this application.

[0075] Example 4: Effect of Ion Pair Concentration Separation and Recovery Rate

[0076] Unlike Example 1, the ion pair concentrations in mobile phase A were 50mM±5mM, 150mM±5mM, and 200mM±5mM TEAB, respectively. The specific conditions and experimental results are shown in Table 4.

[0077]

[0078]

[0079] Compared with Examples 1.3 or 1.4, Examples 4.3 and 4.6 show that the best results are achieved when the ion pair concentration is 100 mM ± 5 mM. At concentrations of 50 mM ± 5 mM or 150 mM ± 5 mM, the purity or recovery rate decreases compared to 100 mM ± 5 mM, but the purity still reaches over 99%. However, the recovery rate at 50 mM ± 5 mM decreases by 4%-5%, which is significant. When the ion pair concentration is 200 mM ± 5 mM, the purity decreases by nearly one percentage point compared to 100 mM ± 5 mM. Example 4.7 shows a significant difference in product purity compared to Examples 1.3 or 1.4.

[0080] Examples 4.4-4.6 show no significant difference in product purity and recovery rate compared to Example 1.

[0081] Based on Example 3, it can be concluded that the separation effect and recovery rate are better when the concentration of TEAA or TEAB is in the range of 100mM to 150mM.

[0082] Example 5: Effect of mobile phase B on separation efficiency and yield

[0083] The difference between Example 5 and Example 1 is that several commonly used reagents, such as ethanol, acetonitrile, and tetrahydrofuran, were not considered. Since tetrahydrofuran can hardly separate the main peak from the impurities, and due to its danger in industrial production, it was not optimized.

[0084] The experimental results for ethanol and acetonitrile are shown in Table 5.

[0085]

[0086] The preparative chromatogram of Example 5.3 is attached. Figure 13 Compare the data from Example 1.3 and Example 5.3, or compare... Figure 4 or Figure 13 It can be seen that when acetonitrile is used as mobile phase B, the elution time of the dGTP main peak is shifted earlier and the tailing is more severe than in Example 1.3, resulting in a weakened separation between the impurities and the main peak and a reduced recovery rate.

[0087] The preparative chromatogram of Example 5.6 is attached. Figure 14 Comparing the data from Example 5.6 and Example 1.3, it can be seen that when ethanol is used as mobile phase B, the purity and recovery rate of dGTP are significantly reduced, with the purity decreasing by 1 percentage point and the yield decreasing by about 5%.

[0088] Based on this, methanol or acetonitrile is the preferred option for mobile phase B, with methanol being the most preferred option.

[0089] Example 6: Effect of pH on separation efficiency and yield

[0090] The preparation conditions for Example 6 were as follows:

[0091] The mobile phase was phase A: ion pairs were prepared using 100 mM ± 5 mM triethylamine carbonate TEAB aqueous solution. The salt used in mobile phase A was 0.2% sodium bicarbonate, and the pH was adjusted to 6.5, 7.5, and 8.0, respectively.

[0092] Phase B: Methanol, 0–20 min, methanol gradient increased from 0% to 30% and maintained at 30% for 10 min, the methanol used was industrial grade, the purified water was grade III water, and the other additives were all analytical grade.

[0093] The mobile phase flow rate was 15 mL / min;

[0094] The equipment uses a dynamic axial compression column DAC-50, and the packing material is C18-100-8.

[0095] The detection wavelength is 254nm;

[0096] The crude dGTP concentration was 35 g / L, and the HPLC purity of the crude product in this experiment was 93%.

[0097] The experimental results are shown in Table 6.

[0098]

[0099] Table 6 shows that there is no significant difference in product yield and recovery rate at pH 6.5 and 8.0. Therefore, both too low and too high pH will cause a decrease in the purity and yield of dGTP. Thus, pH between 7.0 and 7.5 is the preferred pH.

[0100] Example 7: Effect of crude product concentration (i.e., sample loading amount) on separation efficiency and yield.

[0101] The preparation conditions for Example 7 were as follows:

[0102] The mobile phase was phase A: ion pairs were prepared using a 100 mM ± 5 mM triethylamine carbonate TEAB aqueous solution. The salts used in mobile phase A were 0.2% sodium bicarbonate or potassium bicarbonate, and the pH was adjusted to 6.5, 7.5, and 8.0, respectively.

[0103] Phase B: Methanol, 0–20 min, methanol gradient increased from 0% to 30% and maintained at 30% for 10 min, the methanol used was industrial grade, the purified water was grade III water, and the other additives were all analytical grade.

[0104] The mobile phase flow rate was 15 mL / min;

[0105] The equipment uses a dynamic axial compression column DAC-50, and the packing material is C18-100-8.

[0106] The detection wavelength is 254nm;

[0107] Prepare crude dGTP at concentrations of 25 g / L or 45 g / L. The HPLC purity of the crude product in this experiment was 93%.

[0108] The experimental results are shown in Table 7.

[0109]

[0110] First, Example 7 further verified that there was no significant difference between the potassium salt and the sodium salt.

[0111] Secondly, compared with Example 1.3, Examples 7.1-7.2 show that when the crude product concentration, i.e. the sample loading amount, is as high as 45 g / L, the product purity decreases slightly but is still higher than 99%, and the recovery rate does not change significantly.

[0112] Compared with Example 1.3, Examples 7.3-7.4 show that reducing the crude product concentration can further improve the product purity, but the yield is reduced by 3%. Therefore, considering both purity and yield, the crude product concentration should not exceed 45 g / L.

[0113] Example 8: Effect of crude product purity on separation efficiency and yield

[0114] The preparation conditions for Example 8 were as follows:

[0115] The mobile phase was phase A: ion pairs were prepared using 100 mM ± 5 mM triethylamine carbonate TEAB aqueous solution. The salts used in mobile phase A were 0.2% sodium bicarbonate or potassium bicarbonate, and the pH was adjusted to 6.5, 7.5, and 8.0, respectively.

[0116] Phase B: Methanol, 0–20 min, methanol gradient increased from 0% to 30% and maintained at 30% for 10 min, the methanol used was industrial grade, the purified water was grade III water, and the other additives were all analytical grade.

[0117] The mobile phase flow rate was 15 mL / min;

[0118] The equipment uses a dynamic axial compression column DAC-50, and the packing material is C18-100-8.

[0119] The detection wavelength is 254nm;

[0120] The crude dGTP was prepared at a concentration of 35 g / L. The HPLC purity of the crude product in this experiment was 75% and 85%, respectively.

[0121] The experimental results are shown in Table 8.

[0122]

[0123]

[0124] Compared with Examples 8.1-8.2 and 1.3, the crude product concentration can still reach 35 g / L and the crude product purity is 75%. The technical solution of this application can still achieve a product purity of over 99%. The low recovery rate is due to the low purity of the crude product and the high impurity content. This further proves that the technical solution of this application significantly improves product purity, separation and sample loading compared with the prior art.

[0125] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a deoxynucleoside triphosphate monomer, wherein the method is a high-pressure liquid phase preparation method, characterized in that, The high-performance liquid chromatography (HPLC) mobile phase A uses TEAA or TEAB as an ion-pairing reagent while adding an alkali metal salt, with the concentration of the alkali metal salt being 0.2-0.4%. The HPLC mobile phase B is the eluent, and mobile phase B is one or more of methanol, acetonitrile, ethanol, or tetrahydrofuran. The preparative HPLC column is a reverse-phase column. The alkali metal salt is a sodium or potassium salt. The sodium salt is one or more of sodium chloride, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium bicarbonate, sodium carbonate, sodium nitrate, sodium bromide, sodium sulfate, or sodium bisulfate. The potassium salt is one or more of potassium chloride, potassium bicarbonate, potassium carbonate, potassium nitrate, potassium bromide, potassium sulfate, or potassium bisulfate.

2. The method for preparing deoxynucleoside triphosphate monomers according to claim 1, characterized in that, The alkali metal salt is one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate.

3. The method for preparing deoxynucleoside triphosphate monomers according to any one of claims 1 or 2, characterized in that, The concentration of ion pairs in the mobile phase A is between 100 mM ± 5 mM and 150 mM ± 5 mM.

4. The method for preparing deoxynucleoside triphosphate monomers according to claim 3, characterized in that, The mobile phase B is one or more of methanol, acetonitrile, or ethanol.

5. The method for preparing deoxynucleoside triphosphate monomers according to claim 1, characterized in that, The pH of the mobile phase A is adjusted to 7.0-7.5 using carbon dioxide or sodium hydroxide.

6. The method for preparing deoxynucleoside triphosphate monomers according to claim 1, characterized in that, The eluent is used for gradient elution, wherein the gradient is a methanol gradient increasing from 0% to 30±2% and maintaining at 30±2% for more than 10 minutes.

7. The method for preparing deoxynucleoside triphosphate monomers according to claim 1, characterized in that, The prepared liquid column is a dynamic axial compression column DAC-50, the packing material is C18-100-8, and the flow rate of the high-pressure liquid phase is 15±2 mL / min.

8. The method for preparing deoxynucleoside triphosphate monomers according to claim 1, characterized in that, The crude deoxynucleoside triphosphate monomer concentration for loading shall not exceed 45 g / L.

9. The method for preparing deoxynucleoside triphosphate monomers according to claim 1, characterized in that, The purity of the crude deoxynucleoside triphosphate monomer is greater than or equal to 75% and less than 100%.

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