Process for the preparation of cyclic dinucleotides

Abstract

The application belongs to the field of biological medicine engineering, and relates to a preparation method of a cyclic dinucleotide, comprising the following steps: synthesizing the cyclic dinucleotide in a reaction system containing a dinucleotide cyclase and nucleoside triphosphates, centrifuging a reaction product, and obtaining supernatant; and purifying the cyclic dinucleotide from the supernatant by using a macroporous adsorption resin, wherein gradient elution is performed by using a 5-100 mM triethylamine-acetic acid solution as phase A and a mixed solution containing 70-80% phase A and 20-30% methanol as phase B. The macroporous adsorption resin is used to separate and purify the cyclic dinucleotide, the operation is relatively simple, the separation and purification time can be significantly shortened, the separation and purification speed is improved, and the obtained product has high purity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and particularly relates to a method for preparing cyclic dinucleotides. Background Art

[0002] Cyclic dinucleotides (CDNs) are the primary ligands of the STING protein. Binding to STING activates signaling pathways, promoting the expression of various pro-inflammatory cytokines, including type I interferons, and thereby initiating adaptive immune responses. As important targets for antiviral, antibacterial, and anti-tumor immunotherapy, the development of STING agonists has become a research hotspot, with STING agonists, primarily cyclic dinucleotides and their derivatives, gradually entering clinical trials.

[0003] Currently, cyclic dinucleotides are primarily prepared through tissue extraction, chemical synthesis, and enzymatic synthesis. However, these methods are inefficient, complex, time-consuming, and costly to extract, separate, and purify, as well as environmentally unfriendly. While enzymatic synthesis overcomes some of the shortcomings of these two methods, it still needs improvement in terms of reaction yield and purification efficiency.

[0004] For example, the prior art generally uses reversed-phase high performance liquid chromatography (HPLC) to separate and purify cyclic dinucleotides. Taking the enzymatic synthesis of c-di-GMP as an example, it is usually necessary to boil the reaction solution, add acid and alkali, denature the enzyme, precipitate it, centrifuge it, filter it to remove the enzyme, freeze-dry and concentrate it, and then separate and purify it. Among them, the mobile phase used in HPLC generally contains acetonitrile. It can be seen that the separation and purification process of this method is complicated, time-consuming and energy-consuming. This is because the reversed-phase chromatography column has strict requirements on the sample, and impurities that may be deposited on the column must be removed first, otherwise the service life of the chromatographic column will be reduced. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing cyclic dinucleotides, which can simplify the preparation process and improve the purity and yield of cyclic dinucleotides.

[0006] In order to achieve the above-mentioned purpose, the preparation method of the cyclic dinucleotide of the present invention comprises the following steps.

[0007] synthesizing cyclic dinucleotides in a reaction system comprising dinucleotide cyclase and nucleoside triphosphates, and centrifuging the reaction product to obtain a supernatant;

[0008] The cyclic dinucleotide is purified from the supernatant using a macroporous adsorption resin, wherein gradient elution is performed using a 5-100 mM triethylamine-acetic acid solution as phase A and a mixed solution containing 70-80% phase A and 20-30% methanol as phase B.

[0009] In some embodiments, the dinucleotide cyclase is a DncV enzyme, a cyclic GMP-AMP synthase (cGAS), or a cGAS / DncV-like nucleotide transferase (CdnE).

[0010] In some embodiments, the reaction system of the enzymatic reaction comprises: ammonium acetate 200-500 mmol / L (e.g., 300 mmol / L or 400 mmol / L), manganese chloride 20-40 mmol / L (e.g., 25 mmol / L, 30 mmol / L, or 35 mmol / L) or magnesium chloride 20-70 mmol / L (e.g., 30 mmol / L, 40 mmol / L, 50 mmol / L, or 60 mmol / L), DncV enzyme 3-20 μmol / L (e.g., 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 12 μmol / L, 15 μmol / L, 18 μmol / L), GTP and / or ATP 1-20 mmol / L (e.g., 2 mmol / L, 5 mmol / L, 10 mmol / L, or 15 mmol / L).

[0011] In some embodiments, the cyclic dinucleotide is selected from one or more of c-di-AMP, c-di-GMP, 2',3'-cGAMP, and 3',3'-cGAMP.

[0012] In some embodiments, the reaction temperature is 30-37°C.

[0013] In some embodiments, the pH value of the reaction system is 8.7-9.5.

[0014] In some embodiments, the reaction time is 1-3 h.

[0015] In some embodiments, the macroporous adsorption resin is SP207SS resin or SP207 resin of Mitsubishi Chemical.

[0016] In some embodiments, the concentration of the triethylamine-acetic acid solution is preferably 5-100 mM.

[0017] In some embodiments, the purification is performed on an AKTA purifier.

[0018] In some embodiments, when eluting, first flush with 60% A and 40% B for 2-3 column volumes, then flush with 60%-40% A and 40%-60% B for 2-3 column volumes, and finally flush with 40%-0 A and 60%-100% B for 3-4 column volumes.

[0019] In some embodiments, the flow rate at elution is 0.5-2 column volumes / h, for example 1 column volume / h or 1.5 column volumes / h.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The present application uses macroporous adsorption resin to separate and purify cyclic dinucleotides. The raw material to be separated and purified does not need to be treated by removing enzymes, freeze-drying and concentration before purification, and can be directly separated and purified from the enzyme reaction liquid, so the operation is simple. The method of the present application has large column capacity, high recovery efficiency, is easy to linearly scale up and does not easily inactivate biomolecules. Since more raw materials can be treated at one time, the separation and purification time can be significantly reduced, the separation and purification speed can be improved, and the purity of the obtained product is high.

[0022] Compared with the traditional reverse phase HPLC separation and purification technology, the present application simplifies the steps and saves time, while avoiding the large use of toxic organic solvent acetonitrile, eliminating the harm of acetonitrile to operators and the environment, and having a broader industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] The following drawings are only intended to illustrate and explain the present application and do not limit the scope of the present application. Among them:

[0024] Figure 1 Effect of different metal ions on enzymatic reaction in Example 1 of the present application;

[0025] Figure 2A Elution process when the filler is SP207 in Example 2 of the present application;

[0026] Figure 2B Elution process when the filler is SP207SS in Example 2 of the present application;

[0027] Figure 3 Isocratic elution process in Example 2 of the present application;

[0028] Figure 4 Gradient elution process of c-di-GMP under 100 mM TEAA in Example 2 of the present application;

[0029] Figure 5 Gradient elution process of c-di-GMP under 5 mM TEAA in Example 2 of the present application;

[0030] Figure 6 Gradient elution process of c-di-GMP when the organic solvent is acetonitrile in Example 2 of the present application;

[0031] Figure 7Gradient elution procedure for c-di-AMP in Example 2 of the present application with 5 mM TEAA.

[0032] Figure 8 Cellular experimental results in Example 3 of the present application. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with specific examples and with reference to the accompanying drawings.

[0034] The "dinitucleotide cyclase" in the present application refers to an enzyme capable of catalyzing nucleotide triphosphates (such as adenosine triphosphate ATP, guanosine triphosphate GTP, cytidine triphosphate CTP, uridine triphosphate UTP, etc.) to form cyclic dinucleotides. Common dinitucleotide cyclases are DncV enzyme, cyclic GMP-AMP synthase (cGAS) or cGAS / DncV-like nucleotide transferase (CdnE).

[0035] DncV (VC0179) is a dinucleotide cyclase in Vibrio (DncV) derived from Vibrio cholerae, which can use ATP and GTP as substrates to catalyze the synthesis of cyclic adenosine monophosphate-guanosine monophosphate (c-AMP-GMP, cGAMP). The cGAMP contains two 3-5-phosphodiester bonds. DncV has high specificity and efficiency in catalyzing ATP and GTP to form cGAMP. Research literature reports that when equal molar amounts of ATP and GTP are given, DncV almost synthesizes cGAMP with 100% catalytic efficiency, and rarely forms cAMP and cGMP. In addition, DncV can also use GTP and ATP as single substrates to synthesize cyclic di-guanosine monophosphate (c-di-GMP) and cyclic di-adenosine monophosphate (c-di-AMP), respectively.

[0036] Examples of DncV enzyme include, but are not limited to, UniProtKB / Swiss-Prot: Q9KVG7.1, which can be Vibrio cholerae dncV gene of NCBI Gene ID: 2614190 encoding the protein. The DncV enzyme can be obtained by constructing a dinucleotide cyclase V (DncV) recombinant expression vector, inducing expression by isopropy-β-D-thiogalactoside (IPTG), and affinity purification. The amino acid sequence of the DncV enzyme, the encoding gene, and the preparation method are routine techniques in the art (for example, see: Soluble prokaryotic expression of Vibrio cholerae dinucleotide cyclase and biological activity analysis of the expression product, Yang Fan et al., China Veterinary Science, 2021, 51 (12): 1533-1539), which will not be repeated here.

[0037] cGAS (cyclic GMP-AMP synthase) derived from metazoans can synthesize 2',3'-cGAMP (i.e., forming a 2'-5' phosphodiester bond and a 3'-5' phosphodiester bond). These molecules can also activate IFN-I production and response through the STING signaling pathway. In addition, in the exploration of DncV homologs, cGAS / DncV-like nucleotide transferase (CdnE) was found, which also has the function of dinucleotide cyclization. The gene sequence of this molecule is named because it has homologous sequences with cGAS and DncV, and it can catalyze the synthesis of c-UMP-AMP (c-UAMP).

[0038] The cyclic dinucleotide in the present application is a cyclic molecule formed by connecting two nucleoside monophosphates through two phosphodiester bonds, and can be at least one selected from 2',3'-cGAMP, 3',3'-cGAMP, c-di-GMP and c-di-AMP. Among them, the structural formula of cyclic diadenosine monophosphate is:

[0039]

[0040] The structural formula of cyclic diguanosine monophosphate is:

[0041]

[0042] The structural formula of 3',3'-cyclic guanosine monophosphate-adenosine monophosphate is:

[0043]

[0044] The structural formula of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate is:

[0045]

[0046] Example 1 Preparation of cyclic dinucleotides

[0047] The buffer solution system containing DncV enzyme, GTP and metal ions was placed in a water bath for one-step reaction to generate c-di-GMP under appropriate conditions. After the reaction was completed, it was taken out and stood at room temperature for more than 30 min; centrifuged at 12000 x g for 10 min, the supernatant was taken, 500 μl of water for injection was added to the precipitate and mixed evenly by blowing, centrifuged at 12000 x g for 10 min, the supernatant was taken, and the two supernatants were combined together to obtain the crude product of c-di-GMP, and the conversion rate of GTP was about 80-90%. The crude product was further purified to obtain the final product of c-di-GMP with a purity of more than 99%.

[0048] In this example, the inventors studied the factors that may affect the enzymatic reaction conditions, and optimized the enzyme activity, enzyme concentration, reaction temperature, solution pH, metal ion type and concentration, reaction time, etc. Some reagents and consumables in the experiment are shown in Table 1.

[0049] Table 1. Reagents and consumables for experiments

[0050]

[0051] (1) Optimization of enzyme concentration

[0052] In terms of enzyme concentration, the enzymatic reaction is accelerated with the increase of enzyme concentration, and the side reaction is reduced. The commonly used concentration of dinucleotide cyclase is generally 1-20 μM. In this example, we verified the effect of 1, 3, 5, 8 μM DncV enzyme on the enzymatic reaction, and the detection results are shown in Table 2. It can be seen that with the increase of DncV enzyme concentration, the purity of c-di-GMP in the reaction product increases accordingly, but when the DncV enzyme concentration increases to 8 μM, the purity of c-di-GMP tends to be flat, and considering the economic cost, the appropriate enzyme concentration is 3-5 μM, and 3 μM of DncV enzyme is used for the subsequent enzymatic reaction.

[0053] Table 2. Detection results of different enzyme concentrations

[0054]

[0055] (2) Temperature optimization

[0056] Different temperatures will affect the progress of enzymatic reactions. We verified the efficiency of enzymatic reactions at 25°C, 30°C, 33°C, 37°C, and 42°C. The test results are shown in Table 3. The degree of enzymatic reaction is similar at 30-37°C, and the enzyme activity decreases significantly at 42°C.

[0057] Table 3. Effects of different temperatures on enzymatic reactions

[0058]

[0059] (3) Optimization of pH value

[0060] Different pH values ​​will produce differences in the enzymatic activity of the DncV enzyme. We verified the difference in enzyme activity between pH 8.5 and 9.5, and the test results are shown in Table 4. The results show that the DncV enzyme activity is very sensitive to pH value, and the DncV enzyme activity is optimal when the pH value is between 8.7 and 9.5.

[0061] Table 4. Effects of different pH values ​​on enzymatic reactions

[0062]

[0063] (4) Optimization of reaction time

[0064] We verified the effect of different reaction times on the enzymatic reaction, and the test results are shown in Table 5. The results show that the reaction time is sufficient within 1-3 hours, and further extending the reaction time has limited effect on improving the reaction efficiency.

[0065] Table 5 Effect of reaction time on enzymatic reaction

[0066]

[0067] (5) Optimization of metal ions and concentrations

[0068] Different metal ions and concentrations will affect the enzymatic reaction. We verified the effects of magnesium ions and manganese ions on the enzymatic reaction. The test results are as follows: Figure 1 The results show that the catalytic effect of Mn ions is better than that of Mg ions, and 20 mM Mn has the best catalytic effect.

[0069] Based on the above work, we obtained better reaction conditions. The reaction system contains 3 μM DncV enzyme, 300 mM ammonium acetate, 20 mM manganese chloride and 10 mM GTP (the content of each component is the final concentration in the reaction system). The reaction system is placed in a 37°C water bath and incubated for 1-2 hours to obtain c-di-GMP.

[0070] In addition, GTP in the reaction system was replaced by ATP, and c-di-AMP was obtained under the same reaction conditions. When the reaction system contained equimolar amounts of GTP and ATP, 3',3'-cGAMP was obtained.

[0071] Example 2. Purification of cyclic dinucleotides

[0072] (1) Selection of filler

[0073] In terms of the selection of the purification resin filler, we compared two types of Mitsubishi Chemical resins, namely SP207 resin and SP207SS resin. Under the same loading amount, sample volume and flow rate, the elution curves of the two resins were different, as shown in Figure 2A (SP207 resin) and Figure 2B (SP207SS resin). The results showed that both resins could meet the needs of separating c-di-GMP, and the separation degree of SP207SS resin was better.

[0074] (2) Selection of elution method

[0075] In this example, 100 mM triethylamine-acetic acid solution (TEAA) was used for purification column hanging. After loading, there was no change in absorbance during the equilibration process. After column hanging, according to the characteristics of small molecule oligonucleotides with strong polarity, isocratic elution was performed using a TEAA-methanol ion pair system, and different concentrations of methanol could separate different components to achieve the separation of c-di-GMP and impurities, and the purification process is shown in Figure 3 The eluted peaks were collected, dried and sent for HPLC to determine whether the collected eluted peaks contained c-di-GMP and to detect the purity (i.e., the proportion of c-di-GMP in all substances dissolved in the eluent, which is calculated as the peak area ratio in this application), and the experimental results are shown in Table 6.

[0076] Table 6. Isocratic purification results

[0077]

[0078] The results showed that under this condition, gradient elution could purify c-di-GMP, but the purity of c-di-GMP in the final product was only 85.85%, which was relatively low. Subsequently, the isocratic elution was adjusted to gradient elution method, and the purification process is shown in Figure 4 , and the purification results are shown in Table 7. After changing the elution program to gradient elution, the purity of c-di-GMP in the final product was increased to more than 98%, so this purification method is feasible and can obtain high-purity c-di-GMP samples.

[0079] Table 7. 100 mM TEAA gradient purification results

[0080]

[0081] (3) Optimization of TEAA solution concentration

[0082] Considering that 100 mM TEAA can achieve complete column hanging, the inventors also verified whether reducing the concentration of TEAA solution can continue to completely hang the c-di-GMP column and purify and separate it, and designed 50 mM, 25 mM, 10 mM, 5 mM, 2.5 mM, a total of 5 concentrations. The purification results are shown in Table 8. The TEAA concentration is reduced to 5 mM, which can achieve complete column hanging of c-di-GMP (as shown in Figure 5 ), but when reduced to 2.5 mM, a small amount of c-di-GMP exists in the flow-through, and according to the comparison of the peak shape, the separation degree between the main peak and the rest of the impurity peaks is not enough. Therefore, under this condition, about 50% of the recovery rate needs to be reduced to obtain high-purity c-di-GMP.

[0083] Table 8. Purification results of different concentrations of TEAA

[0084]

[0085] In summary, the minimum TEAA concentration required for purifying c-di-GMP is 5 mM, which can ensure that c-di-GMP has a high purity and recovery rate, and as the TEAA concentration decreases (100 mM→5 mM), the main peak of the purification is eluted under the condition of lower concentration of methanol, and baseline separation between the main peak and the impurity peak can be basically achieved.

[0086] (4) Selection of organic solvents

[0087] In addition to methanol, we also investigated the effect of acetonitrile on the elution of c-di-GMP purification, and the acetonitrile purification process is shown in Figure 6 , and the purification results are shown in Table 9. The experimental results prove that acetonitrile can replace methanol for purification and elution, but baseline separation between the main peak and the impurity peak cannot be achieved. Although the purity of the final product c-di-GMP can be maintained at more than 99%, the recovery rate is greatly reduced, only half of that when methanol is eluted.

[0088] Table 9. Acetonitrile purification results

[0089]

[0090] On the basis of the above work, we used the following method to purify c-di-GMP:

[0091] 1. Activating the filler: Soak the SP207SS resin (Mitsubishi Chemical) filler in anhydrous ethanol overnight, and then soak the washed resin in 20% ethanol for long-term sealed storage.

[0092] 2. Column loading: Load the activated resin into the AKTA purification glass column, ensuring that there are no gaps between the fillers, no dryness, etc., and ensuring good sealing.

[0093] 3. Pre-equilibration: Connect the column with the AKTA purifier, set the flow rate to 0.5-2 column volumes / h; first rinse with water for injection for 2-3 column volumes; then rinse with 50 mM TEAA solution for 4-5 column volumes for pre-equilibration.

[0094] 4. Purification process:

[0095] (1) Sample loading: Take 5 ml of the sample to be purified, and load it at a flow rate of 0.5-2 column volumes / h;

[0096] (2) Equilibration: The flow rate is 0.5-2 column volumes / h, and the 5 mM TEAA solution is equilibrated for 4-5 column volumes; collect the eluate when the absorbance rises rapidly, and stop collecting when the absorbance is flat;

[0097] (3) 10% elution: The flow rate is 0.5-2 column volumes / h, and 60% A (5 mM TEAA solution) and 40% B (75% A phase added with 25% methanol) are rinsed for 2-3 column volumes; collect the eluate when the absorbance rises rapidly, and stop collecting when the absorbance is flat;

[0098] (4) 10-15% gradient elution: The flow rate is 0.5-2 column volumes / h, and 60%-40% A and 40% B-60% B are rinsed for 2-3 column volumes; collect the eluate when the absorbance rises rapidly, and stop collecting when the absorbance is flat;

[0099] (5) 15-25% gradient elution: The flow rate is 0.5-2 column volumes / h, and 40%-0 A and 60% B-100% B are rinsed for 3-4 column volumes; collect the eluate when the absorbance rises rapidly, and stop collecting when the absorbance is flat.

[0100] Finally, the collected sample is dried by a rotary evaporator: set the water bath to 66°C, manually adjust the vacuum degree to 0, and set the rotation speed to 120 rpm. Place the sample to be dried into the drying bottle, and dry for 30 min.

[0101] The dried sample is reconstituted with water for injection, and the purity is detected by HPLC, showing a purity of >99.5%.

[0102] The inventors purified c-di-AMP (Example 1) Figure 7 and 3',3'-cGAMP (Example 2) under the same conditions, and the purity of each was 99.3% to 99.6%.

[0103] Comparative Example 1 Purification by preparative HPLC

[0104] A sample of c-di-GMP prepared in Example 1 was purified using preparative HPLC under the following chromatographic conditions:

[0105] Hypersil GOLD™ PREP C18 column, 150 mm in length, 30 mm in internal diameter, and 5 μm in particle size;

[0106] Mobile phase A was 10 mM TEAA solution, pH = 7.0; mobile phase B was acetonitrile solution;

[0107] Gradient elution program:

[0108] From 0 to 18 min, mobile phase A was decreased from 97% to 90%, and mobile phase B was increased from 3% to 10%;

[0109] From 18 to 23 min, mobile phase A was decreased from 90% to 87.5%, and mobile phase B was increased from 10% to 12.5%;

[0110] From 23 to 26 min, mobile phase A was kept at 87.5%, and mobile phase B was kept at 12.5%;

[0111] From 26 to 29 min, mobile phase A was decreased from 87.5% to 0, and mobile phase B was increased from 12.5% to 100%;

[0112] From 29 to 37 min, mobile phase A was kept at 0%, and mobile phase B was kept at 100%;

[0113] From 37 to 37.1 min, mobile phase A was increased from 0% to 97%, and mobile phase B was decreased from 100% to 3%;

[0114] From 37.1 to 52 min, mobile phase A was kept at 97%, and mobile phase B was kept at 3%;

[0115] The flow rate of the mobile phase was 10 to 20 mL / min.

[0116] The product was analyzed by analytical HPLC to determine purity, and the results showed that the purity was 95% to 97%.

[0117] Example 3 Cell experiments

[0118] The c-di-GMP with different doses of 0.225 μg / ml, 2.25 μg / ml, 22.5 μg / ml, 45 μg / ml, 90 μg / ml and 180 μg / ml is incubated with mouse monocyte macrophage RAW246.7 for 24 hours, and then the expression amount of the downstream cytokine IFN-β of the STING channel is detected. The results show that the c-di-GMP prepared by the application has high biological activity. As shown in the following Table 1, the expression amount of IFN-β increases in a dose-dependent manner with the c-di-GMP. Figure 8

[0119] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the application. It should be understood that the above description is only for specific embodiments of the application and is not used to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application should be included in the protection scope of the application.​

Claims

1. A method for preparing a cyclic dinucleotide, characterized in that: The following steps are involved: synthesizing cyclic dinucleotides in a reaction system comprising dinucleotide cyclase and nucleoside triphosphates, and centrifuging the reaction product to obtain a supernatant; Purifying the cyclic dinucleotide from the supernatant using macroporous adsorption resin SP207SS, wherein gradient elution is performed using a 5-100 mM triethylamine-acetic acid solution as phase A and a mixed solution containing 70-80% phase A and 20-30% methanol as phase B; When eluting, first wash with 60% A and 40% B for 2-3 column volumes, then wash with 60% -40% A and 40% -60% B for 2-3 column volumes, and finally wash with 40% -0 A and 60% -100% B for 3-4 column volumes. The cyclic dinucleotide is selected from c-di-GMP or 3',3'-cGAMP.

2. The preparation method according to claim 1, characterized in that The dinucleotide cyclase is a DncV enzyme.

3. The preparation method according to claim 2, characterized in that The reaction system includes: 200-500 mmol / L of ammonium acetate, 3-20 μmol / L of DncV enzyme, 1-20 mmol / L of GTP, and 5-40 mmol / L of manganese chloride or 20-70 mmol / L of magnesium chloride.

4. The preparation method according to claim 2, characterized in that The reaction system includes: 200-500 mmol / L of ammonium acetate, 3-20 μmol / L of DncV enzyme, 1-20 mmol / L of GTP and ATP, and 5-40 mmol / L of manganese chloride or 20-70 mmol / L of magnesium chloride.

5. The preparation method according to claim 1, characterized in that The reaction temperature is 30-37°C.

6. The preparation method according to claim 1, characterized in that The pH value of the reaction system is 8.7-9.

5.

7. The preparation method according to claim 1, characterized in that The reaction time is 1-3h.

8. The preparation method according to claim 1, characterized in that The purification was performed on an AKTA purifier.

9. The preparation method according to claim 1, characterized in that The flow rate during elution was 0.5-2 column volumes / h.

Images