Preparation method of cyclic dinucleotide

Abstract

The invention belongs to the field of biomedical engineering, and relates to a cyclic dinucleotide preparation method, which comprises: synthesizing cyclic dinucleotide in a reaction system containing dinucleotide cyclase and nucleoside triphosphate, and centrifuging the reaction product to obtain a supernatant; and purifying the cyclic dinucleotide from the supernatant by using macroporous adsorption resin, and carrying out gradient elution by using a 5-100 mM triethylamine-acetic acid solution as a phase A and a mixed solution containing 70-80% of the phase A and 20-30% of methanol as a phase B. The cyclic dinucleotide is separated and purified by adopting the macroporous adsorption resin, the operation is relatively simple, the separation and purification time can be remarkably shortened, the separation and purification speed is improved, and the purity of the obtained product is relatively high.

Description

Technical Field

[0001] The 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 main ligands of STING protein. After binding to STING, they can activate signaling pathways, thereby promoting the expression of various pro-inflammatory cytokines such as type I interferon, and then initiating adaptive immune responses. As an important target for antiviral, antibacterial and anti-tumor immunotherapy, the development of STING agonists has become a current research boom, and STING agonists based on cyclic dinucleotides and their derivatives have gradually entered the clinical research stage.

[0003] At present, the preparation of cyclic dinucleotides is mainly through tissue extraction, chemical synthesis and enzymatic synthesis, but the tissue extraction and chemical synthesis methods are inefficient, and the extraction, separation and purification processes are complex, time-consuming, costly and environmentally unfriendly. Although the enzymatic synthesis method overcomes some of the shortcomings of the above two methods, there is still room for 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, filter to remove the enzyme, freeze-dry and concentrate it before separation and purification. 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 chromatographic 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 object, the method for preparing cyclic dinucleotide of the present invention comprises the following steps.

[0007] synthesizing cyclic dinucleotides in a reaction system comprising dinucleotide cyclase and nucleoside triphosphates, centrifuging the reaction product to obtain a supernatant; The cyclic dinucleotide is purified from the supernatant using a macroporous adsorption resin, wherein a 5-100 mM triethylamine-acetic acid solution is used as phase A and a mixed solution containing 70-80% phase A and 20-30% methanol is used as phase B for gradient elution.

[0008] In some embodiments, the dinucleotide cyclase is a DncV enzyme, cyclic GMP-AMP synthase (cGAS), or cGAS / DncV-like nucleotidyltransferase (CdnE).

[0009] In some embodiments, the reaction system of the enzymatic reaction includes: 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).

[0010] 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.

[0011] In some embodiments, the reaction temperature is 30-37°C.

[0012] In some embodiments, the pH value of the reaction system is 8.7-9.5.

[0013] In some embodiments, the reaction time is 1-3 hours.

[0014] In some embodiments, the macroporous adsorption resin is SP207SS resin or SP207 resin of Mitsubishi Chemical.

[0015] In some embodiments, the concentration of the triethylamine-acetic acid solution is preferably 5-100 mM.

[0016] In some embodiments, the purification is performed on an AKTA purifier.

[0017] In some embodiments, during elution, 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.

[0018] In some embodiments, the flow rate during elution is 0.5-2 column volumes / h, such as 1 column volume / h or 1.5 column volumes / h.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses a macroporous adsorption resin to separate and purify cyclic dinucleotides. The raw materials to be separated and purified do not need to be treated by enzyme removal, freeze-drying and concentration before purification, and can be directly separated and purified from the enzyme reaction solution, so the operation is simple. The method of the present invention has a large column capacity, high recovery efficiency, is easy to linearly amplify, and is not easy to inactivate biological molecules. Since more raw materials can be processed at one time, the separation and purification time can be significantly reduced, the separation and purification speed can be increased, and the obtained product has high purity.

[0020] Compared with the traditional reversed-phase HPLC separation and purification technology, the present invention simplifies the steps and saves time, while also avoiding the large-scale use of the toxic organic solvent acetonitrile, eliminating the harm caused by acetonitrile to the operator and the environment, and has a broader industrial application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings are intended only to illustrate and explain the present invention, and are not intended to limit the scope of the present invention. Figure 1 The effects of different metal ions on the enzymatic reaction in Example 1 of the present invention; Figure 2A This is the elution process when the filler is SP207 in Example 2 of the present invention; Figure 2B This is the elution process when the filler is SP207SS in Example 2 of the present invention; Figure 3 This is the isocratic elution process in Example 2 of the present invention; Figure 4 This is the gradient elution process of c-di-GMP at 100 mM TEAA in Example 2 of the present invention; Figure 5 This is the gradient elution process of c-di-GMP at 5 mM TEAA in Example 2 of the present invention; Figure 6 This is the gradient elution process of c-di-GMP when the organic solvent is acetonitrile in Example 2 of the present invention; Figure 7 This is the gradient elution process of c-di-AMP at 5 mM TEAA in Example 2 of the present invention; Figure 8 This is the cell experiment result in Example 3 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0023] The "dinucleotide cyclase" in the present invention refers to an enzyme that can catalyze the synthesis of nucleoside triphosphates (such as adenosine triphosphate ATP, guanosine triphosphate GTP, cytidine triphosphate CTP, uridine triphosphate UTP, etc.) into cyclic dinucleotides. Common dinucleotide cyclases are DncV enzymes, cyclic GMP-AMP synthases (cGAS) or cGAS / DncV-like nucleotidyltransferases (CdnE).

[0024] DncV (VC0179) is a cyclic dinucleotide cyclase (Dinucleotidecyclase in Vibrio, DncV) derived from Vibrio cholerae. It can use ATP and GTP as substrates to catalyze the synthesis of cyclic adenosine monophosphate-guanosine monophosphate (c-AMP-GMP, cGAMP), which contains two 3-5-phosphodiester bonds. DncV catalyzes ATP and GTP to form cGAMP with high specificity and efficiency. Research literature reports that when given equimolar amounts of ATP and GTP, DncV almost 100% synthesizes and catalyzes the formation of cGAMP, while very little cAMP and cGMP are formed. In addition, DncV can also use GTP and ATP as single substrates to synthesize cyclic diguanylate (c-di-GMP) and cyclic diadenylate (c-di-AMP), respectively.

[0025] Examples of DncV enzymes include, but are not limited to, UniProtKB / Swiss-Prot: Q9KVG7.1, and the protein may be encoded by the Vibrio cholerae dncV gene of NCBI gene ID: 2614190. The DncV enzyme can be obtained by constructing a dinucleotide cyclase (DncV) recombinant expression vector, inducing expression with isopropyl-β-D-thiogalactoside (IPTG) and affinity purification. The amino acid sequence, encoding gene, and preparation method of the DncV enzyme are conventional techniques in the art (for example, see: Soluble prokaryotic expression of Vibrio cholerae dinucleotide cyclase and biological activity analysis of its expression product, Yang Fan et al., Chinese Veterinary Science, 2021, 51 (12): 1533-1539), which will not be elaborated here.

[0026] 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) with the same dinucleotide cyclization function was discovered. The gene sequence of this molecule is named because it has homologous sequences with cGAS and DncV, which can catalyze the synthesis of c-UMP-AMP (c-UAMP).

[0027] The cyclic dinucleotide in the present invention is a cyclic molecule formed by two nucleoside monophosphates connected by two phosphodiester bonds, and can be selected from at least one of 2',3'-cGAMP, 3',3'-cGAMP, c-di-GMP and c-di-AMP. Among them, the structural formula of cyclic dimonophosphate adenosine is:

[0028] The structural formula of cyclic guanosine monophosphate is:

[0029] The structural formula of 3',3'-cyclic guanosine monophosphate-adenosine monophosphate is:

[0030] The structural formula of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate is:

[0031] Example 1 Preparation of cyclic dinucleotides The buffer solution system containing DncV enzyme, GTP and metal ions is placed in a water bath, and c-di-GMP can be generated in one step under appropriate conditions. After the reaction is completed, it is taken out and allowed to stand at room temperature for more than 30 minutes; centrifuged at 12000×g for 10 minutes, the supernatant is taken, 500 μl of injection water is added to the precipitate and mixed, centrifuged at 12000×g for 10 minutes, the supernatant is taken, and the two supernatants are combined to obtain the crude product of c-di-GMP. The conversion rate of GTP is approximately 80~90%. The crude product is further purified to obtain a c-di-GMP final product with a purity of more than 99%.

[0032] In this embodiment, the inventors studied the factors that may affect the enzymatic reaction conditions and optimized them from the aspects of enzyme activity, enzyme concentration, reaction temperature, solution pH, metal ion type and concentration, reaction time, etc. Some reagents and consumables used in the experiment are shown in Table 1.

[0033] Table 1. Reagents and consumables for the experiment

[0034] (1) Optimization of enzyme concentration

[0035] In terms of enzyme concentration, the enzymatic reaction is accelerated as the enzyme concentration increases, and the side reactions are reduced. The commonly used concentration of dinucleotide cyclase is generally 1-20 μM. In this embodiment, we verified the effect of 1, 3, 5, and 8 μM DncV enzymes on the enzymatic reaction, and the test results are shown in Table 2. It can be seen that as the concentration of DncV enzyme increases, the purity of c-di-GMP in the reaction product increases accordingly, but when the concentration of DncV enzyme increases to 8 μM, the change in c-di-GMP purity tends to be flat. For economic cost considerations, the appropriate enzyme concentration is 3-5 μM, and 3 μM DncV enzyme is used for enzymatic reactions in subsequent experiments.

[0036] Table 2. Detection results of different enzyme concentrations

[0037] (2) Temperature optimization

[0038] 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 drops significantly at 42°C.

[0039] Table 3. Effects of different temperatures on enzymatic reactions

[0040] (3) Optimization of pH value

[0041] 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 activity of the DncV enzyme is very sensitive to the pH value, and the DncV enzyme activity is optimal when the pH value is between 8.7 and 9.5.

[0042] Table 4. Effects of different pH values ​​on enzymatic reactions

[0043] (4) Optimization of reaction time

[0044] 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 at 1-3 h, and further extending the reaction time has limited effect on improving the reaction efficiency.

[0045] Table 5 Effect of reaction time on enzymatic reaction

[0046] (5) Optimization of metal ions and concentrations

[0047] 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.

[0048] Based on the above work, we obtained better reaction conditions. The reaction system contained 3 μM DncV enzyme, 300 mM ammonium acetate, 20 mM manganese chloride and 10 mM GTP (the content of each component was the final concentration in the reaction system). The reaction system was placed in a 37°C water bath and incubated for 1-2 hours to obtain c-di-GMP.

[0049] In addition, c-di-AMP was obtained under the same reaction conditions by replacing GTP with ATP in the reaction system, and 3',3'-cGAMP was obtained when the reaction system contained equimolar amounts of GTP and ATP.

[0050] Example 2 Purification of cyclic dinucleotides

[0051] (1) Selection of filler In terms of the selection of purification resin fillers, we compared two Mitsubishi Chemical resins, SP207 resin and SP207SS resin. Under the same loading amount, purification sample volume and flow rate, the elution curves between the two are different, such as Figure 2A (SP207 resin) and Figure 2B The results show that both resins can meet the requirements for separation of c-di-GMP, and the separation degree of SP207SS resin is better.

[0052] (2) Selection of elution method

[0053] In this embodiment, 100 mM triethylamine-acetic acid solution (TEAA) was used for purification column hanging. There was no change in absorbance during the equilibrium process after loading. After hanging the column, according to the strong polarity of small molecule oligonucleotides, TEAA-methanol ion pair system was used for isocratic elution. Different concentrations of methanol can separate different components to achieve the separation of c-di-GMP and impurities. The purification process is as follows: Figure 3 The eluted elution peak was collected, dried and sent to HPLC for inspection to determine whether the collected elution peak contained c-di-GMP and to detect its purity (i.e., the proportion of c-di-GMP in all substances dissolved in the eluent, calculated as the peak area ratio in the present invention). The experimental results are shown in Table 6.

[0054] Table 6. Isocratic purification results

[0055] The results showed that c-di-GMP could be purified by gradient elution under this condition, but the purity of c-di-GMP in the final product was only 85.85%, which was relatively low. The isocratic elution was subsequently adjusted to the gradient elution method. The purification process was as follows: Figure 4 The purification results are shown in Table 7. After the elution procedure was changed to gradient elution, the purity of c-di-GMP in the final product was increased to more than 98%, so the purification method was feasible and a high-purity c-di-GMP sample could be obtained.

[0056] Table 7. 100 mM TEAA gradient purification results

[0057] (3) Optimization of TEAA solution concentration

[0058] Considering that 100 mM TEAA can achieve complete column attachment, the inventors also verified whether reducing the concentration of TEAA solution can continue to completely attach c-di-GMP to the column and purify and separate it, and designed 5 concentrations of 50 mM, 25 mM, 10 mM, 5 mM, and 2.5 mM. The purification results are shown in Table 8. When the TEAA concentration is reduced to 5 mM, c-di-GMP can be completely attached to the column (such as Figure 5 However, when the concentration was reduced to 2.5 mM, a small amount of c-di-GMP was present in the flow-through, and the separation between the main peak and other impurity peaks was insufficient according to the comparison of the purified peak shapes. Therefore, under this condition, the recovery rate needs to be reduced by about 50% to obtain high-purity c-di-GMP.

[0059] Table 8. Purification results of different concentrations of TEAA

[0060] In summary, the minimum TEAA concentration required for purification of c-di-GMP is 5 mM, which can ensure that c-di-GMP has a high purity and recovery rate. As the TEAA concentration decreases (100 mM→5 mM), the main peak of purification is eluted under lower methanol concentration conditions, and the baseline separation between the main peak and the impurity peaks can basically be achieved.

[0061] (4) Selection of organic solvent In organic phases other than methanol, we also investigated the effect of acetonitrile on the purification and elution of c-di-GMP. The acetonitrile purification process is as follows: Figure 6 The purification results are shown in Table 9. The experimental results show that acetonitrile can replace methanol for purification and elution, but the 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 above 99%, the recovery rate is greatly reduced, only half of that when eluted with methanol.

[0062] Table 9. Acetonitrile purification results

[0063] Based on the above work, we used the following method to purify c-di-GMP: 1. Filler activation: Soak the SP207SS resin (Mitsubishi Chemical) filler in anhydrous ethanol overnight. After cleaning the resin, soak it in 20% ethanol and seal it for long-term storage.

[0064] 2. Column packing: Load the activated resin into the AKTA purification glass column, ensuring that there are no gaps between the fillers, no drying, etc., and ensure good sealing.

[0065] 3. Pre-equilibrium treatment: Connect the column filled with filler to the AKTA purifier and set the flow rate to 0.5-2 column volumes / h; first flush 2-3 column volumes with injection water; then flush 4-5 column volumes with 50 mM TEAA solution for pre-equilibrium treatment.

[0066] 4. Purification treatment: (1) Sample loading: Take 5 ml of the sample to be purified and load it at 0.5-2 column volumes / h; (2) Equilibration: The flow rate is 0.5-2 column volumes / h, and 5 mM TEAA solution is used to equilibrate for 4-5 column volumes. The eluate is collected when the absorbance rises rapidly, and collection is stopped when the absorbance levels off. (3) 10% elution: flow rate 0.5-2 column volumes / h, 60% A (5 mM TEAA solution) and 40% B (75% A phase plus 25% methanol) for 2-3 column volumes, collect the eluate from the time when the absorbance rises rapidly, and stop collecting when the absorbance levels off; (4) 10-15% gradient elution: flow rate 0.5-2 column volumes / h, 60%-40%A and 40%B-60%B, flush 2-3 column volumes, collect the eluate from the time when the absorbance rises rapidly, and stop collecting when the absorbance levels off; (5) 15-25% gradient elution: flow rate is 0.5-2 column volumes / h, 40%-0 A and 60%B-100%B, flush for 3-4 column volumes, collect the eluate from the time when the absorbance rises rapidly, and stop collecting when the absorbance levels off.

[0067] Finally, the collected samples were dried by rotary evaporation: the water bath was set to 66°C, the vacuum was manually adjusted to 0, and the rotation speed was set to 120 rpm. The collected samples to be dried were placed in a spin-drying bottle and dried for 30 minutes.

[0068] The dried sample was reconstituted with water for injection and tested for purity by HPLC, and the results showed that the purity was >99.5%.

[0069] The inventors tested c-di-AMP ( Figure 7 ) and 3',3'-cGAMP were purified, and their purities could reach 99.3%~99.6%.

[0070] Comparative Example 1 Preparative HPLC Purification

[0071] The c-di-GMP sample prepared in Example 1 was purified by preparative HPLC under the following chromatographic conditions: Hypersil GOLD™ PREP C18 column, column length 150 mm, column inner diameter 30 mm, column particle size 5 μm; Mobile phase A was 10 mM TEAA solution, pH = 7.0; mobile phase B was acetonitrile solution; Gradient elution program: From 0 to 18 min, mobile phase A was reduced from 97% to 90%, and mobile phase B was increased from 3 to 10%; Within 18-23 min, mobile phase A decreased from 90% to 87.5%, and mobile phase B increased from 10% to 12.5%; From 23 to 26 min, mobile phase A was maintained at 87.5% and mobile phase B was maintained at 12.5%; Within 26-29 min, mobile phase A decreased from 87.5% to 0, and mobile phase B increased from 12.5% ​​to 100%; From 29 to 37 min, mobile phase A was maintained at 0% and mobile phase B was maintained at 100%; In 37-37.1 min, mobile phase A increased from 0 to 97%, and mobile phase B decreased from 100% to 3%; From 37.1 to 52 min, mobile phase A was maintained at 97% and mobile phase B was maintained at 3%; The flow rate of the mobile phase is 10~20 mL / min.

[0072] The purity of the product was tested by analytical HPLC, and the results showed that its purity was 95%~97%.

[0073] Example 3 Cell experiment

[0074] Different doses of c-di-GMP, 0.225 μg / ml, 2.25 μg / ml, 22.5 μg / ml, 45 μg / ml, 90 μg / ml and 180 μg / ml, were co-incubated with mouse mononuclear macrophage RAW246.7 for 24 hours, and then the expression of IFN-β, a downstream cytokine of the STING pathway, was detected. The results showed that the c-di-GMP prepared by the present invention has high biological activity. Figure 8 As shown, the expression of IFN-β increased in a c-di-GMP dose-dependent manner.

[0075] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

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, centrifuging the reaction product to obtain a supernatant; The cyclic dinucleotide is purified from the supernatant using a macroporous adsorption resin, wherein a 5-100 mM triethylamine-acetic acid solution is used as phase A and a mixed solution containing 70-80% phase A and 20-30% methanol is used as phase B for gradient elution.

2. The preparation method according to claim 1, characterized in that: The dinucleotide cyclase is a DncV enzyme, cyclic GMP-AMP synthase (cGAS) or cGAS / DncV-like nucleotidyl transferase (CdnE).

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

4. The preparation method according to claim 1, characterized in that: The cyclic dinucleotide is selected from one or more of c-di-AMP, c-di-GMP, 2',3'-cGAMP and 3',3'-cGAMP.

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 macroporous adsorption resin is SP207SS resin or SP207 resin produced by Mitsubishi Chemical.

9. The preparation method according to claim 1, characterized in that: The concentration of the triethylamine-acetic acid solution is 5-100 mM.

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

11. The preparation method according to claim 1, characterized in that: 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.

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

Images