The invention relates to a one-pot method for synthesizing high-purity 5apos; -DMT-2apos, and-DMT-2apos; method for protecting nucleoside by using-TBDMS
In the synthesis of 5'-DMT-2'-TBDMS protective nucleoside, first DMT protects the 5'-position hydroxyl group, then TBDMS protects the 2'-position hydroxyl group, and recrystallizes through diethyl ether/methyl tert-butyl ether, the existing technology is solved. The problems of by-product generation and high cost in the middle have obtained high purity and low cost products, suitable for industrial production.
Patent Information
- Application Number
- CN202311768316.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-21
- Publication Date
- 2024-12-13
AI Technical Summary
In the existing synthesis method of 5'-DMT-2'-TBDMS protected nucleoside, similar activities of hydroxyl groups at positions 2' and 3' lead to the generation of by-products, increasing the difficulty of separation and reducing the purity of the target product. The materials used at the same time High cost.
By using a one-pot synthesis method, DMT protection is performed on the 5' hydroxyl group, and then TBDMS protection is performed on the 2' hydroxyl group, and then recrystallization is performed on the mixed solvent of diethyl ether and methyl tert-butyl ether to obtain high purity 5′-DMT-2′-TBDMS protects nucleoside.
Achieve high purity (not less than 99.0%) products, reduce production costs, and simplify operations, suitable for industrial production.
Smart Images
Figure CN119119139A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biopharmaceutical intermediates, and in particular relates to a method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides through a one-pot process. Background Art
[0002] The 5′ end of most messenger RNA (mRNA) is blocked or capped, which can promote its normal function in cells. With the development and research of this type of RNA drugs, the demand for its synthetic precursor nucleoside monomers in the drug market is also increasing. Since nucleosides carry multiple functional groups, such as amino groups and hydroxyl groups, it is often necessary to protect certain functional groups on the sugar group in order to selectively react at specific sites for efficient RNA synthesis.
[0003] 5′-DMT-2′-TBDMS protected nucleoside is an important nucleoside monomer for RNA synthesis. Currently, Leonid Beigelman's group has reported a method for its synthesis (Tetrahedron Letters 43 (2002) 1983–1985). The specific synthesis route is as follows:
[0004]
[0005] The above method mainly involves first protecting the 3′ and 5′ hydroxyl groups with di-tert-butylsilylbis(trifluoromethanesulfonic acid), then adding a TBDMS protecting group to the 2′ hydroxyl group, then removing the protecting groups of the 3′ and 5′ hydroxyl groups, and finally adding a DMT protecting group. Here, since the activities of the 2′ and 3′ hydroxyl groups are very similar, according to this method, it is inevitable that the TBDMS protecting group on the 2′ hydroxyl group will be transferred to the 3′ hydroxyl group in the last step of the reaction, generating an isomeric by-product, which obviously increases the difficulty of separating the target product and reduces the purity of the target product; at the same time, the price of the material di-tert-butylsilylbis(trifluoromethanesulfonic acid) is relatively high, accounting for the vast majority of the cost of the entire project, and the cost of this method is relatively high. Summary of the invention
[0006] Based on the above technical problems, the present invention provides a one-pot method for synthesizing high-purity 5′-DMT-2′-TBDMS protected nucleoside, which is not only low in cost and high in product purity, but also simple in operation and suitable for industrial production.
[0007] The present invention provides a one-pot method for synthesizing high-purity 5′-DMT-2′-TBDMS protected nucleoside, comprising the following steps:
[0008] S1, sequentially subjecting the 5′-hydroxyl group and the 2′-hydroxyl group on the sugar group of the compound represented by the structural formula I to DMT and TBDMS protecting groups to obtain a crude product of the compound represented by the structural formula II;
[0009] S2, the crude product of the compound represented by structural formula II is recrystallized and purified by using a mixed solvent of diethyl ether and methyl tert-butyl ether to obtain a purified product of the compound represented by structural formula II, which is the high-purity 5′-DMT-2′-TBDMS protected nucleoside;
[0010]
[0011] Wherein, B is a natural or modified nucleoside base.
[0012] Preferably, B is a natural or modified purine or pyrimidine base.
[0013] Preferably, the structural formula of B is one of the following:
[0014]
[0015] Preferably, in step S1, the hydroxyl protecting agent used in the DMT protecting group reaction is 4,4′-dimethoxytriphenylmethane chloride DMTrCl, and the catalyst used in the DMT protecting group reaction is at least one of pyridine or quinoline;
[0016] Preferably, the amount of the hydroxyl protecting agent is 1-1.5 times the molar amount of the compound represented by structural formula I;
[0017] Preferably, the amount of the catalyst used is 1-3 times the molar amount of the compound represented by structural formula I.
[0018] Preferably, in step S1, the temperature of the DMT protecting group reaction is 0-5°C and the time is 1-3h.
[0019] Preferably, in step S1, the hydroxyl protecting agent used in the TBDMS protecting group reaction is tert-butyldimethylsilyl chloride TBDMSCl, and the catalyst used in the TBDMS protecting group reaction is at least one of N-methylimidazole or N-methylmorpholine;
[0020] Preferably, the amount of the hydroxyl protecting agent is 0.9-1.1 times the molar amount of the compound represented by structural formula I;
[0021] Preferably, the amount of the catalyst used is 1-3 times the molar amount of the compound represented by structural formula I.
[0022] Preferably, in step S1, the temperature of the TBDMS protecting group reaction is 15-35°C and the time is 6-24h.
[0023] Preferably, in step S2, the volume ratio of diethyl ether to methyl tert-butyl ether is 3-5:1.
[0024] Preferably, the temperature of the recrystallization purification is 5-15°C and the time is 12-36h.
[0025] Preferably, the purity of the high-purity 5′-DMT-2′-TBDMS protected nucleoside is not less than 99.0%.
[0026] The invention provides a one-pot method for synthesizing high-purity 5′-DMT-2′-TBDMS protected nucleoside. According to the activity difference of three hydroxyl groups on the compound shown in structural formula I, DMT is firstly used to protect the 5′ hydroxyl group, and then TBDMS is used to protect the 2′ hydroxyl group, so as to obtain a crude product of the compound shown in structural formula II, and then recrystallize it through ether / methyl tert-butyl ether to obtain a 5′-DMT-2′-TBDMS protected nucleoside with a purity of not less than 99.0%. Compared with other methods in the prior art, the invention not only improves the problems of high cost and long cycle of the prior art, but also can obtain a product with higher purity. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is the hydrogen spectrum of the compound 5′-DMT-2′-TBDMS-G-iBu described in Example 1;
[0028] Figure 2 This is the high performance liquid chromatogram of the compound 5′-DMT-2′-TBDMS-G-iBu described in Example 1. DETAILED DESCRIPTION
[0029] Hereinafter, the present invention will describe the technical solution in detail through specific embodiments, but it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.
[0030] Example 1
[0031] This example provides a one-pot method for synthesizing high-purity 5′-DMT-2′-TBDMS protected nucleoside, and the specific synthesis method is as follows:
[0032] Compound G-iBu (5.66 mol, 2 kg) and pyridine (49.71 mol, 14 L) were added to a 1 L reaction bottle, and DMTrCl (7.38 mol, 2.5 kg) was added after cooling to 0 °C. After stirring and reacting for 2 h, the raw material was <5% as monitored by HPLC. N-methylimidazole (9.13 mol, 0.75 kg) was added, and then TBDMSCl (5.30 mol, 10.8 kg) was added in batches. The reaction was kept warm at 25 °C for 12 h. The intermediate state was <5% as monitored by HPLC. %; the resulting reaction solution was added to ethyl acetate (20 L), and washed with 8 wt % aqueous citric acid solution (10 L). After separation, the solution was washed with saturated aqueous sodium bicarbonate solution (10 L). After separation, the solution was concentrated to remove the solvent, and a mixed solvent consisting of ether (40 L) and methyl tert-butyl ether (10 L) was added. The mixture was stirred at 10 ° C for 24 h, and then recrystallized, filtered, and dried to obtain compound 5′-DMT-2′-TBDMS-G-iBu (1.50 kg, yield 57.5%, purity 99.8%).
[0033] Among them, the hydrogen spectrum of compound 5′-DMT-2′-TBDMS-G-iBu is referenced Figure 1 Its high performance liquid chromatogram is shown in Figure 2 shown.
[0034]
[0035] Example 2-11
[0036] The preparation method of reference example 1 is different in that, in examples 2-11, the compound 5′-DMT-2′-TBDMS-G-iBu is prepared using different catalysts, recrystallization solvents, and recrystallization solvent ratios of the same equivalent, and the yield and purity are measured. The results are shown in Table 1 below:
[0037] Table 1
[0038]
[0039] It can be seen from Table 1 above that compared with triethylamine, DIPEA or imidazole as a catalyst, when pyridine or N-methylimidazole is used as a catalyst, the yield of the obtained compound 5′-DMT-2′-TBDMS-G-iBu is the highest, and compared with diethyl ether, n-hexane, methyl tert-ether, etc. as recrystallization solvents, the purity of the compound 5′-DMT-2′-TBDMS-G-iBu obtained by recrystallization after diethyl ether / methyl tert-ether is the highest, and when the ratio of the two is 4:1, the purity is optimal.
[0040] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A one-pot method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides, characterized in that: The steps include: S1, sequentially subjecting the 5'-hydroxyl group and the 2'-hydroxyl group on the sugar group of the compound represented by the structural formula I to DMT and TBDMS protecting groups to obtain a crude product of the compound represented by the structural formula II; S2, the crude product of the compound represented by structural formula II is recrystallized and purified by using a mixed solvent of diethyl ether and methyl tert-butyl ether to obtain a purified product of the compound represented by structural formula II, which is the high-purity 5'-DMT-2'-TBDMS protected nucleoside; Wherein, B is a natural or modified nucleoside base.
2. The method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides by one-pot method according to claim 1, characterized in that: B is a natural or modified purine or pyrimidine base.
3. The method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleoside by one-pot method according to claim 1 or 2, characterized in that, The structural formula of B is one of the following:
4. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 3, characterized in that: In step S1, the hydroxyl protecting agent used in the DMT protecting group reaction is 4,4'-dimethoxytriphenylmethane chloride DMTrCl, and the catalyst used in the DMT protecting group reaction is at least one of pyridine or quinoline; Preferably, the amount of the hydroxyl protecting agent is 1-1.5 times the molar amount of the compound represented by structural formula I; Preferably, the amount of the catalyst used is 1-3 times the molar amount of the compound represented by structural formula I.
5. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 4, characterized in that: In step S1, the temperature of the DMT protecting group reaction is 0-5°C and the time is 1-3h.
6. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 5, characterized in that: In step S1, the hydroxyl protecting agent used in the TBDMS protecting group reaction is tert-butyldimethylsilyl chloride TBDMSCl, and the catalyst used in the TBDMS protecting group reaction is at least one of N-methylimidazole or N-methylmorpholine; Preferably, the amount of the hydroxyl protecting agent is 0.9-1.1 times the molar amount of the compound represented by structural formula I; Preferably, the amount of the catalyst used is 1-3 times the molar amount of the compound represented by structural formula I.
7. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 6, characterized in that: In step S1, the temperature of the TBDMS protecting group reaction is 15-35°C and the time is 6-24h.
8. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 7, characterized in that: In step S2, the volume ratio of diethyl ether to methyl tert-butyl ether is 3-5:
1.
9. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 8, characterized in that: The temperature of the recrystallization purification is 5-15°C and the time is 12-36h.
10. A method for synthesizing high-purity 5'-DMT-2'-TBDMS protected nucleosides in one pot according to any one of claims 1 to 9, characterized in that: The purity of the high-purity 5'-DMT-2'-TBDMS protected nucleoside is not less than 99.0%.