Preparation process of tris salt cap composition and tris salt cap composition

Abstract

This application relates to the fields of chemistry and bioengineering, and specifically discloses a preparation process of a Tris salt cap composition and the Tris salt cap composition. The preparation process includes the following steps: (1) preparing a mixed salt form of a cap analog to obtain a mixture, wherein the structure of the cap analog is a compound of formula (I), or its stereoisomer, tautomer, or isotopic variant; (2) eluting the mixture with a Tris-acid aqueous solution, and after desalting and concentration, obtaining a Tris salt cap composition; wherein the pH value of the Tris-acid aqueous solution is 2.5-7.5. In preparing the Tris salt cap composition, this application not only avoids the problem of easy glycosidic bond breakage of the cap analog under strong acid conditions, but also achieves more thorough salt conversion, and can conveniently obtain a Tris salt cap composition with excellent performance.

Description

Technical Field

[0001] This application relates to the fields of chemical and biological engineering, and more specifically, to a process for preparing a Tris salt cap composition and the Tris salt cap composition. Background Technology

[0002] In vitro transcription to synthesize mRNA has become an important tool for introducing exogenous genes to express proteins, and it is widely used in the treatment and prevention of diseases. In the process of in vitro transcription to synthesize mRNA, the chemical capping process is a crucial step. However, the systems used for mRNA capping are relatively complex. For example, the recognition efficiency of capping enzymes is poor for some specific mRNA sequences, resulting in low capping efficiency; some mRNA sequences also have many secondary structures, which can lead to significant steric hindrance during capping, ultimately resulting in low capping efficiency as well.

[0003] Because Tris has good biocompatibility, it exhibits even better biocompatibility with mRNA when transcribing certain specific mRNA sequences, which helps to improve the yield of mRNA transcription and significantly enhances the efficiency of mRNA capping.

[0004] Currently reported techniques mostly involve forming a salt-cap analog complex through the free acid phase and then adjusting the pH. However, because the glycosidic bonds of the cap analogs are easily broken under strong acid conditions, this approach is not conducive to large-scale production. Therefore, there is an urgent need in the field to develop a new method for preparing Tris salt-cap analog compositions. Summary of the Invention

[0005] This application provides a preparation process for a Tris salt-capped composition and a Tris salt-capped composition. In preparing the Tris salt-capped composition, this application not only avoids the problem that the glycosidic bond of the cap analog is easily broken under strong acid conditions, but also achieves more thorough salt conversion, and can conveniently obtain a Tris salt-capped composition with excellent performance.

[0006] In a first aspect, this application provides a preparation process for a Tris salt cap composition, employing the following technical solution:

[0007] A process for preparing a Tris salt cap composition includes the following steps:

[0008] (1) Prepare a mixed salt form of the cap analogue to obtain a mixture, wherein the cap analogue has the structure of compound (I), or its stereoisomer, tautomer or isotopic variant:

[0009]

[0010] in,

[0011] It is a single key or does not exist.

[0012] X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O.

[0013] OCH2CH2 or a three-membered cycloalkyl group;

[0014] R1, R2, R3, and R4 are independently selected from halogen, OH, amino, acetyl, bridged ring, C, and C groups, respectively. 1-3 Alkyl, C 1-3 Alkoxy, C 2-3 alkenyl, C 2-6 alkynyl or C 3-6 cycloalkyl; wherein the C 1-3 Alkyl, C 1-3 The alkoxy and amino groups are not substituted, or are replaced by halogens, OH, or C. 1-3 Alkyl, C 1-3 Alkoxy or acetyl substitution;

[0015] B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases;

[0016] (2) The mixture was eluted with Tris-acid aqueous solution, and after desalting and concentration, a Tris salt cap composition was obtained; wherein the pH value of the Tris-acid aqueous solution was 2.5-7.5.

[0017] Furthermore, the salt in the mixed salt form of the cap analogue includes at least one of sodium salt, magnesium salt, zinc salt, and triethylamine salt.

[0018] Furthermore, the Tris-acid aqueous solution includes at least one of Tris-HCl and Tris-acetic acid.

[0019] Furthermore, the concentration of the Tris-acid aqueous solution is 0.3-3M.

[0020] Furthermore, the pH value of the Tris-acid aqueous solution is 3.5-6.5, preferably 4.5-6.5, and even more preferably 4.5, 5.5 or 6.5.

[0021] Further, in step (2), the mixture is loaded onto an anion exchange chromatography column; gradient elution is performed using phase A, which is purified water, and phase B, which is Tris-acid aqueous solution.

[0022] Furthermore, the packing material of the anion chromatography column includes DEAE Sephadex or 201 anion exchange resin.

[0023] Furthermore, in step (2), the gradient elution range is 15%-45%.

[0024] Furthermore, in step (2), after elution, the components with a purity greater than 98% are collected, desalted by preparative chromatography, and then concentrated.

[0025] Furthermore, the structure of the cap analogue is shown in formula (I-1):

[0026]

[0027] Furthermore, the structure of the cap analogue is shown in formula (I-2):

[0028]

[0029] Secondly, this application provides a Tris salt cap composition, which adopts the following technical solution:

[0030] A Tris salt cap composition prepared by the above-described preparation process. Further, the Tris salt cap composition is in the form of a system of 1 to 7 Tris counterions.

[0031] Furthermore, the Tris salt cap composition is in the form of a system of 2 to 6 Tris counterions.

[0032] In summary, this application has the following beneficial effects:

[0033] This application utilizes Tris-acids as the eluent for ion exchange during the ion purification of crude cap analogs, while also providing Tris. This allows for the convenient and rapid acquisition of cap analog compositions containing varying numbers of cap analogs. Furthermore, it effectively reduces glycosidic bond breakage under strong acid conditions, resulting in improved yield and more thorough salt conversion. Moreover, this application reduces the steps involved in converting to free acid forms, specifically selecting Tris-acids as the eluent for ion exchange, and optimizing elution process parameters, thus simplifying the preparation process. Attached Figure Description

[0034] Figure 1 This is the elution time spectrum from the LCMS analysis of the glycosidic bond breakage in Comparative Example 1.

[0035] Figure 2 This is the abundance spectrum of each peak in the LCMS analysis of the glycosidic bond breaking situation in Comparative Example 1.

[0036] Figure 3 This is the mass spectrum of the substance at the corresponding time (8.316 min) in the LCMS analysis of the glycosidic bond breakage in Comparative Example 1.

[0037] Figure 4 This is the mass spectrum of the substance at the corresponding time (11.924 min) in the LCMS analysis of the glycosidic bond breakage in Comparative Example 1. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Example

[0040] The mixed salt form of the cap analog (I-1) used in Examples 1-6 and Comparative Example 1 was synthesized as follows:

[0041] Intermediate A (30.0 mmol) and intermediate B (20.0 mmol) were suspended in DMSO (210.0 mL), and ZnCl2 (160.0 mmol) was added to the reaction solution under ice bath conditions. After stirring at room temperature for 36 hours, the reaction was terminated with 0.25 M EDTA-2Na (270.0 mmol) solution.

[0042] The above reaction route is shown in the following equation. The mixed salt form of its cap analogues (specifically, a mixed salt form of sodium salt, zinc salt, and triethylamine salt) is denoted as S-1:

[0043]

[0044] The mixed salt form of the cap analogue (I-2) used in Examples 7-8 was synthesized as follows:

[0045] Intermediate C (30.0 mmol) and intermediate B (20.0 mmol) were suspended in DMSO (210.0 mL), and ZnCl2 (160.0 mmol) was added to the reaction solution under ice bath conditions. After stirring at room temperature for 36 hours, the reaction was terminated with 0.25 M EDTA-2Na (270.0 mmol) solution.

[0046] The above reaction route is shown in the following equation. The mixed salt form of its cap analogue (specifically, a mixed salt form of sodium salt, zinc salt, and triethylamine salt) is denoted as S-2:

[0047]

[0048] The following explanation is provided through specific examples.

[0049] Example 1

[0050] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0051] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 2.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 2.5 with 12M concentrated hydrochloric acid.

[0052] S-1 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 2.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted by chromatography, and concentrated to obtain the Tris salt of CAP GAG, in the form of a 1–2 Tris counterion system. The content was determined by UV spectrophotometry, and the overall yield was calculated to be 52.4%.

[0053] Example 2

[0054] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0055] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 3.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 3.5 with 12M concentrated hydrochloric acid.

[0056] S-1 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 3.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted by chromatography, and concentrated to obtain the Tris salt of CAP GAG, in the form of a 2–3 Tris counterion system. The content was determined by UV spectrophotometry, and the overall yield was calculated to be 54.2%.

[0057] Example 3

[0058] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0059] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 4.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 4.5 with 12M concentrated hydrochloric acid.

[0060] S-1 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 4.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted by chromatography, and concentrated to obtain the Tris salt of CAP GAG, in the form of a 3–4 Tris counterion system. The content was determined by UV spectrophotometry, and the overall yield was calculated to be 54.8%.

[0061] Example 4

[0062] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0063] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 5.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 5.5 with 12M concentrated hydrochloric acid.

[0064] S-1 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 5.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted, and concentrated to obtain the Tris salt of CAP GAG, in the form of a 4–5 Tris counterion system. The content was determined using a UV spectrophotometer, and the overall yield was calculated to be 54.9%.

[0065] Example 5

[0066] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0067] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 6.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 6.5 with 12M concentrated hydrochloric acid.

[0068] S-1 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 6.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted by chromatography, and concentrated to obtain the Tris salt of CAP GAG, in the form of a 5–6 Tris counterion system. The content was determined by UV spectrophotometry, and the overall yield was calculated to be 54.7%.

[0069] Example 6

[0070] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0071] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 7.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 7.5 with 12M concentrated hydrochloric acid.

[0072] S-1 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 7.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted by chromatography, and concentrated to obtain the Tris salt of CAP GAG, in the form of a 6–7 Tris counterion system. The content was determined by UV spectrophotometry, and the overall yield was calculated to be 53.4%.

[0073] Example 7

[0074] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0075] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 4.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 4.5 with 12M concentrated hydrochloric acid.

[0076] S-2 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 4.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted, and concentrated to obtain the Tris salt of CAP GAG (ENE), in the form of a 3–4 Tris counterion system. The content was determined using a UV spectrophotometer, and the overall yield was calculated to be 55.0%.

[0077] Example 8

[0078] The Tris salt cap composition provided in this embodiment is prepared as follows:

[0079] Preparation of a 1.0M Tris-HCl aqueous solution with a pH of 5.5: Weigh 1.0 mol of Tris and dissolve it in 1L of purified water, then adjust the pH to 5.5 with 12M concentrated hydrochloric acid.

[0080] S-2 was loaded onto a DEAE Sephadex column. Gradient elution was performed using purified water (phase A) and a 1.0 M Tris-HCl aqueous solution (pH 5.5) as phase B, with a gradient range of 15%–45% (5% increase per 2 cv gradient). Fractions with a purity greater than 98% were collected, preparatively desalted, and concentrated to obtain the Tris salt of CAP GAG (ENE), in the form of a 4–5 Tris counterion system. The content was determined using a UV spectrophotometer, and the overall yield was calculated to be 54.9%.

[0081] Comparative Example

[0082] Comparative Example 1

[0083] S-1 was loaded onto a DEAE Sephadex column. The product was eluted linearly using a 0-1.0 M TEAB eluent, and the eluent with an HPLC purity >98% was collected. The product was then concentrated and desalted to obtain the triethylamine salt form of the cap analogue.

[0084] The triethylamine salt form of the cap analogue was converted into the product in the form of a free acid of the same concentration using a cation exchange resin. 0.2 M Tris base was added dropwise to the product solution until the pH reached 5–5.8, followed by concentration and volume adjustment to obtain the final product, which at this point existed as a system of 4–5 Tris counterions. The content was determined using a UV spectrophotometer, and the overall yield was calculated to be 43.4%.

[0085] Performance testing

[0086] (1) Detection of glycosidic bond breakage

[0087] To detect the glycosidic bond cleavage in Comparative Example 1, S-1 was loaded onto a DEAE Sephadex column. The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. Eluent with an HPLC purity >98% was collected, concentrated, and desalted to obtain the triethylamine salt form of the cap analog. The triethylamine salt form of the cap analog was then converted to a free acid form of the same concentration using a cation exchange resin. Eluents at different pH values ​​(collected at different times) were sent to LC-MS for analysis. When the pH of the effluent was <1, the product was unstable under excessively acidic conditions, resulting in degradation impurities. Specifically... Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown.

[0088] (2) Yield detection

[0089] The content was determined using a UV spectrophotometer, and the total yield of the Tris salt cap composition was calculated. The results are shown in Table 1.

[0090] Table 1. Yield test results for the examples and comparative examples.

[0091] Detection object Yield (%) Example 1 52.4 Example 2 54.2 Example 3 54.8 Example 4 54.9 Example 5 54.7 Example 6 53.4 Example 7 55.0 Example 8 54.9 Comparative Example 1 43.4

[0092] Firstly by Figure 1-4 It can be seen from LCMS analysis that during the conversion of the triethylamine salt form of the cap analog into the product of the same concentration of free acid through cation exchange resin, the glycosidic bond of the cap analog is easily broken under strong acid conditions (pH < 1), resulting in the production of the first base glycosidic bond breakage byproduct. Figure 3 The present application directly uses Tris-acid with a suitable pH value to elute the mixed salt form of the cap analogue. This not only avoids the breakage of glycosidic bonds but also effectively provides Tris, resulting in a more thorough salt conversion. The yield of the Tris salt cap composition is increased from 43.4% to 52.4-54.9%, an increase of 20.7%-26.5%, which is beneficial for scale-up production. Moreover, the present application eliminates the steps of converting the mixed salt form of the cap analogue to the pure triethylamine salt form and then to the free acid form. Instead, it uses Tris-acid as the ion exchange eluent during the ion purification of the crude cap analogue, while simultaneously providing Tris. This simplifies the preparation process and allows for a more convenient and rapid acquisition of cap analogue compositions containing different numbers of caps.

[0093] 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 preparation process for a Tris salt cap composition, characterized in that, Includes the following steps: (1) Prepare a mixed salt form of the cap analogue to obtain a mixture, wherein the cap analogue has the structure of compound (I), or its stereoisomer, tautomer or isotopic variant: in, It is a single key or does not exist; X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O. OCH2CH2 or a three-membered cycloalkyl group; R1, R2, R3, and R4 are independently selected from halogen, OH, amino, acetyl, bridged ring, C, and C groups, respectively. 1-3 Alkyl, C 1-3 Alkoxy, C 2-3 alkenyl, C 2-6 alkynyl or C 3-6 cycloalkyl; wherein the C 1-3 Alkyl, C 1-3 The alkoxy and amino groups are not substituted, or are replaced by halogens, OH, or C. 1-3 Alkyl, C 1-3 Alkoxy or acetyl substitution; B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases; (2) The mixture was eluted with Tris-acid aqueous solution, and after desalting and concentration, a Tris salt cap composition was obtained; wherein the pH value of the Tris-acid aqueous solution was 2.5-7.

5.

2. The preparation process of the Tris salt cap composition according to claim 1, characterized in that, The Tris-acid aqueous solution includes at least one of Tris-HCl and Tris-acetic acid.

3. The preparation process of the Tris salt cap composition according to claim 1, characterized in that, The concentration of the Tris-acid aqueous solution is 0.3-3M.

4. The preparation process of the Tris salt cap composition according to claim 1, characterized in that, The pH value of the Tris-acid aqueous solution is 3.5-6.

5.

5. The preparation process of the Tris salt cap composition according to claim 1, characterized in that, In step (2), the mixture is loaded onto an anion exchange chromatography column; gradient elution is performed using phase A, which is purified water, and phase B, which is Tris-acid aqueous solution.

6. The preparation process of the Tris salt cap composition according to claim 5, characterized in that, The packing material for the anion chromatography column includes DEAE Sephadex or 201 anion exchange resin.

7. The preparation process of the Tris salt cap composition according to claim 5, characterized in that, In step (2), the gradient elution range is 15%-45%.

8. The preparation process of the Tris salt cap composition according to claim 1, characterized in that, The structure of the cap-like object is shown in formula (I-1):

9. The preparation process of the Tris salt cap composition according to claim 1, characterized in that, The structure of the cap-like object is shown in formula (I-2):

10. A Tris salt cap composition prepared by the preparation process of the Tris salt cap composition according to any one of claims 1-9.

11. The Tris salt cap composition according to claim 10, characterized in that, The Tris salt cap composition is in the form of a system of 1 to 7 Tris counterions.

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