Liquid chromatographic analysis method for monitoring dipeptide activated ester reaction

Abstract

The invention provides a liquid chromatographic analysis method for monitoring a synthesis reaction of dipeptide activated ester. According to the method, a cyano column and an organic mobile phase are used, and the synthesis reaction of the dipeptide activated ester can be rapidly, efficiently and stably monitored.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, and in particular to a liquid chromatography analysis method for monitoring a dipeptide activated ester synthesis reaction. Background Art

[0002] Peptides are widely present in organisms, widely participate in and regulate the functional activities of various systems, organs, tissues and cells in the body, and play an important role in life activities. In recent years, peptide drugs have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, and AIDS due to their wide indications, high safety and significant efficacy. At present, with the increasing maturity of biotechnology and peptide synthesis technology, more and more peptide drugs will be developed and used in clinical practice.

[0003] There are two main methods for preparing peptides: chemical synthesis and biosynthesis. Chemical synthesis is more suitable for the rapid acquisition of short peptides, and is divided into liquid phase method and solid phase method. Although the production cost of biosynthesis is generally lower, it is often combined with chemical synthesis when it involves non-natural amino acid sequences. In chemical synthesis, in order to improve the condensation efficiency of amino acids / peptides, the carboxyl group of amino acids / peptides is often activated with N-hydroxysuccinimide (NHS) to form activated esters (OSu), and then connected to the amino groups of other amino acids / peptides.

[0004] As a representative of GLP-1 drugs, semaglutide can significantly reduce the level of glycated hemoglobin (HbA1c) and reduce weight in patients with type 2 diabetes, while greatly reducing the risk of hypoglycemia, so it has a large application market. Due to the presence of a non-natural amino acid, 2-aminoisobutyric acid (Aib), at the second position of the N-terminus of semaglutide, the preparation process often involves the reaction of a dipeptide activated ester (Fmoc-His(Trt)-Aib-OSu) with the 29-peptide main chain. Since the dipeptide activated ester is unstable when exposed to water and easily causes the OSu group to fall off, conventional reversed-phase liquid chromatography analysis methods cannot be used for monitoring the synthesis reaction. Therefore, it is urgent to develop a method for monitoring the synthesis reaction of dipeptide activated esters that is simple to operate and has good separation effects. Summary of the invention

[0005] Therefore, the object of the present invention is to provide a novel dipeptide activated ester synthesis reaction monitoring method.

[0006] In a first aspect of the present invention, there is provided a method for monitoring an activated esterification reaction, comprising the steps of:

[0007] (1) preparing a sample solution;

[0008] (2) subjecting the sample solution to liquid chromatography analysis;

[0009] in,

[0010] In step (1), the sample solution comprises a reaction solution of an activated esterification reaction and an organic solvent; the reaction solution comprises: a reaction raw material, a reaction product, or a combination thereof;

[0011] In step (2), the monitoring conditions of the liquid chromatography analysis include:

[0012] A cyano column is used as a chromatographic column; an organic solvent is used as a mobile phase; and 200-230 nm ultraviolet light is used to detect components.

[0013] In some embodiments, the activated esterification reaction is a dipeptide activated esterification reaction, the reaction raw material is a dipeptide, and the reaction product is a dipeptide activated ester.

[0014] In another preferred embodiment, the activated esterification reaction is shown in the following formula:

[0015]

[0016] The dipeptide (Fmoc-His(Trt)-Aib-OH) reacts with the condensing agent DCC and the activating agent HOSu to generate the dipeptide activated ester (Fmoc-His(Trt)-Aib-OSu).

[0017] In some embodiments, the organic solvent is selected from the group consisting of ethyl acetate, n-hexane, isopropanol, ethanol, acetonitrile, and combinations thereof;

[0018] Furthermore, the mobile phase contains almost no water.

[0019] In another preferred embodiment, the “almost free of water” means that the water content is below 1%, preferably below 0.5%, and more preferably below 0.1%.

[0020] In some embodiments, the mobile phase is a mixed solution of acetonitrile and ethanol;

[0021] Wherein, the volume ratio of acetonitrile to ethanol is 1:1 to 1:30.

[0022] In some embodiments, the volume ratio of acetonitrile to ethanol is 1:5 to 1:20.

[0023] In some embodiments, the mobile phase further comprises 0.01% to 1% of an acid, and the acid is selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid, or a combination thereof.

[0024] In some embodiments, the stationary phase of the chromatographic column is a cyano bonded phase.

[0025] In some embodiments, the monitoring conditions of the liquid chromatography analysis further include:

[0026] The flow rate of the mobile phase is 0.5-1 mL / min;

[0027] Column temperature is 20℃~40℃;

[0028] The wavelength of ultraviolet light is 210-220nm;

[0029] Elution mode: isocratic elution.

[0030] In some embodiments, the method further comprises the steps of:

[0031] (3) determining the reaction progress of the activated esterification reaction by analyzing the results of liquid chromatography;

[0032] If the content of the reaction raw materials is less than 10%, it indicates that the activated esterification reaction is finished.

[0033] In some embodiments, if the content of the reaction raw materials is less than 5%, it indicates that the activated esterification reaction is completed.

[0034] In a second aspect of the present invention, a kit for monitoring an activated esterification reaction is provided, comprising:

[0035] (1) An organic solvent as the mobile phase;

[0036] (2) Chromatographic column;

[0037] in,

[0038] The organic solvent is selected from the following group: ethyl acetate, n-hexane, isopropanol, ethanol, acetonitrile, and combinations thereof; and the organic solvent has a water content of less than 1%, preferably less than 0.5%, and more preferably less than 0.1%.

[0039] The chromatographic column is a cyano column.

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the chromatogram of the reaction solution after the raw material dipeptide reacted with NHS for 1 hour.

[0042] Figure 2 This is the chromatogram of the reaction solution after the raw material dipeptide reacts with NHS for 1 to 7 hours.

[0043] Figure 3 This is the chromatogram of the reaction solution after the raw material dipeptide reacted with NHS for 7 hours.

[0044] Figure 4This is the C8 reverse phase chromatogram of the reaction of the raw dipeptide with NHS for 7 hours. DETAILED DESCRIPTION

[0045] The inventors found that when performing a dipeptide activated ester synthesis reaction, even after a long reaction, a large amount of raw material dipeptide residues could still be observed after separation by conventional column chromatography. This phenomenon is inconsistent with the nature of the dipeptide activated esterification reaction itself. Therefore, the inventors explored the column chromatography monitoring conditions of the reaction system. After extensive and in-depth research, the inventors unexpectedly found that the dipeptide activated ester synthesis reaction system has a good separation effect in a cyano column, and thus further studied it and developed a dipeptide activated ester synthesis monitoring method, which can quickly, efficiently and stably monitor the dipeptide activated ester synthesis reaction. The present invention was completed on this basis.

[0046] the term

[0047] All technical terms used in the present invention, unless otherwise defined, have the meanings commonly understood by ordinary technicians in the fields relevant to the present invention.

[0048] As used herein, the term "activated ester" refers to a class of carboxylic acid derivatives in which the carboxyl group reacts with N-hydroxysuccinimide (NHS) to form an activated ester (OSu) group. The activated ester (OSu) group has the structure:

[0049] As used in the present invention, the term "activated esterification reaction" refers to a process in which a compound having a carboxyl group reacts with N-hydroxysuccinimide (NHS) to form an activated ester (OSu) group.

[0050] As used in the present invention, the term "dipeptide activated esterification reaction" or "dipeptide activated ester synthesis reaction" refers to a process in which a dipeptide compound undergoes an activated esterification reaction as described above.

[0051] As used in the present invention, the term "cyano column" refers to a chromatographic column with a cyano-bonded stationary phase. The stationary phase matrix is ​​spherical porous silica gel of 2-5 μm, and the column size is 250*4.6 mm or 150*4.6 mm.

[0052] Chromatographic columns

[0053] In some embodiments of the present invention, Yuexu Ultimate XB-CN (cyano) chromatographic column is used, which has both normal phase and reverse phase modes. The reversed-phase cyano column has fast elution for hydrophobic molecules, unique selectivity for polar compounds, and good peak shape for separation of strongly alkaline compounds (including ammonium salts). It has different selectivity from reversed-phase columns such as C18 and C8. The cyano column provides different selectivity from reversed-phase columns such as C18, C8 and C4 phenyl. The pH range of use is 1.5-9.0. In normal-phase chromatography, cyano bonded phase can replace silica gel. Bonded normal-phase columns have the characteristics of rapid equilibrium and more consistent surface activity than non-derivatized silica gel.

[0054] Example 1

[0055] Reaction system

[0056] The reaction used for monitoring in the embodiments and comparative examples of the present invention is shown in the following formula:

[0057]

[0058] The dipeptide (Fmoc-His(Trt)-Aib-OH) reacts with the condensing agent DCC and the activating agent HOSu to generate the dipeptide activated ester (Fmoc-His(Trt)-Aib-OSu). Among them, DCC is dicyclohexylcarbodiimide, and the structural formula is:

[0059] HOSu is N-hydroxysuccinimide, with the structural formula:

[0060] Test conditions

[0061] Instrument: Waters e2695, Waters 2489 UV detector

[0062] Chromatographic column: Yuexu cyano column Ultimate XB-CN, 5μm, 250*4.6mm

[0063] Mobile phase: A is acetonitrile containing 0.1% trifluoroacetic acid (TFA), B is ethanol containing 0.1% TFA

[0064] Detection wavelength: 214nm

[0065] Flow rate: 0.8 mL / min

[0066] Column temperature: 30°C

[0067] Injection volume: 5 μL

[0068] Mobile phase A:B = 10:90, isocratic elution for 25 min

[0069] Sample solution preparation: Take 10 μL of the dipeptide activated ester synthesis reaction solution (reaction time is 1, 2, 3, 4, 5, 6, and 7 hours, respectively) into a 1.5 mL centrifuge tube, add 990 μL of acetonitrile, and shake well.

[0070] The test results are detailed in Figures 1 to 3 . Figure 1 Figure 1 is the chromatogram of the reaction solution after 1 hour. In the figure, two chromatographic peaks of dipeptide (Fmoc-His(Trt)-Aib-OH) and dipeptide activated ester (Fmoc-His(Trt)-Aib-OSu) can be clearly monitored, and the separation effect is good. As the reaction time increases, the chromatogram shows that the peak area of ​​dipeptide (10.7min) gradually decreases, and the peak area of ​​dipeptide activated ester (13.0min) gradually increases. Finally, the dipeptide chromatographic peak basically disappears, and the reaction is complete ( Figure 2 , Figure 3 ).

[0071] It can be seen that in the chromatographic analysis using the method of the present invention, the separation effect of the raw material dipeptide and the product dipeptide activated ester is good, which is convenient for monitoring whether the reaction is complete.

[0072] Comparative Example 1

[0073] Instrument: Waters e2695, Waters 2489 UV detector

[0074] Chromatographic column: C8, 5μm, 250*4.6mm

[0075] Mobile phase: A is 10 mM KH2PO4, B is ACN;

[0076] Detection wavelength: 214nm

[0077] Flow rate: 1.0mL / min

[0078] Column temperature: 30°C

[0079] Injection volume: 5 μL

[0080] The elution gradient is shown in Table 1:

[0081] Table 1: Elution gradient used in Comparative Example 1

[0082] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 15 30 70 20 0 100 25 0 100 25.1 80 20 32 80 20

[0083] Sample solution preparation: Take 10 μL of dipeptide activated ester synthesis reaction solution (reaction time is 7 hours) into a 1.5 mL centrifuge tube, add 990 μL acetonitrile, and shake well. Test results are shown in Figure 4 Among them, the pure raw material dipeptide was tested under the same conditions, and its peak was also at about 19.1 minutes, proving that Figure 4 The peak in the middle is the raw material peak.

[0084] Comparative Example 2

[0085] The dipeptide activated ester synthesis reaction solution was tested using the conditions shown in Table 2:

[0086] Table 2: Test conditions for comparative example 2

[0087]

[0088] Instrument: Waters e2695, Waters 2489 UV detector

[0089] Detection wavelength: 214nm

[0090] Flow rate: 1.0mL / min

[0091] Column temperature: 30°C

[0092] Injection volume: 5 μL

[0093] From the test results of Example 1, it can be seen that the reaction should have been completed after 7 hours, and the residual amount of dipeptide is small, but the conventional reverse phase system (Comparative Example 1) and other systems tested by the inventors (Comparative Example 2) were used for chromatographic analysis, and an obvious dipeptide peak was still visible at 7 hours of reaction. It can be seen that the dipeptide activated ester is unstable in conventional liquid chromatography, which will cause the product to degrade and produce a large amount of raw material dipeptide, and the reaction progress cannot be correctly judged.

[0094] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for monitoring an activated esterification reaction, comprising the steps of: (1) preparing a sample solution; (2) subjecting the sample solution to liquid chromatography analysis; in, In step (1), the sample solution comprises a reaction solution of an activated esterification reaction and an organic solvent; the reaction solution comprises: a reaction raw material, a reaction product, or a combination thereof; In step (2), the monitoring conditions of the liquid chromatography analysis include: A cyano column is used as the chromatographic column; an organic solvent is used as the mobile phase; The components were detected using 200-230 nm ultraviolet light.

2. The method according to claim 1, characterized in that The activated esterification reaction is a dipeptide activated esterification reaction, the reaction raw material is a dipeptide, and the reaction product is a dipeptide activated ester. In another preferred embodiment, the activated esterification reaction is shown in the following formula: The dipeptide (Fmoc-His(Trt)-Aib-OH) reacts with the condensing agent DCC and the activating agent HOSu to generate the dipeptide activated ester (Fmoc-His(Trt)-Aib-OSu).

3. The method according to claim 1, characterized in that The organic solvent is selected from the group consisting of ethyl acetate, n-hexane, isopropanol, ethanol, acetonitrile, and combinations thereof; Furthermore, the mobile phase contains almost no water. In another preferred embodiment, the “almost free of water” means that the water content is below 1%, preferably below 0.5%, and more preferably below 0.1%.

4. The method according to claim 1, characterized in that The mobile phase is a mixed solution of acetonitrile and ethanol; Wherein, the volume ratio of acetonitrile to ethanol is 1:1 to 1:

30.

5. The method according to claim 4, characterized in that The volume ratio of acetonitrile to ethanol is 1:5 to 1:

20.

6. The method according to claim 1, characterized in that The mobile phase further comprises 0.01% to 1% of an acid, and the acid is selected from the following group: formic acid, acetic acid, trifluoroacetic acid, or a combination thereof.

7. The method according to claim 1, characterized in that The stationary phase of the chromatographic column is a cyano bonded phase.

8. The method according to claim 1, characterized in that The monitoring conditions of the liquid chromatography analysis also include: The flow rate of the mobile phase is 0.5-1 mL / min; Column temperature is 20℃~40℃; The wavelength of ultraviolet light is 210-220nm; Elution mode: isocratic elution.

9. The method according to claim 1, characterized in that The method further comprises the steps of: (3) determining the reaction progress of the activated esterification reaction by analyzing the results of liquid chromatography; If the content of the reaction raw materials is less than 10%, it indicates that the activated esterification reaction is finished. In another preferred embodiment, if the content of the reaction raw materials is less than 5%, it indicates that the activated esterification reaction is completed.

10. A kit for monitoring an activated esterification reaction, comprising: (1) An organic solvent as the mobile phase; (2) Chromatographic column; in, The organic solvent is selected from the following group: ethyl acetate, n-hexane, isopropanol, ethanol, acetonitrile, and combinations thereof; and the organic solvent has a water content of less than 1%, preferably less than 0.5%, and more preferably less than 0.1%. The chromatographic column is a cyano column.

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