A method for green cleavage of protected peptides from 2-chlorotrityl chloride resin

Abstract

The present invention relates to the technical field of polypeptide synthesis, and particularly relates to a method for green cleavage of protected peptides from 2-chlorotrityl chloride resin. The method of the present invention comprises the following steps: amino acids are sequentially linked according to the polypeptide peptide sequence on the 2-chlorotrityl chloride resin to obtain resin-bound peptide; a cleavage solution is mixed with the resin-bound peptide to effect cleavage of the protected peptide in the resin-bound peptide; wherein the cleavage solution is a weak acid solution. The weak acid solution includes any one of formic acid solution, acetic acid solution or lactic acid solution, all of which are green organic acids, and the solvent of the weak acid solution is also a green solvent. The method of the present invention has high cleavage efficiency, and can maintain the integrity of more than 98% of the peptide chain structure. At the same time, the present invention also utilizes continuous flow to cleave the resin-bound peptide, further improving the cleavage efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and particularly relates to a method for green cleavage of protected peptides from 2-chlorotrityl chloride resin. Background Art

[0002] The synthesis of long polypeptide sequences faces many challenges, especially in the field of chemical synthesis (such as solid-phase synthesis). The core bottleneck in the synthesis of long polypeptide fragments lies in the exponential decay of coupling efficiency in solid-phase synthesis, the solubility barrier caused by hydrophobic aggregation, and the complexity of disulfide bond precise pairing and post-translational modification. In addition, the purification resolution of peptide segments by RP-HPLC decreases, and the sensitivity of mass spectrometry characterization is insufficient, making it difficult to separate impurities and verify the structure.

[0003] To solve the technical bottleneck in long peptide synthesis, the fragment synthesis strategy has shown significant advantages in industrial applications: firstly, by synthesizing short peptides in segments and then connecting them directionally, the length bottleneck of the traditional solid-phase synthesis method can be effectively overcome; secondly, the characteristics of short peptides allow for the precise introduction of post-translational modifications such as phosphorylation and glycosylation at specific sites, avoiding side reaction interference in long-chain synthesis; thirdly, short peptide fragments are more likely to solve the problem of hydrophobic aggregation through solvent optimization or protecting group modification, improving the reaction homogeneity; fourthly, the modular combination mode accelerates the process of polypeptide drug structure optimization, and hundreds of functional variants can be quickly generated from a single fragment library. This technology has now become the mainstream solution for the industrial production of polypeptides with complex structures such as insulin analogs and antimicrobial peptides.

[0004] Although the fragment synthesis technology has significant advantages, it still faces key technical challenges. The core difficulty lies in the separation problem between the resin carrier and the peptide chain during the solid-phase synthesis process. Since the synthesis reaction occurs on the resin surface, it is necessary to ensure that short peptide fragments can be completely cleaved from the solid-phase carrier with a complete structure and high efficiency, while retaining specific active functional groups (such as C-terminal carboxyl or N-terminal amino group) for subsequent fragment connection. There are three main technical obstacles in this process: firstly, traditional strong acid cleavage conditions may cause the cleavage of some sensitive amino acid side chain protecting groups (such as trityl Trt); secondly, incomplete cleavage will leave peptide chain fragments bound to the resin, seriously affecting the efficiency of subsequent ligation reactions. In addition, the shaker or kettle-type resin cleavage process commonly used in traditional industrial production requires a cleavage reaction time of up to 2-3 h, resulting in an extended overall production cycle; on the other hand, after the reaction is completed, it is necessary to go through multiple cumbersome post-treatment processes, including resin filtration, ether precipitation, centrifugal separation and other operation steps. This inefficient process flow not only increases the time cost, but also easily causes product loss and quality fluctuations between batches, severely restricting the efficiency of large-scale production. The current industrial community is actively exploring innovative processes such as continuous flow reactors and automated separation systems in order to break through this production bottleneck. Summary of the Invention

[0005] Objective of the Invention: The technical problem to be solved by the present invention is to provide a method for green cleavage of protected peptides from 2-chlorotrityl chloride resin in view of the defects that the existing industrial trifluoroacetic acid cleavage system (TFA-DCM) affects the cleavage of peptide chains from the resin, and the traditional cleavage process is time-consuming and cumbersome in operation.

[0006] To solve the above technical problems, the present invention discloses a method for green cleavage of protected peptides from 2-chlorotrityl chloride resin. The present invention uses 1% v / v formic acid-DCM, 5% v / v acetic acid-DCM, 5% v / v lactic acid-DCM, and 10% v / v formic acid-anisole to cleave the protected peptide fragments from the 2-CTC resin. And a set of continuous flow circulation cleavage device is built by itself and the cleavage of peptide chains on the resin using continuous flow related technologies is verified. The specific technical solutions are as follows:

[0007] The present invention provides a method for green cleavage of protected peptides from 2-chlorotrityl chloride resin, comprising the following steps: connecting amino acids in sequence according to the polypeptide sequence on the 2-chlorotrityl chloride resin to obtain resin peptides; mixing the cleavage solution with the resin peptides to achieve the cleavage of the protected peptides in the resin peptides; wherein the cleavage solution is a weak acid solution.

[0008] Wherein, the weak acid solution includes any one of formic acid solution, acetic acid solution or lactic acid solution.

[0009] Wherein, for the weak acid solution, the solvent includes any one or a combination of more than one of dichloromethane (DCM), dimethyl carbonate (DMC), p-xylene (PX), toluene (Tol) or anisole (Ano).

[0010] Wherein, for the weak acid solution, the volume concentration of the weak acid solute is 1% - 10%.

[0011] Preferably, the weak acid solution is any one of 1% v / v formic acid-DCM, 5% v / v acetic acid-DCM, 5% v / v lactic acid-DCM or 10% v / v formic acid-Ano.

[0012] Wherein, the dosage of the cleavage solution is calculated as 5 - 15 μL of the cleavage solution is mixed per mg of resin peptides, and preferably 10 - 15 μL of the cleavage solution is mixed per mg of resin peptides.

[0013] Wherein, for the mixing, the mixing method is direct mixing or continuous flow mixing, preferably continuous flow mixing, and the continuous flow mixing is carried out according to the following method: swelling the resin peptides and then loading them into a preparation column, and circulating and pumping in the cleavage solution to achieve mixing.

[0014] Among them, in the continuous flow mixing process, the temperature of the cutting fluid is 40-50 °C, preferably 45 °C.

[0015] Among them, for the swelling treatment, the solvent used is dichloromethane, and the treatment time is 30-60 min.

[0016] Among them, for the cyclic pumping, the cutting fluid flowing through the resin peptide is repeatedly pumped into the preparation column, the pumping speed is 1-5 mL / min, and the pumping time is 30-75 min. Preferably 45-75 min.

[0017] Preferably, the preparation of the resin peptide specifically includes the following steps:

[0018] (1) Swell the 2-chlorotrityl chloride resin in dichloromethane;

[0019] (2) Dissolve the first amino acid Fmoc-AA-OH of the polypeptide peptide sequence in dichloromethane, and add N-N’ diisopropylethylamine to obtain a mixed solution;

[0020] (3) Mix the resin prepared in step (1) and the mixed solution prepared in step (2), carry out the reaction, carry out capping after the reaction ends, and add piperidine solution to remove Fmoc after the capping is completed;

[0021] (4) Repeat steps (2)-(3), and sequentially connect the Fmoc-protected amino acid groups according to the polypeptide peptide sequence to obtain the resin peptide.

[0022] Further preferably, the polypeptide includes but is not limited to any one of Pseudostellaria heterophylla peptide, thymopentin, semaglutide fragment (1-5), and tirzepatide fragment (15-21). Their peptide sequences are as follows:

[0023] Pseudostellaria heterophylla peptide: Fmoc-Gly-Phe-Ile-Pro-Pro-Leu-Gly-OH;

[0024] Thymopentin: Fmoc-Tyr(tBu)-Val-Asp(OtBu)-Lys(Boc)-Arg(Pbf)-OH;

[0025] Semaglutide fragment (1-5): Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH;

[0026] Tirzepatide fragment (15-21): Fmoc-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Aib-Ala-OH.

[0027] Beneficial effects:

[0028] The research data of the present invention show that in the process of solid-phase synthesis of polypeptides, the use of formic acid and acetic acid systems in cleaving protecting peptides exhibits significant advantages compared to traditional trifluoroacetic acid (TFA): not only is the corrosion and bioaccumulative toxicity significantly reduced, but more notably, the application effect of food-grade lactic acid, which is completely green and harmless, in resin peptide cleavage is remarkable. These green organic acids can achieve a cleavage efficiency of over 90% after a mild reaction for 2 hours, while maintaining a peptide chain structural integrity of over 98%, which far exceeds the TFA system (usually accompanied by 5 - 15% cleavage of amino acid protecting groups). In addition, progress has also been made in the green transformation of the solvent system: anisole, as a safe substitute for dichloromethane (DCM), while maintaining similar solubility, has significantly better acute toxicity and environmental impact factors than the traditional DCM solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0030] Figure 1 It is the liquid chromatogram of the cleavage of Fmoc-His(Trt)-O-2-CTC by trifluoroacetic acid (TFA), Figure 1 where A in [figure] is the liquid chromatogram of the first cleavage of Fmoc-His(Trt)-O-2-CTC using 2% v / v TFA-DCM, Figure 1 and B in [figure] is the liquid chromatogram of the cleavage of the resin after the first cleavage using the concentrated acid cutting solution TFA-Tis-H2O (volume ratio 95% TFA: 2.5% Tis: 2.5% H2O) to test the degree of incomplete cleavage on the resin after the first cleavage.

[0031] Figure 2 It is the liquid chromatogram of the cleavage of Fmoc-His(Trt)-O-2-CTC by lactic acid (LA), Figure 2 where A in [figure] is the liquid chromatogram of the first cleavage of Fmoc-His(Trt)-O-2-CTC using 5% v / v LA-DCM, Figure 2 and B in [figure] is to test the degree of incomplete cleavage on the resin after the first cleavage using the concentrated acid cutting solution TFA-Tis-H2O (volume ratio 95% TFA: 2.5% Tis: 2.5% H2O).

[0032] Figure 3 It is the mass spectrum of Fmoc-His-OH.

[0033] Figure 4 It is the 1H NMR spectrum of Fmoc-His-OH.

[0034] Figure 5 It is the mass spectrum of Trt.

[0035] Figure 6 It is the 1H NMR spectrum of Trt.

[0036] Figure 7 It is the physical diagram of the continuous flow reaction device. Description of the attached drawing reference numerals: 1 - pump module (with built-in pressure monitoring), 2 - pipeline, 3 - preparation column, 4 - rotary evaporation flask, 5 - metal sand bath magnetic stirring instrument. Specific implementation mode

[0037] In the following examples, the experimental reagents and instruments are as follows:

[0038] Pure synthetic 2-chlorotrityl chloride resin (2-CTC resin) is used, and its substitution degree is 1.211 mmol / g.

[0039] Trifluoroacetic acid (TFA), trifluoroethanol (TFE), formic acid (FA), acetic acid (AcOH), lactic acid (LA), dimethyl carbonate (DMC), triisopropylsilane (Tis), imidazole, piperidine, ammonia water, toluene and anisole are all of analytical grade.

[0040] Hydrochloric acid, dichloromethane (DCM), absolute ethanol (EtOH), N,N'-dimethylformamide (DMF) and methanol (MeOH) are all of analytical grade.

[0041] Fmoc-protected amino acid (Fmoc-AA-OH), N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBT) and N,N'-diisopropylethylamine (DIEA) are all of analytical grade.

[0042] High-purity nitrogen and high-purity hydrogen; DRX500 400MHz type and ADVANCE Ⅲ 600MHz type nuclear magnetic resonance spectrometers (NMR); Agilent 1260 type liquid chromatography-mass spectrometry combined chromatograph (LC-MS) and Agilent 1260 type high performance liquid chromatograph (HPLC); Wooking K 2025 high performance liquid chromatograph (HPLC); N-1300 type Anke Yq rotary evaporator, OSB-1200 Anke Yq water bath; DLK-2003 rapid low-temperature cooling circulator.

[0043] Example 1 Preparation of resin peptides

[0044] 1. Preparation of single amino acid resin peptides

[0045] Based on research requirements, the present invention first selects single amino acid resin peptides (Fmoc-AA-O-2-CTC) as templates. Considering the quantitative requirements and the characteristics of easy cleavage of amino acid protecting groups, the present invention has screened out two suitable single amino acid resin peptide templates:

[0046] Fmoc-Phe-O-2-CTC: The advantage of this template is the convenience of quantification. It has no side chain protecting groups. After cleavage, the characteristic peaks are monitored by reverse phase high performance liquid chromatography, and the results are more intuitive and clear, which is beneficial for subsequent analysis.

[0047] Fmoc-His(Trt)-O-2-CTC: This template contains the side chain protecting group trityl (Trt), which is extremely sensitive to acids and is prone to cleavage, meeting the considerations of the characteristics of protecting groups in the research.

[0048] The single amino acid resin peptide (Fmoc-AA-O-2-CTC) is prepared by the following method:

[0049] (1) Swell the 2-CTC resin in DCM for 1 h.

[0050] (2) Take Fmoc-AA-OH (herein referring to Fmoc-Phe-OH or Fmoc-His(Trt)-OH) in an amount three times the molar equivalent of the 2-CTC resin, dissolve it with DCM (0.5 mL / 100 mg resin), ultrasonically treat for 5 minutes, and then add DIEA in an amount 2.5 times the molar equivalent of the 2-CTC resin to this solution to obtain a mixed solution.

[0051] (3) Add the mixed solution obtained in step (2) to the swollen resin obtained in step (1), and carry out a gas bubbling reaction in a solid phase reaction kettle for 2 hours.

[0052] (4) After the reaction is completed, wash the unreacted Fmoc-AA-OH with DCM, repeat the washing three times, and finally contract the resin obtained after the reaction with methanol and store it in a dry environment to obtain Fmoc-AA-O-2-CTC. In this example, Fmoc-Phe-O-2-CTC or Fmoc-His(Trt)-O-2-CTC is obtained.

[0053] 2. Preparation of peptide fragment resin peptides

[0054] The present invention not only uses a single amino acid template for testing, but also applies the research results to complete sequence peptide fragments. Screening the sequence peptide template remains crucial and needs to meet multiple conditions: first, an amino acid template without amino acid side chain protection; second, a template covering as many types of amino acid side chain protecting groups as possible; third, a template containing the trityl (Trt) protecting group; fourth, a popular peptide that fits the current research hotspot. Based on the above conditions, the following four peptide fragments were selected:

[0055] Taizishen peptide: This peptide segment does not contain any side chain protecting groups and has a relatively simple structure, which is convenient for observing basic reactions in specific research situations. Its sequence is: Fmoc-Gly-Phe-Ile-Pro-Pro-Leu-Gly-OH.

[0056] Thymopentin: It contains various amino acid side chain protecting groups and can provide diverse samples for studying the roles of different protecting groups in reactions. Its sequence is: Fmoc-Tyr(tBu)-Val-Asp(OtBu)-Lys(Boc)-Arg(Pbf)-OH.

[0057] Semaglutide fragment (1-5): It contains the trityl (Trt) protecting group, which helps to conduct in-depth research on the characteristics of the Trt protecting group. Its sequence is: Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH.

[0058] Tirzepatide fragment (15-21): As a current popular therapeutic drug peptide, its research can closely follow the forefront of pharmaceutical research directions and contribute to the development of related fields. Its sequence is: Fmoc-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Aib-Ala-OH.

[0059] The above peptide fragments were prepared into corresponding resin peptides and obtained according to the following method:

[0060] (1) Swell 2-CTC resin in DCM for 2 h.

[0061] (2)Connect amino acids to the resin in sequence according to the peptide sequence of the peptide fragment as follows: Take the first amino acid Fmoc-AA-OH on the peptide fragment to be synthesized (the first amino acid of the Pseudostellaria heterophylla peptide is Fmoc-Gly-OH, the first amino acid of thymopentin is Fmoc-Tyr(tBu)-OH, the first amino acid of the semaglutide fragment is Fmoc-His(Trt)-OH, and the first amino acid of the tirzepatide fragment is Fmoc-Asp(tBu)-OH). The amount of Fmoc-AA-OH is 3 times the molar equivalent of the 2-CTC resin, and it is dissolved in DCM (0.5 mL / 100 mg resin), ultrasonicated for 5 minutes, and then 2.5 times the molar equivalent of DIEA of the 2-CTC resin is added to this solution to obtain a mixed solution, which is added to the swollen resin obtained in step (1), and bubbled with gas in a solid-phase reaction kettle for 2 hours.

[0062] (3)After the reaction in step (2) is completed, it is blocked with a blocking solution (the volume ratio of DCM: methanol: DIEA is 80:15:5), blocked twice in total, 30 minutes each time. After blocking, it is washed three times with DMF, and then the freshly prepared 20% v / v piperidine-DMF solution is used to remove Fmoc. After that, steps (2) - (3) can be repeated to couple the next Fmoc-AA-OH in sequence according to the peptide fragment sequence. The subsequent peptide sequences of the 4 peptide fragments synthesized in this example are as follows:

[0063] Pseudostellaria heterophylla peptide: Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Gly-OH.

[0064] Thymopentin: Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH.

[0065] Semaglutide fragment (1 - 5): Fmoc-Aib-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH.

[0066] Tirzepatide fragment (15 - 21): Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, Fmoc-Ala-OH.

[0067] Couple Fmoc-AA-OH in sequence according to the above peptide sequence until the desired peptide fragment resin peptide is obtained.

[0068] After the reaction of all the above peptide sequences is completed, the unreacted Fmoc-AA-OH is washed with DCM, and the washing is repeated three times. Finally, methanol is used to shrink the resin, and it is stored in a dry environment to obtain the peptide fragment resin peptide.

[0069] Example 2 Cleavage of resin peptide by traditional method

[0070] Prepare the cleavage solution, add the single amino acid resin peptide (50 mg) prepared in Example 1 into the cleavage solution (0.5 mL), then close the container lid and seal it with a sealing film. Place it in a shaker and shake it at 40 °C for 120 min. After the reaction is completed, use absorbent cotton for solid-liquid separation to separate the filtrate from the resin. Add ammonia water to the filtrate for quenching. Then, use a rotary evaporator, or the airflow generated by a hair dryer, or nitrogen gas flow to dry the cleavage solution to constant weight. Then add 1 mL of methanol to dissolve it fully, and add 100 μL of the internal standard (Fmoc-Ala-OH). The treated solution is monitored and analyzed by reversed-phase high-performance liquid chromatography, and the internal standard peak area V is recorded. 内标 And the characteristic peak area V of the product after 2 h of reaction 2h。

[0071] Monitoring of by-products: For example, Fmoc-His(Trt)-OH, the dropped Trt and Fmoc-His-OH should be in an equal amount of 1:1. However, since the ultraviolet absorption intensity of Trt is different from that of amino acids, only the characteristic peak area of Fmoc-His-OH after dropping the Trt amino acid protecting group is recorded to avoid calculation errors, and the area V is recorded. 2h副。

[0072] Perform secondary cleavage on the resin part obtained by solid-liquid separation with absorbent cotton. In the resin after the primary separation, add the concentrated acid cleavage solution TFA-Tis-H2O (volume ratio 95% TFA: 2.5% Tis: 2.5% H2O). The ratio of the added volume mL of the concentrated acid cleavage solution to the resin mass g is 20:1. Also place it in a shaker and react at 40 °C for 2 hours. After the reaction is completed, filter the solution with absorbent cotton, dry the filtrate to constant weight, and then add the same amount of methanol and internal standard (Fmoc-Ala-OH) as used after the primary cleavage. Also use a reversed-phase high-performance liquid chromatograph, inject the sample according to the above analysis method, monitor the characteristic peaks, and record the internal standard peak area V this time. 内标 And the characteristic peak area V of the product after 2 h of secondary cleavage 2h* . Similarly, record the characteristic peak area V of the by-products after secondary cleavage. 2h副*。

[0073] Based on the data obtained from the above experiments and substituting them into specific formulas, the cleavage efficiency of this system within 2 hours of the first cleavage can be calculated. The calculation formulas are as shown in (1) to (3) below:

[0074]

[0075]

[0076]

[0077] In this study, the reversed-phase high-performance liquid chromatography area normalization method was used to achieve quantitative analysis by monitoring the characteristic peak area, and the internal standard method was introduced to effectively reduce experimental errors. The high-performance liquid chromatography conditions were as follows: mobile phase A: 0.08% TFA methanol, mobile phase B: 0.08% TFA pure water, linear gradient 5% A - 100% A for 10 min, and 100% B for 5 min.

[0078] The present invention screened the cutting fluid to screen green organic acids and green solvents that can dissociate Fmoc-Phe-OH and the template Fmoc-His(Trt)-OH with a protecting group from the 2-CTC resin. The cutting fluid described in the present invention is composed of a green organic acid and a solvent. The green organic acid is any one of formic acid (FA), acetic acid (AcOH), or lactic acid (LA), and the solvent is any one of dichloromethane (DCM), dimethyl carbonate (DMC), p-xylene (PX), toluene (Tol), or anisole (Ano). The formulation of the cutting fluid and its cleavage efficiency are shown in Table 1 and Table 2. At the same time, a 2% v / v trifluoroacetic acid (TFA)-DCM solution was used as the cutting fluid control group. The liquid phase chromatograms of the products obtained by cleaving Fmoc-His(Trt)-O-2-CTC with different cutting fluids are as Figure 1 and Figure 2 shown, Figure 1 in which A is the chromatogram when 2% v / v TFA-DCM is used as the cutting fluid in the control group, Figure 1 in which B is the product chromatogram after the secondary cleavage of the resin obtained by treating with TFA-Tis-H2O (volume ratio 95% TFA: 2.5% Tis: 2.5% H2O) after the first cleavage, Figure 1 in which, the products corresponding to different retention times are:

[0079] 5.668 min: Fmoc-His-OH, 6.864 min: internal standard Fmoc-Ala-OH, 7.584 min: Trt, 8.747 min: Fmoc-His(Trt)-OH.

[0080] Figure 2In which, A is the chromatogram of Fmoc-Phe-O-2-CTC cleaved by 5% v / v LA-DCM, Figure 2 In which, B is the chromatogram of the product after secondary cleavage of the resin obtained by treating the resin after the first cleavage with TFA-Tis-H2O (volume ratio 95% TFA: 2.5% Tis: 2.5% H2O), Figure 2 In it, the products corresponding to different retention times are:

[0081] 5.668 min: Fmoc-His-OH, 6.864 min: internal standard Fmoc-Ala-OH, 7.584 min: Trt, 8.747 min: Fmoc-His(Trt)-OH.

[0082] In addition, the present invention also performs mass spectrometry analysis on Fmoc-His-OH (as Figure 3 shown) and nuclear magnetic resonance hydrogen spectrum analysis, as Figure 4 shown, and the nuclear magnetic resonance hydrogen spectrum information is as follows: 1H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.6 Hz, 2H), 7.72 – 7.55 (m, 4H), 7.42 (t, J = 7.5 Hz, 2H), 7.32 (td, J = 7.5, 3.1 Hz, 2H), 6.86 (s, 1H), 4.23 (td, J = 8.1, 7.0, 4.3 Hz, 4H), 2.99 (dd, J = 14.7, 4.8 Hz, 1H), 2.88 (dd, J = 14.7, 9.1 Hz, 1H).

[0083] Figure 5 And Figure 6 are the mass spectrometry and nuclear magnetic resonance hydrogen spectrum analysis of the Trt group. The nuclear magnetic resonance hydrogen spectrum information of the Trt group is as follows: Trt: 1H NMR (400 MHz, Chloroform-d) δ 7.28 (d, J = 1.4 Hz, 1H), 7.27–7.23 (m, 5H), 7.23–7.15 (m, 3H), 7.14–7.03 (m, 6H), 5.54 (s, 1H).

[0084] Table 1 Template Fmoc-Phe-O-2-CTC Cleavage Test

[0085]

[0086] Table 2 Template Fmoc-His(Trt)-O-2-CTC Cleavage Test

[0087]

[0088] As shown in Table 2, when traditional trifluoroacetic acid (TFA) is used to cleave Fmoc-His (Trt)-O-2-CTC, the protecting groups of amino acid side chains will break, which will seriously damage the integrity of the peptide chain. As expected, the reaction product should be Fmoc-His (Trt)-OH, but due to the breakage of the side chain protecting group, by-products Trt and Fmoc-His-OH will be generated. The generation of these by-products greatly reduces the purity of the target product. The purpose of cleaving the protected peptide fragment is to prepare for the subsequent fragment synthesis. If the purity of the pre-product is poor, it will not only reduce the purity of the subsequent fragment condensation product, but also make the post-treatment process more complicated.

[0089] Then, the complete peptide fragment resin peptides prepared in Example 1 were used for cleavage tests with the above cleavage system. The cleavage method was the same as that of the single amino acid resin peptide described above, and different solvents were used for testing. The complete peptide fragments included: Pseudostellaria heterophylla peptide, semaglutide fragment (1-5), thymopentin, and tirzepatide (15-21). The test results are shown in Table 3.

[0090] Table 3 Peptide Fragment Resin Peptide Cleavage Test

[0091]

[0092] Example 3 Continuous Flow Circulation Cleavage of Resin Peptides

[0093] For the construction of the continuous flow circulation cleavage experimental device in this example, the preparation instrument of Boyun Biotechnology Co., Ltd. was selected. This instrument integrates a pump module for flow rate adjustment, a pressure monitoring module, and an ultraviolet detection module, greatly streamlining the device structure, reducing space occupancy, and improving operation convenience. Each module operates in coordination to ensure the high efficiency of experimental data monitoring and control. The experiment uses a metal bath as the heating module, which can accurately control the temperature and provide a stable thermal environment for the reaction. In addition, a preparative chromatography column (preparation column) is equipped as the reaction module, and the continuous flow reaction device is as shown Figure 7 shown.

[0094] Given that the total volume of the pump body and the pipeline is 20 mL, the amount of cutting fluid used needs to exceed 20 mL. The specific steps are as follows: First, swell 2 g of resin peptide template with DCM (10 mL) for (30 - 60 min), and then load it into the preparation column 3 (10 mm × 100 mm). Take a section of pipeline 2, connect one end to the pump module (with built-in pressure monitoring) 1, and hang the other end vertically into the rotary evaporation flask 4 placed in the metal sand bath magnetic stirring instrument 5 through an iron stand (as Figure 7 shown). Set the temperature of the metal sand bath magnetic stirring instrument 5 to 45 °C, and add 30 mL of formic acid - DCM (volume ratio 1:99) solution (the system to be measured) to the rotary evaporation flask 4. The inlet pipeline of the pump module (with built-in pressure monitoring) 1 is also placed in this rotary evaporation flask 4, and set the flow rate (1 - 5 mL / min) (the pressure should not exceed the upper limit, and the flow rate in this embodiment is 5 mL / min). The operation mode of the entire flow path is that the pump module (with built-in pressure monitoring) 1 pumps the cutting fluid in the rotary evaporation flask 4 to the preparation column 3, and the solution drops back into the rotary evaporation flask 4 after flowing through the preparation column 3, and so on in a cycle for a certain period of time (30 - 120 min). After reacting for 1 h and 2 h with different batches of resin peptides, the solutions are rotary evaporated respectively to remove the solvent, then diluted with methanol, and then monitored by reverse-phase high-performance liquid chromatography, and the corresponding characteristic peak areas are recorded. Similarly, the resin after the first cleavage is subjected to secondary cleavage according to the method described in Example 2, and finally the cleavage efficiency is calculated in the same way as in Example 2. Table 4 shows the cutting of the Fmoc-Phe-OH template using the 1% v / v FA-DCM cutting fluid according to the method described in Example 2, with a cutting temperature of 45 °C and a time of 30 - 120 min. Tables 5 and 6 show the continuous flow cyclic cleavage of the Fmoc-Phe-OH template and the Pseudostellaria heterophylla peptide using the method of the present invention respectively.

[0095] Table 4 Traditional cleavage test of Fmoc-Phe-OH template

[0096]

[0097] Table 5 Continuous flow cleavage test of Fmoc-Phe-OH template

[0098]

[0099] Table 6 Continuous flow cleavage test of Pseudostellaria heterophylla peptide template

[0100]

[0101] In addition, the present invention further confirms that the above cleavage method has significant specificity for 2-CTC resin and poor cutting efficiency for other resins (such as Siber resin).

[0102] The present invention provides an idea and method for green cleavage of protected peptides from 2-chlorotrityl chloride resin. There are many methods and ways to specifically implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A method for green cleavage of protected peptides from 2-chlorotrityl chloride resin, characterized in that, It includes the following steps: On the 2-chlorotrityl chloride resin, amino acids are sequentially linked according to the polypeptide sequence to obtain a resin peptide; a cleavage solution is mixed with the resin peptide to achieve cleavage of the protected peptide in the resin peptide; wherein the cleavage solution is a weak acid solution; Among them, for the mixing, the mixing method is continuous flow mixing, and the continuous flow mixing is carried out according to the following method: the resin peptide is loaded into a preparation column after swelling treatment, and the cleavage solution is pumped in a cycle to achieve mixing; the weak acid solution is selected from any one of formic acid solution, acetic acid solution or lactic acid solution; when the weak acid solution is formic acid solution, its solvent is any one of dichloromethane or anisole, and when the weak acid solution is acetic acid solution or lactic acid solution, its solvent is dichloromethane.

2. The method according to claim 1, wherein In the weak acid solution, the volume concentration of the weak acid solute is 1% - 10%.

3. The method according to claim 1, characterized in that, The dosage of the cleavage solution is calculated as 5 - 15 μL of the cleavage solution is mixed per mg of the resin peptide.

4. The method according to claim 1, wherein During the continuous flow mixing process, the temperature of the cleavage solution is 40 - 50 °C.

5. The method according to claim 1, characterized in that, For the swelling treatment, the solvent used is dichloromethane, and the treatment time is 30 - 60 min.

6. The method according to claim 1, characterized in that For the cyclic pumping, the cleavage solution flowing through the resin peptide is repeatedly pumped into the preparation column, and the pumping speed is 1 - 5 mL / min, and the pumping time is 30 - 75 min.

Images