A method for liquid phase synthesis of telopeptides

Abstract

The application discloses a method for synthesizing telopeptide in liquid phase, which comprises the following steps: sequentially coupling amino acids under the assistance of a carrier PTESE to respectively prepare a first peptide segment, a second peptide segment and a third peptide segment; sequentially coupling amino acids under the assistance of a carrier BTPM to prepare a fourth peptide segment; coupling the first peptide segment and the second peptide segment to obtain a fifth peptide segment; coupling the third peptide segment and the fourth peptide segment to obtain a sixth peptide segment; coupling the fifth peptide segment and the sixth peptide segment to obtain telopeptide; compared with a solid-phase polypeptide synthesis (SPPS) process and a traditional liquid-phase polypeptide synthesis (LPPS) process, the CF-LPPS (carrier-assisted continuous flow liquid-phase polypeptide synthesis) process has obvious advantages in single coupling-deprotection cycle reaction time, reagent equivalent, crude product purity, impurity removal mode and amplification potential.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis technology, specifically relating to a method for liquid-phase synthesis of telpoeptide. Background Technology

[0002] Tirzepatide is a long-acting glucose-dependent insulinotropic peptide receptor / glucagon-like peptide-1 receptor dual agonist. Its backbone consists of 39 amino acid residues, with a fatty acid-modified side chain attached to the lysine residue at position 20. Compared to conventional short peptides, tirzepatide has a longer sequence, containing multiple functional groups such as hydroxyl, carboxyl, amide, and indole, as well as sterically hindered non-natural amino acids like Aib and fatty acid side chains. Therefore, the synthesis of tirzepatide requires addressing challenges such as long-chain peptide coupling efficiency, introduction of sterically hindered amino acids, fatty acid side chain integration, orthogonal compatibility of protecting groups, control of deletion impurities, and large-scale green manufacturing.

[0003] Currently, telpoide and similar GLP-1 long peptide drugs are mainly prepared using solid-phase peptide synthesis. Solid-phase peptide synthesis uses resin as a carrier and achieves peptide chain extension through repeated cycles of "deprotection-washing-coupling-washing," offering advantages such as mature operation and high automation. However, for long-chain complex peptides like telpoide, solid-phase synthesis has significant limitations: Firstly, the peptide chain is fixed inside the resin, and the reaction is greatly affected by resin swelling, pore diffusion, and mass transfer limitations. As the number of synthesis steps increases, incomplete coupling or deprotection at any step can lead to the formation of missing peptides, truncated peptides, duplicate peptides, or residual protecting groups, which accumulate in subsequent steps. Secondly, to improve the coupling conversion rate of long-chain resin-bound peptides, especially the incorporation efficiency of sterically hindered amino acids such as Aib, a large excess of protecting amino acids and a strongly activated condensation system are usually required, leading to increased raw material costs, impurity risks, and waste liquid burden. In addition, solid-phase synthesis relies heavily on solvents or reagents such as DMF, NMP, DCM, piperidine, and TFA, resulting in high material consumption and significant environmental pressure. Furthermore, its intermediates are difficult to sample, characterize, and quality control as effectively as liquid-phase products.

[0004] To overcome the limitations of mass transfer and the inseparability of intermediates in solid-phase synthesis, existing techniques have attempted to prepare long-chain peptides using liquid-phase fragment condensation strategies. Liquid-phase synthesis can first prepare several fully protected peptide fragments, and then obtain the target long peptide through inter-fragment condensation. This strategy has advantages such as homogeneous reaction, characterizable fragments, and controllable local impurities, and is theoretically suitable for the segmented synthesis of long-sequence peptides like telpoide. However, traditional liquid-phase fragment synthesis also faces difficulties: as peptide chain length increases, fully protected peptide fragments have large molecular weights, strong hydrophobicity, and complex conformations, resulting in insufficient solubility and reaction homogeneity in conventional organic solvents; problems such as low reaction rates, decreased terminal group activation efficiency, aggregation, and epimerization may also occur during fragment condensation. In particular, telpoide contains multiple acid-sensitive and fatty acid side-chain protecting groups, and intermediate fragments need to continue condensation while maintaining full protection. How to achieve fragment preparation, separation, and transformation without destroying the side-chain protecting groups is the key to the viability of the liquid-phase route.

[0005] Hydrophobic carrier-assisted liquid-phase peptide synthesis offers a possible solution to the aforementioned problems. This technique involves attaching a hydrophobic carrier to the C-terminus of the peptide chain, allowing peptide intermediates to accumulate in the organic phase. Excess amino acids, condensation agent byproducts, deprotection byproducts, and other small molecule impurities can be removed through extraction, washing, or precipitation, thus combining the advantages of solid-phase synthesis (facilitating impurity removal) and liquid-phase synthesis (facilitating characterization). However, existing hydrophobic carrier-assisted techniques are still difficult to directly apply to complex long peptides like thiopeptides. Some hydrophobic carriers require strong acid conditions for removal, which can easily damage acid-sensitive protecting groups such as tBu, Boc, and Trt. While some silicon-based carriers can be removed with fluorine reagents, they may have limitations in removal efficiency, fluorine reagent dosage, carrier UV response, and process monitoring during long peptide fragment preparation. A single carrier system also struggles to simultaneously achieve mild removal at the fragment stage, complete deprotection, protecting group compatibility, and purification convenience.

[0006] Continuous flow reaction technology boasts high mass and heat transfer efficiency, precise residence time control, good safety, and ease of modular scale-up, and has been applied to processes such as amide bond formation, protecting group removal, hydrogenation reactions, and online extraction. For peptide synthesis, continuous flow technology holds promise for shortening coupling and deprotection times, reducing reagent equivalents, and achieving process integration through continuous extraction, membrane separation, and fixed-bed catalysis. However, existing continuous flow peptide synthesis methods are mostly concentrated in solid-phase systems, still struggling to address resin mass transfer limitations and the inability to adequately characterize intermediates. Existing continuous flow liquid-phase synthesis methods are mostly limited to short peptides or single-step reactions, and a systematic process encompassing "coupling—deprotection—impurity removal—phase separation—fragmentation—fragment assembly" suitable for the preparation of the full-sequence thiopeptide has not yet been established.

[0007] In summary, existing telpoeptide synthesis technologies still have the following shortcomings: traditional solid-phase synthesis consumes a large amount of reagents and solvents, accumulates significant missing impurities, and has insufficient quality control of intermediates; traditional liquid-phase fragment synthesis is limited by the solubility, separation and purification, and compatibility of protecting groups of long peptide fragments; existing hydrophobic carrier-assisted technologies cannot simultaneously meet the requirements of mild deprotection at the fragment stage and final complete deprotection; existing continuous flow technologies have not yet achieved a systematic integration of carrier-assisted continuous liquid-phase preparation, online impurity removal, fragment assembly, and final deprotection for the entire telpoeptide sequence.

[0008] Therefore, there is an urgent need in this field to develop a liquid-phase synthesis method suitable for telpoide, which can prepare key fully protected peptide fragments while maintaining the stability of side chain protecting groups. By employing orthogonal carrier design, continuous flow coupling / deprotection, online extraction and separation, and fragment condensation strategies, the method can reduce impurity accumulation and material consumption in the stepwise synthesis of long chains, improve process controllability, crude peptide quality, and scale-up applicability, thereby providing a new technical route for the green and efficient manufacturing of telpoide and similar long-acting peptide drugs. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a method for liquid-phase synthesis of telpoeptide, which addresses the shortcomings of the prior art.

[0010] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0011] A method for liquid-phase synthesis of telpoeptide includes the following steps:

[0012] Step 1: With the assistance of the vector PTESE, amino acids are coupled to obtain a fully protected first peptide fragment, a fully protected second peptide fragment with the N-terminal protecting group removed, and a fully protected third peptide fragment, respectively; with the assistance of the vector BTPM, amino acids are coupled sequentially to obtain a fully protected fourth peptide fragment with the N-terminal protecting group removed.

[0013] Step 3: After removing the PTESE vector from the fully protected fifth peptide fragment, couple it with the fully protected sixth peptide fragment after removing the N-terminal protecting group to obtain the fully protected telpoeptide. Remove the BTPM vector and all protecting groups to obtain telpoeptide.

[0014] Wherein, the first peptide fragment is positions 1 to 14 of the telposide backbone sequence from the N-terminus to the C-terminus; the second peptide fragment includes positions 15 to 21 of the telposide backbone sequence from the N-terminus to the C-terminus and a telposide side chain coupled to the lysine residue at position 20 of the telposide backbone sequence via an amide bond; the third peptide fragment is positions 22 to 29 of the telposide backbone sequence from the N-terminus to the C-terminus; and the fourth peptide fragment is positions 30 to 39 of the telposide backbone sequence from the N-terminus to the C-terminus.

[0015] The chemical structural formula of the carrier PTESE is shown in Formula I:

[0016]

[0017] Formula I.

[0018] The carrier PTESE was synthesized according to the following method:

[0019] Methyl phenylacetate (30.0 g, 200 mmol) was added to a pre-dried three-necked flask equipped with a magnetic stirrer under nitrogen protection at room temperature. 400 mL of anhydrous acetonitrile (MeCN) was added to dissolve it, followed by the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.5 eq, 45.7 g, 300 mmol). The mixture was stirred at room temperature for 10 min. Then, 4-acetamidobenzenesulfonyl azide (1.5 eq, 72.1 g, 300 mmol) was added. The reaction mixture was stirred at room temperature for 2 h in the dark. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was dissolved in 400 mL of ethyl acetate (EA), and the reaction mixture was transferred to a separatory funnel and washed three times with an equal volume of saturated NaCl aqueous solution. The organic phase was dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated to dryness. The residue was purified by rapid column chromatography (ethyl acetate: petroleum ether = 1:12, v / v) to give an orange-red oily compound 3 (32.5 g, yield 92.2%).

[0020] Under nitrogen protection, copper(I) tetraacetonitrile hexafluorophosphate (0.06 eq, 4.1 g, 11.1 mmol) was added to a pre-dried three-necked flask equipped with a magnetic stirrer, and dissolved in 50 mL of anhydrous DCM. Triethylsilane (3 eq, 64.3 g, 553.2 mmol) was then added. The reaction mixture was cooled to -40 °C, and an anhydrous DCM solution (350 mL) of compound 3 (32.5 g, 184.4 mmol) was slowly added dropwise. After the addition was complete, the temperature was raised to -10 °C, and stirring was continued for 4 h. After the reaction was complete, the solvent was evaporated to dryness. The residue was purified by rapid column chromatography (ethyl acetate: petroleum ether = 1:40, v / v) to give a colorless oily compound 4 (43.1 g, yield 88.4%).

[0021] Under nitrogen protection, compound 4 (43.1 g, 163.0 mmol) obtained in the previous step was added to a pre-dried three-necked flask equipped with a magnetic stirrer, and dissolved in 100 mL of anhydrous tetrahydrofuran (THF). The reaction system was cooled to 0 °C, and a THF solution of AlLiH4 (1 eq, 65.2 mL, 2.5 M, 163 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and the reaction was continued for 2 h. After the reaction was complete, the reaction solution was cooled to 0 °C under nitrogen protection and stirred thoroughly. First, 3.6 mL of water was added dropwise very slowly with vigorous stirring. After the addition was complete, 3.6 mL of 150 g / L NaOH aqueous solution was slowly added, and finally 10.8 mL of water was added. After the addition was complete, the ice bath was removed, and the mixture was warmed to room temperature and stirred for 15–30 min until a white granular suspension was formed. The suspension was filtered through a diatomaceous earth filter, the filter cake was washed with solvent, the organic phases were combined, and the solvent was evaporated to dryness. The residue was dissolved in 100 mL of EA and transferred to a separatory funnel. The mixture was washed twice with equal volumes of saturated NH4Cl aqueous solution and once with saturated NaCl aqueous solution. The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated to dryness. The residue was purified by rapid column chromatography (EA:PE = 1:16, v / v) to give a colorless oily compound 5 (36.2 g, yield 94.2%), i.e., the carrier PTESE.

[0022] The synthetic route of the carrier PTESE is as follows: Figure 1 As shown.

[0023] The chemical structural formula of the carrier BTPM is shown in Formula II:

[0024]

[0025] Formula II.

[0026] The carrier BTPM and its preparation method have been disclosed in patent CN 118561901 A.

[0027] The chemical structural formula of the telpoeptide side chain is shown in Formula III:

[0028]

[0029] Formula III.

[0030] In step 1, the fully protected first peptide fragment, the fully protected second peptide fragment with the N-terminal protecting group removed, the fully protected third peptide fragment, and the fully protected fourth peptide fragment with the N-terminal protecting group removed are all synthesized using a continuous flow peptide liquid phase synthesis system.

[0031] The continuous flow polypeptide liquid phase synthesis system includes a coupling unit, an Fmoc deprotection unit, a Cbz deprotection unit, a continuous extraction unit, and a liquid-liquid separator; the coupling unit is equipped with a first microchannel reactor; the Fmoc deprotection unit is equipped with a second microchannel reactor; the Cbz deprotection unit is equipped with a fixed-bed reaction column and a T-type gas-liquid mixer; and the continuous extraction unit is equipped with a third microchannel reactor.

[0032] The outlet of the first microchannel reactor is connected to the inlet of the third microchannel reactor; the outlet of the third microchannel reactor is connected to the inlet of the liquid-liquid separator; the outlet of the liquid-liquid separator is connected to the inlet of the second microchannel reactor, the inlet of the T-type gas-liquid mixer, and the inlet of the first microchannel reactor, respectively; the outlet of the T-type gas-liquid mixer is connected to the inlet of the fixed-bed reaction column; the outlet of the fixed-bed reaction column and the outlet of the second microchannel reactor are respectively connected to the inlet of the third microchannel reactor.

[0033] In some embodiments, the coupling unit is further provided with two plunger pumps, a pressure sensor, a flow meter, and a temperature sensor. The plunger pumps are used to pump reaction raw materials into the first microchannel reactor. The pressure sensor, flow meter, and temperature sensor are used to monitor the pressure in the first microchannel reactor, the flow rate of the raw materials pumped into the first microchannel reactor, and the temperature in the first microchannel reactor in real time, respectively.

[0034] In some embodiments, the Fmoc deprotection unit is further provided with two plunger pumps, a pressure sensor, a flow meter, and a temperature sensor. The plunger pumps are used to pump the reaction raw materials into the second microchannel reactor. The pressure sensor, flow meter, and temperature sensor are used to monitor the pressure in the second microchannel reactor, the flow rate of the reaction raw materials pumped into the second microchannel reactor, and the temperature in the second microchannel reactor in real time, respectively.

[0035] In some embodiments, the Cbz deprotection unit is further provided with a constant flow pump and a pressure sensor, which are used to pump the reaction raw materials into the fixed bed reaction column and monitor the pressure inside the fixed bed reaction column, respectively.

[0036] In some embodiments, the continuous extraction unit is further provided with two pumps for pumping extraction feedstock into the third microchannel reactor.

[0037] In some embodiments, the liquid-liquid separator is a Corning Zaiput high-efficiency liquid-liquid separator SEP-10, which is provided with a hydrophobic PTFE membrane.

[0038] The method for synthesizing the fully protected first peptide fragment, the fully protected second peptide fragment (with the N-terminal protecting group removed), or the fully protected third peptide fragment includes the following steps:

[0039] Step a: Dissolve the carrier PTESE and the amino acid unit corresponding to the first position of the C-terminus of the peptide fragment sequence in 2-methyltetrahydrofuran (2-MeTHF), add a condensing agent, and carry out a first condensation reaction to obtain a first condensation reaction solution. Extract, collect the first organic phase, separate and purify to obtain the conjugate X-AA-PTESE of the amino acid unit and the carrier PTESE. Dissolve it in ethyl acetate to obtain an X-AA-PTESE solution. Pump the X-AA-PTESE solution and the first lysis buffer into the second microchannel reactor of the Fmoc deprotection unit to carry out a first deprotection reaction to obtain a first deprotection reaction solution. Pump the first deprotection reaction solution and the extractant into the third microchannel reactor of the continuous extraction unit to carry out a first continuous extraction to obtain a first extract. Pump the extract into the liquid-liquid separator for liquid-liquid phase separation to obtain a second organic phase.

[0040] Step b: The solution of the second organic phase and the amino acid unit corresponding to the second position of the C-terminus of the peptide fragment sequence is pumped into the first microchannel reactor of the coupling unit to carry out the second condensation reaction, and the second condensation reaction solution is obtained. The second condensation reaction solution and the extractant are pumped into the third microchannel reactor of the continuous extraction unit to carry out the second continuous extraction, and the second extract is obtained. The extract is then pumped into the liquid-liquid separator for liquid-liquid phase separation to obtain the third organic phase.

[0041] Step c: The third organic phase and the first pyrolysis solution are pumped into the second microchannel reactor of the Fmoc deprotection unit to carry out the second deprotection reaction, and the second deprotection reaction solution is obtained. The second deprotection reaction solution and the extractant are pumped into the third microchannel reactor of the continuous extraction unit to carry out the third continuous extraction, and the third extract is obtained. The extract is then pumped into the liquid-liquid separator for liquid-liquid phase separation to obtain the fourth organic phase.

[0042] Step d: Replace the second organic phase in step b with the fourth organic phase, and replace the solution of the amino acid unit corresponding to the second position of the C-terminus of the peptide fragment sequence in step b with a solution of the amino acid unit corresponding to the next position after the C-terminus of the peptide fragment sequence. Repeat steps b and c until all amino acid units corresponding to the peptide fragment sequence are sequentially coupled. In the process of synthesizing the fully protected first or third peptide fragment, when the amino acid unit corresponding to the next position after the C-terminus of the peptide fragment sequence is the last amino acid unit corresponding to the C-terminus of the peptide fragment sequence, only step b is executed, and step c is not executed. Finally, a fully protected second peptide fragment with the N-terminal protecting group removed is obtained in the fourth organic phase, or a fully protected first or third peptide fragment is obtained in the third organic phase.

[0043] Wherein, the amino acid unit is a protected amino acid with a free α-carboxyl group; except for the amino acid unit corresponding to the first position of the N-terminus of the fully protected first peptide fragment sequence, whose α-amino group is Boc and the amino acid unit corresponding to the first position of the N-terminus of the fully protected third peptide fragment sequence, whose α-amino group is Cbz, the α-amino group of all other amino acid units is Fmoc.

[0044] In the process of synthesizing the fully protected second peptide fragment after removing the N-terminal protecting group, the amino acid unit corresponding to the second position of the C-terminus of the peptide fragment sequence is Fmoc-Lys[C20-Y-Glu(Z)-AEEA-AEEA]-OH, where Y is the protecting group of eicosanoic acid and Z is the protecting group of Glu.

[0045] In some embodiments, during the synthesis of a fully protected second peptide fragment with the N-terminal protecting group removed, the amino acid unit corresponding to the second C-terminal position of the peptide fragment sequence is Fmoc-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-OH. The Fmoc-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-OH is commercially available.

[0046] The solution of the amino acid unit is prepared by dissolving the amino acid unit in an EA / DMSO solution obtained by mixing ethyl acetate and dimethyl sulfoxide at a volume ratio of 4:1, adding a condensing agent for pre-activation for 5-20 min. Except for the amino acid unit corresponding to the 3rd position of the C-terminus of the first peptide fragment sequence, which uses N,N-diisopropylethylamine and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate as the condensing agent, the other amino acid units use 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl). The molar ratio of the amino acid unit, 1-hydroxybenzotriazole, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1~1.1):(1~ 1.1); The molar ratio of the amino acid unit corresponding to the 3rd position of the C-terminus of the first peptide fragment sequence, N,N-diisopropylethylamine, and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:3:1.2; The solid-liquid ratio of the amino acid unit to the EA / DMSO solution is 4~5 mmol:20 mL.

[0047] In some embodiments, when preparing a solution of the amino acid unit corresponding to the second C-terminus of the second peptide fragment sequence, the pre-activation time is 20 min.

[0048] In step a, the condensing agent is 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; the molar ratio of the carrier PTESE, amino acid unit, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.2:0.1:1.2; the solid-liquid ratio of the carrier PTESE to the 2-methyltetrahydrofuran is 0.3 mol:1 L; the first condensation reaction is carried out under the following conditions: stirring at 50°C for 2 hours; the extraction uses a 50% extractant. The volume ratio of the extractant to the first condensation reaction solution is 1:1. The separation and purification include the following steps: the first organic phase is dried with anhydrous sodium sulfate and filtered, the filtrate is collected, the organic solvent 2-methyltetrahydrofuran is removed by rotary evaporation, and the conjugate is purified by column chromatography to obtain the conjugate X-AA-PTESE. The concentration of X-AA-PTESE in the X-AA-PTESE solution is 0.195 M.

[0049] In step a, the first lysis buffer is prepared by dissolving sodium 3-mercapto-1-propanesulfonate (Mps) in a solution of diethylamine (DEA); the diethylamine solution has a volume fraction of 20%, and its solvent is a mixture of ethyl acetate and dimethyl sulfoxide (DMSO) in a volume ratio of 3:1; the solid-liquid ratio of sodium 3-mercapto-1-propanesulfonate to the diethylamine solution is 11.7 mmol : 20 mL.

[0050] In step a, the flow rates of the X-AA-PTESE solution and the first lysis buffer pumped into the second microchannel reactor are both 10.0 mL / min; in the second microchannel reactor, the residence time of the first deprotection reaction is 9.0 s, and the reaction temperature of the first deprotection reaction is 40 °C; the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the first deprotection reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the first continuous extraction is 12 s; and the flow rate of the first extractant pumped into the liquid-liquid separator is 15 mL / min.

[0051] In step b, during the coupling of the amino acid unit corresponding to the third position of the C-terminus of the first peptide fragment sequence, the flow rate of the second organic phase and the solution of the amino acid unit pumped into the first microchannel reactor is 1.5 mL / min. In the first microchannel reactor, the residence time of the second condensation reaction is 60 s, and the reaction temperature is 40°C. During the coupling of the amino acid unit corresponding to the second position of the C-terminus of the second peptide fragment sequence, the flow rate of the second organic phase and the solution of the amino acid unit pumped into the first microchannel reactor is 4 mL / min. In the first microchannel reactor, the residence time of the second condensation reaction is 22.5 s, and the reaction temperature is 40°C. Except for the amino acid unit corresponding to the third position of the C-terminus of the first peptide fragment sequence and the amino acid unit corresponding to the second position of the C-terminus of the second peptide fragment sequence, during the coupling of other amino acid units, the flow rate of the second organic phase and the solution of the amino acid unit pumped into the first microchannel reactor is 5 mL / min. In the first microchannel reactor, the residence time of the second condensation reaction is 18.0 s, and the reaction temperature is 40°C.

[0052] In step b, the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the second condensation reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the second continuous extraction is 12 s; and the flow rate of the second extract pumped into the liquid-liquid separator is 15 mL / min.

[0053] In step c, the flow rates of the third organic phase and the first pyrolysis solution pumped into the second microchannel reactor are both 10.0 mL / min; in the second microchannel reactor, the residence time of the second deprotection reaction is 9.0 s, and the reaction temperature of the second deprotection reaction is 40 °C; the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the second deprotection reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the third continuous extraction is 12 s; and the flow rate of the third extractant pumped into the liquid-liquid separator is 15 mL / min.

[0054] In some embodiments, a method for separating and purifying a fully protected second peptide fragment (with N-terminal protecting group removed) from a fourth organic phase includes the following steps: removing the organic solvent from the organic phase by rotary evaporation, purifying the remaining material by rapid column chromatography, using a mixture of dichloromethane and methanol at different concentration gradients as the eluent (the volume ratio of dichloromethane to methanol is (20~40):1), and rotary evaporating the eluent containing the target material to obtain a fully protected second peptide fragment containing N-terminal protecting group removed.

[0055] In some embodiments, a method for separating and purifying a fully protected first or third peptide fragment from a third organic phase containing the first or third peptide fragment includes the following steps: removing the organic solvent from the third organic phase by rotary evaporation, thereby obtaining the fragment.

[0056] Step 1, the method for synthesizing the fully protected fourth peptide fragment with the N-terminal protecting group removed, includes the following steps:

[0057] Step i: The solution of the BTPM carrier and the solution of the amino acid unit corresponding to the first C-terminus of the fourth peptide fragment sequence are pumped into the first microchannel reactor of the coupling unit to carry out the third condensation reaction, obtaining the third condensation reaction solution. The third condensation reaction solution and the extractant are pumped into the third microchannel reactor of the continuous extraction unit for the fourth continuous extraction, obtaining the fourth extract. The extract is then pumped into the liquid-liquid separator for separation to obtain the fifth organic phase. Formic acid (FA) is added to the extract to obtain an acidic solution of the fifth organic phase, which is then passed through a T-type gas-liquid mixer. The liquid inlet of the device is pumped into the T-type gas-liquid mixer, where it mixes with hydrogen gas introduced into the T-type gas-liquid mixer through the gas inlet. The resulting gas-liquid mixture is then pumped through the liquid outlet of the T-type gas-liquid mixer into the fixed-bed reaction column of the Cbz deprotection unit for the third deprotection reaction, yielding the third deprotection reaction liquid. The third deprotection reaction liquid and the extractant are then pumped into the third microchannel reactor of the continuous extraction unit for the fifth continuous extraction, yielding the fifth extract. This extract is then pumped into the liquid-liquid separator for separation, yielding the sixth organic phase.

[0058] Step ii: Replace the ethyl acetate solution of the BTPM carrier in step i with the sixth organic phase obtained in step i, and replace the solution of the amino acid unit corresponding to the first position of the C-terminus of the fourth peptide fragment sequence in step i with the solution of the amino acid unit corresponding to the next position after the C-terminus of the fourth peptide fragment sequence. Repeat step i until all the corresponding amino acid units of the fourth peptide fragment sequence are coupled sequentially, and finally obtain the fully protected fourth peptide fragment with the N-terminal protecting group removed in the sixth organic phase.

[0059] In step i, the solution of the carrier BTPM is in the form of ethyl acetate with a concentration of 0.2 M; the amino acid unit is a protected amino acid with a free α-carboxyl group; and the protecting group of the α-amino group of the amino acid unit is Cbz.

[0060] In step i, the solution of the amino acid unit is prepared by dissolving the amino acid unit in an EA / DMSO solution obtained by mixing ethyl acetate and dimethyl sulfoxide at a volume ratio of 4:1, adding 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for pre-activation for 5-20 min, and then obtaining the solution. The molar ratio of the amino acid unit, 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1-1.1):(1-1.1). The solid-liquid ratio of the amino acid unit to the EA / DMSO solution is 4-5 mmol:20 mL.

[0061] In step i, the flow rates of the BTPM carrier solution and the amino acid unit solution pumped into the first microchannel reactor are both 5 mL / min; in the first microchannel reactor, the residence time of the third condensation reaction is 18.0 s, and the reaction temperature of the condensation reaction is 40℃; the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the third condensation reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the fourth continuous extraction is 12 s; the flow rate of the fourth extractant pumped into the liquid-liquid separator is 15 mL / min; the amount of formic acid added is controlled so that the volume fraction of formic acid in the acidic solution of the fifth organic phase is 5%; the hydrogen pressure in the fixed-bed reaction column is 1.0 MPa; the flow rate of the gas-liquid mixture pumped into the fixed-bed reaction column is 5 mL / min. The fixed-bed reaction column is filled with Pd / C catalyst; the residence time of the third deprotection reaction in the fixed-bed reaction column is 56.5 s, and the temperature of the third deprotection reaction is 50℃; the flow rates of the third deprotection reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; the residence time of the fifth continuous extraction in the third microchannel reactor is 12 s; the flow rate of the fifth extractant pumped into the liquid-liquid separator is 15 mL / min.

[0062] In some embodiments, a method for separating and purifying a fully protected fourth peptide fragment (with N-terminal protecting group removed) from a sixth organic phase includes the following steps: removing the organic solvent from the organic phase by rotary evaporation, purifying the remaining material by rapid column chromatography, using a mixture of dichloromethane and methanol at different concentration gradients as the eluent (the volume ratio of dichloromethane to methanol is (20~40):1), and rotary evaporating the eluent containing the target material to obtain a fully protected fourth peptide fragment (with N-terminal protecting group removed).

[0063] In some embodiments, the sequence of the fully protected first peptide fragment is:

[0064] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-PTESE.

[0065] In some embodiments, the sequence of the fully protected second peptide fragment is:

[0066] Fmoc-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-Ala-PTESE.

[0067] In some embodiments, the sequence of the fully protected third peptide fragment is:

[0068] Cbz-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-PTESE.

[0069] In some embodiments, the sequence of the fully protected fourth peptide fragment is:

[0070] Cbz-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-BTPM.

[0071] In some embodiments, the method for removing the vector PTESE from the fully protected first peptide fragment, fully protected third peptide fragment, or fully protected fifth peptide fragment includes the following steps:

[0072] The fully protected peptide fragment was dissolved in dichloromethane (DCM), and tetrabutylammonium fluoride trihydrate (TBAF·3H2O) was added. The resulting mixture was stirred at room temperature for 0.5 to 1 h. The resulting reaction solution was dissolved in ethyl acetate after removing the dichloromethane by rotary evaporation to obtain the first mixture. This mixture was extracted with saturated NaCl aqueous solution, and the organic phase was collected. A settling agent was added to the organic phase for sedimentation. The precipitate was collected and dried under vacuum to obtain the first, third, or fifth peptide fragments with the PTESE carrier removed.

[0073] The molar ratio of the fully protected peptide fragment to the tetrabutylammonium fluoride trihydrate is 1:2; the precipitant used when removing the carrier PTESE from the fully protected first peptide fragment or the fully protected fifth peptide fragment is methyl tert-butyl ether (MTBE); and the precipitant used when removing the fully protected third peptide fragment is methanol (MeOH).

[0074] Step 2, the method for synthesizing a fully protected fifth peptide fragment using a fully protected first peptide fragment (with the PTESE vector removed) and a fully protected second peptide fragment (with the N-terminal protecting group removed), or the method for synthesizing a fully protected sixth peptide fragment using a fully protected third peptide fragment (with the PTESE vector removed) and a fully protected fourth peptide fragment (with the N-terminal protecting group removed), or the method for synthesizing a fully protected telpolide using a fully protected fifth peptide fragment (with the PTESE vector removed) and a fully protected sixth peptide fragment (with the N-terminal protecting group removed), includes the following steps:

[0075] The fully protected first, third, or fifth peptide fragments, after removing the PTESE carrier, were dissolved in a mixture of tetrahydrofuran (THF) and dimethyl sulfoxide. N,N-diisopropylethylamine (DIEA) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were added. The resulting mixture was stirred at 40°C for 30 min. A tetrahydrofuran solution containing the fully protected second, fourth, or sixth peptide fragments, after removing the N-terminal protecting group, was added to the resulting reaction solution. The mixture was stirred at 40°C for 2 h to obtain a reaction solution containing a fully protected fifth peptide fragment, a fully protected sixth peptide fragment, or a fully protected telpoeptide. After separation and purification, the fully protected fifth peptide fragment, the fully protected sixth peptide fragment, or the fully protected telpoeptide was obtained.

[0076] The volume ratio of tetrahydrofuran to dimethyl sulfoxide in the mixture of tetrahydrofuran and dimethyl sulfoxide is 1:1; the molar ratio of the fully protected first, third, or fifth peptide fragments, N,N-diisopropylethylamine, and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate after removing the PTESE carrier is 1:3:1.2.

[0077] The amount of tetrahydrofuran solution added to the fully protected second, fourth, or sixth peptide fragment after removing the N-terminal protecting group is controlled such that the molar ratio of the fully protected first peptide fragment after removing the PTESE carrier to the fully protected second peptide fragment after removing the N-terminal protecting group is (1 ~ 1.1): (1 ~ 1.1), or the molar ratio of the fully protected third peptide fragment after removing the PTESE carrier to the fully protected fourth peptide fragment after removing the N-terminal protecting group is (1 ~ 1.1): (1 ~ 1.1), or the molar ratio of the fully protected fifth peptide fragment after removing the PTESE carrier to the fully protected sixth peptide fragment after removing the N-terminal protecting group is (1 ~ 1.1): (1 ~ 1.1).

[0078] In some embodiments, the solid-liquid ratio of the fully protected first, third, or fifth peptide fragments deprotected from the PTESE carrier to the mixture of tetrahydrofuran and dimethyl sulfoxide is 3.25 mmol : 10 mL; the concentration of the tetrahydrofuran solution of the fully protected second, fourth, or sixth peptide fragments deprotected from the N-terminal protecting group is 0.3 M.

[0079] In some embodiments, the method for separating and purifying the fully protected fifth peptide fragment or the fully protected telpoeptide from the reaction solution containing the fully protected fifth peptide fragment, the fully protected sixth peptide fragment, or the fully protected telpoeptide comprises the following steps: rotary evaporation of the reaction solution to remove the organic solvent therein, dissolving the remaining substance in ethyl acetate, and continuously extracting the resulting solution with 2.5% ammonia water in the third microchannel reactor, followed by liquid-liquid separation to obtain a seventh organic phase, which is then continuously extracted with 100 g / L sodium sulfate aqueous solution in the third microchannel reactor to obtain an eighth organic phase containing the fully protected fifth peptide fragment, the fully protected sixth peptide fragment, or the fully protected telpoeptide, followed by rotary evaporation to remove the organic solvent, thereby obtaining the fully protected fifth peptide fragment or the fully protected telpoeptide; wherein the liquid-to-solid ratio of the amount of ethyl acetate used to the fully protected first, third, or fifth peptide fragment after removal of the PTESE carrier is 15 mL : 3.25 mmol.

[0080] In some embodiments, the preparation method of the N-terminal deprotected sixth peptide fragment includes the following steps: adding formic acid to the eighth organic phase containing the fully protected sixth peptide fragment to obtain an acid solution of the eighth organic phase; pumping this solution into the T-type gas-liquid mixer through the inlet of the T-type gas-liquid mixer; mixing the solution with hydrogen gas introduced into the T-type gas-liquid mixer through the gas inlet of the T-type gas-liquid mixer; pumping the resulting gas-liquid mixture into the fixed-bed reaction column of the Cbz deprotection unit through the outlet of the T-type gas-liquid mixer for a third deprotection reaction to obtain a third deprotection reaction solution; pumping the third deprotection reaction solution and extractant into the third microchannel reactor of the continuous extraction unit for a fifth continuous extraction to obtain a fifth extract; pumping the extract into the liquid-liquid separator for separation to obtain a sixth organic phase containing the N-terminal deprotected sixth peptide fragment; evaporating the extract; and purifying the residual material by rapid column chromatography, with the eluent being a mixture of dichloromethane and methanol at different concentration gradients (the volume ratio of dichloromethane to methanol is (20~40): ). 1) The eluent containing the target substance is rotary evaporated to obtain a fully protected sixth peptide fragment containing the N-terminal protecting group removed; wherein, the amount of formic acid added is controlled so that the volume fraction of formic acid in the acid solution of the eighth organic phase is 5%.

[0081] The method for removing the carrier BTPM and all protecting groups from the fully protected telpoeptide includes the following steps:

[0082] The fully protected telpoeptide was added to an acid hydrolysate at 0°C under a nitrogen atmosphere and stirred at room temperature until the reaction was complete. After the reaction was completed, methyl tert-butyl ether was added to the resulting reaction solution for precipitation. The precipitate was collected and dried under vacuum to obtain telpoeptide.

[0083] The acid hydrolysate is a mixture of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanethiol), triisopropylsilane, and phenol in a volume ratio of 92.5:2.5:2.5:2.5.

[0084] All of the above reactions were monitored to be complete by HPLC.

[0085] The synthetic route of the telpoeptide of the present invention is as follows: Figure 2 As shown.

[0086] Beneficial effects:

[0087] (1) This invention employs an orthogonal hydrophobic carrier strategy combining PTESE and BTPM to prepare telpoeptide. PTESE can be gently removed at the fragmentation stage using fluorine reagents, thereby obtaining a fully protected peptide fragment that can continue to participate in fragment condensation without destroying acid-sensitive side chain protecting groups such as tBu, Boc, and Trt. BTPM can be retained until the final acid hydrolysis step and removed together with the side chain protecting groups, which is beneficial for the segmented construction and final assembly of the fully protected telpoeptide fragment. This carrier combination can balance the compatibility of protecting groups at the fragmentation stage, fragment solubility, and the convenience of final deprotection.

[0088] (2) This invention combines carrier-assisted liquid-phase synthesis with continuous flow coupling, Fmoc deprotection, Cbz fixed-bed hydrogenolysis, continuous extraction, and liquid-liquid membrane separation to form a continuous operation process of "coupling—deprotection—impurity removal—phase separation—further extension". Compared with traditional solid-phase synthesis, this method reduces resin pore diffusion and mass transfer limitations; compared with conventional liquid-phase synthesis, this method can remove excess amino acids, condensing agent byproducts, deprotection byproducts, and inorganic salts in a timely manner through online extraction and membrane separation, reducing the burden of intermediate separation and purification.

[0089] (3) This invention reduces the risk of accumulation of impurities such as missing peptides, truncated peptides, and residual protecting groups during the linear stepwise synthesis of long chains by splitting the telpopeptide backbone into multiple fully protected fragments and pre-introducing fatty acid side chains into key fragments. Each fragment is in a liquid or near-homogeneous system, which facilitates process monitoring and quality confirmation by methods such as HPLC and MS, and helps to improve the quality controllability of key intermediates and final crude peptides.

[0090] (4) This invention uses solvent systems such as ethyl acetate, ethyl acetate / dimethyl sulfoxide, and 2-methyltetrahydrofuran, and reduces the consumption of hazardous solvents through continuous extraction and solvent recovery. Compared with the traditional solid-phase peptide synthesis process that uses large amounts of DMF, NMP, DCM and piperidine, this invention can reduce the amount of high-concern solvents used, reduce waste liquid burden, and improve process quality intensity and environmental factors.

[0091] (5) The process parameters of the present invention, including flow rate, temperature, residence time, extraction ratio, hydrogen pressure and fixed bed residence time, can be quantitatively controlled. The microchannel reactor, fixed bed reaction column, liquid-liquid membrane separator and pumping system used can be modularly scaled up, which is suitable for the continuous, green and large-scale preparation of telpopeptide and similar long-acting peptide drugs.

[0092] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0093] Figure 1This is a synthetic route diagram for PTESE, the carrier used in this invention.

[0094] Figure 2 This is a synthetic route diagram of the telpoeptide of the present invention.

[0095] Figure 3 The figures shown are actual images of the continuous flow liquid-phase polypeptide synthesis apparatus used in Examples 1 to 4 of the present invention; wherein, figure a is an actual image of the coupling unit or Fmoc deprotection unit, figure b is an actual image of the Cbz deprotection unit, and figure c is an actual image of the continuous flow extractor and liquid-liquid separator.

[0096] Figure 4 This is a schematic diagram of the continuous flow liquid-phase polypeptide synthesis process of the present invention.

[0097] Figure 5 The image shows the MS spectrum of compound 34 synthesized in Example 1.

[0098] Figure 6 The image shows the HRMS spectrum of compound 35 synthesized in Example 1.

[0099] Figure 7 The image shows the MS spectrum of compound 37 synthesized in Example 2.

[0100] Figure 8 The image shows the MS spectrum of compound 42 synthesized in Example 3.

[0101] Figure 9 The image shows the HRMS spectrum of compound 46 synthesized in Example 4.

[0102] Figure 10 The MS spectrum of compound 47 synthesized in Example 5 is shown.

[0103] Figure 11 The MS spectrum of compound 48 synthesized in Example 5 is shown.

[0104] Figure 12 The image shows the MS spectrum of the telpoeptide synthesized in Example 5. Detailed Implementation

[0105] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0106] The following examples use a physical diagram of the continuous flow liquid-phase peptide synthesis apparatus. Figure 3 As shown.

[0107] Figure 4 This is a schematic diagram of the continuous flow liquid-phase polypeptide synthesis process of the present invention.

[0108] The liquid-liquid separator used in the following examples is the Corning Zaiput SEP-10 high-efficiency liquid-liquid separator, which is equipped with a hydrophobic PTFE membrane.

[0109] The parameters of the microchannel reactor, fixed-bed reaction column, and liquid-liquid separator used in the following embodiments are shown in Table 1.

[0110] Table 1. Parameters of microchannel reactors, fixed-bed reaction columns, and liquid-liquid separators

[0111]

[0112] Example 1 Synthesis of the first peptide fragment

[0113] The carrier PTESE (0.9 g, 4 mmol) and Fmoc-Leu-OH (1.2 eq, 1.7 g, 6 mmol) were added to a 100 mL reaction flask and dissolved in 15 mL of anhydrous 2-MeTHF. DMAP (0.1 eq) and EDC·HCl (1.2 eq) were then added sequentially. The reaction mixture was stirred at 50 °C for 2 h, and the reaction was monitored to completion by HPLC. The reaction solution was transferred to a separatory funnel and washed three times with an equal volume of 50 g / L K₂CO₃ aqueous solution. The solution was dried over anhydrous Na₂SO₄, filtered, and the organic phase was evaporated to dryness. The residue was purified by rapid column chromatography to give a colorless oily compound, Fmoc-Leu-PTESE 31 (2.6 g, yield 96.9%).

[0114] Accurately weigh Fmoc-Leu-PTESE (2.3 g, 3.9 mmol) and dissolve it in 20 mL of EA. Separately, dissolve Mps (3 eq, 2.1 g, 11.7 mmol) in 20 mL of an EA / DMSO (3:1, v / v) mixture containing 20% ​​(v / v) DEA. Simultaneously pump both mixtures into the Fmoc deprotection unit at a flow rate of 10.0 mL / min. Combine the two solutions and pump them into the second microchannel glass reactor (40 °C, residence time 9.0 s) of the Fmoc deprotection unit at a total flow rate of 20.0 mL / min to carry out the deprotection reaction and obtain a deprotection reaction solution containing H-Leu-PTESE. Subsequently, the deprotected reaction solution and 80 mL of 50 g / L K2CO3 aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was introduced into a liquid-liquid membrane separator at a total flow rate of 15 mL / min for online liquid-liquid separation. The separated organic phase was regarded as solution A. Separately, Fmoc-Aib-OH (1.1 eq, 1.42 g, 4.3 mmol) was dissolved in 20 mL of EA / DMSO (4:1, v / v), and HOBt (1.1 eq, 0.6 g, 4.3 mmol) and EDC·HCl (1.1 eq, 0.8 g, 4.3 mmol) were added sequentially for pre-activation for 5 min, which was regarded as solution B. Solutions A and B were simultaneously pumped into the first microchannel glass reactor of the coupling unit at a flow rate of 5 mL / min. After the two solutions merged, they were passed through the first microchannel glass reactor (40℃, residence time 18.0 s) at a total flow rate of 10 mL / min to undergo a condensation reaction, yielding a condensation reaction solution containing Fmoc-Aib-Leu-PTESE. Next, the condensation reaction solution and 80 mL of 50 g / L K₂CO₃ aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was fed into a liquid-liquid membrane separator at a total flow rate of 15 mL / min to achieve online liquid-liquid separation. After 12 cycles, the fully protected first peptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-PTESE was finally obtained, designated as compound 34 (6.9 g, yield 74.6%, HPLC purity 95%).Finally, the obtained compound 34 was treated in DCM with TBAF·3H2O (2 eq, 1.9 g, 6 mmol) for 30 min, the solvent was evaporated, and 20 mL of EA was added to dissolve and dilute the mixture. The reaction solution was pumped into a continuous extraction unit with 40 mL of saturated NaCl aqueous solution in the same manner. After extraction, MTBE was added for precipitation, the precipitate was collected by centrifugation, and dried under vacuum to obtain compound 34 without the PTESE support, which was designated as compound 35 (6.1 g, yield 72%, HPLC purity 94%).

[0115] Compound 34 was analyzed by MS: MS (ESI-TOF) m / z : [M-tBu+2H] 2+ Calcd for [C 119 H 192 N 14 O 27 Si] / 21139.18, found: 1139.21, see detailed MS spectrum. Figure 5 .

[0116] Compound 35 was analyzed by HRMS:

[0117] HRMS (ESI-TOF) m / z: [M-tBu-H] - Calcd for C 105 H 168 N 14 O 27 Si 2057.2303, found: 2057.1495;

[0118] HRMS (ESI-TOF) m / z: [M-tBu-2H] 2- Calculated for [C 105 H 167 N 14 O 27 Si] / 2 1028.5415, found: 1028.0472. See the detailed HRMS spectrum for details. Figure 6 .

[0119] Example 2 Synthesis of the second peptide fragment

[0120] The carrier PTESE (0.9 g, 4 mmol) and Fmoc-Ala-OH (1.2 eq, 1.5 g, 4.8 mmol) were added to a 100 mL reaction flask and dissolved in 15 mL of anhydrous 2-MeTHF. DMAP (0.1 eq) and EDC·HCl (1.2 eq) were added sequentially. The reaction mixture was stirred at 50 °C for 2 h, and the reaction was monitored to completion by HPLC. The reaction solution was transferred to a separatory funnel and washed three times with an equal volume of 50 g / L K₂CO₃ aqueous solution. The solution was dried over anhydrous Na₂SO₄, filtered, and the organic phase was evaporated to dryness. The residue was purified by rapid column chromatography to give a colorless oily compound, Fmoc-Ala-PTESE (2.1 g, yield 96.4%).

[0121] Accurately weigh Fmoc-Ala-PTESE (2.1 g, 3.9 mmol) and dissolve it in 20 mL of EA. Separately, dissolve Mps (3 eq, 2.1 g, 11.7 mmol) in 20 mL of an EA / DMSO (3:1, v / v) mixture containing 20% ​​(v / v) DEA. Simultaneously pump both mixtures into the Fmoc deprotection unit at a flow rate of 10.0 mL / min. Combine the two solutions and pump them into the second microchannel glass reactor (40 °C, residence time 9.0 s) of the Fmoc deprotection unit at a total flow rate of 20.0 mL / min to carry out the deprotection reaction and obtain a deprotection reaction solution containing H-Ala-PTESE. Subsequently, the deprotected reaction solution and 80 mL of 50 g / L K2CO3 aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was introduced into a liquid-liquid membrane separator at a total flow rate of 15 mL / min for online liquid-liquid separation. The separated organic phase was regarded as solution A. Separately, Fmoc-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]OH (1.1 eq, 5.3 g, 4.3 mmol) was dissolved in 20 mL of EA / DMSO (4:1, v / v), and HOBt (1.1 eq, 0.6 g, 4.3 mmol) and EDC·HCl (1.1 eq, 0.8 g, 4.3 mmol) were added sequentially for pre-activation for 20 min, which was regarded as solution B. Solutions A and B were simultaneously pumped into the first microchannel glass reactor of the coupling unit at a flow rate of 4 mL / min. After the two solutions were combined, they underwent a condensation reaction at a total flow rate of 8 mL / min through the microchannel glass reactor (40℃, residence time 22.5 s) to obtain a condensation reaction solution containing the compound Fmoc-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-Ala-PTESE. Next, the condensation reaction solution and 80 mL of 50 g / L K2CO3 aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was introduced into a liquid-liquid membrane separator at a total flow rate of 15 mL / min to achieve online liquid-liquid separation. After five cycles, the fully protected second peptide fragment Fmoc-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-Ala-PTESE was finally obtained, denoted as compound 36.Finally, the obtained compound 36 was deprotected in the Fmoc deprotection unit. The deprotected reaction solution was then extracted in a continuous extraction unit. The resulting extract was separated into liquid and liquid phases by a liquid-liquid membrane separator. The resulting organic phase was dried by rotary evaporation and then purified by rapid column chromatography to obtain compound 36 without the N-terminal protecting group Fmoc, which was designated as compound 37 (6.4 g, yield 71.4%, HPLC purity 98%).

[0122] Compound 37 was analyzed by MS: MS (ESI-TOF) m / z: [M+2H] 2+ Calcd for [C 120 H 194 N 13 O 25 Si] / 2 1125.20, found: 1125.2, see detailed MS spectrum. Figure 7 .

[0123] Example 3 Synthesis of the third peptide fragment

[0124] The carrier PTESE (0.9 g, 4 mmol) and Fmoc-Gly-OH (1.2 eq, 1.8 g, 6 mmol) were added to a 100 mL reaction flask and dissolved in 15 mL of anhydrous 2-MeTHF. DMAP (0.1 eq) and EDC·HCl (1.2 eq) were added sequentially. The reaction mixture was stirred at 50 °C for 2 h, and the reaction was monitored to completion by HPLC. The reaction solution was transferred to a separatory funnel and washed three times with an equal volume of 50 g / L K₂CO₃ aqueous solution. The solution was dried over anhydrous Na₂SO₄, filtered, and the organic phase was evaporated to dryness. The residue was purified by rapid column chromatography to give a colorless oily compound, Fmoc-Gly-PTESE 38 (2.0 g, yield 96.7%).

[0125] Accurately weigh Fmoc-Leu-PTESE (2.0 g, 3.9 mmol) and dissolve it in 20 mL of EA. Separately, dissolve Mps (3 eq, 2.1 g, 11.7 mmol) in 20 mL of an EA / DMSO (3:1, v / v) mixture containing 20% ​​(v / v) DEA. Simultaneously pump both mixtures into the Fmoc deprotection unit at a flow rate of 10.0 mL / min. Combine the two solutions and pump them into the second microchannel glass reactor (40 °C, residence time 9.0 s) of the Fmoc deprotection unit at a total flow rate of 20.0 mL / min to carry out the deprotection reaction and obtain a deprotection reaction solution containing H-Gly-PTESE. Subsequently, the deprotected reaction solution and 80 mL of 50 g / L K₂CO₃ aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was introduced into a liquid-liquid membrane separator at a total flow rate of 15 mL / min for online liquid-liquid separation. The separated organic phase was regarded as solution A. Separately, Fmoc-Ala-OH (1.1 eq, 1.3 g, 4.3 mmol) was dissolved in 20 mL of EA / DMSO (4:1, v / v), and HOBt (1.1 eq, 0.6 g, 4.3 mmol) and EDC·HCl (1.1 eq, 0.8 g, 4.3 mmol) were added sequentially for pre-activation for 10 min, which was regarded as solution B. Solutions A and B were simultaneously pumped into the first microchannel glass reactor of the coupling unit at a flow rate of 5 mL / min. After the two solutions were combined, they were passed through the first microchannel glass reactor (40°C, residence time 18.0 s) at a total flow rate of 10 mL / min to undergo a condensation reaction, yielding a condensation reaction solution containing Fmoc-Ala-Gly-PTESE. Next, the condensation reaction solution and 40 mL of 50 g / L K₂CO₃ aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was fed into a liquid-liquid membrane separator at a total flow rate of 15 mL / min to achieve online liquid-liquid separation. After 6 operating cycles, the fully protected third peptide fragment Cbz-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-PTESE was finally obtained, designated as compound 41 (5.8 g, yield 88.6%).Finally, the obtained compound 41 was treated in DCM with TBAF·3H2O (2 eq, 2.2 g, 7 mmol) for 30 min, the solvent was evaporated, and 20 mL of EA was added to dissolve and dilute the mixture. The reaction solution was pumped into a continuous extraction unit with 40 mL of saturated NaCl aqueous solution in the same manner. After extraction, MeOH was added to precipitate the mixture, the precipitate was collected by centrifugation, and dried under vacuum to obtain compound 41 without the PTESE support, which was designated as compound 42 (4.6 g, yield 81.3%, HPLC purity 99%).

[0126] Compound 42 was analyzed by MS: MS (ESI-TOF) m / z: [MH] - Calcd forC 79 H 97 N 10 O 14 1409.72, found: 1409.42, see detailed mass spectrum. Figure 8 .

[0127] Example 4 Synthesis of the fourth peptide fragment

[0128] Accurately weigh BTPM (2.1 g, 4 mmol) and dissolve it in 20 mL of EA, treating this as solution A. Separately weigh Cbz-Ser(tBu)-OH (1.2 eq, 1.42 g, 4.8 mmol) and dissolve it in 20 mL of EA / DMSO (4:1, v / v). Add HOBt (5.28 mmol) and EDC·HCl (5.28 mmol) sequentially for pre-activation for 10 min, treating this as solution B. Pump both solutions A and B simultaneously into the coupling unit at a flow rate of 5 mL / min. After the two solutions are combined, pump them into the first microchannel glass reactor (40℃, residence time 18.0 s) of the coupling unit at a total flow rate of 10 mL / min to carry out the condensation reaction and obtain the condensation reaction solution containing Cbz-Ser(tBu)-BTPM. Next, the condensation reaction solution and 80 mL of 50 g / L K2CO3 aqueous solution were simultaneously pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. After 12 s of extraction, the extract was introduced into a liquid-liquid membrane separator at a total flow rate of 15 mL / min for online liquid-liquid separation. FA (FA added at a volume ratio of 5:95 to the organic phase) was added to the organic phase obtained from the liquid-liquid separation and then introduced into the T-type gas-liquid mixer of the Cbz deprotection unit along with hydrogen for gas-liquid mixing. The resulting gas-liquid mixture was pumped into a fixed-bed reaction column filled with Pd / C catalyst (50℃, residence time 56.5 s, hydrogen pressure set to 1.0 MPa) at a flow rate of 5.00 mL / min for hydrogen deprotection reaction, yielding a deprotected reaction solution containing H-Ser(tBu)-BTPM. Subsequently, the deprotected reaction solution and 80 mL of 50 g / L K2CO3 aqueous solution were pumped into the third microchannel glass reactor of the continuous extraction unit at flow rates of 5 mL / min and 10 mL / min, respectively, for continuous extraction. Continuous extraction was performed by pumping the extract at flow rates of 10 mL / min and 10 mL / min into the third microchannel glass reactor of the continuous extraction unit. After 12 s of extraction, the extract was introduced into a liquid-liquid membrane separator at a total flow rate of 15 mL / min for online liquid-liquid separation. The organic phase was then used for coupling of the next amino acid. After multiple coupling-deprotection operations, a fully protected fourth peptide fragment, Cbz-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-BTPM, was obtained, denoted as compound 45. The final deprotection unit operation was then performed to obtain compound 45 without the N-terminal protecting group Cbz, denoted as compound 46 (4.7 g, yield 77.5%, HPLC purity 99%).

[0129] Compound 46 was analyzed by HRMS: HRMS (ESI-TOF) m / z: [M+H] + Calcd forC 79H 131 N 11 O 15 Si21531.9398, found: 1531.9322, see detailed HRMS spectrum. Figure 9 .

[0130] Example 5 Synthesis of telpolide

[0131] (1) Synthesis of a fully protected sixth peptide fragment with the N-terminal Cbz protecting group removed

[0132] a. Compound 42 (3.25 mmol) obtained in Example 3 was dissolved in 10 mL of THF / DMSO (1:1, v / v), followed by the addition of DIEA (3 eq, 1.26 g, 9.75 mmol) and HATU (1.2 eq, 1.5 g, 3.9 mmol). The reaction mixture was stirred at 40 °C for 30 min, and then compound 46 (3.0 mmol) dissolved in 10 mL of THF was added. The reaction mixture was stirred at 40 °C for another 2 h. After the reaction was completed, a reaction solution containing a fully protected sixth peptide fragment was obtained. The resulting reaction solution was evaporated to dryness, and the residue was dissolved in 15 mL of EA. Continuous extraction was performed in a continuous extraction unit using 2.5% (v / v) ammonia water (the continuous extraction method was the same as in Example 1). After liquid-liquid separation, the resulting organic phase was pumped back into the continuous extraction unit and continuously extracted using 100 g / L Na2SO4 aqueous solution (the continuous extraction method was the same as in Example 1). After liquid-liquid separation, an organic phase containing a fully protected sixth peptide fragment was obtained.

[0133] b. Add formic acid (FA added in a volume ratio of 5:95 to the organic phase) to the organic phase containing the fully protected sixth peptide fragment obtained in step a. Perform hydrogen deprotection reaction and subsequent continuous extraction and liquid-liquid separation in the same manner as in Example 4 to obtain an organic phase containing the fully protected sixth peptide fragment with the N-terminal Cbz protecting group removed. Dry the phase by rotary evaporation, and purify the residue by rapid column chromatography to obtain the fully protected sixth peptide fragment with the N-terminal Cbz protecting group removed, designated as compound 47 (5.8 g, single-step yield 69.2%, HPLC purity 92%).

[0134] Compound 47 was analyzed by MS: MS (ESI-TOF) m / z: [M-tBu+Na+H] 2+ Calcd for [C 146 H 211 N 21 O 26 Si₂Na] / ₂ 1376.80, found: 1376.92, see detailed MS spectrum. Figure 10 .

[0135] (2) Synthesis of the fifth peptide fragment after removal of the PTESE carrier

[0136] c. Compound 35 obtained in Example 1 and compound 37 obtained in Example 2 were coupled, extracted and liquid-liquid separated according to the method in step a, and finally an organic phase containing a fully protected fifth peptide fragment (denoted as compound 48) was obtained.

[0137] d. The organic phase containing the fully protected fifth peptide fragment obtained in step c was evaporated by rotary evaporation, and the carrier PTESE was removed therein according to the method in Example 1 to obtain the fifth peptide fragment without carrier PTESE, denoted as compound 49 (5.5 g, 80.4%, HPLC purity 91%).

[0138] Compound 48 was analyzed by MS: MS (ESI-TOF) m / z: [M+5H] 5+ Calculated for [C 215 H 351 N 27 O 51 [ / 5825.50, found: 825.49, see detailed MS spectrum] Figure 11 .

[0139] (3) Synthesis of telpoeptide

[0140] Compound 47 (5.8 g) obtained in Example (1) and compound 49 (5.5 g) obtained in Example (2) were coupled, extracted, and separated by liquid-liquid method according to step a. The resulting organic phase containing fully protected telpoeptide was evaporated to dryness, yielding the fully protected telpoeptide. This was then added to an acid hydrolysis solution (TFA / DODT / TIS / PhOH = 92.5 / 2.5 / 2.5 / 2.5, v / v / v / v) under nitrogen protection at 0°C. The mixture was stirred at room temperature until the reaction was complete. After the reaction, pre-chilled MTBE was added for precipitation. The white solid was collected by centrifugation and dried under vacuum to obtain crude telpoeptide, designated as compound 50 (5.3 g, total yield 27.5%, HPLC purity 82.4%).

[0141] telpolide was analyzed by MS: MS (ESI-TOF) m / z: [M+4H] 4+ Calculated for [C 225 H 352 N 48 O 68 [ / 41204.36, found: 1204.69, see detailed mass spectrum] Figure 12 .

[0142] Example 6: Comparison of SPPS, LPPS, and CF-LPPS processes for synthesizing telpolide

[0143] To objectively evaluate the advantages and limitations of the CF-LPPS process proposed in this invention, a systematic comparison was made between the traditional SPPS process, the LPPS process, and the CF-LPPS process of this invention from multiple dimensions, including reaction time, reagent equivalent, solvent type, impurity removal method, yield, crude peptide purity, equipment complexity, and scale-up potential. The results are shown in Table 2.

[0144] The data in Table 2 show that the results indicate that the CF-LPPS process of this invention is not simply a replacement of solid-phase synthesis with liquid-phase synthesis, but rather a novel integrated route suitable for the preparation of long-chain complex peptides of telpoeptide through a combination of processes including "carrier-assisted liquid-phase synthesis, continuous flow reaction, online extraction membrane separation, and fragment condensation assembly".

[0145] In terms of reaction efficiency, the single coupling-deprotection cycle time for conventional SPPS is approximately 1.3 h, for conventional LPPS it is approximately 2 h, while the CF-LPPS process of this invention takes approximately 0.5 h, significantly shortening the single-cycle operation time. This demonstrates that continuous flow microreactors have significant advantages in mass transfer, heat transfer, and residence time control, enhancing the coupling reaction between protected amino acids and carrier-bound peptide fragments and improving the process efficiency of deprotection. Compared to conventional batch liquid-phase synthesis, continuous flow systems can reduce human operational variations and improve the stability and repeatability of the reaction process by precisely controlling the flow rate, reaction temperature, and residence time.

[0146] From the perspective of reagent consumption, traditional SPPS typically requires approximately three times the equivalent of protective amino acids or condensing agents to compensate for the limitations of mass transfer within the resin and incomplete reactions. In contrast, the reagent equivalent in the CF-LPPS process of this invention is approximately 1.1 times. This result indicates that in a carrier-assisted liquid-phase system, the reaction substrate is in a liquid or near-homogeneous state, resulting in significantly better reaction accessibility than in a resin solid-phase system. Furthermore, the condensation reaction efficiency is high under continuous flow conditions, eliminating the need for a large excess of reagent to drive the reaction. Therefore, this invention can significantly reduce the usage of protective amino acids, condensing agents, and related reagents, which has significant cost control implications for high-value long-chain peptide drugs such as telpolide.

[0147] In terms of impurity removal methods, SPPS relies on resin washing and filtration, while traditional LPPS often employs batch extraction or sedimentation separation. However, the CF-LPPS of this invention utilizes continuous extraction combined with liquid-liquid membrane separation to achieve online separation of coupling byproducts, deprotection byproducts, and peptide intermediates bound to the carrier by excess small molecule reagents. This process feature is a key technical difference between this invention and traditional liquid-phase peptide synthesis. Continuous extraction and membrane separation not only reduce batch transfers and manual operations but also enable the "coupling—deprotection—impurity removal—phase separation—further extension" process to form a continuous closed loop, providing a feasible pathway for the continuous liquid-phase synthesis of long peptide fragments.

[0148] From a product quality perspective, the crude peptide purity of telpoide prepared by traditional SPPS is 41.2%, while the crude peptide purity of telpoide obtained by the CF-LPPS process of this invention reaches 82.4%. This significant increase in crude peptide purity indicates that this invention, through fragmented synthesis and quality control of key intermediates, effectively reduces the impact of missing peptides, truncated peptides, and accumulated impurities on the final product quality in traditional linear solid-phase synthesis. For 39-peptides like telpoide, the difficulty of subsequent preparation and purification is closely related to the crude peptide purity. Higher crude peptide purity is more conducive to reducing the chromatographic load of preparation, reducing the number of purification steps, increasing the recovery rate of the target peak, and reducing the overall cost in industrial production. It should be noted that the overall yield of the CF-LPPS process of this invention is 27.5%, lower than the 65.1% of the SPPS process. This result reflects that the fragment condensation strategy still has room for optimization in terms of condensation efficiency, solubility, conformational matching, and purification losses during long fragment docking. However, from an industrialization perspective, the production of telpoeptide depends not only on the overall yield but also on multiple indicators such as crude peptide purity, impurity profile complexity, feedstock equivalent, solvent recyclability, scale-up stability, and post-processing burden. This invention achieves significantly higher crude peptide purity with lower reagent equivalents and a greener solvent system, demonstrating its outstanding comprehensive advantages in high-quality, green, and continuous manufacturing. With further optimization of fragment condensation conditions, solvent systems, activation methods, and online purification strategies, there is still room for further improvement in the overall yield.

[0149] CF-LPPS significantly outperforms SPPS in terms of crude product purity, reagent consumption, solvent greenness, and scale-up potential. It achieves higher purity and lower material consumption with a partial yield, making it more suitable for large-scale green production of long peptides. Although SPPS equipment is simple, it has the lowest purity and scale-up potential.

[0150] Table 2. Comprehensive comparison of three peptide synthesis processes

[0151]

[0152] Note: aDefined as the time cycle of a single coupling-deprotection cycle for synthesizing telpolide (4 mmol), including the time for washing / extraction and other operations; b SPPS and CF-LPPS represent the crude peptide yields of telposide (4 mmol) synthesized using linear synthesis and fragment condensation strategies, respectively. No comparable data for the entire process were available for conventional LPPS. c SPPS and CF-LPPS represent the crude peptide HPLC purity of telpolide (4 mmol) synthesized using linear synthesis and fragment condensation strategies, respectively. LPPS was not included in the comparison.

[0153] Example 7: Evaluation of the green chemical properties of telpoeptide synthesized from SPPS and CF-LPPS

[0154] Using the synthesis of 4 mmol of telpoeptide as a baseline, the green chemistry attributes of the conventional SPPS process and the CF-LPPS process of this invention were compared. Based on the 12 fundamental principles of green chemistry, indicators such as process quality intensity (PMI), environmental factor E-Factor, and water consumption (WC) were selected to systematically evaluate the sustainability potential of the two processes within the framework of green chemistry. This paper defines a specific indicator, hazardous solvent consumption (HSC, according to GSK guidelines), to quantify the total amount of hazardous solvents used per unit product (see Table 3). It should be noted that the EA used in CF-LPPS has a boiling point of 76.5℃ and can be recovered by distillation with a recovery rate of approximately 90%, effectively reducing net solvent consumption. The system boundary is defined as the process from coupling the first amino acid to cleavage to obtain the crude peptide. Solid-phase synthesis was performed linearly using the standard SPPS procedure; while the CF-LPPS strategy first rapidly prepared four fully protected fragments, and then assembled the complete telpoeptide through pairwise docking. The material inputs and related yields and purities of the two processes are shown in Table 4.

[0155] The results showed that, based on the synthesis of 4 mmol of telpolide, the conventional SPPS process had a hazardous solvent consumption of 46587.2, an environmental factor of 10105.4, and a process mass intensity of 10106.4; while the CF-LPPS process of this invention reduced hazardous solvent consumption to 2411.0, the environmental factor to 1762.6, and the process mass intensity to 1763.6. These data indicate that this invention is significantly superior to the conventional SPPS process in terms of hazardous solvent consumption, waste generation, and material input per unit product.

[0156] Of particular note is that the CF-LPPS process of this invention uses a solvent system such as ethyl acetate / dimethyl sulfoxide to replace the high-concern solvents such as N,N-dimethylformamide that are widely used in traditional SPPS. Furthermore, ethyl acetate has the advantages of a moderate boiling point, easy recovery, and good industrial availability, and can be further reduced in net solvent consumption through distillation. Although a certain amount of aqueous phase is introduced into the CF-LPPS process for continuous extraction, this aqueous phase is mainly used to remove small molecule byproducts and inorganic salt impurities. Compared with the large amount of organic waste liquid in traditional SPPS, its overall environmental burden is still significantly reduced.

[0157] Table 3 Comparison of green chemical properties of SPPS and CF-LPPS

[0158]

[0159] Table 4 Comparison of input materials for the synthesis of telpolide using SPPS and CF-LPPS

[0160]

[0161] Continued from Table 4

[0162]

[0163] The Chinese names of the English abbreviations used in this application specification are shown in Table 5.

[0164] Table 5. Chinese names corresponding to English abbreviations

[0165]

[0166] This invention provides a method and approach for the liquid-phase synthesis of telpolide. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for liquid-phase synthesis of telpoeptide, characterized in that, Includes the following steps: Step 1: With the assistance of the vector PTESE, amino acids are sequentially coupled to obtain a fully protected first peptide fragment, a fully protected second peptide fragment with the N-terminal protecting group removed, and a fully protected third peptide fragment, respectively; with the assistance of the vector BTPM, amino acids are sequentially coupled to obtain a fully protected fourth peptide fragment with the N-terminal protecting group removed. Step 2: Remove the PTESE vector from the fully protected first peptide fragment and couple it with the fully protected second peptide fragment after removing the N-terminal protecting group to obtain the fully protected fifth peptide fragment; remove the PTESE vector from the fully protected third peptide fragment and couple it with the fully protected fourth peptide fragment after removing the N-terminal protecting group to obtain the fully protected sixth peptide fragment. Step 3: After removing the PTESE vector from the fully protected fifth peptide fragment, couple it with the fully protected sixth peptide fragment after removing the N-terminal protecting group to obtain the fully protected telpoeptide. Remove the BTPM vector and all protecting groups to obtain telpoeptide. Wherein, the first peptide fragment is positions 1 to 14 of the telposide backbone sequence from the N-terminus to the C-terminus; the second peptide fragment includes positions 15 to 21 of the telposide backbone sequence from the N-terminus to the C-terminus and a telposide side chain coupled to the lysine residue at position 20 of the telposide backbone sequence via an amide bond; the third peptide fragment is positions 22 to 29 of the telposide backbone sequence from the N-terminus to the C-terminus; and the fourth peptide fragment is positions 30 to 39 of the telposide backbone sequence from the N-terminus to the C-terminus. The chemical structural formula of the carrier PTESE is shown in Formula I: Formula I; The chemical structural formula of the carrier BTPM is shown in Formula II: Formula II.

2. The method according to claim 1, characterized in that, In step 1, the fully protected first peptide fragment, the fully protected second peptide fragment with the N-terminal protecting group removed, the fully protected third peptide fragment, and the fully protected fourth peptide fragment with the N-terminal protecting group removed are all synthesized using a continuous flow peptide liquid phase synthesis system. The continuous flow polypeptide liquid phase synthesis system includes a coupling unit, an Fmoc deprotection unit, a Cbz deprotection unit, a continuous extraction unit, and a liquid-liquid separator; the coupling unit is equipped with a first microchannel reactor; the Fmoc deprotection unit is equipped with a second microchannel reactor; the Cbz deprotection unit is equipped with a fixed-bed reaction column and a T-type gas-liquid mixer; and the continuous extraction unit is equipped with a third microchannel reactor. The outlet of the first microchannel reactor is connected to the inlet of the third microchannel reactor; the outlet of the third microchannel reactor is connected to the inlet of the liquid-liquid separator; the outlet of the liquid-liquid separator is connected to the inlet of the second microchannel reactor, the inlet of the T-type gas-liquid mixer, and the inlet of the first microchannel reactor, respectively; the outlet of the T-type gas-liquid mixer is connected to the inlet of the fixed-bed reaction column; the outlet of the fixed-bed reaction column and the outlet of the second microchannel reactor are respectively connected to the inlet of the third microchannel reactor.

3. The method according to claim 2, characterized in that, In step 1, the method for synthesizing the fully protected first peptide fragment, the fully protected second peptide fragment (with the N-terminal protecting group removed), or the fully protected third peptide fragment includes the following steps: Step a: Dissolve the carrier PTESE and the amino acid unit corresponding to the first position of the C-terminus of the peptide fragment sequence in 2-methyltetrahydrofuran, add a condensing agent, and carry out a first condensation reaction to obtain a first condensation reaction solution. Extract, collect the first organic phase, separate and purify to obtain the conjugate X-AA-PTESE of the amino acid unit and the carrier PTESE. Dissolve it in ethyl acetate to obtain an X-AA-PTESE solution. Pump the X-AA-PTESE solution and the first lysis buffer into the second microchannel reactor of the Fmoc deprotection unit to carry out a first deprotection reaction to obtain a first deprotection reaction solution. Pump the first deprotection reaction solution and the extractant into the third microchannel reactor of the continuous extraction unit to carry out a first continuous extraction to obtain a first extract. Pump the extract into the liquid-liquid separator for liquid-liquid phase separation to obtain a second organic phase. Step b: The solution of the second organic phase and the amino acid unit corresponding to the second position of the C-terminus of the peptide fragment sequence is pumped into the first microchannel reactor of the coupling unit to carry out the second condensation reaction, and the second condensation reaction solution is obtained. The second condensation reaction solution and the extractant are pumped into the third microchannel reactor of the continuous extraction unit to carry out the second continuous extraction, and the second extract is obtained. The extract is then pumped into the liquid-liquid separator for liquid-liquid phase separation to obtain the third organic phase. Step c: The third organic phase and the first pyrolysis solution are pumped into the second microchannel reactor of the Fmoc deprotection unit to carry out the second deprotection reaction, and the second deprotection reaction solution is obtained. The second deprotection reaction solution and the extractant are pumped into the third microchannel reactor of the continuous extraction unit to carry out the third continuous extraction, and the third extract is obtained. The extract is then pumped into the liquid-liquid separator for liquid-liquid phase separation to obtain the fourth organic phase. Step d: Replace the second organic phase in step b with the fourth organic phase, and replace the solution of the amino acid unit corresponding to the second position of the C-terminus of the peptide fragment sequence in step b with a solution of the amino acid unit corresponding to the next position after the C-terminus of the peptide fragment sequence. Repeat steps b and c until all amino acid units corresponding to the peptide fragment sequence are sequentially coupled. During the synthesis of the fully protected first or third peptide fragment, if the amino acid unit corresponding to the next position after the C-terminus of the peptide fragment sequence is the last amino acid unit corresponding to the C-terminus of the peptide fragment sequence, only step b is performed, and step c is not performed. Finally, a fully protected second peptide fragment with the N-terminal protecting group removed is obtained in the fourth organic phase, or a fully protected first or third peptide fragment is obtained in the third organic phase. Wherein, the amino acid unit is a protected amino acid with a free α-carboxyl group; except for the amino acid unit corresponding to the first position of the N-terminus of the fully protected first peptide fragment sequence, whose α-amino group is Boc and the amino acid unit corresponding to the first position of the N-terminus of the fully protected third peptide fragment sequence, whose α-amino group is Cbz, the α-amino group of other amino acid units is Fmoc. In the process of synthesizing the fully protected second peptide fragment after removing the N-terminal protecting group, the amino acid unit corresponding to the second position of the C-terminus of the peptide fragment sequence is Fmoc-Lys[C20-Y-Glu(Z)-AEEA-AEEA]-OH, where Y is the protecting group of eicosanoic acid and Z is the protecting group of Glu. The solution of the amino acid unit is prepared by dissolving the amino acid unit in an EA / DMSO solution obtained by mixing ethyl acetate and dimethyl sulfoxide at a volume ratio of 4:1, adding a condensing agent for pre-activation for 5-20 min. Except for the amino acid unit corresponding to the 3rd position of the C-terminus of the first peptide fragment sequence, which uses N,N-diisopropylethylamine and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate as condensing agents, the other amino acid units use 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as condensing agents. The molar ratio of the amino acid unit, 1-hydroxybenzotriazole, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1~1.1):(1~ 1.1); The molar ratio of the amino acid unit corresponding to the 3rd position of the C-terminus of the first peptide fragment sequence, N,N-diisopropylethylamine, and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:3:1.2; The solid-liquid ratio of the amino acid unit to the EA / DMSO solution is 4~5 mmol:20 mL.

4. The method according to claim 3, characterized in that, In step a, the condensing agent is 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; the molar ratio of the carrier PTESE, amino acid unit, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.2:0.1:1.2; the first condensation reaction is carried out under the following conditions: stirring at 50°C for 2 h; the extractant used for extraction is a 50 g / L potassium carbonate aqueous solution; the concentration of X-AA-PTESE in the X-AA-PTESE solution is 0.195 M; the first lysis buffer is prepared by dissolving sodium 3-mercapto-1-propanesulfonate in a solution of diethylamine; the diethylamine solution has a volume fraction of 20%, and its solvent is ethyl acetate and dimethyl sulfoxide in a volume ratio of 3: A mixed solution was obtained by mixing the following: the solid-liquid ratio of the sodium 3-mercapto-1-propanesulfonate solution to the diethylamine solution was 11.7 mmol : 20 mL; the flow rate of the X-AA-PTESE solution and the first lysis buffer pumped into the second microchannel reactor was 10.0 mL / min; in the second microchannel reactor, the residence time of the first deprotection reaction was 9.0 s, and the reaction temperature of the first deprotection reaction was 40 °C; the extractant was a 50 g / L potassium carbonate aqueous solution; the flow rates of the first deprotection reaction solution and the extractant pumped into the third microchannel reactor were 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the first continuous extraction was 12 s; the flow rate of the first extractant pumped into the liquid-liquid separator was 15 mL / min. And / or, in step b, during the coupling of the amino acid unit corresponding to the 3rd position of the C-terminus of the first peptide fragment sequence, the flow rate of the second organic phase and the solution of the amino acid unit pumped into the first microchannel reactor is 1.5 mL / min, the residence time of the second condensation reaction in the first microchannel reactor is 60 s, and the reaction temperature of the second condensation reaction is 40°C; during the coupling of the amino acid unit corresponding to the 2nd position of the C-terminus of the second peptide fragment sequence, the flow rate of the second organic phase and the solution of the amino acid unit pumped into the first microchannel reactor is 4 mL / min, the residence time of the second condensation reaction in the first microchannel reactor is 22.5 s, and the reaction temperature of the second condensation reaction is 40°C; except for the amino acid unit corresponding to the 3rd position of the C-terminus of the first peptide fragment sequence and the amino acid unit corresponding to the 2nd position of the C-terminus of the second peptide fragment sequence, during the coupling of other amino acid units, the flow rate of the second organic phase and the solution of the amino acid unit pumped into the first microchannel reactor is 5 mL / min, and the residence time of the second condensation reaction in the first microchannel reactor is 18.0 s. The reaction temperature of the second condensation reaction is 40°C; the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the second condensation reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; the residence time of the second continuous extraction in the third microchannel reactor is 12 s; the flow rate of the second extractant pumped into the liquid-liquid separator is 15 mL / min. And / or, in step c, the flow rates of the third organic phase and the first pyrolysis solution pumped into the second microchannel reactor are both 10.0 mL / min; in the second microchannel reactor, the residence time of the second deprotection reaction is 9.0 s, and the reaction temperature of the second deprotection reaction is 40 °C; the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the second deprotection reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the third continuous extraction is 12 s; and the flow rate of the third extractant pumped into the liquid-liquid separator is 15 mL / min.

5. The method according to claim 2, characterized in that, Step 1, the method for synthesizing the fully protected fourth peptide fragment with the N-terminal protecting group removed, includes the following steps: Step i: The solution of the BTPM carrier and the solution of the amino acid unit corresponding to the first C-terminus of the fourth peptide fragment sequence are pumped into the first microchannel reactor of the coupling unit to carry out the third condensation reaction, obtaining the third condensation reaction solution. The third condensation reaction solution and the extractant are pumped into the third microchannel reactor of the continuous extraction unit for the fourth continuous extraction, obtaining the fourth extract. The extract is then pumped into the liquid-liquid separator for separation to obtain the fifth organic phase. Formic acid is added to the extract to obtain an acidic solution of the fifth organic phase, which is then passed through a T-type gas-liquid mixer. The liquid is pumped into the T-type gas-liquid mixer through the inlet and mixed with hydrogen gas introduced into the T-type gas-liquid mixer through the gas inlet. The resulting gas-liquid mixture is pumped into the fixed-bed reaction column of the Cbz deprotection unit through the liquid outlet of the T-type gas-liquid mixer to carry out the third deprotection reaction, obtaining the third deprotection reaction liquid. The third deprotection reaction liquid and the extractant are pumped into the third microchannel reactor of the continuous extraction unit for the fifth continuous extraction, obtaining the fifth extract. The extract is then pumped into the liquid-liquid separator for separation to obtain the sixth organic phase. Step ii: Replace the ethyl acetate solution of the BTPM carrier in step i with the sixth organic phase obtained in step i, and replace the solution of the amino acid unit corresponding to the first position of the C-terminus of the fourth peptide fragment sequence in step i with the solution of the amino acid unit corresponding to the next position after the C-terminus of the fourth peptide fragment sequence. Repeat step i until all the corresponding amino acid units of the fourth peptide fragment sequence are coupled sequentially, and finally obtain the fully protected fourth peptide fragment with the N-terminal protecting group removed in the sixth organic phase.

6. The method according to claim 5, characterized in that, In step i, The solution of the BTPM carrier is in the form of ethyl acetate with a concentration of 0.2 M; the amino acid unit is a protected amino acid with a free α-carboxyl group; and the protecting group of the α-amino group of the amino acid unit is Cbz. The solution of the amino acid unit is prepared by dissolving the amino acid unit in an EA / DMSO solution obtained by mixing ethyl acetate and dimethyl sulfoxide at a volume ratio of 4:1, adding 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for pre-activation for 5-20 min; the molar ratio of the amino acid unit, 1-hydroxybenzotriazole, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1-1.1):(1-1.1); the solid-liquid ratio of the amino acid unit to the EA / DMSO solution is 4-5 mmol: 20 mL. The flow rates of the BTPM carrier solution and the amino acid unit solution pumped into the first microchannel reactor are both 5 mL / min; in the first microchannel reactor, the residence time of the third condensation reaction is 18.0 s, and the reaction temperature of the condensation reaction is 40 °C; the extractant is a 50 g / L potassium carbonate aqueous solution; the flow rates of the third condensation reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; in the third microchannel reactor, the residence time of the fourth continuous extraction is 12 s; the flow rate of the fourth extractant pumped into the liquid-liquid separator is 15 mL / min; the amount of formic acid added is controlled so that the volume fraction of formic acid in the acidic solution of the fifth organic phase is 5%; the hydrogen pressure in the fixed-bed reaction column is 1.0 MPa; the flow rate of the gas-liquid mixture pumped into the fixed-bed reaction column is 5 mL / min; the fixed-bed reaction column is filled with a Pd / C catalyst; in the fixed-bed reaction column, the residence time of the third deprotection reaction is 56.5 s. The temperature of the third deprotection reaction is 50°C; the flow rates of the third deprotection reaction solution and the extractant pumped into the third microchannel reactor are 5 mL / min and 10 mL / min, respectively; the residence time of the fifth continuous extraction in the third microchannel reactor is 12 s; and the flow rate of the fifth extractant pumped into the liquid-liquid separator is 15 mL / min.

7. The method according to any one of claims 1 to 6, characterized in that, The sequence of the fully protected first peptide fragment is as follows: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-PTESE; The sequence of the fully protected second peptide fragment is as follows: Fmoc-Asp(tBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys[C20-OtBu-Glu(OtBu)-AEEA-AEEA]-Ala-PTESE; The sequence of the fully protected third peptide fragment is as follows: Cbz-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-PTESE; The sequence of the fully protected fourth peptide fragment is as follows: Cbz-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-BTPM.

8. The method according to claim 1, characterized in that, The method for removing the vector PTESE from the fully protected first peptide fragment, fully protected third peptide fragment, or fully protected fifth peptide fragment includes the following steps: The fully protected peptide fragment was dissolved in dichloromethane, and tetrabutylammonium fluoride trihydrate was added. The resulting mixture was stirred at room temperature for 0.5 to 1 h. The resulting reaction solution was dissolved in ethyl acetate after removing the dichloromethane by rotary evaporation to obtain the first mixture. This mixture was extracted with saturated NaCl aqueous solution, and the organic phase was collected. A settling agent was added to the mixture for sedimentation, and the precipitate was collected and dried under vacuum to obtain the fully protected first, third, or fifth peptide fragments after removing the PTESE carrier. The molar ratio of the fully protected peptide fragment to the tetrabutylammonium fluoride trihydrate is 1:2; the precipitant used when removing the carrier PTESE from the fully protected first peptide fragment or the fully protected fifth peptide fragment is methyl tert-butyl ether; and the precipitant used when removing the fully protected third peptide fragment is methanol.

9. The method according to claim 1, characterized in that, A method for synthesizing a fully protected fifth peptide fragment using a fully protected first peptide fragment (without the PTESE vector) and a fully protected second peptide fragment (without the N-terminal protecting group), or a method for synthesizing a fully protected sixth peptide fragment using a fully protected third peptide fragment (without the PTESE vector) and a fully protected fourth peptide fragment (without the N-terminal protecting group), or a method for synthesizing a fully protected telpolide using a fully protected fifth peptide fragment (without the PTESE vector) and a fully protected sixth peptide fragment (without the N-terminal protecting group), comprises the following steps: The fully protected first, third, or fifth peptide fragments, after removing the PTESE carrier, were dissolved in a mixture of tetrahydrofuran and dimethyl sulfoxide. N,N-diisopropylethylamine and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate were added. The resulting mixture was stirred at 40°C for 30 min. A tetrahydrofuran solution containing the fully protected second, fourth, or sixth peptide fragments, after removing the N-terminal protecting group, was added to the resulting reaction solution. The resulting mixture was stirred at 40°C for 2 h to obtain a reaction solution containing a fully protected fifth peptide fragment, a fully protected sixth peptide fragment, or a fully protected telpoeptide. After separation and purification, the fully protected fifth peptide fragment, the fully protected sixth peptide fragment, or the fully protected telpoeptide was obtained. The volume ratio of tetrahydrofuran to dimethyl sulfoxide in the mixture of tetrahydrofuran and dimethyl sulfoxide is 1:1; the molar ratio of the fully protected first, third, or fifth peptide fragments, N,N-diisopropylethylamine, and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate after removing the PTESE carrier is 1:3:1.

2.

10. The method according to claim 1, characterized in that, Step 3, the method for removing the carrier BTPM and all protecting groups from the fully protected telpoeptide, includes the following steps: The fully protected telpoeptide was added to the acid hydrolysate at 0°C under a nitrogen atmosphere and stirred at room temperature. After the reaction was completed, methyl tert-butyl ether was added to the resulting reaction solution for precipitation. The precipitate was collected and dried under vacuum to obtain telpoeptide. The acid hydrolysate is a mixture of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanethiol), triisopropylsilane, and phenol in a volume ratio of 92.5:2.5:2.5:2.5.

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