Compound for polypeptide liquid phase synthesis carrier and application thereof

Abstract

The invention discloses a compound for a polypeptide liquid phase synthesis carrier and application of the compound, and belongs to the technical field of polypeptide liquid phase synthesis carriers. The compound contains a ferrocene structure, and the structural formula of the compound is # imgabs0 #. The compound is used as a polypeptide liquid phase synthesis carrier, not only shows excellent performance in the aspects of improving the operation efficiency, reducing the emulsification phenomenon, conveniently monitoring the reaction process and the like, but also has the advantages of economical efficiency and sustainable use, can remarkably improve the efficiency and controllability of the polypeptide liquid phase synthesis process, and has extremely high practicability.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide liquid phase synthesis carriers, and in particular to a compound used as a polypeptide liquid phase synthesis carrier and application thereof. Background Art

[0002] Chemical peptide synthesis involves chemically condensing amino acids to form peptide bonds, which are then gradually extended to form peptide chains. Chemical peptide synthesis primarily involves solid-phase and liquid-phase methods.

[0003] The main steps of solid-phase synthesis are: first, the amino group of the amino acid is protected with a protecting agent such as Fmoc or Boc, then the carboxyl end of the first amino acid is bound to the resin, and the protecting agent on the amino end is removed. The second amino acid is then attached, and this step is repeated to condense the desired amino acid sequence in a certain order. Finally, the peptide is cleaved from the resin under acidic conditions and purified to obtain the desired peptide. Solid-phase synthesis has certain advantages in speed and a pseudo-dilution effect, which can effectively reduce the problem of amino acid racemization. Therefore, solid-phase synthesis is currently the most commonly used method for peptide synthesis. However, due to the high cost of solid-phase synthesis carriers and the large amount of reagents used for washing operations during the synthesis process, solid-phase synthesis has certain limitations.

[0004] Conventional liquid-phase synthesis methods couple amino acids in a specific solvent to produce the corresponding peptides. This is generally a homogeneous reaction with good reactivity, requiring only equivalent amounts or a slight excess of reagents. Intermediates can be further purified to the required purity through washing, crystallization, chromatography, or preparative chromatography. Conventional liquid-phase synthesis offers the advantages of cost-effectiveness and flexibility, but it is labor-intensive and has the disadvantage of being difficult to synthesize with long peptide chains.

[0005] In recent years, to address the high cost of solid-phase synthesis carriers, liquid-phase synthesis of peptides has been developed by combining solid-phase synthesis methods with liquid-phase synthesis carriers with specific structures. This significantly improves the purity of liquid-phase synthesized peptides or their intermediates, effectively reduces the racemization of amino acids, and reduces the production cost of peptide drugs while improving production efficiency. However, this synthesis method also suffers from the problem that the crystal precipitation state deteriorates with the increase in peptide chains, making washing difficult and time-consuming, and sometimes causing inadequate washing, which is not conducive to the production of high-quality peptides.

[0006] For example, Ajinomoto Co., Ltd.'s liquid-phase synthesis carrier, AJIPHASE, is primarily composed of benzene rings and aliphatic chains. It can be dissolved in solution for amino acid coupling. After the reaction, the carrier can be precipitated using a highly polar solvent, enabling the synthesis of long peptide chains. This offers the advantages of cost-effectiveness and flexibility. However, AJIPHASE forms a paste after peptide chain precipitation, making filtration difficult and requiring lengthy washing times. This can easily lead to inadequate washing, resulting in high impurity levels and low peptide purity. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a compound used as a carrier for polypeptide liquid phase synthesis with good crystal precipitation state, easy washing, low cost and high synthesis efficiency and its application.

[0008] In order to achieve the above object, the present invention first provides a compound for use as a carrier for liquid phase synthesis of polypeptides, which contains a ferrocene structure, and its structure is shown in formula (I):

[0009]

[0010] (I)

[0011] in:

[0012] X is selected from OH, NH2, halogen, and 5- to 8-membered aromatic ring.

[0013] R is selected from C1-C100 alkyl, C2-C100 alkenyl, C2-C100 alkynyl, C3-C100 cycloalkyl, C3-C100 cycloalkyl substituted with C1-C100 alkyl, C3-C100 cycloalkyl substituted with C1-C50 alkoxy, and substituted C1-C100 alkyl;

[0014] Y is selected from H, phenyl, halogenated phenyl, aliphatic chain substituted phenyl, and nitrobenzene.

[0015] As a further improvement of the above compound: the structural formula of the compound is:

[0016] .

[0017] As a further improvement of the above compound: the structural formula of the compound is:

[0018]

[0019] in:

[0020] R1 is selected from C1-C100 alkyl, C2-C100 alkenyl, C2-C100 alkynyl, C3-C100 cycloalkyl, C3-C100 cycloalkyl substituted with C1-C100 alkyl, C3-C100 cycloalkyl substituted with C1-C50 alkoxy, and substituted C1-C100 alkyl.

[0021] As a further improvement of the above compound: the compound is selected from the following structural formula:

[0022]

[0023] .

[0024] As a further improvement of the above compound: the structural formula of the compound is:

[0025]

[0026] in:

[0027] R1 is selected from C1-C100 alkyl, C2-C100 alkenyl, C2-C100 alkynyl, C3-C100 cycloalkyl, C3-C100 cycloalkyl substituted with C1-C100 alkyl, C3-C100 cycloalkyl substituted with C1-C50 alkoxy, and substituted C1-C100 alkyl;

[0028] R2 is selected from OH, NH2, halogen, C1~C100 alkyl, C2~C100 alkenyl, C2~C100 alkynyl, C3~C100 cycloalkyl, C3~C100 cycloalkyl substituted with C1~C100 alkyl, C3~C100 cycloalkyl substituted with C1~C50 alkoxy, and substituted C1~C100 alkyl.

[0029] As a further improvement of the above compound: the structural formula of the compound is:

[0030] .

[0031] As a further improvement of the above compound: the compound is selected from the following structural formula:

[0032]

[0033] .

[0034] To achieve the above objectives, the present invention further provides the use of the compound described in the first aspect as a carrier in the liquid phase synthesis of polypeptides. Preferably, the polypeptide is Gly-Ala-Pro-Pro-Pro-Ser-NH2 or Fmoc-Tyr(tBu)-Phe-Ser(tBu)-Ala-Pro-Gly-OH. A coupling precursor connected to the carboxyl end of an amino acid at the functional group of the compound represented by formula (I) is used as the starting material. After deprotection, a single amino acid residue or polypeptide (dipeptide, tripeptide, etc.) is condensed, and a similar cycle is repeated until the synthesis of the last amino acid is completed. Finally, the corresponding target peptide is obtained by cleavage of the liquid phase synthesis carrier and removal of the protecting group.

[0035] It has been verified that the compound containing a ferrocene structure of the present invention has the following advantages when used as a polypeptide liquid phase synthesis carrier (hereinafter referred to as a ferrocene carrier) in polypeptide liquid phase synthesis:

[0036] (1) Significantly improve operational efficiency: In peptide liquid-phase synthesis, traditional carriers tend to form lumps or paste-like gels during the precipitation stage, increasing the difficulty and time of filtration and washing. The present invention uses a compound containing a ferrocene structure as a carrier component, effectively avoiding the formation of paste-like gel crystals, significantly improving the operational efficiency of the filtration and washing steps, and making the entire synthesis process smoother and more efficient.

[0037] (2) Reduce emulsification: In the presence of long fatty chains, emulsification often occurs due to their lipophilicity, which in turn affects the efficiency of liquid separation or filtration. The ferrocene carrier of the present invention can significantly reduce the probability of emulsification, effectively avoiding the difficulties in liquid separation or filtration caused by emulsification, ensuring smooth operation, thereby reducing the potential risk of experimental failure and improving the reliability and repeatability of the overall experiment.

[0038] (3) Convenient monitoring of reaction progress: The ferrocene carrier of the present invention exhibits highly pronounced reaction-indicating properties on thin-layer chromatography (TLC), with the carrier's color points clearly visible under natural light. This feature allows operators to conveniently monitor reaction progress, particularly in complex synthetic systems, allowing for rapid determination of reaction completion. This significantly improves experimental convenience and accuracy, reducing errors and time waste.

[0039] (4) Economical and convenient for recycling and reuse: The raw materials required for the synthesis of the ferrocene carrier of the present invention are extensive and low in cost, and can be reused through simple recycling and treatment methods after synthesis, which reduces production costs and has high economic and environmental protection. This makes the carrier have a good cost-effectiveness in practical applications, can greatly reduce the use of monomer materials, reagents and solvents, and is suitable for promotion and application in large-scale polypeptide synthesis processes.

[0040] In summary, the present invention uses a compound containing a ferrocene structure as a carrier for peptide liquid-phase synthesis, which not only exhibits excellent performance in improving operating efficiency, reducing emulsification, and facilitating monitoring of reaction progress, but also has the advantages of economy and sustainable use. It can significantly improve the efficiency and controllability of the peptide liquid-phase synthesis process and has strong practicality.

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The drawings that constitute part of this invention are intended to assist in understanding the invention. The contents provided in the drawings and their related descriptions in the present invention may be used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0043] Figure 1 This is a physical photo of intermediate 4c.

[0044] Figure 2 This is a photo of the reaction between intermediate 4c and 0.5% TFA / TCM followed by separation using an aqueous solution. DETAILED DESCRIPTION

[0045] The present invention is described clearly and completely below with reference to the accompanying drawings. A person skilled in the art will be able to implement the present invention based on these descriptions. Before describing the present invention with reference to the accompanying drawings, it should be noted that:

[0046] The technical solutions and technical features provided in each part of the present invention, including the following description, may be combined with each other unless there is any conflict.

[0047] In addition, the embodiments of the present invention described below are generally only part of the embodiments of the present invention, rather than all of the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making any creative efforts should fall within the scope of protection of the present invention.

[0048] Regarding the terms and units in the present invention: The terms "include", "have" and any variations thereof in the description and claims of the present invention and the related parts are intended to cover non-exclusive inclusions.

[0049] In this specification and the following examples, the substances represented by the following abbreviations are:

[0050] DMF: N,N-dimethylformamide;

[0051] DCM: dichloromethane;

[0052] THF: tetrahydrofuran;

[0053] 1-Bromooctadecane: 1-bromooctadecane.

[0054] In this specification and the following examples, the following terms have the following meanings:

[0055] Room temperature: refers to 20-25℃.

[0056] Substitution: refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0057] The minimum and maximum carbon atom content of a hydrocarbon group is indicated by a prefix. For example, the prefix (C1-Cb) alkyl indicates any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1-C20 alkyl refers to a straight or branched chain alkyl group containing from 1 to 20 carbon atoms.

[0058] Alkyl: refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl -CH3, ethyl -CH2CH3, and methylene -CH2-; the alkyl group can also be part of other groups, such as C1~C6 alkoxy and C1~C6 alkylamino.

[0059] Alkoxy: refers to an alkyl group connected to an oxygen atom to form a substituent, for example, methoxy is -OCH3.

[0060] Cycloalkyl: refers to a saturated or partially saturated cyclic group having multiple carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, bridged, and spiro ring systems).

[0061] 5- to 8-membered aromatic ring / group: refers to an aromatic single ring or multiple cyclic groups that do not contain heteroatoms and are composed of C atoms.

[0062] Alkenyl: includes straight-chain or branched alkenyl.

[0063] Alkynyl: includes straight-chain or branched alkynyl groups.

[0064] Example 1

[0065] The synthesis of compound A1 (1-(3-(docosanyloxy)phenyl)-1'-aminoferrocene) containing a ferrocene structure and used as a carrier for peptide liquid phase synthesis is as follows:

[0066]

[0067]

[0068] Step 110, Synthesis of Intermediate 1b (m-Anisylbenzoyl chloride)

[0069] To a 50 mL flask, add 30 mL of DCM and 4 mL of DMF, followed by 5 g of starting material 1a (3-methoxybenzoic acid). Slowly add thionyl chloride (1.5 eq) dropwise, and reflux for 4 h to obtain intermediate 1b. After rotary evaporation, the product can be directly used in the next step without further purification.

[0070] Step 120, Synthesis of Intermediate 1c ((4-methoxyphenyl)ferrocene)

[0071] To a 100-mL single-necked flask, add 11.6 g of aluminum chloride and 40 mL of dichloromethane. Slowly add 4.5 g of 3-methoxybenzoyl chloride or 4-methoxybenzoyl chloride in an ice bath. Stir the reaction for 30 minutes, then slowly add 4.1 g of ferrocene. After 12 hours at 0°C, quench the reaction by adding 20 mL of water. Wash the reaction mixture three times with saturated sodium bicarbonate and dichloromethane. Combine the organic layers, dry over anhydrous sodium sulfate, and column chromatography with a 9:1 ratio of petroleum ether to ethyl acetate as the eluent to obtain intermediate 1c.

[0072] 1 H-NMR (400MHz, Chloroform-d): δ7.73 (ddd, J=8.4, 2.2, 1.0Hz, 1H), 7.41-7.34 (m, 2H), 6.95 (dd d, J=8.4, 1.8, 0.9Hz, 1H), δ4.21 (s, 5H), 3.80 (s, 3H), 2.18-2.13 (m, 2H), 1.87 (d, J=5.2Hz, 2H).

[0073] Step 130, Synthesis of Intermediate 1d ((4-hydroxyphenyl)ferrocene)

[0074] To a solution of compound 1c in dichloromethane (30 mL) was added 1 M BBr (2 eq) in dichloromethane dropwise at 0° C., and the mixture was stirred at room temperature for 12 h. After completion of the reaction as monitored by TLC, the reaction mixture was washed with saturated aqueous sodium bicarbonate solution, and the organic extract was dried over anhydrous sodium sulfate, filtered, and evaporated to afford intermediate 1d.

[0075] 1H-NMR (400MHz, Chloroform-d): δ7.69 (ddd, J=7.6, 2.2, 1.2Hz, 1H), 7.36 (t, J=8.4Hz, 1H), 7.20 (t, J= 2.4Hz, 1H), 6.87 (ddd, J=8.4, 2.1, 1.1Hz, 1H), δ4.21 (s, 5H), 2.18-2.13 (m, 2H), 1.87 (d, J=5.4Hz, 2H).

[0076] Step 140, Synthesis of Intermediate 1e (4-(docosanyloxycarbonyl)phenyl]ferrocene)

[0077] 35 mmol of intermediate 1d was dissolved in 140 mL of DMF at room temperature, and then 71 mmol of potassium carbonate and 71 mmol of 1-bromooctadecane were added. The mixture was stirred at 80°C for 15 h, and then water and dichloromethane were added for extraction. The mixture was dried and concentrated to obtain a crude product. Methanol was added to the crude product and filtered to obtain intermediate 1e.

[0078] 1 H-NMR (400MHz, Chloroform-d): δ7.73 (ddd, J=8.4, 2.2, 1.0Hz, 1H), 7.40-7.33 (m, 2H), 6.93 (ddd, J=8.4, 1.7, 0.9Hz, 1H), δ4.21 (s, 5H), 4.04-3. 98(m, 2H), 2.18-2.13(m, 2H), 1.90-1.85(m, 2H), 1.75(tt, J=7.6, 6.3Hz, 2H), 1.44(dq, J=7.6, 7.0Hz, 2H), 1.35-1.23(m, 35H), 0.94-0.86(m, 3H).

[0079] Step 150, Synthesis of Intermediate 1f (1'-(Acetyl)-1-{[4-(Docosanyloxy)phenyl]carbamoyl}ferrocene)

[0080] 10 g (16.27 mmmol) of intermediate le was dissolved in DCM, and 1 g (1.5 eq, 25 mmol) of formamide was added. The mixture was heated to 170 °C and reacted for 18 h. After the reaction was complete, the mixture was cooled and added to acetonitrile solution. The precipitated solid was filtered, washed, and dried to obtain intermediate 1f, which was directly used in the next step without further purification.

[0081] Step 160, Synthesis of Compound A1

[0082] Intermediate 1f was dissolved in THF, and 50 mL of a 2 mol aqueous lithium hydroxide solution was added. The mixture was stirred for 2 h, and the organic phase was separated and concentrated. Methanol was added to precipitate the mixture, which was then washed with acetonitrile and dried to obtain 8 g of compound A1 with a yield of 81%.

[0083] 1 H-NMR (400MHz, Chloroform-d): δ7.31-7.25(m, 2H), 6.90-6.83(m, 2H), δ4.21(s, 5H), 4.12(td, J=5.6, 0.7Hz, 1H), 4.01(t , J=6.4Hz, 2H), 1.97-1.93(m, 2H), 1.80-1.68(m, 6H), 1.44(dq, J=7.6, 7.0Hz, 2H), 1.35-1.23(m, 35H), 0.94-0.86(m, 3H).

[0084] According to the above synthetic ideas, compounds A2-A8 can be synthesized by simple changes.

[0085] Example 2

[0086] The synthesis of compound B1 (1-(4'-chloro-2-fluoro-4-docosanyloxybiphenyl)ferrocene) containing a ferrocene structure and used as a carrier for peptide liquid phase synthesis is as follows:

[0087]

[0088] Step 210, Synthesis of Intermediate 2e (1-(4'-hydroxy-2-fluoro-4-docosanyloxybiphenyl)ferrocene)

[0089] 5 g of intermediate 1d was dissolved in THF, placed in an ice bath under nitrogen protection, and then 4 eq, 65.3 ml of 3-fluorophenylmagnesium bromide (1 mmol / L) were gradually added. The mixture was then gradually returned to room temperature and stirred for 4 h. After the reaction was completed, aqueous ammonium chloride was added to the reaction solution for quenching. The mixture was then extracted with ethyl acetate and water, and the organic phase was dried and concentrated to obtain a crude product. The crude product was purified by flash column chromatography to obtain 6.1 g of intermediate 2e in a yield of 93%.

[0090] 1H-NMR (400MHz, Chloroform-d): δ7.95 (s, 1H), 7.36 (td, J=7.6, 5.0Hz, 1H), 7.29-7.22 (m, 2H), 7.18-7.13 (m, 1H), 7.13-7.03(m, 2H), 6.78-6.72(m, 2H), 4.89(s, 1H), δ4.21(s, 5H), 2.16-2.11(m, 2H), 1.87–1.83(m, 2H).

[0091] Step 220, Synthesis of Intermediate 2f (1-(4'-hydroxy-2-fluoro-4-docosanyloxybiphenyl)ferrocene)

[0092] 6 g of intermediate 2e was dissolved in 50 mL of DMF at room temperature. 4 eq (8.24 g) of potassium carbonate and 1.2 eq (7.5 g) of 1-bromodocosane were added, and the mixture was stirred at 80°C for 15 h. After the reaction, water and dichloromethane were added to the reaction solution for extraction. The mixture was dried and concentrated to obtain a crude product. The crude product was slurried with methanol and filtered to obtain 9.17 g of intermediate 2f in a yield of 96.5%.

[0093] 1 H-NMR (400MHz, Chloroform-d): δ7.36 (td, J=7.6, 5.0Hz, 1H), 7.30-7.18 (m, 3H), 7.13-7.03 (m, 2H), 6.92-6.82 (m, 2H), 4.89 (s, 1H), δ4.21 (s, 5H), 4.01(t, J=6.4Hz, 2H), 2.16-2.11(m, 2H), 1.87-1.83(m, 2H), 1.80-1.71(m , 2H), 1.44 (dq, J=7.6, 7.0Hz, 2H), 1.35-1.23 (m, 35H), 0.94-0.86 (m, 3H).

[0094] Step 230, synthesis of compound B1:

[0095] 2 g of intermediate 2f was added to chloroform, and after dissolution and clarification, 0.8 mL of acetyl chloride solution was added dropwise. After the reaction was completed, the product was spin-dried and acetonitrile was added to precipitate a solid to obtain compound B1.

[0096] 1H-NMR (400MHz, Chloroform-d): δ7.36 (td, J=7.6, 5.0Hz, 1H), 7.30-7.18 (m, 3H), 7.13-7.03 (m, 2H), 6.92-6.82 (m, 2H), 4.89 (s, 1H), δ4.21 (s, 5H), 4.01(t, J=6.4Hz, 2H), 2.16-2.11(m, 2H), 1.87-1.83(m, 2H), 1.80-1.71(m , 2H), 1.44 (dq, J=7.6, 7.0Hz, 2H), 1.35-1.23 (m, 35H), 0.94-0.86 (m, 3H).

[0097] According to the above synthetic ideas, compounds B2-B8 can be synthesized by simple changes.

[0098] Example 3

[0099] Compound A1 of Example 1 was used to synthesize the polypeptide Gly-Ala-Pro-Pro-Pro-Ser-NH2. The specific steps are as follows:

[0100] Step 310, synthesis of intermediate 3a

[0101]

[0102] 3a

[0103] A mixture of 2.3 g of Fmoc-Ser(tBu)-OH (1.2 eq), 2 mL of DIC (2 eq), and 2 g of HOBT (1.2 eq) was added to a chloroform solution to dissolve, followed by the addition of 3.1 g, 5 mmol, of compound A1. The mixture was stirred for 2 h and monitored by TLC. 400 mL of methanol solution was added to the solution to precipitate a solid, which was filtered under reduced pressure to obtain a solid. The solid was washed with 200 mL of acetonitrile solution and filtered under reduced pressure to obtain intermediate 3a.

[0104] 1H-NMR (400MHz, Chloroform-d): δ7.82 (dd, J=7.6, 1.4Hz, 2H), 7.64 (dq, J=7.6 , 0.8Hz, 2H), 7.46-7.35 (m, 4H), 6.87 (dd, J=7.2, 1.2Hz, 2H), 4.39-4.30 (m, 3H) , δ4.21 (s, 5H), 4.01 (t, J=6.4Hz, 2H), 2.08-2.03 (m, 2H), 1.80-1.71 (m, 4H), 1. 44(dq, J=8.0, 7.0Hz, 2H), 1.35-1.23(m, 38H), 1.21(s, 9H), 0.91-0.87(m, 3H).

[0105] Step 320: Synthesis of Intermediate 3b

[0106]

[0107] 3b

[0108] The intermediate 3a obtained in the previous step was added to a 20% DBU solution in chloroform, stirred at room temperature for 40 min, monitored by TLC. After the reaction was complete, 200 mL of methanol solution was added to precipitate a solid, which was filtered under reduced pressure and washed with ACN solution. The solid was filtered under reduced pressure to obtain a solid. 2.2 g of Fmoc-Pro-OH (1.2 eq), 2 mL of DIC (2 eq), 2 g of HOBT (1.2 eq), and the solid obtained by filtration under reduced pressure were added to the chloroform solution and dissolved. The mixture was stirred for 2 h and monitored by TLC. 400 mL of methanol solution was added to the solution to precipitate a solid. The solid was filtered under reduced pressure to obtain a solid, which was washed with 200 mL of acetonitrile solution and filtered under reduced pressure to obtain intermediate 3b.

[0109] Step 330: Synthesis of Intermediate 3c

[0110]

[0111] 3c

[0112] The intermediate 3b obtained in the previous step was added to a 20% DBU solution in chloroform, stirred at room temperature for 40 minutes, monitored by TLC, and after the reaction was complete, 200 mL of methanol solution was added to precipitate a solid, filtered under reduced pressure, washed with ACN solution, and filtered under reduced pressure to obtain a solid. 2.2 g of Fmoc-Pro-OH (1.2 eq), 2 mL of DIC (2 eq), 2 g of HOBT (1.2 eq), and the solid obtained by filtration under reduced pressure were added to the chloroform solution and dissolved. The mixture was stirred for 2 hours and monitored by TLC. 400 mL of methanol solution was added to the solution to precipitate a solid, filtered under reduced pressure to obtain a solid, washed with 200 mL of acetonitrile solution, and filtered under reduced pressure to obtain a solid. Fmoc-Pro-OH was replaced with different amino acid raw materials, and step 320 was repeated 12 times to obtain intermediate 3c.

[0113] 1 H-NMR (400MHz, Chloroform-d): δ7.85(d, J=8.4Hz, 1H), 7.76(d, J=8.4Hz, 1H), 7.65(d, J=8.4Hz, 1H), 7.28(d, J=16.8Hz, 1H), 7.19(d , J=9.2Hz, 1H), 6.87 (dd, J=7.2, 1.2Hz, 2H), 4.76 (t, J=6.4Hz, 2H), 4.61 (dddt, J=14.8, 5.9, 4.0, 1.8Hz, 2H), 4.44-4.37 (m, 2H), δ4.21 (s, 5H), 4.01 (t, J=6.4Hz, 2H), 3.73-3.50 (m, 9H), 2.22-2.08 (m, 3H), 2.08-2.00 (m, 2H), 2.00-1.92 (m, 4H), 1.92-1.89 (m, 2H), 1.87 (t ddd, J=9.2, 5.6, 2.8, 1.1Hz, 3H), 1.80-1.71 (m, 4H), 1.44 (dq, J=7.6, 7.0Hz, 2H), 1.35-1.23 (m, 40H), 1.21 (s, 9H), 0.93-0.85 (m, 3H).

[0114] Step 340: Synthesis of polypeptide Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0115]

[0116] Intermediate 3c was added to a mixed solution consisting of 94% TFA, 2% TIS, 2% H2O, and 2% EDT by volume, respectively. The mixture was stirred at room temperature for 2 h. The resulting product was added with glacial ether to precipitate a solid, which was repeatedly washed and purified by flash column liquid chromatography to obtain the polypeptide Gly-Ala-Pro-Pro-Pro-Ser-NH2 with a yield of 81% and a crude purity of 91.3%.

[0117] 1 H-NMR (400MHz, Chloroform-d): δ7.85 (d, J=8.4Hz, 1H), 7.70 (d, J=9.6Hz, 1H), 4.76 (t, J=6.0Hz, 2H), 4.61 (dddt, J=14.8, 5.9, 4.0, 1.8Hz, 2H), 4.44-4.38 (m, 1H), 4.34 -4.23 (m, 2H), δ4.21 (s, 5H), 4.07 (t, J=5.4Hz, 1H), 3.93 (ddd, J=12.4, 5.6, 4.6Hz, 1 H), 3.73-3.50(m, 9H), 2.22-2.07(m, 3H), 2.03-1.82(m, 9H), 1.27(d, J=6.4Hz, 3H).

[0118] Example 4

[0119] The polypeptide Fmoc-Tyr(tBu)-Phe-Ser(tBu)-Ala-Pro-Gly-OH was synthesized using compound B1 of Example 2. The specific steps are as follows:

[0120] Step 410, synthesis of intermediate 4a

[0121]

[0122] 4a

[0123] A mixture of 2.3 g of Fmoc-Gly-OH (1.2 eq) and 2 mL of DIPEA (2 eq) was added to a chloroform solution to dissolve, followed by the addition of 3.9 g (5 mmol) of compound A1. The mixture was stirred for 3 h and monitored by TLC. 400 mL of methanol solution was added to the solution to precipitate a solid, which was filtered under reduced pressure to obtain a solid. The solid was washed with 200 mL of acetonitrile solution and filtered under reduced pressure to obtain intermediate 4a.

[0124] 1H-NMR (400MHz, Chloroform-d): δ7.82 (dd, J=7.6, 1.4Hz, 2H), 7.64 (dq, J=7.6, 0.8Hz, 2H), 7.46-7.35 (m, 5H), 7.28-7.14(m, 4H), 7.11-7.03(m, 1H), 6.92-6.84(m, 2H), 6.33(t, J=5.6Hz, 1H), 5.02-4.96(m, 1H), 4. 38(d, J=4.8Hz, 2H), δ4.21(s, 5H), 4.02(dt, J=12.8, 6.2Hz, 4H), 2.14-2.09(m, 2H), 1.89-1.84(m, 2H), 1.8 0-1.71 (m, 2H), 1.44 (dq, J=7.6, 7.0Hz, 2H), 1.35-1.26 (m, 33H), 1.26 (d, J=2.4Hz, 3H), 0.94-0.86 (m, 3H).

[0125] Step 420, synthesis of intermediate 4b

[0126]

[0127] 4b

[0128] Intermediate 4a was added to a 20% DBU solution in chloroform, stirred at room temperature for 40 min, monitored by TLC. After the reaction was complete, 200 mL of methanol solution was added to precipitate a solid, which was filtered under reduced pressure and washed with ACN solution. The solid was filtered under reduced pressure to obtain a solid. 2.2 g of Fmoc-Pro-OH (1.2 eq), 2 mL of DIC (2 eq), and 2 g of HOBT (1.2 eq) were added and the solid obtained by filtration under reduced pressure was dissolved in chloroform solution, stirred thoroughly for 2 h, monitored by TLC, and 400 mL of methanol solution was added to the solution to precipitate a solid. The solid was filtered under reduced pressure to obtain a solid, washed with 200 mL of acetonitrile solution, and filtered under reduced pressure to obtain intermediate 4b.

[0129] Step 430, synthesis of intermediate 4c

[0130]

[0131] 4c

[0132] Intermediate 4b was added to a 20% DBU solution in chloroform, stirred at room temperature for 40 minutes, monitored by TLC, and after the reaction was complete, 200 mL of methanol solution was added to precipitate a solid, filtered under reduced pressure, washed with ACN solution, and filtered under reduced pressure to obtain a solid. 2.2 g of Fmoc-Ala-OH (1.2 eq), 2 mL of DIC (2 eq), 2 g of HOBT (1.2 eq), and the solid obtained by filtration under reduced pressure were added to the chloroform solution and dissolved. The mixture was stirred for 2 hours and monitored by TLC. 400 mL of methanol solution was added to the solution to precipitate a solid, filtered under reduced pressure to obtain a solid, washed with 200 mL of acetonitrile solution, and filtered under reduced pressure to obtain a solid. Fmoc-Pro-OH was replaced with different amino acid raw materials, and steps 420 were repeated 12 times to obtain intermediate 4c.

[0133] 1 H-NMR (400MHz, Chloroform-d): δ7.90 (dd, J=24.4, 8.6Hz, 2H), 7.82 (dd, J=7.6, 1.4Hz, 2H), 7.6 7-7.61(m, 3H), 7.46-7.35(m, 4H), 7.31-7.24(m, 6H), 7.24-7.17(m, 2H), 7.16-7.10(m, 2H), 6.8 5-6.76 (m, 3H), 6.73 (ddd, J=8.4, 2.2, 1.2Hz, 1H), 6.62 (t, J=2.4Hz, 1H), 6.16 (d, J=8.8Hz, 1H), 4.98-4.94(m, 1H), 4.51-4.28(m, 7H), δ4.21(s, 5H), 4.06-3.94(m, 4H), 3.85(dd, J=12.8, 4.1Hz , 1H), 3.68-3.55(m, 3H), 3.52(dddd, J=12.0, 4.7, 3.2, 1.4Hz, 1H), 3.04-2.99(m, 1H), 2.92(ddt , J=14.4, 6.3, 0.9Hz, 1H), 2.76 (ddt, J=14.4, 6.2, 0.8Hz, 1H), 2.67 (ddt, J=14.4, 6.3, 0.9Hz, 1H ), 2.28-2.23 (m, 2H), 2.08 (dddd, J=12.4, 7.1, 5.5, 3.7Hz, 1H), 1.98 (ddtd, J=12.8, 7.2, 5.4, 3. 6Hz, 1H), 1.92–1.71 (m, 6H), 1.44 (dq, J=7.6, 7.0Hz, 2H), 1.35-1.19 (m, 58H), 0.94-0.86 (m, 3H).

[0134] Figure 1This is a photo of the intermediate 4c. Figure 1 It can be seen that the intermediate 4c obtained by direct drying after filtration under reduced pressure is in powder form, indicating that it is easy to filter.

[0135] Step 440, synthesize the peptide Fmoc-Tyr(tBu)-Phe-Ser(tBu)-Ala-Pro-Gly-OH

[0136]

[0137] Intermediate 4c was added to a 0.5% TFA / TCM solution and stirred at room temperature for 2 h. After the reaction was completed as monitored by TLC, the mixture was separated using an aqueous solution. The organic phase was collected and concentrated to dryness, and then an acetonitrile solution was added. The mixture was filtered three times. The filtrate was concentrated and purified by flash column liquid chromatography to obtain the polypeptide Fmoc-Tyr(tBu)-Phe-Ser(tBu)-Ala-Pro-Gly-OH with a yield of 83% and a crude purity of 93.2%.

[0138] 1 H-NMR (400MHz, Chloroform-d): δ8.12 (t, J=6.8Hz, 1H), 7.95-7.86 (m, 2H), 7.8 6-7.79(m, 2H), 7.76(d, J=8.8Hz, 1H), 7.64(dq, J=7.2, 0.8Hz, 2H), 7.46-7.35(m , 4H), 7.26(pd, J=4.4, 1.9Hz, 5H), 7.16-7.10(m, 2H), 6.85-6.78(m, 2H), 6.16( d, J=8.8Hz, 1H), 4.99-4.93 (m, 1H), 4.47 (dt, J=8.8, 6.4Hz, 1H), 4.43-4.28 (m, 6 H), δ4.21(s, 5H), 3.94-3.81(m, 3H), 3.68–3.57(m, 2H), 3.56-3.48(m, 1H), 3.0 1(ddt, J=13.6, 6.2, 0.7Hz, 1H), 2.92 (ddt, J=13.6, 6.2, 0.8Hz, 1H), 2.76 (ddt, J =13.6, 6.2, 0.7Hz, 1H), 2.67 (ddt, J=13.6, 6.2, 0.8Hz, 1H), 2.14-2.04 (m, 1H), 1 .98(ddtd, J=12.4, 7.1, 5.5, 3.6Hz, 1H), 1.92-1.80(m, 2H), 1.26-1.19(m, 21H).

[0139] Figure 2This is a photo of the reaction of intermediate 4c with 0.5% TFA / TCM and separation using aqueous solution. Figure 2 As shown, the organic phase did not emulsify, and the interface between the organic phase and the aqueous phase was clear, making it easy to separate or filter.

[0140] The above describes the relevant contents of the present invention. Based on this description, a person skilled in the art will be able to implement the present invention. Based on the above contents of the present invention, all other embodiments obtained by a person skilled in the art without making any creative efforts should fall within the scope of protection of the present invention.

Claims

1. A compound for use as a carrier for liquid-phase synthesis of polypeptides, comprising a ferrocene structure, the structure of which is shown in formula (I): (I) in: X is selected from OH, NH2, halogen, and 5- to 8-membered aromatic ring. R is selected from C1-C100 alkyl, C2-C100 alkenyl, C2-C100 alkynyl, C3-C100 cycloalkyl, C3-C100 cycloalkyl substituted with C1-C100 alkyl, C3-C100 cycloalkyl substituted with C1-C50 alkoxy, and substituted C1-C100 alkyl; Y is selected from H, phenyl, halogenated phenyl, aliphatic chain substituted phenyl, and nitrobenzene.

2. The compound according to claim 1, wherein: The structural formula of the compound is: 。 3. The compound according to claim 2, wherein: The structural formula of the compound is: in: R1 is selected from C1-C100 alkyl, C2-C100 alkenyl, C2-C100 alkynyl, C3-C100 cycloalkyl, C3-C100 cycloalkyl substituted with C1-C100 alkyl, C3-C100 cycloalkyl substituted with C1-C50 alkoxy, and substituted C1-C100 alkyl.

4. The compound according to claim 3, wherein: The compound is selected from the following structural formula: 。 5. The compound according to claim 1, wherein: The structural formula of the compound is: in: R1 is selected from C1-C100 alkyl, C2-C100 alkenyl, C2-C100 alkynyl, C3-C100 cycloalkyl, C3-C100 cycloalkyl substituted with C1-C100 alkyl, C3-C100 cycloalkyl substituted with C1-C50 alkoxy, and substituted C1-C100 alkyl; R2 is selected from OH, NH2, halogen, C1~C100 alkyl, C2~C100 alkenyl, C2~C100 alkynyl, C3~C100 cycloalkyl, C3~C100 cycloalkyl substituted with C1~C100 alkyl, C3~C100 cycloalkyl substituted with C1~C50 alkoxy, and substituted C1~C100 alkyl.

6. The compound according to claim 5, characterized in that: The structural formula of the compound is: 。 7. The compound according to claim 6, wherein: The compound is selected from the following structural formula: 。 8. Use of the compound according to any one of claims 1 to 7 as a carrier in liquid phase synthesis of polypeptides.

9. The use according to claim 8, characterized in that: The polypeptide is Gly-Ala-Pro-Pro-Pro-Ser-NH2 or Fmoc-Tyr(tBu)-Phe-Ser(tBu)-Ala-Pro-Gly-OH.

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