Preparation method of brain-targeting peptide angiopep-2 modified with cholesterol

Abstract

The present application relates to the technical field of polypeptide compounds, and in particular to a preparation method of a cholesterol-modified brain-targeting peptide Angiopep-2, comprising: (a) using a solid-phase synthesis method, coupling each Fmoc-protected amino acid to a resin in order from the C-terminal to the N-terminal of the peptide sequence of the brain-targeting peptide Angiopep-2, and removing a Fmoc protecting group to obtain a peptide resin; (b) performing a first condensation reaction on the peptide resin with succinic anhydride, then performing a second condensation reaction with cholesterol, and performing a cleavage treatment; when coupling the first Thr in the peptide sequence, the Fmoc-protected amino acid used is at least one of Fmoc-N(Dmb)-Thr(tBu)-OH and Fmoc-N(Hmb)-Thr(tBu)-OH. The preparation method can significantly reduce the cost of raw materials while improving the yield and purity of the cholesterol-modified brain-targeting peptide Angiopep-2.

Description

Technical Field

[0001] This invention relates to the field of polypeptide compound technology, and in particular to a method for preparing a cholesterol-modified brain-targeting peptide, Angiopep-2. Background Technology

[0002] Angiopep-2 is a short peptide composed of 19 amino acids with high affinity for brain penetration. It can be specifically recognized and bound by low-density lipoprotein receptor-associated protein 1 (LRP-1), forming internalized transport vesicles through cell membrane invagination, thereby initiating cell internalization and penetrating the blood-brain barrier (BBB) ​​into the brain. Therefore, it is often used to modify targeted drug delivery systems to enhance BBB penetration. To further enhance the targeted delivery efficiency of Angiopep-2 and optimize the physicochemical properties of the drug carrier, cholesterol can be modified onto Angiopep-2. On the one hand, the hydrophobicity of cholesterol can enhance the interaction with the BBB endothelial cell membrane, assisting Angiopep-2 to penetrate the BBB more efficiently through LRP-1-mediated engulfmentation. On the other hand, the hydrophobic properties of cholesterol allow Angiopep-2 to spontaneously assemble into micelles or nanoparticles in aqueous solution, improving drug loading capacity.

[0003] Currently, the preparation of cholesterol-modified brain-targeting peptide Angiopep-2 faces the problem of excessively high raw material costs. This key factor leads to a significant increase in the cost of its mass production, which severely limits the clinical application of this modified peptide.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing cholesterol-modified brain-targeting peptide Angiopep-2. The synthetic route of this invention can not only reduce the cost of raw materials, but also significantly improve the yield and purity of cholesterol-modified brain-targeting peptide Angiopep-2.

[0006] To achieve the above-mentioned objectives of the present invention, a first aspect of the present invention provides a method for preparing the cholesterol-modified brain-targeting peptide Angiopep-2, comprising the following steps: (a) Using a solid-phase synthesis method, each Fmoc-protected amino acid is coupled to the resin sequentially from the C-terminus to the N-terminus according to the peptide sequence of the brain-targeting peptide Angiopep-2, and the Fmoc protecting group is removed to obtain the peptide resin. (b) The peptide resin undergoes a first condensation reaction with succinic anhydride, then a second condensation reaction with cholesterol, followed by cleavage. The peptide sequence of the brain-targeting peptide Angiopep-2 is as follows: NH2-Thr1 -Phe 2 -Phe 3 -Tyr 4 -Gly 5 -Gly 6 -Ser 7 -Arg 8 -Gly 9 -Lys 10 -Arg 11 -Asn 12 -Asn 13 -Phe 14 -Lys 15 -Thr 16 -Glu 17 -Glu 18 -Tyr 19 -OH; The sequence of the cholesterol-modified brain-targeting peptide is as follows: Cholesteryl hemisuccinate-Thr 1 -Phe 2 -Phe 3 -Tyr 4 -Gly 5 -Gly 6 -Ser 7 -Arg 8 -Gly 9 -Lys 10 -Arg 11 -Asn 12 -Asn 13 -Phe 14 -Lys 15 -Thr 16 -Glu 17 -Glu 18 -Tyr 19 -OH; When the first Thr in the coupled peptide sequence is used, the amino acid protected by Fmoc is at least one of Fmoc-N(Dmb)-Thr(tBu)-OH and Fmoc-N(Hmb)-Thr(tBu)-OH.

[0007] In a specific embodiment of the present invention, when the first Thr in the coupled peptide sequence is used, the amino acid protected by Fmoc is Fmoc-N(Dmb)-Thr(tBu)-OH.

[0008] In a specific embodiment of the present invention, in the first condensation reaction, the peptide resin reacts with the succinic anhydride under the action of DIEA. Further, the molar ratio of the peptide resin, the succinic anhydride, and the DIEA is 1:(2-4):(3-6).

[0009] In a specific embodiment of the present invention, in the second condensation reaction, the peptide resin after the first condensation reaction reacts with the cholesterol under the action of a condensing agent, wherein the condensing agent comprises Oxyma / DMAP / DIC. Further, in the second condensation reaction, the molar ratio of the peptide resin after the first condensation reaction to the cholesterol is 1:(4-6); the molar ratio of the cholesterol to Oxyma, DMAP, and DIC is (4-6):(4-6):(0.05-0.2):(4-6).

[0010] In a specific embodiment of the present invention, the cutting fluid used in the cutting process includes TFA, DCM, DODT, TIS, and H2O. Further, the cutting fluid includes TFA, DCM, DODT, TIS, and H2O in a volume ratio of (68–72):(14–16):(4–6):(4–6):(4–6).

[0011] In a specific embodiment of the present invention, the ratio of the cutting fluid to the peptide resin after the second condensation reaction is (5-15) mL: 1g.

[0012] In a specific embodiment of the present invention, the temperature of the cutting process is 20-25°C, and the cutting process time is 2-3 hours.

[0013] In a specific embodiment of the present invention, the amino acids protected by each Fmoc are: Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ty ... c-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-G ly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-N(Dmb)-Thr(tBu)-OH.

[0014] In a specific embodiment of the present invention, each coupling includes a deprotection reaction and a coupling reaction; the deprotection reaction is carried out using a deprotection reagent, which includes an aprotic organic solvent containing an organic base, wherein the organic base includes Pip, and the aprotic organic solvent includes at least one of DCM, DMF, and NMP.

[0015] In a specific embodiment of the present invention, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid to the resin after the deprotection reaction is (2-4):1.

[0016] In a specific embodiment of the present invention, the condensing agent used in the coupling reaction includes at least one of Oxyma / DIC and PyBOP / DIEA.

[0017] In a specific embodiment of the present invention, the coupling reaction time is 1 to 1.5 hours; the coupling reaction temperature is 20 to 30°C.

[0018] In a specific embodiment of the present invention, the resin includes at least one of 2-CTC resin and Wang resin.

[0019] In a specific embodiment of the present invention, the method further includes: collecting the solution after the cutting process, adding a poor solvent to precipitate the solution, collecting the solid and washing it; the poor solvent includes at least one of MTBE and diethyl ether.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: In the preparation method of cholesterol-modified brain-targeting peptide Angiopep-2 of the present invention, cholesterol and succinic anhydride are used as raw materials, and Fmoc-N(Dmb)-Thr(tBu)-OH and / or Fmoc-N(Hmb)-Thr(tBu)-OH are used as raw materials for the first Thr. This method can significantly reduce the cost of raw materials while improving the yield and purity of cholesterol-modified brain-targeting peptide Angiopep-2. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The HPLC chromatogram of the crude product obtained by the method for preparing cholesterol-modified brain-targeting peptide Angiopep-2 provided in Example 1 of this invention; Figure 2 The HPLC chromatogram of the crude product obtained by the method for preparing cholesterol-modified brain-targeting peptide Angiopep-2 provided in Comparative Example 1. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0024] The Chinese meanings of some of the English abbreviations involved in this invention are shown in Table 1.

[0025] Table 1 Meaning of English Abbreviations

[0026] Currently, the synthetic route for preparing the cholesterol-modified brain-targeting peptide Angiopep-2 typically involves synthesizing Angiopep-2 using conventional methods, followed by a reaction with cholesterol succinate. However, this synthetic route faces a significant cost bottleneck—the market price of cholesterol succinate, a key raw material, is as high as approximately 150 yuan / gram, resulting in a substantial increase in overall production costs and severely restricting the large-scale preparation and clinical application of this modified peptide.

[0027] This invention proposes a stepwise synthesis strategy after preparing the brain-targeting peptide Angiopep-2, reacting the peptide sequentially with low-cost succinic anhydride (market price approximately 0.5 RMB / g) and cholesterol (market price approximately 10 RMB / g). This process route significantly reduces raw material costs by replacing expensive cholesterol succinate, providing a practical solution for the large-scale industrial production of cholesterol-modified peptides. Through extensive research, the inventors discovered a key technical bottleneck in the stepwise reaction route with succinic anhydride and cholesterol: after the peptide reacts with succinic anhydride to generate a succinylated intermediate, during the reaction with cholesterol, the activated succinic acid carboxyl group preferentially reacts with the imino group of the preceding (adjacent) amide bond to form a succinimide ring structure. This side reaction not only significantly reduces the yield of the target product but also results in the crude product containing multiple components such as cyclized byproducts and unreacted intermediates, greatly increasing the difficulty and cost of subsequent purification processes.

[0028] Based on this, in the synthetic route of the present invention, the brain-targeting peptide Angiopep-2 is coupled to Thr. 1 When using Fmoc-N(Dmb)-Thr(tBu)-OH and / or Fmoc-N(Hmb)-Thr(tBu)-OH as reactants, the intramolecular cyclization reaction of activated succinic acid with adjacent amide bonds is effectively avoided, ensuring the smooth condensation of the carboxyl group of succinic acid with cholesterol. This not only reduces the cost of raw materials but also significantly improves the yield of the target product.

[0029] The first aspect of this invention provides a method for preparing the cholesterol-modified brain-targeting peptide Angiopep-2, comprising the following steps: (a) Using a solid-phase synthesis method, each Fmoc-protected amino acid is coupled to the resin sequentially from the C-terminus to the N-terminus according to the peptide sequence of the brain-targeting peptide Angiopep-2, and the Fmoc protecting group is removed to obtain the peptide resin. (b) The peptide resin undergoes a first condensation reaction with succinic anhydride, followed by a second condensation reaction with cholesterol, and then cleavage. The peptide sequence of the brain-targeting peptide Angiopep-2 is as follows: NH2-Thr 1 -Phe 2 -Phe 3 -Tyr 4 -Gly 5 -Gly 6 -Ser 7 -Arg 8 -Gly 9 -Lys 10 -Arg 11 -Asn 12 -Asn 13 -Phe 14 -Lys 15 -Thr 16 -Glu 17 -Glu 18 -Tyr 19 -OH; The sequence of the cholesterol-modified brain-targeting peptide is as follows: Cholesteryl hemisuccinate-Thr 1 -Phe 2 -Phe 3 -Tyr 4 -Gly 5 -Gly 6 -Ser 7 -Arg 8 -Gly 9 -Lys10 -Arg 11 -Asn 12 -Asn 13 -Phe 14 -Lys 15 -Thr 16 -Glu 17 -Glu 18 -Tyr 19 -OH; When the first Thr in the coupled peptide sequence is used, the amino acid protected by Fmoc is at least one of Fmoc-N(Dmb)-Thr(tBu)-OH and Fmoc-N(Hmb)-Thr(tBu)-OH.

[0030] The inventors of this invention discovered in their research that reacting peptides with succinate and cholesterol separately resulted in crude products with complex compositions, low purity, and low yield. This invention uses Fmoc-N(Dmb)-Thr(tBu)-OH and / or Fmoc-N(Hmb)-Thr(tBu)-OH as reactants to prepare peptides, effectively avoiding the intramolecular cyclization reaction between activated succinic acid and adjacent amide bonds, ensuring the smooth condensation of the carboxyl group of succinic acid with cholesterol. This not only reduces raw material costs but also significantly improves the yield of the target product.

[0031] The structural formulas of Fmoc-N(Dmb)-Thr(tBu)-OH and Fmoc-N(Hmb)-Thr(tBu)-OH are as follows:

[0032] The synthetic routes for Fmoc-N(Dmb)-Thr(tBu)-OH and Fmoc-N(Hmb)-Thr(tBu)-OH can be found below:

[0033] Taking Fmoc-N(Dmb)-Thr(tBu)-OH as an example, its synthetic route is explained as follows: Equimolar amounts of 2-amino-3-hydroxybutyric acid, 2,4-dimethoxybenzyl bromide (DmbBr), and potassium carbonate (the molar amount of potassium carbonate being 1.1 times the molar amount of DmbBr) were heated to reflux in a suitable solvent (such as acetone) for 6–8 h. The reaction was stopped after TLC monitoring showed the reaction was complete. The mixture was extracted with dichloromethane and saturated brine, and the organic phase was collected. After vacuum distillation, column chromatography was used to separate intermediate I. Equimolar amounts of intermediate I, 9-fluorenyl chloroformate (FmocCl), and potassium carbonate (the molar amount of potassium carbonate being 1.1 times the molar amount of FmocCl) were heated to reflux in a suitable solvent (such as acetone) for 2–4 h. The reaction was stopped after TLC monitoring showed the reaction was complete. The mixture was extracted with dichloromethane and saturated brine, and the organic phase was collected. After vacuum distillation, column chromatography was used to separate intermediate II. Intermediate II was dissolved in an appropriate amount of dichloromethane. Isobutylene (molar ratio of isobutylene to intermediate II was 1:1) was introduced under an acidic catalyst (such as p-toluenesulfonic acid, with a molar ratio of 1:1 to intermediate II) at 5–10 °C. The reaction was then carried out at 15–20 °C for 6–8 h. After vacuum distillation, Fmoc-N(Dmb)-Thr(tBu)-OH was obtained by column chromatography.

[0034] The preparation of Fmoc-N(Hmb)-Thr(tBu)-OH follows the same method as described above, except that an equimolar amount of intermediate I' is used instead of intermediate I. The preparation of intermediate I' includes: dissolving equimolar amounts of 2-amino-3-hydroxybutyric acid and 4-methoxysalicylaldehyde in a suitable solvent (such as isopropanol), then adding sodium cyanoborohydride (0.2 times the molar amount of 2-amino-3-hydroxybutyric acid), stirring the mixture at 27±2℃ for 10–12 h, stopping the reaction when TLC monitoring showed completion; removing the solvent by vacuum distillation, adding water to the remaining material, extracting with ethyl acetate, collecting the organic phase, and separating it by column chromatography after vacuum distillation to obtain intermediate I'.

[0035] The product was characterized by mass spectrometry and verified to be the target product.

[0036] In a specific embodiment of the present invention, when conjugating Thr at the first position of the peptide sequence, the amino acid protected by Fmoc is Fmoc-N(Dmb)-Thr(tBu)-OH. Using Fmoc-N(Dmb)-Thr(tBu)-OH not only better avoids the intramolecular cyclization reaction between the activated succinic acid and the adjacent amide bond, but also ensures the smooth progress of subsequent reaction steps, improving the purity of the crude product and the product yield.

[0037] In a specific embodiment of the present invention, in the first condensation reaction, the peptide resin and succinic anhydride react under the action of DIEA. Further, the molar ratio of peptide resin, succinic anhydride and DIEA is 1:(2-4):(3-6).

[0038] In some embodiments, in the first condensation reaction, the molar ratio of peptide resin to succinic anhydride can be a range of 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:3.8, 1:4, or any two of these; the molar ratio of peptide resin to DIEA can be a range of 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, or any two of these.

[0039] In practice, during the first condensation reaction, a small amount of DMF (the specific amount of DMF is not limited, as long as it can dissolve the succinic anhydride used) is added to the peptide resin to dissolve the succinic anhydride. Then, DIEA is added, and the reaction is carried out at 20–30°C for 0.5–2 hours. The resin is then washed with a solvent; the solvent used for washing may include, but is not limited to, DMF. Specifically, the reaction time can be adjusted according to the Kaiser test results. When the Kaiser test result is negative, the reaction is considered complete. In different embodiments, the time of the first condensation reaction can be 0.5 hours, 1 hour, 1.5 hours, 2 hours, or any combination thereof.

[0040] In a specific embodiment of the present invention, in the second condensation reaction, the peptide resin after the first condensation reaction reacts with cholesterol under the action of a condensing agent, which includes Oxyma / DMAP / DIC. Further, in the second condensation reaction, the molar ratio of the peptide resin after the first condensation reaction to cholesterol is 1:(4-6); the molar ratio of cholesterol to Oxyma, DMAP, and DIC is (4-6):(4-6):(0.05-0.2):(4-6).

[0041] In some embodiments, during the second condensation reaction, the molar ratio of the peptide resin to cholesterol after the first condensation reaction can be a range of 1:4, 1:4.2, 1:4.5, 1:4.8, 1:5, 1:5.2, 1:5.5, 1:5.8, 1:6, or any two of these; the molar ratio of cholesterol to Oxyma can be 4:4, 4:5, 4:6, 5:4, 5:6, 6:4, 6:5, or any two of these. The range of the two components; the molar ratio of cholesterol to DMAP can be 4:0.05, 4:0.1, 4:0.2, 5:0.05, 5:0.1, 5:0.2, 6:0.05, 6:0.1, 6:0.2, or any two of these components; the molar ratio of cholesterol to DIC can be 4:4, 4:5, 4:6, 5:4, 5:6, 6:4, 6:5, or any two of these components.

[0042] In practice, during the second condensation reaction, a small amount of DMF (the specific amount of DMF is not limited, as long as it can dissolve the cholesterol and Oxyma) is added to the peptide resin after the first condensation reaction. Then, DIC and DMAP are added, and the reaction is carried out at 20–30°C for 24–30 hours. The resin is then washed with a solvent; the washing process includes washing with DCM 2–3 times, followed by washing with MTBE 2–3 times, and then drying. Specifically, the endpoint of the second condensation reaction can be confirmed through a small-scale cutting test.

[0043] In a specific embodiment of the present invention, the cutting fluid used in the cutting process includes TFA, DCM, DODT, TIS, and H2O. Further, the cutting fluid includes TFA, DCM, DODT, TIS, and H2O in a volume ratio of (68–72):(14–16):(4–6):(4–6):(4–6).

[0044] In some embodiments, the volume ratio of TFA to H2O in the cutting fluid can be 68:4, 68:5, 68:6, 70:4, 70:5, 70:6, 72:4, 72:5, 72:6, or any combination thereof; the volume ratio of DCM to H2O can be 14:4, 14:5, 14:6, 15:4, 15:5, 15:6, or 16: The volume ratio of DODT to H2O can be 4:4, 4:5, 4:6, 5:4, 5:6, 6:4, 6:5, or any two of these ratios; the volume ratio of TIS to H2O can be 4:4, 4:5, 4:6, 5:4, 5:6, 6:4, 6:5, or any two of these ratios. This invention uses Fmoc-N(Dmb)-Thr(tBu)-OH and / or Fmoc-N(Hmb)-Thr(tBu)-OH as reactants, simultaneously coupling succinic anhydride and cholesterol stepwise. Combined with a cutting fluid that has undergone cleavage treatment, this effectively removes protecting groups and loadings, while avoiding interference with the modifying agent, significantly improving yield and purity.

[0045] In some embodiments, the cutting fluid comprises TFA, DCM, DODT, TIS, and H2O in a volume ratio of 70:15:5:5:5.

[0046] In a specific embodiment of the present invention, the ratio of the cutting fluid to the peptide resin after the second condensation reaction is (5-15) mL: 1g.

[0047] Here, (5-15) mL:1g means that the amount of cutting fluid used can be 5-15 mL relative to 1 g of peptide resin after the second condensation reaction. This is a limitation on the ratio of cutting fluid to peptide resin after the second condensation reaction, not a limitation on the amount of each component. In some embodiments, the amount of cutting fluid used relative to 1 g of peptide resin after the second condensation reaction can be a range of 5 mL, 8 mL, 10 mL, 12 mL, 15 mL, or any combination thereof.

[0048] In a specific embodiment of the present invention, the cutting temperature is 20–25°C, and the cutting time is 2–3 hours. In actual operation, after the cutting process, filtration is performed, and the filtrate is collected.

[0049] In specific embodiments of the present invention, the amino acids protected by each Fmoc are: Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc -Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-G ly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-N(Dmb)-Thr(tBu)-OH.

[0050] In a specific embodiment of the present invention, each coupling reaction includes a deprotection reaction and a coupling reaction; the deprotection reaction is carried out using a deprotection reagent, which includes an aprotic organic solvent containing an organic base, the organic base including Pip, and the aprotic organic solvent including at least one of DCM, DMF and NMP.

[0051] In some embodiments, the organic base in the deprotecting agent has a volume fraction of 15% to 25%. Further, the deprotecting agent is a DMF solution containing Pip, with a Pip volume fraction of 20%.

[0052] In a specific embodiment of the present invention, the deprotection reaction time is 0.5 to 1 hour, and the deprotection reaction temperature is 20 to 30°C.

[0053] In a specific embodiment of the present invention, the ratio of the deprotecting agent to the resin is (5-15) mL: 1 g. Furthermore, after the deprotection reaction, a washing step is included; the washing solvent is DMF, and the washing is performed 4-6 times; the amount of washing solvent can be adjusted according to conventional methods.

[0054] In some embodiments, the amount of deprotecting agent used relative to 1g of resin can be 5mL, 8mL, 10mL, 12mL, 15mL, or any combination thereof.

[0055] In practice, the deprotection reaction includes: placing the resin in a deprotection reagent, mixing at 20-30°C for 0.5-1 h, and then washing with a solvent; the solvent used for washing may include, but is not limited to, DMF.

[0056] In a specific embodiment of the present invention, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid to the resin after the deprotection reaction is (2-4):1.

[0057] In some embodiments, during the coupling reaction, the molar ratio of the Fmoc-protected amino acid to the deprotected resin can be 2:1, 2.2:1, 2.5:1, 2.8:1, 3:1, 3.2:1, 3.5:1, 3.8:1, 4:1, or any combination thereof.

[0058] In a specific embodiment of the present invention, the condensing agent used in the coupling reaction includes at least one of Oxyma / DIC and PyBOP / DIEA.

[0059] In practice, the coupling reaction includes: dissolving the Fmoc-protected amino acid and Oxyma in a small amount of DMF (the specific amount of DMF is not limited, as long as it can dissolve the Fmoc-protected amino acid and Oxyma) to obtain an amino acid solution; cooling the amino acid solution to 0–5°C; adding DIC for activation for 5–10 min to obtain an activated amino acid solution; adding the activated amino acid solution to the deprotected resin and reacting at 20–30°C; washing the resin with a solvent after the reaction is complete; washing includes washing with DMF 2–4 times. Specifically, the coupling reaction time can be adjusted according to the Kaiser test results. When the Kaiser test result is negative, the reaction is considered complete.

[0060] In a specific embodiment of the present invention, the coupling reaction time is 1 to 1.5 hours; the coupling reaction temperature is 20 to 30°C.

[0061] In a specific embodiment of the present invention, the removal of Fmoc protection is carried out using a deprotecting reagent; the deprotecting reagent includes an aprotic organic solvent containing an organic base, the organic base including Pip, and the aprotic organic solvent including at least one of DCM, DMF, and NMP. Further, the reaction time for removing the Fmoc protecting group is 0.5–1 h, and the reaction temperature is 20–30 °C.

[0062] In practice, the reaction conditions for removing the Fmoc protecting group are the same as those described above for the deprotection reaction.

[0063] In a specific embodiment of the present invention, the resin comprises 2-CTC resin. Further, the degree of resin substitution is 0.3 to 1 mmol / g, such as 0.3 mmol / g, 0.35 mmol / g, 0.4 mmol / g, 0.45 mmol / g, 0.5 mmol / g, 0.6 mmol / g, 0.8 mmol / g, 1 mmol / g, or any combination thereof.

[0064] In some embodiments, when the resin is coupled to the first Fmoc-protected amino acid, the resin, Fmoc-Tyr(tBu)-OH, DIEA, and solvent are mixed and reacted for 2–3 hours, followed by methanol capping. The solvent includes, but is not limited to, DCM. The ratio of DCM to resin is (5–15) mL:1 g, and the ratio of methanol to resin is (0.8–1.2) mL:1 g.

[0065] In a specific embodiment of the present invention, the method further includes: collecting the solution after the cutting process, adding a poor solvent to precipitate the solution, collecting the solid and washing it; the poor solvent includes MTBE.

[0066] In a specific embodiment of the present invention, the method further includes purifying the solid precipitated from the unsuitable solvent. Further, the purification includes, but is not limited to, HPLC separation and purification. Specifically, reversed-phase chromatography is used to purify the solid. Further, in the reversed-phase chromatography, mobile phase A is an aqueous solution of TFA with a volume fraction of 0.1%, and mobile phase B is acetonitrile.

[0067] In a specific embodiment of the present invention, in reversed-phase chromatography, gradient elution includes increasing the volume fraction of mobile phase B from 20% to 50% within 60 min.

[0068] Example 1 This embodiment provides a method for preparing the cholesterol-modified brain-targeting peptide Angiopep-2, including the following steps: (1) 4.08 g of 2-CTC resin (substitution degree of 0.98 mmol / g) was added to a sand core reactor, along with 2 eq of Fmoc-Tyr(tBu)-OH, 6 eq of DIEA and DCM (the ratio of DCM to resin was 10 mL: 1 g). The mixture was then mixed and reacted at 25 °C for 2.5 h. Methanol (the ratio of methanol to resin was 1 mL: 1 g) was then added, and the mixture was mixed and reacted for another 30 min. The mixture was then filtered, and the resin was washed 5 times with DCM to obtain Fmoc-Tyr(tBu)-CTC resin.

[0069] (2) Take Fmoc-Tyr(tBu)-CTC resin, add deprotection reagent (the deprotection reagent is a DMF solution of PiP with a volume fraction of 20%), the ratio of deprotection reagent to Fmoc-Tyr(tBu)-CTC resin is 10mL: 1g, mix and react at 25℃ for 30min, filter, wash the resin with DMF 6 times to obtain the deprotected resin.

[0070] (3) Weigh 3 eq of Fmoc-Glu(OtBu)-OH and 3 eq of Oxyma, dissolve them in a small amount of DMF to obtain an amino acid solution, cool the amino acid solution to 0-5℃, add 3 eq of DIC to activate for 10 min to obtain an amino acid activated solution; slowly add the amino acid activated solution to the aforementioned deprotected resin, and react at 25℃ for 1 h. After the reaction is complete, the Kaiser test result is negative, filter, and wash the resin 4 times with DMF.

[0071] (4) Repeat steps (2) and (3) to couple the corresponding Fmoc-protected amino acids according to the peptide chain sequence of the brain-targeting peptide Angiopep-2 until the coupling is completed; each Fmoc-protected amino acid, Oxyma, and DIC is 3 equivalents of the resin. The amino acids protected by Fmoc, in sequence, are: Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, and Fmoc-N(Dmb)-Thr(tBu)-OH.

[0072] (5) Add a deprotecting agent (a 20% DMF solution of PiP) to the resin obtained in step (4). The ratio of the deprotecting agent to the resin obtained in step (4) is 10 mL: 1 g. Mix and react at 25 °C for 30 min. Filter and wash the resin with DMF 6 times to obtain the deprotected resin.

[0073] (6) Weigh 3 eq of succinic anhydride, dissolve it in a small amount of DMF and add it to the deprotected resin obtained in step (5), then add 6 eq of DIEA and react at 25°C for 1 h. After the reaction is complete, the Kaiser test result is negative. Filter the resin and wash it with DMF 4 times.

[0074] (7) Weigh 5 eq cholesterol and 5 eq Oxyma, dissolve them in a small amount of DMF and add them to the resin obtained in step (6), then add 5 eq DIC and 0.1 eq DMAP and react at 25°C for 24 h; filter, wash twice with DCM, then wash twice with MTBE and dry.

[0075] (8) Prepare a cutting solution with a volume ratio of TFA, DCM, DODT, TIS, and H2O of 70:15:5:5:5. Add the solution to the dried resin obtained in step (7), with a ratio of 8 mL to 1 g of cutting solution to dried resin. Shake and react at 20°C for 3 h. Filter the resin, collect the filtrate, add 8 times the volume of pre-cooled MTBE (≤-10°C) to the filtrate for precipitation, collect the solid, and wash the solid twice with MTBE to obtain the crude cholesterol-modified brain-targeting peptide Angiopep-2. The HPLC chromatogram of the crude product is shown below. Figure 1 As shown, the yield of the crude product was 111.43%, and the HPLC purity was 61.3% (the peak position of the target substance is indicated by the arrow in the figure).

[0076] Example 2 This embodiment refers to the preparation method of Embodiment 1, the only difference being that: Fmoc-N(Dmb)-Thr(tBu)-OH in step (4) is replaced with an equal amount of Fmoc-N(Hmb)-Thr(tBu)-OH.

[0077] The yield of the crude product obtained in step (8) was 108.20%, and the HPLC purity was 45.2%.

[0078] Example 3 This embodiment refers to the preparation method of Embodiment 1, the only difference being that the composition of the cutting fluid is different in step (8).

[0079] In this embodiment, the cutting fluid is prepared according to a volume ratio of TFA, DCM, DODT, TIS, and H2O of 68:14:6:6:6.

[0080] The yield of the crude product obtained in step (8) was 109.67%, and the HPLC purity was 60.9%.

[0081] Example 4 This embodiment refers to the preparation method of Embodiment 1, the only difference being that the composition of the cutting fluid is different in step (8).

[0082] In this embodiment, the cutting fluid is prepared according to a volume ratio of TFA, DCM, DODT, TIS, and H2O of 72:16:4:4:4.

[0083] The crude product obtained in step (8) had a yield of 114.54% and an HPLC purity of 60.7%.

[0084] Example 5 This embodiment refers to the preparation method of Embodiment 1, the only difference being that the composition of the cutting fluid is different in step (8).

[0085] In this embodiment, the cutting fluid is prepared according to a volume ratio of TFA, DCM, DODT, and TIS of 75:15:5:5.

[0086] The yield of the crude product obtained in step (8) was 110.33%, and the HPLC purity was 57.2%.

[0087] Example 6 This embodiment refers to the preparation method of Embodiment 1, the only difference being that the composition of the cutting fluid is different in step (8).

[0088] In this embodiment, the cutting fluid is prepared according to a volume ratio of TFA, DCM, EDT, and TIS of 75:15:5:5.

[0089] The yield of the crude product obtained in step (8) was 112.56%, and the HPLC purity was 58.2%.

[0090] Example 7 This embodiment refers to the preparation method of Example 1, the only difference being that the amount of materials used in steps (6) and (7) is different; the specific differences are as follows: In step (6) of this embodiment, the amount of succinic anhydride is 2 eq and the amount of DIEA is 4 eq; in step (7), the amount of cholesterol is 4 eq, the amount of Oxyma is 4 eq, the amount of DIC is 4 eq, and the amount of DMAP is 0.05 eq.

[0091] The yield of the crude product obtained in step (8) was 109.45%, and the HPLC purity was 59.2%.

[0092] Example 8 This embodiment refers to the preparation method of Example 1, the only difference being that the amount of materials used in steps (6) and (7) is different; the specific differences are as follows: In step (6) of this embodiment, the amount of succinic anhydride is 4 eq and the amount of DIEA is 6 eq; in step (7), the amount of cholesterol is 6 eq, the amount of Oxyma is 6 eq, the amount of DIC is 6 eq and the amount of DMAP is 0.2 eq.

[0093] The yield of the crude product obtained in step (8) was 113.65%, and the HPLC purity was 55.4%.

[0094] Comparative Example 1 Comparative Example 1 uses the same preparation method as Example 1, except that Fmoc-N(Dmb)-Thr(tBu)-OH in step (4) is replaced with an equal amount of Fmoc-Thr(tBu)-OH.

[0095] The HPLC chromatogram of the crude product obtained in step (8) is as follows: Figure 2 As shown, the crude product yield was 108.53%, the HPLC purity was 12.9% (the peak position of the target substance is indicated by the arrow in the figure), and the crude product contained 42.7% succinimide ring structure.

[0096] In the preparation method of cholesterol-modified brain-targeting peptide Angiopep-2 of the present invention, cholesterol and succinic anhydride are used as raw materials, and Fmoc-N(Dmb)-Thr(tBu)-OH and / or Fmoc-N(Hmb)-Thr(tBu)-OH are used as raw materials for the first Thr. This method can significantly reduce the cost of raw materials while improving the yield and purity of cholesterol-modified brain-targeting peptide Angiopep-2.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing the cholesterol-modified brain-targeting peptide Angiopep-2, characterized in that, Includes the following steps: (a) Using a solid-phase synthesis method, each Fmoc-protected amino acid is coupled to the resin sequentially from the C-terminus to the N-terminus according to the peptide sequence of the brain-targeting peptide Angiopep-2, and the Fmoc protecting group is removed to obtain the peptide resin. (b) The peptide resin undergoes a first condensation reaction with succinic anhydride, then a second condensation reaction with cholesterol, followed by cleavage. The peptide sequence of the brain-targeting peptide Angiopep-2 is as follows: NH2-Thr 1 Phe 2 Phe 3 Tyr 4 Gly 5 Gly 6 Ser 7 Arg 8 Gly 9 Lys 10 Arg 11 Asn 12 Asn 13 Phe 14 Lys 15 Thr 16 Glu 17 Glu 18 Tyr 19 -OH; The sequence of the cholesterol-modified brain-targeting peptide is as follows: Cholesteryl hemisuccinate Thr 1 Phe 2 Phe 3 Tyr 4 Gly 5 Gly 6 Ser 7 Arg 8 Gly 9 Lys 10 Arg 11 Asn 12 Asn 13 Phe 14 Lys 15 Thr 16 Glu 17 Glu 18 Tyr 19 -OH; When the first Thr in the coupled peptide sequence is used, the amino acid protected by Fmoc is at least one of Fmoc-N(Dmb)-Thr(tBu)-OH and Fmoc-N(Hmb)-Thr(tBu)-OH; In step (a), the resin is 2-CTC resin; In the second condensation reaction, the peptide resin after the first condensation reaction reacts with the cholesterol under the action of a condensing agent, which includes Oxyma, DMAP, and DIC. The cutting fluid used in the cutting process includes TFA, DCM, DODT, TIS, and H2O. Each coupling reaction includes a deprotection reaction and a coupling reaction; the deprotection reaction is carried out using a deprotection reagent, which includes an aprotic organic solvent containing an organic base, wherein the organic base includes Pip, and the aprotic organic solvent includes at least one of DCM, DMF, and NMP; The coupling reaction uses at least one of the following condensing agents: a combination of Oxyma and DIC, or a combination of PyBOP and DIEA.

2. The preparation method according to claim 1, characterized in that, When the first Thr in the coupled peptide sequence is used, the amino acid protected by Fmoc is Fmoc-N(Dmb)-Thr(tBu)-OH.

3. The preparation method according to claim 1, characterized in that, In the first condensation reaction, the peptide resin reacts with the succinic anhydride under the action of DIEA.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the peptide resin, the succinic anhydride and the DIEA is 1:(2-4):(3-6).

5. The preparation method according to claim 1, characterized in that, In the second condensation reaction, the molar ratio of the peptide resin after the first condensation reaction to the cholesterol is 1:(4-6).

6. The preparation method according to claim 1, characterized in that, The molar ratio of cholesterol to Oxyma, DMAP and DIC is (4-6): (4-6): (0.05-0.2): (4-6).

7. The preparation method according to claim 1, characterized in that, The cutting fluid comprises TFA, DCM, DODT, TIS and H2O in a volume ratio of (68-72):(14-16):(4-6):(4-6):(4-6).

8. The preparation method according to claim 1, characterized in that, The ratio of the cutting fluid to the peptide resin after the second condensation reaction is (5-15) mL: 1 g.

9. The preparation method according to claim 1, characterized in that, The cutting process is carried out at a temperature of 20–25°C for 2–3 hours.

10. The preparation method according to claim 1, characterized in that, The amino acids protected by each Fmoc are respectively: Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr( tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg( Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH and Fmoc-N(Dmb)-Thr(tBu)-OH.

11. The preparation method according to claim 1, characterized in that, In the coupling reaction, the molar ratio of the Fmoc-protected amino acid to the resin is (2-4):

1.

12. The preparation method according to claim 1, characterized in that, The coupling reaction takes 1 to 1.5 hours and is carried out at a temperature of 20 to 30°C.

13. The preparation method according to claim 1, characterized in that, Also includes: Collect the solution after the cutting process, add a poor solvent to precipitate the solid, collect the solid and wash it; The undesirable solvents include at least one of MTBE and diethyl ether.

Images