Cysteine derivatives and their use in the preparation of cyclic peptides
By using cysteine derivatives as a scaffold, the problem of low yield in the olefin metathesis reaction during stapling peptide synthesis was solved, achieving high yield and stability of cyclic peptides, which are suitable for large-scale production and cell targeting.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU SHENGNUO BIOPHARM
- Filing Date
- 2022-07-21
- Publication Date
- 2026-05-29
AI Technical Summary
Existing peptide synthesis strategies suffer from low yields in olefin metathesis reactions, which negatively impacts product yield and makes them unsuitable for large-scale production.
Cysteine derivatives were used as scaffolds to prepare cyclic peptides via solid-phase peptide synthesis. Amino acid derivatives were used to improve the stable conformation of the cyclic peptides and enhance the stability and affinity of the α-helix structure.
It improves the synthesis yield of cyclic peptides, making them suitable for large-scale production, and enhances the stability and cell membrane penetration ability of cyclic peptides.
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Abstract
Description
Technical Field
[0001] This invention relates to a cysteine derivative and its application in the preparation of cyclic peptides. Background Technology
[0002] Stapling peptides are chemically modified cyclic peptides developed based on the requirement that polypeptides form α-helices to cross the cell membrane and enter the cell. Many biological processes are regulated through protein-protein interactions, such as viral self-assembly, cell growth, division, and differentiation. However, the protein-protein interface is usually too large, making it difficult for small molecule drugs to target and specifically block these interactions, thus hindering therapeutic efficacy. Protein drugs, because they struggle to cross the cell membrane, also cannot directly target intracellular interactions. Therefore, researchers have sought new drug molecules that overcome these limitations, enabling them to both cross the cell membrane and specifically target protein-protein interactions.
[0003] Studies have shown that peptides with α-helical structures and high positive charges can cross cell membranes. Therefore, researchers have developed α-helical structures using disulfide bonds and intramolecular amide bonds as scaffolds; however, these scaffolds are not stable under physiological conditions. In 2000, Verdine et al. developed a method using carbon-carbon bonds as a scaffold to stabilize the α-helical structure of peptides. Peptides obtained by this method are called stapled peptides. Stapled peptides have advantages such as a higher degree of α-helix structure, stronger affinity, ability to cross cell membranes, resistance to protease hydrolysis, and a long half-life in vivo.
[0004] The conventional strategy for synthesizing staple peptides involves introducing two non-natural amino acids containing α-methyl and α-olefin groups during solid-phase peptide chain synthesis. These two non-natural amino acids then undergo an olefin metathesis reaction (RCM) to cyclize and form a stable α-helical conformation, thus synthesizing the staple peptide. However, the low yield of the olefin metathesis reaction, coupled with the need for hydrogenation processes, significantly impacts the product yield.
[0005] This invention provides a cysteine derivative, offering a new solution for stapled peptides, while significantly increasing the yield of cyclic peptides and making it more suitable for large-scale production. Summary of the Invention
[0006] This invention first provides a cysteine derivative:
[0007]
[0008] The cysteine in structure I is either L-cysteine or D-cysteine;
[0009] In structure I, m1 is an integer from 2 to 10;
[0010] In structure I, m2 is an integer from 2 to 10;
[0011] In structure I, R stands for Teoc or Troc.
[0012] The aforementioned amino acid derivatives are used to prepare cyclic peptides with stable conformations. Detailed Implementation
[0013] This invention discloses an amino acid derivative and its application in the preparation of cyclic peptides. Those skilled in the art can refer to this document and appropriately modify the relevant parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method of this invention has been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0014] The Chinese names corresponding to the English abbreviations used in this invention are shown in the table below:
[0015]
[0016] Example 1: Preparation of Compound 1
[0017]
[0018] The chemical reaction formula is as follows:
[0019]
[0020] 1. Preparation of Intermediate 1
[0021] Take 0.8 mol of raw material 1 and 1 mol of raw material 2, dissolve them in an appropriate amount of DMF, add 1 mol of pyridine, stir and react overnight at 60°C, evaporate the DMF to dryness, dissolve the residue in 2 L of 10% TFA / DCM solution and stir and react for 1 hour, evaporate the TFA / DCM solution to dryness, dissolve the residue in ethyl acetate, filter out the residue, wash the filtrate 6 times with saturated saline, evaporate the filtrate to dryness to obtain intermediate 1.
[0022] 2. Preparation of Intermediate 2
[0023] Intermediate 1 was dissolved in 60% dioxane aqueous solution, 1 mol NaHCO3 aqueous solution was added, and 1 mol Teoc-OSu was added with stirring. The reaction was stirred at room temperature for 6 hours, the solvent was evaporated, the residue was extracted with ethyl acetate, washed 5 times with saturated brine, the organic phase was dried with anhydrous sodium sulfate, the organic phase was collected by filtration, and the solvent was evaporated to obtain intermediate 2.
[0024] 3. Preparation of intermediate 3
[0025] Intermediate 2 was dissolved in ethyl acetate, the residue was filtered off, and the filtrate was washed 6 times alternately with saturated saline and 10% sodium carbonate solution. The filtrate was then evaporated to dryness to obtain intermediate 3.
[0026] 4. Preparation of intermediate 4
[0027] Intermediate 3 was dissolved in DCM, and 1 mol of bromoacetic acid and 1 mol of DIC were added. The mixture was stirred for 6 hours, the solvent was evaporated, and then DCM was added again to dissolve it. The mixture was then evaporated again. This process was repeated 3 times to obtain intermediate 4.
[0028] 5. Preparation of intermediate 5
[0029] Intermediate 4 was dissolved in 50% tetrahydrofuran aqueous solution, and 1 mol of cysteine was added with stirring. Sodium carbonate solution was added with stirring to maintain pH 8.0. The reaction was continued with stirring for 3 hours. The pH was adjusted to 4.0 with 10% hydrochloric acid solution. The solvent was evaporated to dryness, the residue was dissolved in ethyl acetate, washed 6 times with saturated brine, and the solvent was evaporated to dryness to obtain intermediate 5.
[0030] 6. Preparation of Compound 1
[0031] Intermediate 5 was dissolved in 50% tetrahydrofuran aqueous solution, and 1 mol of NaHCO3 aqueous solution was added. 1 mol of Fmoc-OSu was added while stirring, and the reaction was stirred at room temperature for 6 hours. The pH was adjusted to 3.5 with 1% saline solution. The crude product was filtered and recrystallized from n-hexane-ethyl acetate to obtain compound 1.
[0032] Example 2 Preparation of other compounds
[0033] The following compounds were prepared using the method described above:
[0034]
[0035] Example 3: Preparation of cyclic peptide AOD243907
[0036]
[0037] The preparation method includes: preparing peptide resin by solid-phase peptide synthesis, then acid hydrolyzing the peptide resin to obtain crude product, and finally purifying the crude product to obtain pure product.
[0038] 1. Synthesis of peptide resins
[0039] Using Rink Amide BHHA resin as the carrier resin, peptide resins were prepared by sequentially coupling the peptides with the corresponding protected amino acids through de-Fmoc protection and coupling reactions.
[0040] (1) Access the first protected amino acid in the main chain
[0041] Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution for later use.
[0042] Take 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.4 mmol / g), protect it with 20% PIP / DMF solution for 25 minutes, wash and filter to obtain Fmoc-free resin.
[0043] The activated solution of the first protected amino acid was added to the resin that had been de-Fmoc-treated, and the coupling reaction was carried out for 60–300 minutes. After filtration and washing, a resin containing one protected amino acid was obtained.
[0044] (2) Integrating main chain protected amino acids
[0045] Using the same method as described above for inserting the first protected amino acid into the main chain, the corresponding protected amino acids of the respective polypeptide sequences are sequentially inserted to obtain a resin containing main chain amino acids.
[0046] The protecting amino acid corresponding to the first Tyr is Boc-Tyr(tBu).
[0047] The protecting amino acid corresponding to Cys at position 16 is Fmoc-Cys (S-allyl acetate).
[0048] The protected amino acid corresponding to Cys at position 20 is compound 1.
[0049] (3) Cycloning
[0050] Take 5 mmol of tetrakis(triphenylphosphine)palladium and 50 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, remove the Alloc and All protecting groups from the resin containing the main chain amino acid for 8 hours, filter and wash to obtain the Alloc and All-free resin for later use.
[0051] Take 0.03 mol HOBt and dissolve it in an appropriate amount of DMF; take another 0.03 mol DIC and dissolve it in an appropriate amount of DMF; add it to the resin that has been de-Alloced and de-Alled while stirring, and perform the coupling reaction for 60-300 minutes. Filter and wash to obtain the cyclized resin.
[0052] (4) Add side-chain protected amino acids or monoprotected fatty acids
[0053] Take the above cyclized resin, protect it with 1 mol / L tetrabutylammonium fluoride / tetrahydrofuran solution for 36 hours, wash and filter to obtain Teoc-free resin.
[0054] Using the same method as described above for adding the first protected amino acid to the main chain, the corresponding protected amino acids and monoprotected fatty acids of the side chains are sequentially added to obtain peptide resin.
[0055] 2. Preparation of crude product
[0056] Take the above peptide resin and add a lysis reagent with a volume ratio of TFA:water:EDT = 95:5:5 (10 mL of lysis reagent per gram of resin). Stir well and react at room temperature for 3 hours. Filter the reaction mixture using a sintered glass funnel, collect the filtrate, wash the resin three times with a small amount of TFA, combine the filtrates and concentrate under reduced pressure. Add anhydrous diethyl ether to precipitate the precipitate, wash the precipitate three times with anhydrous diethyl ether, and dry under vacuum to obtain an off-white powder, which is the crude product.
[0057] 3. Preparation of pure products
[0058] Take the above crude product, add water and stir, adjust the pH to 8.0 with ammonia until completely dissolved, filter the solution through a 0.45μm mixed microporous membrane, and purify for later use;
[0059] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18 column, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. The chromatographic column was 30 mm * 250 mm, and the flow rate was 20 mL / min. Gradient elution was used, and the sample was injected repeatedly for purification. The crude product solution was loaded into the chromatographic column, and the mobile phase was started for elution. The main peak was collected, and after acetonitrile was removed, the purified intermediate concentrate was obtained.
[0060] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane and set aside. High-performance liquid chromatography (HPLC) was used for salt exchange. The mobile phase system was 1% acetic acid / water solution-acetonitrile. The chromatographic packing material was a 10 μm reversed-phase C18 column (30 mm * 250 mm) with a flow rate of 20 mL / min (the flow rate can be adjusted according to different column specifications). Gradient elution and cyclic loading were used. The sample was loaded into the column, the mobile phase was started for elution, chromatograms were collected, and changes in absorbance were observed. The salt-exchanged main peak was collected and its purity was determined using analytical liquid chromatography. The salt-exchanged main peak solutions were combined, concentrated under reduced pressure to obtain a pure acetic acid aqueous solution, and then freeze-dried to obtain pure peptides.
[0061] Example 4: Determination of the activity of cyclic peptide AOD243907GLP-1
[0062] 1. Measurement Method
[0063] GLP-1R, upon stimulation by its specific agonist, activates the intracellular adenylate cyclase pathway, increases cAMP levels, and ultimately leads to insulin production and release. Cell lines stably transfected with GLP-1R were stimulated with the analyte, resulting in a rapid increase in intracellular cAMP levels. The relative light units (RLU) after each dose of stimulation were measured using a chemiluminescence method, and the EC50 of the agonist was calculated. This activity assay is currently a widely used method for detecting GLP-1 receptor agonist activity both domestically and internationally.
[0064] Using the CHO-K1 cell line stably expressing GLP-1R, the stable cells were stimulated with different concentrations of agonists. The EC50 value of the agonist was calculated by measuring the relative light units after each dose of stimulation.
[0065] 2. Measurement Results
[0066] The measurement results are shown in the table below:
[0067]
[0068] Example 5: Determination of the activity of cyclic peptide AOD243907GIP
[0069] 1. Measurement Method
[0070] GIPR, when stimulated by its specific agonist, activates the intracellular adenylate cyclase pathway, increases cAMP levels, and ultimately leads to insulin production and release. Cell lines stably transfected with GIPR were stimulated with the analyte, resulting in a rapid increase in intracellular cAMP levels. The relative light units (RLU) after each dose of stimulation were measured using a chemiluminescence method, and the EC50 of the agonist was calculated. This activity assay is currently a widely used method for detecting GIP receptor agonist activity both domestically and internationally.
[0071] Using the CHO-K1 cell line that stably expresses GIPR, the stable cells were stimulated with different concentrations of agonists. The EC50 value of the agonist was calculated by measuring the relative light units after each dose of stimulation.
[0072] 2. Measurement Results
[0073] The measurement results are shown in the table below:
[0074]
Claims
1. A cysteine derivative having structural formula I: ， The cysteine in structure I is either L-cysteine or D-cysteine; In structure I, m1 is an integer from 2 to 10; In structure I, m2 is an integer from 2 to 10; In structure I, R stands for Teoc or Troc.
2. The cysteine derivative according to claim 1, used to prepare a cyclic peptide with a stable conformation.