Method for solid phase synthesis of leu-enkephalin by Fmoc method
By using a mixed solution of imidazole and tricyclohexylphosphine as a deprotecting agent for Fmoc, the problem of tyrosine side chain instability was solved, enabling the efficient synthesis of leucine enkephalin, improving product purity and safety, and reducing production costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YANTAI UNIV
- Filing Date
- 2023-06-29
- Publication Date
- 2026-07-24
AI Technical Summary
In the existing Fmoc method for synthesizing leucene, the protecting group of the tyrosine side chain is unstable, leading to frequent side reactions that affect synthesis efficiency and purity. Furthermore, traditional deprotecting agents such as piperidine have safety and cost issues.
A mixed solution of imidazole and tricyclohexylphosphine was used as the deprotecting agent for Fmoc, and tetrahydrofuran was used as the solvent to avoid protecting the tyrosine side chain. The stability of imidazole and the catalytic effect of tricyclohexylphosphine were utilized to promote the reaction and remove byproducts.
This method achieves efficient synthesis of leuconephrine, increases product purity to 86%, reduces subsequent purification costs and difficulties, and is superior to traditional methods in terms of operational safety and economy.
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Figure CN116554270B_ABST
Abstract
Description
Technical Field:
[0001] This invention relates to the preparation of polypeptide drugs, specifically to a method for solid-phase synthesis of human proendorphin, belonging to the field of polypeptide solid-phase synthesis technology. Background technology:
[0002] Leu-enkephalin is an endogenous endorphin composed of five amino acids with the sequence H-tyrosine-glycine-phenylamine-leucine-NH2 (H-Tyr-Gly-Gly-Phe-Leu-NH2). It belongs to the endorphin family and is primarily found in the central nervous system of humans and various animals, particularly in the forebrain region. Leu-enkephalin possesses analgesic and sedative effects, producing its pharmacological effects through binding to μ- and δ-opioid receptors. It is a natural analgesic involved in regulating pain transmission and emotional responses. The synthesis and release of human pre-enkephalin are regulated by multiple factors, including neurotransmitters, stress, and pain stimuli. Its presence in the nervous system plays a crucial role in pain regulation and emotional stability. Studies have shown that leu-enkephalin is associated with analgesic drugs and drug abuse, and its regulatory function is significant for understanding pain mechanisms and drug abuse behavior.
[0003] Solid-phase synthesis of peptides with a 9-fluorenylmethoxycarbonyl protecting group (Fmoc) offers advantages in terms of speed, efficiency, and controllability. It is widely used in drug development, biomedical research, and bioengineering. Through rational design and optimization of reaction conditions, high-yield, high-purity peptide synthesis can be achieved, providing a feasible route for the synthesis of complex peptide compounds and drugs. The Fmoc solid-phase peptide synthesis method is characterized by its simplicity, mild reaction conditions, and high synthesis efficiency, making it the most commonly used method for chemical peptide synthesis. Fmoc deprotection requires not only an appropriately basic deprotecting agent to remove the 9-H adjacent to the Fmoc fluorene ring system, but also sufficient nucleophilicity to remove the resulting dibenzo-rich ene byproduct, while maintaining a mild deprotection environment to ensure the safety of the peptide's side-chain protecting group. Conventional Fmoc deprotection uses a 20-50% piperidine dimethylformamide (DMF) solution.
[0004] Enkephalin has been successfully synthesized via the Fmoc solid-phase method. Almost all reported methods use Fmoc-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)) as the starting material because piperidine is highly basic, easily causing the unprotected phenolic side chain of tyrosine in enkephalin to form phenolate ions, leading to acylation side reactions. Experiments have shown that, under basic conditions with a certain concentration of piperidine, the alkyl protecting group of the phenolic side chain of tyrosine is far less stable than the protecting groups of other amino acids (such as serine or threonine) (Albert Isidro-Llobet et al. Chemical Reviews, 2009, Vol. 109, No. 6). Tyrosine, as the first amino acid in the solid-phase synthesis of enkephalin, requires the most deprotection steps in the entire synthesis process. Using low concentrations of piperidine, although reducing basicity and side reactions, will also significantly reduce nucleophilicity, affecting the deprotection efficiency of Fmoc and the removal of the byproduct dibenzo-fullene, and even leading to deprotection failure. There are currently no successful examples of solid-phase synthesis of leucine enkephalin without protecting the tyrosine side chain of Fmoc.
[0005] Phosphorus atoms have a much greater delocalization of lone pair electrons than nitrogen atoms, making them less susceptible to steric hindrance from neighboring small and medium-sized groups. Most phosphine compounds have lower relative basicity than their nitrogen-containing organic base counterparts, yet often exhibit superior nucleophilicity. Trialkylphosphine compounds are frequently used as catalysts in conjunction with nucleophiles, significantly enhancing the nucleophilicity of many weak nitrogen-containing organic bases. For example, trialkylphosphine's nucleophilicity causes electron cloud shift upon attacking a double bond, catalyzing amine addition reactions; it can catalyze coupling reactions of unsaturated hydrocarbons with aromatic amines; and it is also the most commonly used catalyst for 1,4-addition nitrogen heterocyclic reactions. To date, there are no reports of peptide synthesis methods using phosphine compounds and organic bases as deprotecting agents for Fmoc. However, this "weak base, strong nucleophilic" catalytic property of trialkylphosphine is precisely what is most needed for deprotection in the chemical synthesis of leucene. We believe that in the solid-phase synthesis of leucene, with trialkylphosphine as a catalyst, a preferred weak organic base as a deprotecting agent for Fmoc may perform as well as or even better than traditional strong bases such as piperidine, thereby achieving the solid-phase synthesis of leucene without the need to protect the tyrosine side chain of Fmoc. Summary of the Invention:
[0006] The purpose of this invention is to provide a method for solid-phase synthesis of leucine enkephalin using the Fmoc method.
[0007] The technical solution adopted in this invention is:
[0008] A method for solid-phase synthesis of leucine enkephalin using the Fmoc method, wherein the Fmoc deprotecting agent is a mixed solution of imidazole and tricyclohexylphosphine.
[0009] Furthermore, the solvent for the above-mentioned imidazole and tricyclohexylphosphine mixed solution is tetrahydrofuran (THF).
[0010] Furthermore, the volumetric molar concentration of imidazole in the above-mentioned mixed solution of imidazole and tricyclohexylphosphine is 1.00 mol / L.
[0011] Furthermore, the volumetric molar concentration of tricyclohexylphosphine in the above-mentioned imidazole and tricyclohexylphosphine mixed solution is 0.02 mol / L.
[0012] A method for solid-phase synthesis of leucine enkephalin using the Fmoc method, wherein the first amino acid tyrosine is used as a starting material by Fmoc-Tyr-OH without the need for a side-chain protecting group.
[0013] The aforementioned Fmoc deprotection process using a mixed solution of imidazole and tricyclohexylphosphine may be as follows:
[0014]
[0015] R represents any resin-loaded polypeptide moiety except for the N-terminal amino group, and the generated... It is (9-fluorenylmethyl)-1-imidazole.
[0016] Compared with methods using piperidine deprotectants, the innovation and advantages of the solid-phase synthesis method for human proendorphins described in this invention are as follows:
[0017] 1. Imidazole is a mild and stable organic weak base. It is more stable than piperidine in physicochemical properties at room temperature and has low toxicity, low volatility and low corrosiveness.
[0018] It is safer to use; imidazole is a solid white crystal at room temperature, and compared to liquid piperidine which has a certain viscosity and corrosiveness, imidazole is easier to transport and store; imidazole itself is also cheaper than piperidine.
[0019] 2. The catalyst used in this invention, tricyclohexylphosphine, has a large ligand cone angle (170°), which allows it to act as an excellent nucleophilic catalyst to promote the chemical removal of the byproduct dibenzofuran by imidazole, rapidly forming the dibenzofuran-imidazole adduct, thereby driving the entire reaction forward. Tricyclohexylphosphine requires only one-fiftieth the imidazole equivalent to effectively catalyze the reaction, exhibits moderate basicity, and has almost no impact on the stability of the entire deprotection system.
[0020] 3. This invention achieves solid-phase synthesis of leucine enkephalin without any side-chain protecting groups using a solution of imidazole and tricyclohexylphosphine mixed with tetrahydrofuran, and avoids the generation of tyrosine side-chain acylation byproducts caused by the traditional piperidine method. The purity of the crude leucine enkephalin obtained using this new method reaches 86%, significantly reducing the cost and difficulty of subsequent purification. This demonstrates the superiority of this method in solid-phase synthesis of leucine enkephalin and has significant reference value for the production and preparation of other peptides.
[0021] 4. The leuconephrine synthesis method proposed in this invention has no significant difference in operation steps and process flow from the conventional Fmoc synthesis method. No additional training is required for existing operators, and there is no additional requirement to update existing equipment. Attached Figure Description
[0022] Figure 1 Fmoc standard curve of deprotected Fmoc-alanine with a THF solution of 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine.
[0023] Figure 2 HPLC comparison of peptide products from Examples 2 (solid-phase synthesis of leucine enkephalin using piperidine as the deprotecting agent (tyrosine side chain without protecting group)), 3 (solid-phase synthesis of leucine enkephalin using piperidine as the deprotecting agent (tyrosine side chain with protecting group)), and 4 (solid-phase synthesis of leucine enkephalin using a mixed solution of imidazole and tricyclohexylphosphine as the deprotecting agent (tyrosine side chain without protecting group)). Detailed implementation method:
[0024] The experimental materials and reagents used in the following embodiments can all be obtained commercially or through known experimental methods. The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments 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.
[0025] Example 1. Deprotection method testing and preparation of Fmoc standard curve
[0026] Step 1. Using Fmoc-L-Ala as a standard, accurately weigh 62.22 mg of Fmoc-L-Ala and treat it with 2.0 mL of THF solution containing 1.00 mol / L imidazole (136.2 mg) and 0.02 mol / L tricyclohexylphosphine (11.2 mg) for 15 minutes. Take 1.0 mL of the solution and separate the products by thin-layer chromatography, comparing the results with the alanine standard to determine the reaction endpoint; characterize the product alanine ([M+H) by mass spectrometry.+ =90.0513) and byproduct 1-((9H-fluorene-9-yl)methyl)-1H-imidazolium ([M+H] + =247.1194). Take 0.1 mL of the remaining 1.0 mL solution and dilute to 10.0 mL.
[0027] Step 2. Using the solution diluted in Step 1, further dilute to prepare standard solutions with concentrations of 27, 45, 68, 87, 105, and 126 μmol / L. The absorbance of the THF solution blank sample (λ = 290 nm) measured using a UV-Vis spectrophotometer was 0.23, 0.43, 0.51, 0.60, 0.72, and 0.88, respectively.
[0028] Step 3. Construct the standard curve for Fmoc, with the linear regression equation being Y = 0.0067X + 0.0237 (X-axis: concentration (μmol / L), Y-axis: absorbance A, linear correlation coefficient: R² = 0.9988). Figure 1 ).
[0029] Example 2: Solid-phase synthesis of leucine enkephalin using piperidine as the deprotecting agent (tyrosine side chain without protecting group).
[0030] Step 1, Preparation of Preloaded Resin: Weigh 200.4 mg of Wang resin (loading capacity 0.82 mmol / g) and place it in a syringe reactor equipped with a sieve plate. Swell the resin sequentially with 5 mL of dichloromethane (DCM) and 5 mL of dimethylformamide (DMF). Completely dissolve 99.5 mg of Fmoc-L-Tyr-OH (1.5 equivalents) and 128.3 mg of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP, 1.5 equivalents) in 5 mL of DMF, add 85.9 μL of N,N-diisopropylethylamine (DIPEA, 3.0 equivalents), transfer to the reactor, mix thoroughly with the swollen resin, and shake to react for 40 minutes. After the reaction is complete, filter the resin. Add a DCM solution containing 31.0 μL of acetic anhydride (2.0 equivalents) and 57.3 μL of DIPEA (2.0 equivalents). Stirring at room temperature for another 30 minutes to cap unreacted hydroxyl groups on the resin. Then, filter the resin and wash it sequentially with DMF three times, DCM twice, DMF twice again, and finally DCM three times. Vacuum dry the resin.
[0031] Step 2. Removal of the Fmoc protecting group preloaded with Fmoc-L-Tyr from the resin: Add 5 mL of 20% piperidine DMF solution, shake for 10 minutes at room temperature, and then filter. Compare the absorbance of the deprotected sample with a blank sample of 20% (v / v) piperidine DMF solution (λ = 290 nm) to confirm the reaction progress. Take a very small amount of resin for ninhydrin detection; a deep blue color indicates complete deprotection.
[0032] Step 3. Sequentially load amino acids Gly, Phe, and Leu, and their Fmoc for deprotection: Wash the pre-loaded resin 5 times with DMF; mix 73.2 mg Fmoc-L-Gly-OH (1.5 equivalents), 85.9 μL DIPEA (3.0 equivalents), and 128.3 mg PyBOP (1.5 equivalents), dissolve in 5 mL DMF, transfer to the reactor, mix thoroughly with the resin, and shake for 40 minutes. After the reaction is complete, filter the resin. Wash 3 times with DMF, 2 times with DCM, and then wash 3 times with DMF again. Add 5 mL of 20% piperidine DMF solution, shake for 10 minutes at room temperature, and then filter. Compare with the blank sample of 20% (v / v) piperidine DMF solution (λ = 290 nm), measure the absorbance of the deprotected sample to confirm the reaction progress, and take a very small amount of resin for ninhydrin detection; a deep blue color indicates complete deprotection. Repeat the above steps, loading the remaining amino acids L-Gly, L-Phe, and L-Leu onto the resin sequentially using 73.2 mg Fmoc-L-Gly-OH, 95.3 mg Fmoc-L-Phe-OH, and 87.0 mg Fmoc-L-Leu-OH as reactants. Ninhydrin assay showed that complete deprotection was achieved with 20% piperidine DMF solution after loading of each amino acid. After loading and deprotection of all amino acids, the resin was washed three times with DMF, twice with DCM, three times with DMF, and then five times with DCM. The resin was then lyophilized.
[0033] Step 4. Peptide cleavage from resin: Add 95% trifluoroacetic acid (TFA) aqueous solution to the lyophilized resin, shake and react for 1 hour, filter the resin and collect the liquid product. Remove most of the TFA by rotary evaporation, precipitate the remaining solution (about 1 mL) in 50 mL of cold diethyl ether, wash three times with diethyl ether, centrifuge and lyophilize to obtain 63.1 mg of crude leucine enkephalin (HPLC purity 26.17%).
[0034] Step 5. Peptide Purification: The crude leucine enkephalin was dissolved in a small amount of 90% acetonitrile (2 mL), centrifuged, and filtered through a 0.2 μm filter membrane. Purification was then performed by injecting the solution into a preparative RP-HPLC column. The mobile phase consisted of 0.1% TFA in water and acetonitrile. The flow rate was 10 mL / min, the gradient was 30%-50% (100 min), and the wavelength was set to 220 nm. After separation and lyophilization, 15.2 mg of leucine enkephalin product was obtained (final yield 16.7%, HPLC purity 91%).
[0035] Example 3. Solid-phase synthesis of leucine enkephalin using piperidine as the deprotecting agent (tyrosine side chain with protecting group)
[0036] Step 1, Preparation of Preloaded Resin: Weigh 200.4 mg of Wang resin (loading 0.82 mmol / g) and place it in a syringe reactor equipped with a sieve plate. Swell the resin sequentially with 5 mL of DCM and 5 mL of DMF. Completely dissolve 113.06 mg of Fmoc-L-Tyr(tBu)-OH (1.5 equivalents) and 128.3 mg of PyBOP (1.5 equivalents) in 5 mL of DMF, add 85.9 μL of DIPEA (3.0 equivalents), transfer to the reactor, mix thoroughly with the swollen resin, and shake to react for 40 minutes. After the reaction is complete, filter the resin. Add a DCM solution containing 31.0 μL of acetic anhydride (2.0 equivalents) and 57.3 μL of DIPEA (2.0 equivalents). Stir for another 30 minutes at room temperature to cap unreacted hydroxyl groups on the resin. Subsequently, the filter resin was washed three times with DMF, twice with DCM, twice with DMF again, and finally three times with DCM. The resin was then vacuum dried.
[0037] Step 2. Remove the Fmoc protecting group preloaded with Fmoc-L-Tyr(tBu) from the resin: Add 5 mL of 20% piperidine DMF solution, shake for 10 minutes at room temperature, and then filter. Compare the absorbance of the deprotected sample with a blank sample of 20% (v / v) piperidine DMF solution (λ = 290 nm) to confirm the reaction progress. Take a very small amount of resin for ninhydrin detection; a deep blue color indicates complete deprotection.
[0038] Step 3. Sequentially load amino acids Gly, Phe, and Leu, and their Fmoc for deprotection: Wash the pre-loaded resin 5 times with DMF; mix 73.2 mg Fmoc-L-Gly-OH (1.5 equivalents), 85.9 μL DIPEA (3.0 equivalents), and 128.3 mg PyBOP (1.5 equivalents), dissolve in 5 mL DMF, transfer to the reactor, mix thoroughly with the resin, and shake for 40 minutes. After the reaction is complete, filter the resin. Wash 3 times with DMF, 2 times with DCM, and then wash 3 times with DMF again. Add 5 mL of 20% piperidine DMF solution, shake for 10 minutes at room temperature, and then filter. Compare with the blank sample of 20% (v / v) piperidine DMF solution (λ = 290 nm), measure the absorbance of the deprotected sample to confirm the reaction progress, and take a very small amount of resin for ninhydrin detection; a deep blue color indicates complete deprotection. Repeat the above steps, loading the remaining amino acids L-Gly, L-Phe, and L-Leu onto the resin sequentially using 73.2 mg Fmoc-L-Gly-OH, 95.3 mg Fmoc-L-Phe-OH, and 87.0 mg Fmoc-L-Leu-OH as reactants. Ninhydrin assay showed that complete deprotection was achieved with 20% piperidine DMF solution after loading of each amino acid. After loading and deprotection of all amino acids, the resin was washed three times with DMF, twice with DCM, three times with DMF, and then five times with DCM. The resin was then lyophilized.
[0039] Step 4. Peptide cleavage from resin: Add 95% TFA aqueous solution to the lyophilized resin, shake and react for 1 hour, filter the resin and collect the liquid product. Remove most of the TFA by rotary evaporation, precipitate the remaining solution (about 1 mL) in 50 mL of cold diethyl ether and wash three times with diethyl ether. After centrifugation and lyophilization, 111.7 mg of crude leucine enkephalin (HPLC purity 54.20%) was obtained.
[0040] Step 5. Peptide Purification: The crude enkephalin was dissolved in a small amount of 90% acetonitrile (2 mL), centrifuged, and filtered through a 0.2 μm filter membrane. Purification was then performed by injecting the solution into a preparative RP-HPLC column. The mobile phase consisted of 0.1% TFA in water and acetonitrile. The flow rate was 10 mL / min, the gradient was 30%-50% (100 min), and the wavelength was set to 220 nm. After separation and lyophilization, 58.5 mg of enkephalin product was obtained (actual yield 61.7% relative to the theoretical resin yield, HPLC purity 96%).
[0041] Example 4. Solid-phase synthesis of leucine enkephalin using a mixed solution of imidazole and tricyclohexylphosphine as the deprotecting agent (tyrosine side chain without protecting group).
[0042] Step 1, Preparation of Preloaded Resin: Weigh 200.2 mg of Wang resin (loading 0.82 mmol / g) and place it in a syringe reactor with a sieve plate. Swell the resin sequentially with 5 mL of DCM and 5 mL of DMF. Completely dissolve 99.5 mg of Fmoc-L-Tyr-OH (1.5 equivalents) and 128.3 mg of PyBOP (1.5 equivalents) in 5 mL of DMF, add 85.9 μL of DIPEA (3.0 equivalents), transfer to the reactor, mix thoroughly with the swollen resin, and shake to react for 40 minutes. After the reaction is complete, filter the resin. Add a DCM solution containing 31.0 μL of acetic anhydride (2.0 equivalents) and 57.3 μL of DIPEA (2.0 equivalents). Stir for another 30 minutes at room temperature to cap unreacted hydroxyl groups on the resin. Subsequently, wash the filtered resin sequentially with DMF 3 times, DCM 2 times, DMF 2 times, and finally DCM 3 times. Vacuum drying of resin.
[0043] Step 2. Removal of the Fmoc protecting group preloaded with Fmoc-L-Tyr from the resin: Add a THF solution containing 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine, shake for 10 minutes at room temperature, and then filter. Compare the deprotected sample with a blank sample containing 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine in THF solution (λ = 290 nm), measure the absorbance of the deprotected sample to confirm the reaction progress, and take a very small amount of resin for ninhydrin detection; a deep blue color indicates complete deprotection.
[0044] Step 3. Sequentially load amino acids Gly, Phe, and Leu, and deprotect them with Fmoc: Wash the pre-loaded resin 5 times with DMF; mix 73.2 mg Fmoc-L-Gly-OH (1.5 equivalents), 85.9 μL DIPEA (3.0 equivalents), and 128.3 mg PyBOP (1.5 equivalents), dissolve in 5 mL DMF, transfer to the reactor, mix thoroughly with the resin, and shake for 40 minutes. After the reaction is complete, filter the resin. Wash 3 times with DMF, 2 times with DCM, and then 3 times with THF. Add 5 mL of THF solution containing 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine, shake for 10 minutes at room temperature, and then filter. A blank sample (λ = 290 nm) containing 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine in THF solution was compared with the deprotected sample. The absorbance of the deprotected sample was measured to confirm the reaction progress. A very small amount of resin was tested for ninhydrin; a deep blue color indicated complete deprotection. The above steps were repeated, with the remaining amino acids L-Gly, L-Phe, and L-Leu loaded onto the resin sequentially using 73.2 mg Fmoc-L-Gly-OH, 95.3 mg Fmoc-L-Phe-OH, and 87.0 mg Fmoc-L-Leu-OH as reactants. Ninhydrin detection showed that each amino acid could achieve complete deprotection after loading with THF solution containing 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine. After loading and deprotection of all amino acids, the resin was washed 3 times with DMF, 2 times with DCM, 3 times with DMF, and then 5 times with DCM. The resin was then lyophilized.
[0045] Step 4. Peptide cleavage from resin: Add 95% TFA aqueous solution to the lyophilized resin, shake and react for 1 hour, filter the resin and collect the liquid product. Remove most of the TFA by rotary evaporation, precipitate the remaining solution (about 1 mL) in 50 mL of cold diethyl ether, wash three times with diethyl ether, centrifuge and lyophilize to obtain 90.5 mg of crude leucine enkephalin (HPLC purity 85.43%).
[0046] Step 5. Peptide Purification: The crude leuconephrine was dissolved in a small amount of 90% acetonitrile (2 mL), centrifuged, and filtered through a 0.2 μm filter membrane. Purification was performed by injecting the solution into a preparative RP-HPLC column. The mobile phase was 0.1% TFA in water and acetonitrile. The flow rate was 10 mL / min, gradient 30%-50% (100 min), and the wavelength was set to 220 nm. After separation and lyophilization, 60.2 mg of leuconephrine product was obtained (actual yield 63.5% relative to the theoretical yield of the resin, HPLC purity 97%). Even without side chain protecting groups, the actual yield of Example 3 was nearly four times higher than that of Example 1, and the yield and purity of the final product were comparable to or even higher than those of Example 2 using conventional methods requiring additional side chain protecting groups. Figure 2 ).
[0047] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of raw materials for the product of the present invention, addition of auxiliary components, selection of specific methods, etc., are all included within the scope of protection and disclosure of the present invention.
Claims
1. A method for solid-phase synthesis of leucine endorphins using the Fmoc method, characterized in that: In this method, the Fmoc deprotecting agent is a tetrahydrofuran solution containing 1.00 mol / L imidazole and 0.02 mol / L tricyclohexylphosphine, and the amino acids used do not have any side chain protecting groups.