Preparation method of acetyl tetrapeptide-3
By employing a multi-step synthesis method, the problem of insufficient preparation of acetyl tetrapeptide-3 was solved, achieving high yield and high purity of acetyl tetrapeptide-3, meeting the needs of cosmetic and hair repair.
Patent Information
- Application Number
- CN202511340546.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-18
- Publication Date
- 2026-02-06
AI Technical Summary
There are few literature reports on the synthesis of acetyl tetrapeptide-3 in the existing technology, and there is a lack of effective preparation methods.
Acetyl tetrapeptide-3 was synthesized through a series of steps, including the condensation reaction of hydroxyl and carboxyl groups, the involvement of amine nucleophiles, and a cleavage process, using specific compounds and solvents.
Achieving high yield and high purity of acetyl tetrapeptide-3 has been accomplished, meeting the needs of cosmetic and hair repair applications.
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Figure CN121471300A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological peptide synthesis, and particularly relates to a preparation method of acetyl tetrapeptide-3. BACKGROUND
[0002] Acetyl tetrapeptide-3 is a cosmetic polypeptide containing four amino acids, which can rapidly repair hair follicle cells, stimulate natural growth of eyebrows and hair, effectively reduce hair loss caused by aging, and make eyebrows and hair more dense, natural, soft and tight. Acetyl tetrapeptide-3 can accelerate the synthesis of extracellular matrix proteins such as laminin, collagen III and VII through fibroblasts; directly act on the tissue around the hair follicle to increase the volume and length of the hair follicle; repair the epidermis-dermis junction tissue (DEJ) and promote the fixation of hair in the hair follicle.
[0003] At present, there are very few literatures reported on the synthesis of acetyl tetrapeptide-3. Therefore, it is necessary to provide a preparation method of acetyl tetrapeptide-3. SUMMARY
[0004] Therefore, it is necessary to provide a preparation method of acetyl tetrapeptide-3.
[0005] A preparation method of acetyl tetrapeptide-3, comprising the following steps:
[0006] Step one, dissolving HOAc and a compound containing an active hydroxyl group in a first organic solvent, adding a carboxyl activating agent at 0-10 DEG C, uniformly mixing, naturally increasing to room temperature to make the hydroxyl group and the carboxyl group condense, separating Ac-O-Z after sufficient reaction, wherein the compound containing an active hydroxyl group is denoted as HO-Z;
[0007] Step two, uniformly mixing Ac-O-Z, H-Lys(Boc)-OH, a first amine nucleophile, water and a second organic solvent at 0-10 DEG C, then naturally increasing to room temperature, separating Ac-Lys(Boc)-OH after sufficient reaction;
[0008] Step three, uniformly mixing Ac-Lys(Boc)-OH, H-Gly-OH, the carboxyl activating agent, a second amine nucleophile, the compound containing an active hydroxyl group and the first organic solvent at 0-10 DEG C, then naturally increasing to room temperature, separating Ac-Lys(Boc)-Gly-OH after sufficient reaction;
[0009] Step four, mixing Fmoc-His(Trt)-OH, H-Lys(Boc)-NH2, the carboxyl activating agent, the second amine nucleophile, the active hydroxyl-containing compound and the first organic solvent uniformly at 0-10°C, then naturally rising to room temperature, separating after sufficient reaction to obtain Fmoc-His(Trt)-Lys(Boc)-NH2;
[0010] Step five, mixing the Fmoc-His(Trt)-Lys(Boc)-NH2, the basic nucleophile and the first organic solvent uniformly at 0-10°C, then naturally rising to room temperature, separating after sufficient reaction to obtain H-His(Trt)-Lys(Boc)-NH2;
[0011] Step six, mixing the Ac-Lys(Boc)-Gly-OH, the H-His(Trt)-Lys(Boc)-NH2, the carboxyl activating agent, the second amine nucleophile, the active hydroxyl-containing compound and the first organic solvent uniformly at 0-10°C, then naturally rising to room temperature, separating after sufficient reaction to obtain Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2;
[0012] Step seven, mixing the Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 and the cleavage solution uniformly at 0-10°C, then naturally rising to room temperature, separating after sufficient reaction to obtain the required acetyl tetrapeptide-3, the structural formula of which is Ac-Lys-Gly-His-Lys-NH2.
[0013] In one embodiment, the active hydroxyl-containing compound is selected from at least one of HOBT, HOPFP, HOSu, HOAT, OXyma and HONB.
[0014] In one embodiment, the first amine nucleophile is selected from at least one of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, lithium hydroxide, DIEA, triethylamine and pyridine;
[0015] The second amine nucleophile is selected from at least one of DIEA, 2,4,6-trimethylpyridine, triethylamine and N-methylmorpholine;
[0016] The basic nucleophile is selected from at least one of diethylamine, piperidine, piperazine, cyclohexylamine, 1-methylpiperazine and anhydrous piperazine;
[0017] The first organic solvent is selected from at least one of DCM, DMF, THF, acetonitrile, DMOS, NMP, 2-THF and EA;
[0018] The second organic solvent is selected from at least one of THF, acetonitrile and NMP, DMF, DMSO, 1,4-dioxane.
[0019] In one embodiment, the carboxyl activating agent is selected from at least one of EDC.HCl, DIC, DCC, HBTU, TBTU, HATU, HCTU, PyBOP and PyAOP.
[0020] In one embodiment, in step two, the volume ratio of the water and the second organic solvent is 1:0.5-2.
[0021] In one embodiment, in step one, the molar ratio of the HOAc, the active hydroxyl group-containing compound and the carboxyl activating agent is 1:1-1.5:1-1.5;
[0022] The operation of separating Ac-O-Z after sufficient reaction is as follows: after sufficient reaction, the reaction solution is filtered, the solid is washed with the first organic solvent, the filtrate is combined and concentrated under reduced pressure to obtain Ac-O-Z;
[0023] In step two, the molar ratio of the Ac-O-Z, the H-Lys(Boc)-OH and the first amine nucleophile is 1:0.9-1.4:2-5;
[0024] The operation of separating Ac-Lys(Boc)-OH after sufficient reaction is as follows: after sufficient reaction, the reaction solution is concentrated under reduced pressure, washed with hydrochloric acid, and then extracted, concentrated and filtered in sequence, and the obtained solid is dried to obtain Ac-Lys(Boc)-OH;
[0025] In step three, the molar ratio of the Ac-Lys(Boc)-OH, the H-Gly-OH, the carboxyl activating agent, the second amine nucleophile and the active hydroxyl group-containing compound is 1:0.9-1.3:1-1.5:2-5:1-1.5;
[0026] The operation of separating Ac-Lys(Boc)-OH after sufficient reaction is as follows: after sufficient reaction, the reaction solution is settled in water, and the solid is precipitated, filtered and then washed with 10% potassium hydrogen sulfate aqueous solution and purified water in sequence once, the washed solid is dissolved with the first organic solvent, and the organic phase is reserved after standing and layering;
[0027] In step four, the molar ratio of the Fmoc-His(Trt)-OH, the H-Lys(Boc)-NH2, the carboxyl activating agent, the second amine nucleophile and the active hydroxyl group-containing compound is 1:0.9-1.3:1-1.5:2-5:1-1.5;
[0028] The operation for separating Fmoc-His(Trt)-Lys(Boc)-NH2 after sufficient reaction is as follows: after sufficient reaction, the reaction solution is settled in water to precipitate solid, the obtained solid is filtered, and then washed once with 10% potassium bisulfate aqueous solution and purified water, respectively; the washed solid is dissolved with the first organic solvent, and then separated by standing; and the organic phase is reserved.
[0029] In one embodiment, in step five, the ratio of Fmoc-His(Trt)-Lys(Boc)-NH2 to the basic nucleophile is 0.4 mol: 0.3 L to 0.6 L.
[0030] The operation for separating H-His(Trt)-Lys(Boc)-NH2 after sufficient reaction is as follows: after sufficient reaction, the reaction solution is concentrated under reduced pressure to an oil, the oil is stirred and dissolved with the first organic solvent until clear, and then MTBE is continuously added until solid is precipitated; the solid is filtered, and then washed once with MTBE; and the solid is dried to obtain H-His(Trt)-Lys(Boc)-NH2.
[0031] In one embodiment, in step six, the molar ratio of Ac-Lys(Boc)-Gly-OH, H-His(Trt)-Lys(Boc)-NH2, the carboxyl activating agent, the second amine nucleophile and the compound containing active hydroxyl group is 1: 0.9 to 1.3: 1 to 1.5: 2 to 5: 1 to 1.5.
[0032] The operation for separating Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 after sufficient reaction is as follows: after sufficient reaction, the reaction solution is settled in water to precipitate solid, the obtained solid is filtered, and then washed once with 10% potassium bisulfate aqueous solution, 5% sodium carbonate solution and water, respectively; the washed product is dissolved with the first organic solvent, and then separated by standing; the organic phase is added into a mixture of ethyl acetate and MTBE in a volume ratio of 1:1 to precipitate solid, the filtered solid is washed with a mixture of ethyl acetate and MTBE in a volume ratio of 4:1, and then dried to obtain Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2.
[0033] In one embodiment, in step seven, the cleavage solution is a mixture of TFA, Tis and H2O in a volume ratio of 95: 1.5 to 4: 1.5 to 4.
[0034] In one embodiment, in step seven, the ratio of Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 to the cleavage solution is 379.3 g: 1 L to 2 L.
[0035] The operation of separating the acetyl tetrapeptide-3 after the sufficient reaction is as follows: after the sufficient reaction, the reaction solution is concentrated under reduced pressure to half, the concentrated reaction solution is added into isopropyl ether to precipitate solid, the filtered solid is washed by slurry with a TFA and acetonitrile mixture with a volume ratio of 1:9, and then washed by slurry with acetonitrile twice, and the washed product is dried to obtain the acetyl tetrapeptide-3.
[0036] The application provides a novel preparation method of acetyl tetrapeptide-3, and the acetyl tetrapeptide-3 prepared by the preparation method of acetyl tetrapeptide-3 has high yield and high purity. BRIEF DESCRIPTION OF DRAWINGS
[0037] Figure 1 The flow chart of the preparation method of acetyl tetrapeptide-3 is shown.
[0038] Figure 2 The detection result chart of mass spectrometry detection of the product in Example 1 is shown.
[0039] Figure 3 The detection result chart of HPLC detection of the product in Example 1 is shown. DETAILED DESCRIPTION
[0040] In order to make the purpose, technical scheme and advantages of the application clearer, the embodiments of the application will be further described below with reference to the drawings.
[0041] In combination with Figure 1 The application discloses a preparation method of acetyl tetrapeptide-3, and the preparation method comprises the following steps.
[0042] S10, dissolving Ac and a compound containing active hydroxyl in a first organic solvent, adding a carboxyl activator at 0-10 DEG C, uniformly mixing, naturally increasing to room temperature to make the hydroxyl and the carboxyl condense, separating Ac-O-Z after sufficient reaction.
[0043] The compound containing active hydroxyl is denoted as HO-Z.
[0044] The compound containing active hydroxyl contains active hydroxyl, drives the amide bond formation through the generation of an activated ester intermediate, and also serves as a condensing agent additive to inhibit racemization, improve coupling efficiency and reduce by-products.
[0045] The carboxyl activator activates the carboxyl (-COOH) to catalyze the dehydration condensation of the carboxyl in HOAc (acetic acid) and the active hydroxyl to form an ester bond.
[0046] Preferably, in the embodiment, the compound containing active hydroxyl is selected from at least one of HOBT, HOPFP, HOSu, HOAT, OXyma and HONB.
[0047] wherein the HOSU can capture the O-acyl isourea to produce stable HOSU ester.
[0048] Specifically, the compound containing active hydroxyl group is HOSu, and the product obtained by S10 is Ac-OSu; the compound containing active hydroxyl group is HOBT, and the product obtained by S10 is Ac-OBT.
[0049] Preferably, in the embodiment, the carboxyl activating agent is selected from at least one of EDC.HCl, DIC, DCC, HBTU, TBTU, HATU, HCTU, PyBOP and PyAOP.
[0050] wherein the DCC can activate the carboxyl group to generate O-acyl isourea.
[0051] Preferably, in the embodiment, the first organic solvent is selected from at least one of DCM, DMF, THF, acetonitrile, DMOS, NMP, 2-THF and EA.
[0052] Preferably, in S10, the molar ratio of HOAc, the compound containing active hydroxyl group and the carboxyl activating agent is 1:1-1.5:1-1.5.
[0053] More preferably, in S10, the molar ratio of HOAc, the compound containing active hydroxyl group and the carboxyl activating agent is 1:1.2:1.2.
[0054] Preferably, in S10, the operation of separating Ac-O-Z after sufficient reaction is as follows: after sufficient reaction, the reaction solution is filtered, the solid is washed with the first organic solvent, and the filtrate is combined and concentrated under reduced pressure to obtain Ac-O-Z.
[0055] Preferably, in S10, the reaction time for sufficient reaction is 5h.
[0056] S20, uniformly mixing Ac-O-Z, H-Lys(Boc)-OH, the first amine nucleophile, water and the second organic solvent at 0-10℃, then naturally rising to room temperature, and separating Ac-Lys(Boc)-OH after sufficient reaction.
[0057] The first amine nucleophile can enhance the nucleophilicity of the amine, promote nucleophilic addition, and in addition, the first amine nucleophile also plays a role in neutralizing the by-product acid and maintaining the reaction balance.
[0058] Preferably, in the embodiment, the first amine nucleophile is selected from at least one of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, lithium hydroxide, DIEA, triethylamine and pyridine.
[0059] Preferably, in the present embodiment, the second organic solvent is selected from at least one of THF, acetonitrile and NMP, DMF, DMSO, 1,4-dioxane
[0060] Preferably, in S20, the volume ratio of water and the second organic solvent is 1:0.5-2.
[0061] More preferably, in S20, the volume ratio of water and the second organic solvent is 1:1.
[0062] Preferably, in S20, the molar ratio of Ac-O-Z, H-Lys(Boc)-OH and the first amine nucleophile is 1:0.9-1.4:2-5.
[0063] More preferably, in S20, the molar ratio of Ac-O-Z, H-Lys(Boc)-OH and the first amine nucleophile is 1:1.1:3.
[0064] Preferably, in S20, the operation of separating Ac-Lys(Boc)-OH after sufficient reaction is as follows: after sufficient reaction, the reaction solution is concentrated under reduced pressure, washed with hydrochloric acid (preferably 3N), and then extracted, concentrated and filtered in sequence, and the obtained solid is dried to obtain Ac-Lys(Boc)-OH.
[0065] Preferably, in S20, the time for sufficient reaction is 2h.
[0066] S30, Ac-Lys(Boc)-OH, H-Gly-OH, a carboxyl activating agent, a second amine nucleophile, a compound containing an active hydroxyl group and a first organic solvent are mixed uniformly at 0-10°C, then naturally raised to room temperature, and Ac-Lys(Boc)-Gly-OH is separated after sufficient reaction.
[0067] The second amine nucleophile can enhance the nucleophilicity of the amine, promote nucleophilic addition, and in addition, the second amine nucleophile also plays a role in neutralizing the by-product acid and maintaining the reaction balance.
[0068] Preferably, in S30, the second amine nucleophile is selected from at least one of DIEA, 2,4,6-trimethylpyridine, triethylamine and N-methyl morpholine.
[0069] More preferably, in S30, the compound containing an active hydroxyl group is selected from HOBT. HOBT contains an active hydroxyl group, and drives the formation of an amide bond by generating an activated ester intermediate.
[0070] More preferably, in S30, the carboxyl activating agent is EDC.HCl. EDC.HCl can catalyze the dehydrating condensation of the amino group of an amino acid and the carboxyl group to form a peptide bond.
[0071] Preferably, in S30, the molar ratio of Ac-Lys(Boc)-OH, H-Gly-OH, carboxyl activating agent, second amine nucleophile and active hydroxyl group-containing compound is 1:0.9-1.3:1-1.5:2-5:1-1.5.
[0072] More preferably, in S30, the molar ratio of Ac-Lys(Boc)-OH, H-Gly-OH, carboxyl activating agent, second amine nucleophile and active hydroxyl group-containing compound is 1:1.05:1.2:3:1.2.
[0073] Preferably, in S30, the operation of separating Ac-Lys(Boc)-OH after sufficient reaction is as follows: after sufficient reaction, the reaction solution is settled in water, and solid is precipitated, the obtained solid is washed once with 10% potassium hydrogen sulfate aqueous solution and purified water in turn, the washed solid is dissolved with the first organic solvent, and the organic phase is reserved after standing and separating.
[0074] Preferably, in S30, the reaction time of sufficient reaction can be determined according to actual conditions. For example, the reaction can be completed until HPLC detection of Ac-Lys(Boc)-OH is less than or equal to 0.2%.
[0075] S40, Fmoc-His(Trt)-OH, H-Lys(Boc)-NH2, carboxyl activating agent, second amine nucleophile, active hydroxyl group-containing compound and first organic solvent are mixed uniformly at 0-10°C, and then naturally increased to room temperature, and Fmoc-His(Trt)-Lys(Boc)-NH2 is separated after sufficient reaction.
[0076] More preferably, in S40, the active hydroxyl group-containing compound is selected from HOBT. HOBT contains active hydroxyl group, and drives amide bond formation through the generation of activated ester intermediate.
[0077] More preferably, in S40, the carboxyl activating agent is EDC.HCl. EDC.HCl can catalyze the dehydrating condensation of amino group of amino acid and carboxyl group to form peptide bond.
[0078] More preferably, in S40, the second amine nucleophile is DIEA. DIEA can enhance the nucleophilicity of amine, promote nucleophilic addition, and in addition, DIEA also plays a role in neutralizing byproduct acid and maintaining reaction balance.
[0079] Preferably, in S40, the molar ratio of Fmoc-His(Trt)-OH, H-Lys(Boc)-NH2, carboxyl activating agent, second amine nucleophile and active hydroxyl group-containing compound is 1:0.9-1.3:1-1.5:2-5:1-1.5.
[0080] Preferably, in S40, the molar ratio of Fmoc-His(Trt)-OH, H-Lys(Boc)-NH2, the carboxyl activating agent, the second amine nucleophile and the compound containing active hydroxyl is 1:1.05:1.2:3:1.2.
[0081] Preferably, in S40, the operation of separating Fmoc-His(Trt)-Lys(Boc)-NH2 after sufficient reaction is as follows: after sufficient reaction, the reaction solution is settled in water, and the solid is precipitated, and the solid obtained after filtration is washed once with 10% potassium hydrogen sulfate aqueous solution and purified water, respectively, the washed solid is dissolved with the first organic solvent, and the organic phase is reserved after standing and layering.
[0082] Preferably, in S40, the reaction time of sufficient reaction can be determined according to actual conditions. For example, it can be that the reaction is carried out until HPLC detection of Fmoc-His(Trt)-OH is less than or equal to 0.2%, indicating that the reaction is complete.
[0083] S50, Fmoc-His(Trt)-Lys(Boc)-NH2, a basic nucleophile and a first organic solvent are mixed uniformly at 0-10°C, and then naturally raised to room temperature, and H-His(Trt)-Lys(Boc)-NH2 is separated after sufficient reaction.
[0084] The basic nucleophile is deprotected by β-elimination reaction.
[0085] Preferably, in S50, the basic nucleophile is selected from at least one of diethylamine, piperidine, piperazine, cyclohexylamine, 1-methylpiperazine and anhydrous piperazine.
[0086] Preferably, in S50, the ratio of Fmoc-His(Trt)-Lys(Boc)-NH2 and the basic nucleophile is 0.4 mol: 0.3 L-0.6 L.
[0087] More preferably, in S50, the ratio of Fmoc-His(Trt)-Lys(Boc)-NH2 and the basic nucleophile is 0.4037 mol: 0.4 L.
[0088] Preferably, in S50, the operation of separating H-His(Trt)-Lys(Boc)-NH2 after sufficient reaction is as follows: after sufficient reaction, the reaction solution is concentrated to an oil under reduced pressure, the first organic solvent is added to stir, dissolve and clarify, and MTBE is continuously added until the solid is precipitated, and the solid is filtered and washed once with MTBE, and H-His(Trt)-Lys(Boc)-NH2 is obtained after drying.
[0089] Preferably, in S50, the reaction time for sufficient reaction can be determined according to actual conditions. For example, the reaction can be until HPLC detection of Fmoc-His(Trt)-Lys(Boc)-NH2 is ≤ 0.1%, indicating that the reaction is complete.
[0090] S60, Ac-Lys(Boc)-Gly-OH obtained in S30, H-His(Trt)-Lys(Boc)-NH2 obtained in S50, a carboxyl activating agent, a second amine nucleophile, a compound containing an active hydroxyl group, and a first organic solvent are mixed uniformly at 0-10°C, then naturally raised to room temperature, and after sufficient reaction, Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 is obtained by separation.
[0091] Preferably, in S60, the molar ratio of Ac-Lys(Boc)-Gly-OH, H-His(Trt)-Lys(Boc)-NH2, a carboxyl activating agent, a second amine nucleophile, and a compound containing an active hydroxyl group is 1:0.9-1.3:1-1.5:2-5:1-1.5.
[0092] More preferably, in S60, the molar ratio of Ac-Lys(Boc)-Gly-OH, H-His(Trt)-Lys(Boc)-NH2, a carboxyl activating agent, a second amine nucleophile, and a compound containing an active hydroxyl group is 1:1.05:1.2:3:1.2.
[0093] Preferably, in S60, after sufficient reaction, Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 is obtained by separation by the following operation: after sufficient reaction, the reaction solution is settled in water, and the solid is precipitated, the obtained solid is washed with 10% potassium hydrogen sulfate aqueous solution, 5% sodium carbonate solution, and water in sequence, the washed product is dissolved with a first organic solvent, and after standing and layering, the organic phase is added to a mixture of ethyl acetate and MTBE in a volume ratio of 1:1 to precipitate the solid, and the filtered solid is washed with a mixture of ethyl acetate and MTBE in a volume ratio of 4:1, and after drying, Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 is obtained.
[0094] Preferably, in S60, the reaction time for sufficient reaction can be determined according to actual conditions. For example, the reaction can be until HPLC detection of Ac-Lys(Boc)-Gly-OH is ≤ 0.2%, indicating that the reaction is complete.
[0095] S70, uniformly mixing Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 and the cleavage solution at 0-10℃, then naturally increasing to room temperature, separating the acetyl tetrapeptide-3 after sufficient reaction.
[0096] Preferably, in S70, the cleavage solution is a mixture of TFA, Tis and H2O with a volume ratio of 95:1.5-4:1.5-4.
[0097] Tis and water are used as side chain protection group capturing agents, and TFA realizes efficient removal of the Boc protecting group through protonation, generation of t-butyl cation, decarboxylation reaction and release of free amine.
[0098] More preferably, in S70, the cleavage solution is a mixture of TFA, Tis and H2O with a volume ratio of 95:2.5:2.5.
[0099] Preferably, in S70, the ratio of Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 to the cleavage solution is 379.3g:1-2L.
[0100] More preferably, in S70, the ratio of Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 to the cleavage solution is 379.3g:1.5L.
[0101] Preferably, in S70, the operation of separating the acetyl tetrapeptide-3 after sufficient reaction is as follows: after sufficient reaction, the reaction solution is concentrated under reduced pressure to half, the concentrated reaction solution is added to isopropyl ether to precipitate the solid, the filtered solid is washed with a mixture of TFA and acetonitrile with a volume ratio of 1:9, then washed twice with acetonitrile, and the washed product is dried to obtain the acetyl tetrapeptide-3.
[0102] The present application provides a new preparation method of acetyl tetrapeptide-3, and the acetyl tetrapeptide-3 prepared by the preparation method of acetyl tetrapeptide-3 of the present application has high yield and high purity.
[0103] The following is a specific embodiment.
[0104] Example 1
[0105] 1. Synthesis of Ac-OSu
[0106] 1.1) The feeding table is shown in Table 1.
[0107] Table 1
[0108] Material name Molecular weight g / mol Moles mmol Equivalent Charge g HOAc 60.05 390 1.0 23.42 HOSu 115.09 468 1.2 53.86 DCC 206.33 468 1.2 96.56 DCM 1.4L
[0109] 1.2) Specific experimental operation
[0110] Into a 2L single-mouth flask, add DCM (1L), acetic acid (23.42g, 1.0eq), HOSu (53.86g, 1.2eq) successively, stir to dissolve and control the temperature at 5±5℃, add DCC (96.56g, 1.2eq), stir for 3-5min, stir at this temperature for 30min, naturally increase the temperature to room temperature and react for 5h.
[0111] Filter the reaction solution, wash the solid with DCM (0.2L) twice, combine the filtrate, and concentrate under reduced pressure to obtain an oil 57.6g, with a yield of 94%, and the solid is Ac-OSu.
[0112] 2, Synthesis of Ac-Lys(Boc)-OH
[0113] 2.1) The feeding table is shown in Table 2 below.
[0114] Table 2
[0115]
[0116]
[0117] 2.2) Specific experimental operation
[0118] Into a 2L single-mouth flask, add THF (0.65L), purified water (0.65L), H-Lys(Boc)-OH (99.43g, 1.1eq) successively, stir to dissolve and control the temperature at 5±5℃, add Ac-OSu (57.6g, 1.0eq), stir for 3-5min, stir at this temperature for 30min, naturally increase the temperature to room temperature and react for 2h, and HPLC detection Ac-OSu≤0.2% indicates that the reaction is complete.
[0119] Concentrate the reaction solution under reduced pressure, wash with 3N hydrochloric acid, extract, concentrate, filter, and vacuum dry the solid to obtain a solid 99.78g, with a yield of 94.3%, and the solid is Ac-Lys(Boc)-OH.
[0120] 3, Synthesis of Ac-Lys(Boc)-Gly-OH
[0121] 3.1) The feeding table is shown in Table 3 below.
[0122] Table 3
[0123] Material name Molecular weight g / mol Moles mmol Equivalent Charge g Ac-Lys(Boc)-OH 288.34 367 1.0 99.78 HOBT 135.12 440.4 1.2 59.50 EDC.HCl 191.7 440.4 1.2 84.42 H-Gly-OH 75.07 385.35 1.05 28.93 DIEA 129.24 1101 3.0 142.3 DMF 1.02L DCM 1.2L Purified water 6L
[0124] 3.2) Specific experimental operation
[0125] Add DMF (0.65L), Ac-Lys(Boc)-OH (99.78g, 1.0eq), and HOBT (59.50g, 1.2eq) sequentially to a 2L single-necked flask. Stir to dissolve and clarify, and control the temperature at 5±5℃. Add EDC.HCl (84.42g, 1.2eq) and H-Gly-OH (28.93g, 1.05eq). Stir at this temperature for 5min. Add DIEA (142.3g, 3.0eq) dropwise. Allow the mixture to cool to room temperature naturally until HPLC detection shows Ac-Lys(Boc)-OH ≤ 0.2%, indicating that the reaction is complete.
[0126] The reaction solution settled into purified water (6L), precipitating a large amount of solid. After filtration, the solid was washed once with 10% potassium hydrogen sulfate aqueous solution and purified water. The solid was then directly dissolved in DCM (1.2L), allowed to stand and separate into layers, and the organic phase was retained. The organic phase was the DCM solution of Ac-Lys(Boc)-Gly-OH.
[0127] 4. Synthesis of Fmoc-His(Trt)-Lys(Boc)-NH2
[0128] 4.1) The material feeding table is shown in Table 4 below.
[0129] Table 4
[0130] Material name Molecular weight g / mol Moles mmol Equivalent Charge g Fmoc-His(Trt)-OH 619.71 403.7 1.0 250.17 HOBT 135.12 484.4 1.2 65.4 EDC.HCl 191.7 484.4 1.2 92.86 [H-Lys(Boc)-NH2] 281.78 423.9 1.05 119.4 DIEA 129.24 1211.1 3.0 156.5 DMF 1.02L DCM 1.2L Purified water 6L
[0131] 4.2) Specific experimental procedures
[0132] Add DMF (0.65L), Fmoc-His(Trt)-OH (250.17g, 1.0eq), and HOBT (65.4g, 1.2eq) sequentially to a 2L single-necked flask. Stir to dissolve and clarify, and control the temperature at 5±5℃. Add EDC.HCl (92.86g, 1.2eq) and H-Lys(Boc)-NH2 (119.4g, 1.05eq). Stir at this temperature for 5min. Add DIEA (156.5g, 3.0eq) dropwise. Allow the mixture to rise naturally to room temperature. The reaction is complete when HPLC detection shows Fmoc-His(Trt)-OH ≤ 0.2%.
[0133] The reaction solution settled into purified water (6L), precipitating a large amount of solid. After filtration, the solid was washed once with 10% potassium hydrogen sulfate aqueous solution and purified water. The solid was then directly dissolved in DCM (1.2L), allowed to stand and separate into layers, and the organic phase was retained. The organic phase was the DCM solution of Fmoc-His(Trt)-Lys(Boc)-NH2.
[0134] 5. Synthesis of H-His(Trt)-Lys(Boc)-NH2
[0135] 5.1) The feeding table is shown in Table 5 below.
[0136] Table 5
[0137]
[0138] 5.2) Specific experimental operation
[0139] To the Fmoc-His(Trt)-Lys(Boc)-NH2 solution in DCM from the previous step, diethylamine (0.4 L) was added and the reaction was allowed to proceed until HPLC detection of Fmoc-His(Trt)-Lys(Boc)-NH2 was <0.1%, indicating that the reaction was complete.
[0140] The reaction solution was concentrated under reduced pressure to an oil, DCM (0.5 L) was added and stirred to dissolve and clarify. A large amount of solid was precipitated upon addition to MTBE (4 L), the solid was filtered and the solid was further slurried with MTBE (0.5 L) once, and dried under vacuum to obtain the product 320.5 g, with a yield of 93.7%. The product was H-His(Trt)-Lys(Boc)-NH2.
[0141] 6. Synthesis of Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2
[0142] 6.1) The feeding table is shown in Table 6 below.
[0143] Table 6
[0144]
[0145]
[0146] 6.2) Specific experimental operation
[0147] Into a 2 L single-necked flask, DMF (1.4 L), Ac-Lys(Boc)-Gly-OH (126.76 g, 1.0 eq), HOBT (54.5 g, 1.2 eq) were sequentially added, stirred to dissolve and clarify and the temperature was controlled at 5±5°C, EDC.HCl (80.0 g, 1.2 eq), H-His(Trt)-Lys(Boc)-NH2 (240.7 g, 1.05 eq) were added, and the reaction was stirred at this temperature for 5 min, DIEA (142.3 g, 3.0 eq) was added dropwise, and the reaction was allowed to proceed to room temperature, and the reaction was allowed to proceed until HPLC detection of Ac-Lys(Boc)-Gly-OH was <0.2%, indicating that the reaction was complete.
[0148] The reaction solution was settled in purified water (9 L), and a large amount of solid was precipitated. The solid was filtered, and the solid was washed with 10% potassium hydrogen sulfate aqueous solution, 5% sodium carbonate solution, and purified water in sequence.
[0149] The solid was directly dissolved with DCM (0.7 L), and the solution was allowed to stand to separate into layers. A large amount of solid was precipitated in ethyl acetate / MTBE (V / V=1 / 1, 5.6 L), and the solid was filtered. The solid was washed with ethyl acetate / MTBE (V / V=4 / 1, 1 L) in sequence, and the solid was vacuum dried to obtain the product 314.8 g, with a yield of 90% and a purity of 98.5%. The product was Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2.
[0150] 7. Synthesis of Ac-Lys-Gly-His-Lys-NH2
[0151] 7.1) The feeding table is shown in Table 7.
[0152] Table 7
[0153]
[0154]
[0155] 7.2) Specific experimental operation
[0156] The cleavage solution was prepared according to TFA:Tis:H2O=95:2.5:2.5 (5 vol, 1.5 L), and the temperature was controlled at 5±5 ℃.
[0157] Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 (314.8 g) was added to the above cleavage solution, and the reaction was performed at 5±5 ℃ for 30 min, and then the reaction was performed at room temperature for 1.5 h. HPLC detection showed that the content of Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 was less than 0.1%, and the reaction was completed.
[0158] The reaction solution was concentrated to half under reduced pressure, and the cleavage solution was settled in isopropyl ether (5.6 L) to precipitate a large amount of solid. The solid was filtered, and the solid was washed with TFA:acetonitrile=1:9 (1 L) twice and acetonitrile (1 L) twice. The solid was vacuum dried to obtain the product 139.4 g.
[0159] The obtained crude peptide product was subjected to mass spectrometry detection, and the purified fine peptide was subjected to high performance liquid chromatography (HPLC) detection, and the following products were obtained, respectively: Figure 2 and Figure 3 .
[0160] Binding Figure 2 It can be seen that the crude peptide product obtained is Ac-Lys-Gly-His-Lys-NH2.
[0161] Binding Figure 3 It can be seen that the purity of the refined peptide product is 99.18%.
[0162] The total yield is 70.0%.
[0163] In the description of the embodiments of the present application, it should be noted that the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the embodiments of the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.
[0164] In the description of the embodiments of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "connected", "connected" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or it can be integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in the embodiments of the present application can be understood according to the specific circumstances.
[0165] In the embodiments of the present application, unless otherwise explicitly specified and limited, the first feature is "on" or "under" the second feature, which can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature "above", "above" and "above" the second feature can be that the first feature is directly above or obliquely above the second feature, or it can only mean that the horizontal height of the first feature is higher than that of the second feature. The first feature "below", "below" and "below" the second feature can be that the first feature is directly below or obliquely below the second feature, or it can only mean that the horizontal height of the first feature is less than that of the second feature.
[0166] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.
[0167] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. A method for preparing acetyl tetrapeptide-3, characterized in that, Includes the following steps: Step 1: Dissolve HOAc and the compound containing active hydroxyl groups in a first organic solvent. Add a carboxyl activator at 0℃~10℃, mix evenly, and then allow to rise naturally to room temperature to allow the hydroxyl and carboxyl groups to undergo a condensation reaction. After the reaction is complete, separate to obtain Ac-OZ. The compound containing active hydroxyl groups is denoted as HO-Z. Step 2: Mix Ac-OZ, H-Lys(Boc)-OH, the first amine nucleophile, water, and the second organic solvent evenly at 0℃~10℃, then allow it to rise naturally to room temperature. After the reaction is complete, separate Ac-Lys(Boc)-OH. Step 3: Mix Ac-Lys(Boc)-OH, H-Gly-OH, the carboxyl activator, the second amine nucleophile, the compound containing the active hydroxyl group, and the first organic solvent at 0℃~10℃ until homogeneous, then allow to rise naturally to room temperature. After the reaction is complete, separate to obtain Ac-Lys(Boc)-Gly-OH. Step 4: Mix Fmoc-His(Trt)-OH, H-Lys(Boc)-NH2, the carboxyl activator, the second amine nucleophile, the compound containing the active hydroxyl group, and the first organic solvent at 0℃~10℃ until homogeneous, then allow to rise naturally to room temperature. After the reaction is complete, separate to obtain Fmoc-His(Trt)-Lys(Boc)-NH2. Step 5: Mix Fmoc-His(Trt)-Lys(Boc)-NH2, basic nucleophile and the first organic solvent at 0℃~10℃ until homogeneous, then allow to rise naturally to room temperature. After the reaction is complete, separate to obtain H-His(Trt)-Lys(Boc)-NH2. Step 6: Mix the Ac-Lys(Boc)-Gly-OH, the H-His(Trt)-Lys(Boc)-NH2, the carboxyl activator, the second amine nucleophile, the compound containing the active hydroxyl group, and the first organic solvent at 0℃~10℃ until homogeneous, then allow it to naturally rise to room temperature. After the reaction is complete, separate to obtain Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2. Step 7: Mix Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 and the lysis buffer evenly at 0℃~10℃, then allow to rise naturally to room temperature. After sufficient reaction, separate to obtain the desired acetyl tetrapeptide-3. The structural formula of acetyl tetrapeptide-3 is Ac-Lys-Gly-His-Lys-NH2.
2. The method for preparing acetyl tetrapeptide-3 according to claim 1, characterized in that, The compound containing an active hydroxyl group is selected from at least one of HOBT, HOPFP, HOSu, HOAT, OXyma, and HONB.
3. The method for preparing acetyl tetrapeptide-3 according to claim 1, characterized in that, The first amine nucleophile is selected from at least one of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, lithium hydroxide, DIEA, triethylamine, and pyridine; The second amine nucleophile is selected from at least one of DIEA, 2,4,6-trimethylpyridine, triethylamine, and N-methylmorpholine; The basic nucleophile is selected from at least one of diethylamine, piperidine, piperazine, cyclohexylamine, 1-methylpiperazine, and anhydrous piperazine; The first organic solvent is selected from at least one of DCM, DMF, THF, acetonitrile, DMOS, NMP, 2-THF, and EA; The second organic solvent is selected from at least one of THF, acetonitrile, NMP, DMF, DMSO, and 1,4-dioxane.
4. The method for preparing acetyl tetrapeptide-3 according to any one of claims 1 to 3, characterized in that, The carboxyl activator is selected from at least one of EDC.HCl, DIC, DCC, HBTU, TBTU, HATU, HCTU, PyBOP, and PyAOP.
5. The method for preparing acetyl tetrapeptide-3 according to claim 4, characterized in that, In step two, the volume ratio of water to the second organic solvent is 1:0.5 to 2.
6. The method for preparing acetyl tetrapeptide-3 according to claim 4, characterized in that, In step one, the molar ratio of HOAc, the compound containing active hydroxyl groups, and the carboxyl activator is 1:1 to 1.5:1 to 1.5; The procedure for separating Ac-OZ after a complete reaction is as follows: after a complete reaction, the reaction solution is filtered, the solid is washed with the first organic solvent, the filtrates are combined and concentrated under reduced pressure to obtain Ac-OZ; In step two, the molar ratio of Ac-OZ, H-Lys(Boc)-OH, and the first amine nucleophile is 1:0.9-1.4:2-5; The procedure for separating Ac-Lys(Boc)-OH after a full reaction is as follows: after a full reaction, the reaction solution is concentrated under reduced pressure, washed with hydrochloric acid, and then extracted, concentrated and filtered in sequence. The solid obtained is Ac-Lys(Boc)-OH after drying. In step three, the molar ratio of Ac-Lys(Boc)-OH, H-Gly-OH, the carboxyl activator, the second amine nucleophile, and the compound containing the active hydroxyl group is 1:0.9-1.3:1-1.5:2-5:1-1.5; The procedure for separating Ac-Lys(Boc)-OH after a full reaction is as follows: after a full reaction, the reaction solution is precipitated into water to precipitate the solid. The solid obtained after filtration is washed once with a 10% potassium hydrogen sulfate aqueous solution and purified water. The washed solid is dissolved in the first organic solvent, allowed to stand and separate into layers, and the organic phase is retained. In step four, the molar ratio of Fmoc-His(Trt)-OH, H-Lys(Boc)-NH2, the carboxyl activator, the second amine nucleophile, and the compound containing the active hydroxyl group is 1:0.9-1.3:1-1.5:2-5:1-1.5; The procedure for separating Fmoc-His(Trt)-Lys(Boc)-NH2 after a full reaction is as follows: after a full reaction, the reaction solution is settled in water to precipitate the solid. The solid obtained after filtration is washed once with 10% potassium hydrogen sulfate aqueous solution and purified water. The washed solid is dissolved in the first organic solvent, allowed to stand and separate into layers, and the organic phase is retained.
7. The method for preparing acetyl tetrapeptide-3 according to claim 4, characterized in that, In step five, the ratio of Fmoc-His(Trt)-Lys(Boc)-NH2 to the basic nucleophile is 0.4 mol: 0.3 L to 0.6 L; The procedure for separating H-His(Trt)-Lys(Boc)-NH2 after a complete reaction is as follows: after a complete reaction, the reaction solution is concentrated under reduced pressure to an oily state, the first organic solvent is added and stirred to dissolve and clarify, MTBE is added until a solid precipitates, the mixture is filtered, the solid is pulped once more with MTBE, and dried to obtain H-His(Trt)-Lys(Boc)-NH2.
8. The method for preparing acetyl tetrapeptide-3 according to claim 4, characterized in that, In step six, the molar ratio of Ac-Lys(Boc)-Gly-OH, H-His(Trt)-Lys(Boc)-NH2, the carboxyl activator, the second amine nucleophile, and the compound containing the active hydroxyl group is 1:0.9-1.3:1-1.5:2-5:1-1.5; The procedure for separating Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 after a complete reaction is as follows: After a complete reaction, the reaction solution is allowed to settle in water, and a solid is precipitated. The solid obtained after filtration is washed sequentially with a 10% potassium bisulfate aqueous solution, a 5% sodium carbonate solution, and water. The washing product is dissolved in the first organic solvent, allowed to stand and separate into layers, and the organic phase is added to a mixture of ethyl acetate and MTBE with a volume ratio of 1:1 to precipitate a solid. The filtered solid is then slurried with a mixture of ethyl acetate and MTBE with a volume ratio of 4:1, and dried to obtain Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2.
9. The method for preparing acetyl tetrapeptide-3 according to claim 4, characterized in that, In step seven, the pyrolysis solution is a mixture of TFA, Tis and H2O in a volume ratio of 95:1.5 to 4:1.5 to 4.
10. The method for preparing acetyl tetrapeptide-3 according to claim 9, characterized in that, In step seven, the ratio of Ac-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-NH2 to the lysis solution is 379.3g: 1L~2L; The procedure for separating the desired acetyl tetrapeptide-3 after a complete reaction is as follows: after a complete reaction, the reaction solution is concentrated to half under reduced pressure. The concentrated reaction solution is then added to isopropyl ether to precipitate a solid. The filtered solid is washed with a mixture of TFA and acetonitrile at a volume ratio of 1:
9. The mixture is then washed twice with acetonitrile. After drying the washing product, the desired acetyl tetrapeptide-3 is obtained.