CN111732628B - Synthetic method of GHK tripeptide - Google Patents
Synthetic method of GHK tripeptide Download PDFInfo
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- CN111732628B CN111732628B CN202010593858.9A CN202010593858A CN111732628B CN 111732628 B CN111732628 B CN 111732628B CN 202010593858 A CN202010593858 A CN 202010593858A CN 111732628 B CN111732628 B CN 111732628B
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Abstract
The invention discloses a method for synthesizing GHK tripeptide, which comprises the steps of firstly respectively protecting glycine, lysine and histidine to obtain protected glycine, lysine and histidine; protected glycine reacts with N-hydroxysuccinimide, protected histidine and N-hydroxysuccinimide in sequence to obtain Boc-Gly-His (Trt) -OSu, then reacts with protected lysine to obtain protected tripeptide, and finally reacts with trifluoroacetic acid to obtain GHK tripeptide. The method provided by the invention avoids palladium carbon hydrogen for deprotection, has simple, stable and safe operation steps, avoids the method of ion exchange for trifluoroacetic acid removal or chromatographic synthesis compared with the prior art, avoids the use of resin, obtains GHK tripeptide by using common economic reagents such as methanol, ethyl acetate and the like for trifluoroacetic acid removal, greatly reduces the cost, and can be used for large-scale production.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to a synthetic method of GHK tripeptide.
Background
Tripeptide-1 (GHK) is an endogenous collagen tripeptide, is present in interstitial fluid such as plasma, can regulate skin regeneration, enhance differentiation and proliferation ability of epidermal basal cells, promote wound healing and tissue regeneration, increase collagen and mucopolysaccharide, stimulate decorin synthesis, promote blood vessel growth and nerve growth, and has antioxidant and anti-inflammatory effects.
The current synthesis method of GHK is mainly classical solid phase synthesis. Solid phase synthesis is to extend protected amino acids from C end to N end one by one, and then the GHK product with the purity higher than 90 percent is prepared by condensation, deprotection, recondensation, resin cutting, centrifugation, freeze drying and high performance liquid phase. The resin needed in the method has over-expensive market price and small exchange activity, and the condensing agent and alkali used in the synthesis process are obviously excessive, so that the cost of the GHK raw material is increased, and the method is not suitable for large-scale production in factories, therefore, the research on the process method for reducing the production cost of the GHK tripeptide and ensuring the stability and safety of the GHK synthesis process has important significance.
Xiongying et al disclosed a synthesis method of GHK tripeptide (xiamen university, 2007 thesis), which uses glycine, lysine and histidine as raw materials to prepare N-Boc-Gly-OH, His (trt) and lys (Boc), respectively, then sequentially synthesize N-Boc-Gly-His (trt) -OH, then synthesize N-Boc-Gly-His (trt) -lys (Boc) -OH, and then remove the protection to obtain GHK tripeptide. In addition, the final product is trifluoroacetate of GHK tripeptide, which is not a GHK tripeptide product and does not meet the actual requirement.
Disclosure of Invention
The invention aims to provide a synthesis method of GHK tripeptide, which is safe and stable, reduces the cost, is simple to operate and can be produced in a large scale.
In order to solve the above problems, the present invention provides a method for synthesizing GHK tripeptide, comprising: firstly, respectively protecting glycine and lysine by using di-tert-butyl dicarbonate to obtain Boc-Gly-OH and Boc-Lys-OH, and then protecting histidine by using triphenylchloromethane to obtain Trt-His-OH; and then reacting Boc-Gly-OH with N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide to obtain Boc-Gly-OSu, then reacting the Boc-Gly-OSu with Trt-His-OH to obtain Boc-Gly-His (Trt) -OH, then reacting the Boc-Gly-His (Trt) -OSu with N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide to obtain Boc-Gly-His (Trt) -OSu, then reacting the Boc-Gly-His (Trt) -OSu with protected lysine to obtain protected tripeptide, and finally reacting the protected tripeptide with trifluoroacetic acid to obtain the GHK tripeptide.
The method specifically comprises the following steps:
(1) under the action of sodium hydroxide, glycine and Boc anhydride react in 1, 4-dioxane, and Boc-Gly-OH is obtained through post-treatment after the reaction is finished;
(2) under the action of triethylamine, reacting histidine with dimethyldichlorosilane in dichloromethane, adding dichloromethane solution containing triphenylchloromethane and triethylamine for continuous reaction after the reaction is finished, and purifying Trt-His-OH after the reaction is finished;
(3) under the action of sodium bicarbonate and copper sulfate pentahydrate, lysine or salt thereof reacts with Boc-anhydride in a mixed solvent of acetone and water, and after the reaction is finished, Boc-Lys-OH is obtained through purification treatment;
(4) reacting Boc-Gly-OH obtained in the step (1) with Hosu in 1, 4-dioxane under the action of DCC, and purifying after the reaction is finished to obtain Boc-Gly-OSu;
(5) reacting Boc-Gly-OSu in the step (4) and Trt-His-OH in the step (2) in DMF (dimethyl formamide) under the action of DIPEA, and purifying after the reaction is finished to obtain Boc-Gly-His (Trt) -OH;
(6) reacting Boc-Gly-His (Trt) -OH in the step (5) with Hosu in dioxane under the action of DCC, and performing post-treatment after the reaction is finished to obtain Boc-Gly-His (Trt) -OSu;
(7) reacting Boc-Lys-OH in the step (3) with Boc-Gly-His (Trt) -OSu in the step (6) in a mixed solvent of tetrahydrofuran and water under the action of sodium bicarbonate, and purifying after the reaction is finished to obtain Boc-Gly-His (Trt) -Lys (Boc) -OH;
(8) under the action of triethylsilane, reacting Boc-Gly-His (Trt) -Lys (Boc) -OH in the step (7) with trifluoroacetic acid in dichloromethane, and purifying after the reaction is finished to obtain GHK tripeptide trifluoroacetate;
(9) and (3) dissolving the GHK tripeptide trifluoroacetate obtained in the step (8) in a mixed solution of ethyl acetate and methanol, adding diethylamine until white precipitate is separated out, performing suction filtration, collecting a filter cake, and performing purification treatment to obtain the GHK tripeptide.
In the step (1), the reaction temperature is room temperature, and the reaction time is 12-24 hours;
the post-treatment comprises the following steps:
concentrating, adjusting pH of the water phase to 2-4 with HCl aqueous solution, extracting with ethyl acetate, drying, filtering, concentrating the filtrate at room temperature, and separating and purifying the concentrate to obtain Boc-Gly-OH.
In the step (1), the separation and purification process is as follows:
and adding dichloromethane to dissolve the concentrate, then adding petroleum ether to perform crystallization, performing suction filtration, and drying to obtain Boc-Gly-OH.
In the step (2), the purification treatment comprises:
after the reaction is finished, adding ethyl acetate and water, stirring, carrying out suction filtration, adding ethyl acetate into a filter cake, carrying out oil bath reflux under a high-temperature condition, closing heating, carrying out suction filtration, adding a large amount of aqueous solution containing triethylamine into the filter cake, stirring at room temperature, carrying out suction filtration, adding a mixed solution of tetrahydrofuran and water in a volume ratio of 1:1 into the filter cake, refluxing under a high-temperature condition until the mixture is completely clear, then crystallizing, filtering and drying to obtain Trt-His-OH.
In the step (3), the purification treatment comprises:
adding a mixed solution of ethyl acetate and water in a volume ratio of 1:2 into the reaction solution, stirring, performing suction filtration, adding 8-hydroxyquinoline and distilled water into the filter cake until the system is clarified, continuing stirring for 10-12h, and separating and purifying to obtain Boc-Lys-OH;
the treatment process does not need resin to adsorb copper, so that the cost is reduced, and the operation is simpler.
In the step (4), the volume-mass ratio of the dioxane to the Dcc is 4.5-5.5: 1.
In the step (5), the purification treatment comprises:
diluting with water, adjusting pH to 3-4 with citric acid solution, stirring, filtering, dissolving the filter cake with ethyl acetate, washing with water, collecting the organic layer, drying, concentrating, adding methyl tert-butyl ether and ethyl acetate to the clear solution, adding petroleum ether, precipitating solid, filtering, and drying to obtain Boc-Gly-His (Trt) -OH.
In the step (6), the post-processing includes: after the reaction is completed, the reaction solution is filtered, the filtrate is concentrated, and then the reaction solution is dissolved by tetrahydrofuran and directly enters the next step.
In the step (7), the post-processing comprises:
removing an organic layer by rotary evaporation, standing, performing suction filtration on a supernatant, dissolving the bottom precipitate for multiple times by using water and performing suction filtration, collecting a filtrate, adjusting the pH to 3-4 by using a citric acid solution, stirring, performing suction filtration, collecting a filter cake, dissolving the filter cake by using ethyl acetate, washing by using water, collecting an organic phase, drying, filtering, concentrating the filtrate to obtain a white solid, and then recrystallizing by using dichloromethane and petroleum ether to obtain Boc-Gly-His (Trt) -Lys (Boc) -OH.
In the step (8), the reaction temperature is room temperature, and the reaction time is 12-24 hours.
In the step (9), the post-treatment is to pulp with ethyl acetate, filter, collect filter cakes and obtain the GHK tripeptide by spin drying.
More specifically, the method provided by the invention comprises the following steps:
(1) synthesis of Boc-Gly-OH
Mixing glycine, 1, 4-dioxane and NaOH solution, adding Boc anhydride under the stirring condition, reacting at room temperature for 24 hours, concentrating at 30-50 ℃, adjusting the pH of a water phase to 2-4 by using HCl aqueous solution, extracting by using ethyl acetate, drying, filtering, concentrating a filtrate at room temperature, and finally separating and purifying a concentrate to obtain Boc-Gly-OH. The chemical equation is as follows:
(2) synthesis of Trt-His-OH
Taking dichloromethane as a solvent, adding histidine, adding dichloromethane solution containing dimethyldichlorosilane and triethylamine under the condition of stirring at room temperature, carrying out oil bath reflux reaction for 6-10h under the condition of high temperature, naturally cooling to room temperature after heating, adding dichloromethane solution containing triphenylchloromethane and triethylamine, and stirring at room temperature. Then adding ethyl acetate and purified water, and stirring at room temperature. And then carrying out suction filtration, adding ethyl acetate into the filter cake, carrying out oil bath reflux under a high-temperature condition, closing heating, carrying out suction filtration, adding a large amount of aqueous solution containing triethylamine into the filter cake, stirring for 1-3h at room temperature, carrying out suction filtration, adding a mixed solution of tetrahydrofuran and purified water in a volume ratio of 1:1 into the filter cake, refluxing to be completely clear under a high-temperature condition, and carrying out separation and purification to obtain Trt-His-OH. The chemical equation is as follows:
(3) synthesis of Boc-Lys-OH
The lysine hydrochloride is completely dissolved in distilled water, sodium bicarbonate and copper sulfate pentahydrate are added and stirred, and then Boc-anhydride and acetone are added and stirred for 24 hours. And then adding a mixed solution of ethyl acetate and distilled water in a volume ratio of 1:2 into the system, stirring for 1-3h, after suction filtration, adding 8-hydroxyquinoline and distilled water into the filter cake until the system becomes turbid, continuing stirring for 10-12h, and separating and purifying to obtain Boc-Lys-OH. The chemical equation is as follows:
(4) synthesis of Boc-Gly-OSu
And (2) completely dissolving the Boc-Gly-OH in the step (1) into 1, 4-dioxane, adding a mixed solution of Hosu and Dcc with the volume-mass ratio of 4.5-5.5:1, stirring at room temperature for 12-24h, adding glacial acetic acid, continuously stirring, and separating and purifying to obtain Boc-Gly-OSu. The chemical equation is as follows:
(5) synthesis of Boc-Gly-His (Trt) -OH
And (3) adding DMF, DIPEA and Trt-His-OH in the step (2) into Boc-Gly-OSu in the step (4), stirring at room temperature for 2-4h, then adding distilled water, adjusting the pH to 2-4 by using a citric acid solution, continuously stirring, separating and purifying to obtain Boc-Gly-His (Trt) -OH. The chemical equation is as follows:
(6) synthesis of Boc-Gly-His (Trt) -OSu
And (3) adding a mixed solution of 1, 4-dioxane, Hosu and Dcc in a volume-mass ratio of 8-9:1 of 1, 4-dioxane to Boc-Gly-His (Trt) -OH in the step (5), stirring at room temperature for 3-5h, carrying out suction filtration, and concentrating the filtrate to obtain Boc-Gly-His (Trt) -OSu. The chemical equation is as follows:
(7) synthesis of Boc-Gly-His (Trt) -Lys (Boc) -OH
And (3) adding a tetrahydrofuran solution containing the dipeptide ester in the step (6) into Boc-Lys-OH in the step (3), then adding a mixed solution of sodium bicarbonate and water in a mass-volume ratio of 1:16-18, stirring at room temperature overnight, judging that the reaction is complete by TLC (a developing solvent is a mixed solution of dichloromethane and methanol), performing rotary evaporation to remove an organic layer until a jelly appears at the bottom of a bottle, stopping the rotary evaporation, and standing. And standing, carrying out suction filtration on the supernatant, dissolving the residual bottom precipitate with water, carrying out suction filtration (stopping the step until the precipitate is not reduced any more), collecting filtrate, adjusting the pH to 3-4 with a citric acid solution, stirring for half an hour, carrying out suction filtration, collecting a filter cake, dissolving the filter cake with ethyl acetate, washing with water, collecting an organic phase, drying and filtering, concentrating the filtrate to obtain a white solid, and separating and purifying to obtain Boc-Gly-His (Trt) -Lys (Boc) -OH. The chemical equation is as follows:
(8) synthesis of GHK tripeptide trifluoroacetate
And (2) adding dichloromethane into Boc-Gly-His (Trt) -Lys (Boc) -OH in the step (7) to completely dissolve the GHK tripeptide trifluoroacetate, adding a mixed solution of trifluoroacetic acid and triethylsilane in a volume ratio of 9:1, stirring at room temperature overnight, concentrating at high temperature, adding water and a mixed solution of ethyl acetate in a volume ratio of (1:1) to dissolve solids, washing and extracting with ethyl acetate, collecting an aqueous phase, and performing rotary evaporation to obtain the GHK tripeptide trifluoroacetate. The chemical equation is as follows:
(9) synthesis of GHK tripeptide
Adding ethyl acetate and methanol into GHK tripeptide trifluoroacetate in the step (8) in a volume ratio of 2: 1, adding diethylamine till white precipitate is separated out, filtering, collecting filter cake, and purifying to obtain GHK tripeptide. The chemical equation is as follows:
compared with the prior art, the invention has the beneficial effects that:
(1) the synthetic method of the invention does not need resin at all, and the used condensing agent and alkali have less dosage, thus effectively reducing the cost of the whole process;
(2) the synthesis method of the invention has simpler post-treatment, only comprises the conventional steps of washing, extraction and crystallization, and is convenient for industrialization.
Drawings
Fig. 1 is a liquid chromatogram of the GHK tripeptide synthesized in example 1.
Detailed Description
The invention is further illustrated with reference to the following figures and examples, but is not limited thereto.
Example 1
1. Synthesis of Boc-Gly-OH
Glycine (2g, 26.64mmol) was mixed with 1, 4-dioxane (50ml), NaOH solution (16ml,31.97mmol), Boc anhydride (6.97g,31.97mmol) was added with stirring, reaction was carried out at room temperature for 12h, then concentration was carried out at 30-50 ℃, the concentrate was washed with ethyl acetate, the aqueous phase was adjusted to PH 2-4 with aqueous HCl and then extracted three to four times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated at 35-40 ℃ until the product was oil-like. Adding dichloromethane to dissolve the mixture into a liquid state, dropwise adding 50ml of petroleum ether under the stirring condition, stirring until a product is separated out, performing suction filtration and drying to obtain Boc-Gly-OH, wherein TLC detection shows that the yield is 95%.
2. Synthesis of Trt-His-OH
4g (25.81mmol) of L-histidine were weighed, 70ml of anhydrous dichloromethane were added, a solution of 5.4ml (43.87mmol) of dimethyldichlorosilane in 10ml of anhydrous dichloromethane was added with stirring at room temperature, and the reaction was refluxed in an oil bath at 65 ℃ for 6 hours. 13ml (92.91mmol) of triethylamine was added, the mixture was refluxed for 4 hours, then the heating was turned off, the mixture was cooled naturally to 30 to 40 ℃ and a solution of 7.2661g (26.06mmol) of triphenylchloromethane in 10ml of anhydrous dichloromethane was added, and then 3.6ml (25.55mmol) of triethylamine was added, and the mixture was stirred at 30 ℃ for 2 hours. Then 80ml of ethyl acetate is added, 7.2ml of purified water is added dropwise, 40ml of ethyl acetate is added until the system can be stirred smoothly, and the mixture is stirred for 2 hours at the temperature of 30 ℃. And (2) carrying out suction filtration, adding 120ml of ethyl acetate into the filter cake, carrying out oil bath reflux for 1h at the temperature of 90 ℃, closing and heating, naturally cooling to room temperature, carrying out suction filtration, adding 140ml of triethylamine aqueous solution containing 2.4ml into the filter cake, stirring for 2h at the room temperature, carrying out suction filtration, adding tetrahydrofuran into the filter cake: 140ml of purified water is added in a ratio of 1:1, and after refluxing at 80 ℃ until the mixture is completely clear, heating and stirring are stopped, and the mixture is crystallized at room temperature. And (4) performing suction filtration for multiple times, collecting a filter cake, drying to obtain Trt-His-OH, and detecting by TLC to show a point, wherein the yield is 95%.
3. Synthesis of Boc-Lys-OH
5g (36.95mmol) of lysine hydrochloride was taken, 50ml of water was added and dissolved, 5.40g (64.28mmol) of sodium bicarbonate was added and stirred for 5-10min, 4.39g (17.56mmol) of copper sulfate pentahydrate was added and the system turned blue, 5.40g (64.28mmol) of sodium bicarbonate was added and stirred for 5-10min, 10.8g (49.48mmol) of Boc-anhydride was added and 55ml of acetone was added and stirred for 24 h. And then adding 300ml of ethyl acetate and 600ml of water into the system, stirring for 2h, carrying out suction filtration, collecting a filter cake, adding 2.74g (18.87mmol) of 8-hydroxyquinoline, adding water under the stirring condition until the system starts to stir, gradually changing the solution from blue to green, keeping the system viscous, continuing stirring for 12h, adding 500ml of water, carrying out suction filtration, collecting a filtrate, and carrying out spin drying on the filtrate to obtain Boc-Lys-OH with the purity of 92% and the yield of 80%.
4. Synthesis of Boc-Gly-OSu
3.5036g (20mmol) of Boc glycine is taken, 40ml of 1, 4-dioxane is added, Hosu2.5320g (22mmol) is added, 10ml of 1, 4-dioxane dissolved in Dcc 4.9519g (24mmol) is added, stirring is carried out for 16h at the room temperature of 30 ℃, then 1ml of glacial acetic acid is added, stirring is continued for 1h, suction filtration is carried out, filtrate is concentrated to be dry at the temperature of 45 ℃, 20ml of isopropanol is added for pulping, suction filtration is carried out, a filter cake is dried to obtain Boc-Gly-OSu, TLC detection shows that one point is obtained, and the yield is 90%.
5. Synthesis of Boc-Gly-His (Trt) -OH
6.807g (25mmol) of Boc glycine ester is taken, 80ml of DMF is added, 4.4ml (25mmol) of DIPEA and 10.313g (25mmol) of Trt histidine are added, the mixture is stirred for 2-3h at room temperature, 375ml of water is then added, the pH is adjusted to 3-4 by using a citric acid solution, the stirring is continued for 15min, the suction filtration is carried out, the filter cake is collected and dissolved by adding ethyl acetate, the washing is carried out by water, the organic layer is collected, the drying is carried out by anhydrous sodium sulfate, the filtration is carried out, the filtrate is concentrated (T ═ 45 ℃), 20ml of methyl tert-butyl ether is added, 3-4ml of ethyl acetate is added until the system is clear, 60ml of petroleum ether is added, a large amount of solid is separated out, the stirring is carried out for 15min, the suction filtration is carried out, the filter cake is collected, and the filter cake is dried by spinning to obtain Boc-Gly-His- (Trt) -OH with the purity of 94% and the yield of 85%.
6. Synthesis of Boc-Gly-His (Trt) -OSu
8.3196g (15mmol) of dipeptide is taken, 40ml of 1, 4-dioxane is added, Hosu2.0716(18mmol) is added, 40ml of Dcc 4.6424g (22.5mmol) dissolved in 1, 4-dioxane is added, stirring is carried out at room temperature for 4-5h, liquid phase monitoring is started after 4h until dipeptide reaction is completed, suction filtration is carried out, filtrate is concentrated (T ═ 45 ℃), tetrahydrofuran (80ml) is used for dissolving after concentration is completed, and then the mixture is put into the next reaction.
7. Synthesis of Boc-Gly-His (Trt) -Lys (Boc) -OH
4.4268g (18mmol) of Boc lysine was added to 80ml of a tetrahydrofuran solution containing 15mmol of dipeptide ester, 4.5365g (54mmol) of sodium bicarbonate was added to the solution, 80ml of water was added thereto, the mixture was stirred overnight at room temperature, the reaction was judged to be complete by TLC (developing solvent dichloromethane: methanol: 9: 1), the organic layer was removed by rotary evaporation (T: 45 ℃ C.), the bottom of the flask appeared to be a gel, and the flask was left to stand after the rotary evaporation was stopped. Standing, carrying out suction filtration on the supernatant, dissolving the precipitate at the bottom of the residue with water (the precipitate has low solubility, and needs to be dissolved with water for multiple times until the precipitate is not reduced), using 800ml of water for total use, carrying out suction filtration again, collecting filtrate, adjusting the pH to 3-4 with citric acid solution, stirring for 15min, carrying out suction filtration, collecting a filter cake, dissolving the filter cake with 225ml of ethyl acetate, washing with water, collecting an organic phase, drying with anhydrous sodium sulfate, filtering, and concentrating the filtrate to obtain a white solid. Adding a proper amount of dichloromethane (15-20ml), then adding a large amount of petroleum ether (150-200ml), precipitating white solid, stirring for 15min, performing suction filtration, and spin-drying a filter cake to obtain Boc-Gly-His (Trt) -Lys (Boc) -OH with the purity of 97 percent and the yield of 80 percent.
8. Synthesis of GHK tripeptide trifluoroacetate
The protected tripeptide 6.2624g (8mmol) was taken, dissolved in 48ml of dichloromethane, and 48ml of trifluoroacetic acid and 2.56ml of triethylsilane were added thereto, stirred at room temperature overnight, and concentrated at 60 ℃. Water (35ml) and ethyl acetate (30ml) were added to dissolve the solid, the extract was washed with ethyl acetate, the aqueous phase was collected and rotary evaporated to give the GHK tripeptide trifluoroacetate salt in 99% purity and 98% yield.
9. Synthesis of GHK tripeptide
7.4819g tripeptide trifluoroacetate is taken, 100ml of methanol is added, 200ml of ethyl acetate is added, the mixture is dissolved, 8ml of diethylamine is added, white precipitate is separated out, the mixture is filtered, a filter cake is collected, the filter cake is pulped for 30min by using ethyl acetate (proper amount, 50ml), the mixture is filtered, the filter cake is collected and is dried by screwing to obtain GHK tripeptide, an HPLC (high performance liquid chromatography) map is shown in figure 1, the purity is 96%, and the yield is 83%.
Example 2
In the synthesis of Boc-Gly-OH of this example step 1,
the other procedure was the same as in step 1 of example 1 except for replacing 1, 4-dioxane with acetone to give Boc-Gly-OH in a yield of 72%. The other steps were the same as in example 1 to obtain GHK tripeptide.
Example 3
In the synthesis of GHK tripeptide trifluoroacetate of this example, 6.2624g (8mmol) of protected tripeptide was taken and 12mL of trifluoroacetic acid, 2.56mL of triethylsilane and 2.56mL of water were added thereto, and the mixture was stirred at room temperature overnight and concentrated at 60 ℃. Water (70ml) and ethyl acetate (60ml) were added to dissolve the solid, and the other steps of this step were the same as in example 1 to obtain the GHK tripeptide trifluoroacetate salt in a yield of 81%. The other steps were the same as in example 1 to obtain GHK tripeptide.
Claims (8)
1. A synthetic method of GHK tripeptide is characterized by comprising the following steps:
(1) under the action of sodium hydroxide, glycine and Boc anhydride react in 1, 4-dioxane, and Boc-Gly-OH is obtained through post-treatment after the reaction is finished;
(2) under the action of triethylamine, reacting histidine with dimethyldichlorosilane in dichloromethane, adding dichloromethane solution containing triphenylchloromethane and triethylamine for continuous reaction after the reaction is finished, and purifying Trt-His-OH after the reaction is finished;
(3) under the action of sodium bicarbonate and copper sulfate pentahydrate, lysine or salt thereof reacts with Boc-anhydride in a mixed solvent of acetone and water, and after the reaction is finished, Boc-Lys-OH is obtained through purification treatment;
(4) reacting Boc-Gly-OH obtained in the step (1) with Hosu in 1, 4-dioxane under the action of DCC, and purifying after the reaction is finished to obtain Boc-Gly-OSu;
(5) reacting Boc-Gly-OSu in the step (4) and Trt-His-OH in the step (2) in DMF (dimethyl formamide) under the action of DIPEA, and purifying after the reaction is finished to obtain Boc-Gly-His (Trt) -OH;
(6) reacting Boc-Gly-His (Trt) -OH in the step (5) with Hosu in dioxane under the action of DCC, and performing post-treatment after the reaction is finished to obtain Boc-Gly-His (Trt) -OSu;
(7) reacting Boc-Lys-OH in the step (3) with Boc-Gly-His (Trt) -OSu in the step (6) in a mixed solvent of tetrahydrofuran and water under the action of sodium bicarbonate, and purifying after the reaction is finished to obtain Boc-Gly-His (Trt) -Lys (Boc) -OH;
(8) 6.2624g and 8mmol of protected tripeptide are taken, 48ml of dichloromethane is added for dissolution, 48ml of trifluoroacetic acid and 2.56ml of triethylsilane are added for stirring at room temperature overnight, concentration is carried out, T is 60 ℃, 35ml of water and 30ml of ethyl acetate are added for dissolution of solid, ethyl acetate is used for washing and extraction, water phase is collected, and rotary evaporation is carried out to obtain GHK tripeptide trifluoroacetate with the purity of 99 percent and the yield of 98 percent;
(9) 7.4819g tripeptide trifluoroacetate is taken, 100ml of methanol is added, 200ml of ethyl acetate is added for dissolution, 8ml of diethylamine is added, white precipitate is separated out, the filtration is carried out, a filter cake is collected, the filter cake is pulped for 30min by 50ml of ethyl acetate, the filtration is carried out, the filter cake is collected and is dried by spinning to obtain the GHK tripeptide with the purity of 96 percent and the yield of 83 percent.
2. The method for synthesizing the GHK tripeptide according to claim 1, wherein in the step (1), the reaction temperature is room temperature and the reaction time is 12-24 hours;
the post-treatment comprises the following steps:
concentrating, adjusting pH of the water phase to 2-4 with HCl aqueous solution, extracting with ethyl acetate, drying, filtering, concentrating the filtrate at room temperature, and separating and purifying the concentrate to obtain Boc-Gly-OH.
3. The method for synthesizing the GHK tripeptide according to claim 1, wherein the purification process in the step (2) comprises:
after the reaction is finished, adding ethyl acetate and water, stirring, carrying out suction filtration, adding ethyl acetate into a filter cake, carrying out oil bath reflux under a high-temperature condition, carrying out suction filtration after heating is closed, adding a large amount of aqueous solution containing triethylamine into the filter cake, stirring at room temperature, carrying out suction filtration, adding a mixed solution of tetrahydrofuran and water in a volume ratio of 1:1 into the filter cake, refluxing under a high-temperature condition until the mixture is completely clarified, and carrying out separation and purification to obtain Trt-His-OH.
4. The method for synthesizing the GHK tripeptide according to claim 1, wherein the purification process in the step (3) comprises:
adding a mixed solution of ethyl acetate and water in a volume ratio of 1:2 into the reaction solution, stirring, performing suction filtration, adding 8-hydroxyquinoline and distilled water into the filter cake until the system begins to be clear, continuing stirring for 10-12h, and separating and purifying to obtain Boc-Lys-OH.
5. The method for synthesizing GHK tripeptide according to claim 1, wherein in the step (4), the volume-mass ratio of dioxane to Dcc is 4.5-5.5: 1.
6. The method for synthesizing the GHK tripeptide according to claim 1, wherein the purification process in the step (5) comprises:
diluting with water, adjusting pH to 3-4 with citric acid solution, stirring, filtering, dissolving the filter cake with ethyl acetate, washing with water, collecting the organic layer, drying, concentrating, adding methyl tert-butyl ether and ethyl acetate to the clear solution, adding petroleum ether, precipitating solid, filtering, and drying to obtain Boc-Gly-His (Trt) -OH.
7. The method for synthesizing the GHK tripeptide according to claim 1, wherein the post-processing in step (6) comprises: after the reaction is completed, the reaction solution is filtered, the filtrate is concentrated, and then the reaction solution is dissolved by tetrahydrofuran and directly enters the next step.
8. The method for synthesizing the GHK tripeptide according to claim 1, wherein the post-processing in step (7) comprises:
removing an organic layer by rotary evaporation, standing, performing suction filtration on a supernatant, dissolving the bottom precipitate for multiple times by using water and performing suction filtration, collecting a filtrate, adjusting the pH to 3-4 by using a citric acid solution, stirring, performing suction filtration, collecting a filter cake, dissolving the filter cake by using ethyl acetate, washing by using water, collecting an organic phase, drying, filtering, concentrating the filtrate to obtain a white solid, and then recrystallizing by using dichloromethane and petroleum ether to obtain Boc-Gly-His (Trt) -Lys (Boc) -OH.
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