CN111004304A - Liquid phase synthesis method of biotin tripeptide-1 - Google Patents
Liquid phase synthesis method of biotin tripeptide-1 Download PDFInfo
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- CN111004304A CN111004304A CN201911412735.4A CN201911412735A CN111004304A CN 111004304 A CN111004304 A CN 111004304A CN 201911412735 A CN201911412735 A CN 201911412735A CN 111004304 A CN111004304 A CN 111004304A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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Abstract
The invention discloses a liquid-phase synthesis method of biotin tripeptide-1, belonging to the technical field of biological medicine preparation methods and comprising the following steps: preparation of Biotin-N-hydroxysuccinimide ester, preparation of Biotin-Gly-OH, preparation of Biont-GHK (Boc) -OH, preparation of Biotin tripeptide-1, and vacuum drying to obtain Biotin tripeptide-1. The invention has the beneficial effects that: according to the biotin tripeptide-1 liquid phase synthesis method, the final product can be purified by a crystallization method, so that the final liquid phase purification required in solid phase synthesis is successfully avoided, the production cost is reduced, and the batch production capacity is increased.
Description
Technical Field
The invention belongs to the technical field of biological medicine preparation methods, and particularly relates to a biotin tripeptide-1 liquid-phase synthesis method.
Background
In 1988 the FDA approved 5% of minoxidil for hair loss treatment and in 1997 the FDA approved 1mg of finasteride as an oral drug for treating hair loss, both of which have been the strongest combinations for treating hair loss for many years. They cannot be added to cosmetics as an active ingredient of a medicine. The most active peptide for hair growth in cosmetics is Biotin-GHK. For hair shaft growth, the milodel effect was comparable at 2ppm concentration (58% increase) and increased 121 at 5ppm concentration (Sederma data) over the blank.
In the prior art, biotin tripeptide-1 is synthesized by a solid phase method, firstly, an amino acid with an amino group protected by a blocking group is covalently connected to a solid phase carrier, the blocking group of the amino group is removed under the action of trifluoroacetic acid, so that a first amino acid is connected to the solid phase carrier, then, a carboxyl group of a second amino acid with an amino group blocked is activated, and the second amino acid with the activated carboxyl group is reacted with the amino group of the first amino acid connected to the solid phase carrier to form a peptide bond, so that a dipeptide with a blocking group is generated on the solid phase carrier; repeating the peptide bond forming reaction to make the peptide chain grow from C end to N end until reaching the required peptide chain length, finally removing the protecting group X, and hydrolyzing the ester bond between the peptide chain and the solid phase carrier by HF to obtain the synthesized peptide.
The main problems of solid phase synthesis are that intermediate heteropeptide on a solid phase carrier cannot be separated and is finally purified by preparing a liquid phase, so that the product is more impure and difficult to purify due to amplification, and the problems of high cost, large environmental pollution and small single-batch secondary yield exist.
Disclosure of Invention
The invention aims to provide a liquid phase synthesis method of biotin tripeptide-1, which solves the problems.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention relates to a biotin tripeptide-1 liquid phase synthesis method, which comprises the following steps:
1) preparation of biotin-N-hydroxysuccinimide ester: adding tetrahydrofuran, biotin, N-hydroxysuccinimide and 1, 3-dicyclohexylcarbodiimide in a reaction device in sequence under stirring, heating for reflux reaction, filtering, and concentrating the filtrate under reduced pressure to obtain a white solid;
2) preparation of Biotin-Gly-OH: adding water, tetrahydrofuran, the biotin-N-hydroxysuccinimide ester obtained in the step 1) and glycine into a reaction device in sequence under stirring, adjusting the pH value to 8.5 by using a 30% sodium hydroxide solution, reacting at room temperature, adjusting the pH value to 2 after the reaction is finished to separate out a large amount of solid, filtering, leaching a filter cake by using water, and drying in vacuum to obtain a white solid;
3) preparation of Biont-GHK (Boc) -OH: adding tetrahydrofuran and the Biontin-Gly-OH obtained in the step 2) in a reaction device in sequence under stirring, cooling in an ice bath, adding N, N' -carbonyldiimidazole, dripping the material into a tetrahydrofuran solution of His-Lys (Boc) -OH and triethylamine when the reaction solution does not bubble any more, reacting at room temperature after dripping, concentrating under reduced pressure after the reaction is finished to obtain an oily substance, adding water and ethyl acetate, adjusting the pH value to be 2.0-2.5 by using a hydrochloric acid solution, and washing an organic phase by using brine; drying with anhydrous magnesium sulfate, and concentrating under reduced pressure to obtain yellow oily substance;
4) preparation of biotin tripeptide-1: and (2) sequentially adding 4M hydrochloric acid and the Biotin-GHK (Boc) -OH obtained in the step 3) in a reaction device under stirring, reacting at room temperature, dropwise adding acetonitrile into the feed liquid after the reaction is finished, standing for growing crystals, filtering after 24 hours, and drying in vacuum to obtain the Biotin tripeptide-1.
The reflux reaction time in the step 1) is controlled to be 1-3 hours.
In the step 2), the reaction temperature is controlled to be 25 +/-5 ℃, and the reaction time is 1-2 hours.
In the step 3), the reaction temperature is controlled to be 30 +/-5 ℃, and the reaction time is 2-3 hours.
In the step 4), the reaction temperature is controlled to be 25 +/-5 ℃, the reaction time is 1-2 hours, and the crystal growing temperature is controlled to be 5 +/-3 ℃.
The invention has the beneficial effects that: according to the biotin tripeptide-1 liquid phase synthesis method, the final product can be purified by a crystallization method, so that the final liquid phase purification required in solid phase synthesis is successfully avoided, the production cost is reduced, and the batch production capacity is increased.
Detailed Description
Example 1
A liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
1) preparation of biotin-N-hydroxysuccinimide ester:
in a 250ml three-necked flask equipped with a thermometer and a stirrer, 100ml of tetrahydrofuran, 20g (81.9mmol) of biotin, 10.4g (90.09mmol) of N-hydroxysuccinimide, and 20.27g (98.24mmol) of 1, 3-dicyclohexylcarbodiimide were sequentially added under stirring, and a reflux reaction was carried out for 2 hours, followed by filtration, and the filtrate was concentrated under reduced pressure to obtain 27.2g of a white solid with a yield of 97.3%;
2) preparation of Biotin-Gly-OH:
in a 500ml three-necked flask equipped with a thermometer and a stirrer, 100ml of water, 100ml of tetrahydrofuran, 15g (43.94mmol) of biotin-N-hydroxysuccinimide ester and 3.96g (52.73mmol) of glycine were sequentially added under stirring, the pH was adjusted to 8.5 with a 30% sodium hydroxide solution and maintained at 8.5, and after 1.5 hours at 25 ℃, a large amount of solid was precipitated by adjusting the pH to 2, and the cake was filtered, rinsed with 2ml of water and dried to obtain 12.9g of a white solid with a molar yield of 97.4%;
3) preparation of Biont-GHK (Boc) -OH:
in a 500ml three-necked flask equipped with a thermometer and a stirrer, 50ml of tetrahydrofuran and 16.59mmol of biotin-Gly-OH 5g were sequentially added under stirring, 2.82g (17.42mmol) of N, N' -carbonyldiimidazole was added under cooling in an ice bath, and when the reaction solution did not bubble any more, the solution was dropped into a tetrahydrofuran solution of His-lys (boc) -oh7.63g (19.91mmol) and 2.01g (19.91mmol) of triethylamine, and after completion of the reaction at 30 ℃ for 2 hours, the solution was concentrated under reduced pressure to obtain an oil, 20ml of water and 50ml of ethyl acetate were added, pH 2.3 was adjusted with 6M hydrochloric acid, and the organic phase was washed with 15ml of brine; 5g of anhydrous magnesium sulfate was added thereto, the mixture was stirred for half an hour, filtered, and concentrated under reduced pressure to give 10.9g of a yellow oil in a molar yield of 98.5%
4) Preparation of biotin tripeptide-1:
30ml of 4mol/L hydrochloric acid and 30ml of Biotin-GHK (Boc) -OH5g (7.50mmol) are sequentially added into a 500ml three-necked flask with a thermometer and a stirrer under stirring, the mixture reacts for 1 hour at 25 ℃, 30ml of acetonitrile is dropwise added into the feed liquid, the mixture stands for crystal growing at 5 ℃, the mixture is filtered after 24 hours, and the product with the purity of 98 percent is obtained by vacuum drying, wherein the molar yield is 95.1 percent.
Example 2
A liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
1) preparation of biotin-N-hydroxysuccinimide ester:
100ml of tetrahydrofuran, 20g (81.9mmol) of biotin, 10.4g (90.09mmol) of N-hydroxysuccinimide and 20.27g (98.24mmol) of 1, 3-dicyclohexylcarbodiimide were sequentially added to a 250ml three-necked flask equipped with a thermometer and a stirrer under stirring, and subjected to reflux reaction for 2.5 hours, filtration and concentration of the filtrate under reduced pressure to obtain 27.1g of a white solid with a molar yield of 96.9%;
2) preparation of Biotin-Gly-OH:
in a 500ml three-necked flask equipped with a thermometer and a stirrer, 100ml of water, 100ml of tetrahydrofuran, 15g (43.94mmol) of biotin-N-hydroxysuccinimide ester and 3.96g (52.73mmol) of glycine were sequentially added under stirring, the pH was adjusted to 8.5 with a 30% sodium hydroxide solution, the pH was maintained at 8.5, after reacting at 25 ℃ for 2 hours, a large amount of solid was precipitated by adjusting the pH to 2, the reaction mixture was filtered, and the filter cake was rinsed with 2ml of water and dried to obtain 12.8g of a white solid with a molar yield of 96.7%;
3) preparation of Biont-GHK (Boc) -OH:
in a 500ml three-necked flask equipped with a thermometer and a stirrer, 50ml of tetrahydrofuran and 16.59mmol of biotin-Gly-OH 5g were sequentially added under stirring, 2.82g (17.42mmol) of N, N' -carbonyldiimidazole was added under cooling in an ice bath, and when the reaction solution did not bubble any more, the solution was dropped into a tetrahydrofuran solution of His-lys (boc) -oh7.63g (19.91mmol) and 2.01g (19.91mmol) of triethylamine, and after completion of the reaction at 30 ℃ for 2.5 hours, the solution was concentrated under reduced pressure to obtain an oil, 20ml of water and 50ml of ethyl acetate were added, pH was adjusted to 2.3 with 6M hydrochloric acid, and the organic phase was washed with 15ml of brine; adding 5g anhydrous magnesium sulfate, stirring for half an hour, filtering, and concentrating under reduced pressure to obtain 10.6g yellow oil with molar yield of 95.8%;
4) preparation of biotin tripeptide-1:
30ml of 4mol/L hydrochloric acid and 30ml of Biotin-GHK (Boc) -OH5g (7.50mmol) are sequentially added into a 500ml three-necked flask with a thermometer and a stirrer under stirring, the mixture reacts for 1.5 hours at 25 ℃, 30ml of acetonitrile is dropwise added into the feed liquid, the mixture stands for crystal growth at 3 ℃, the mixture is filtered after 24 hours, and the product with the purity of 98 percent is obtained after vacuum drying, wherein the molar yield is 97.3 percent.
Example 3
A liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
1) preparation of biotin-N-hydroxysuccinimide ester:
100ml of tetrahydrofuran, 20g (81.9mmol) of biotin, 10.4g (90.09mmol) of N-hydroxysuccinimide and 20.27g (98.24mmol) of 1, 3-dicyclohexylcarbodiimide were sequentially added to a 250ml three-necked flask equipped with a thermometer and a stirrer under stirring, and subjected to reflux reaction for 3 hours, filtration and concentration of the filtrate under reduced pressure to obtain 27.0g of a white solid with a molar yield of 96.7%;
2) preparation of Biotin-Gly-OH:
in a 500ml three-necked flask equipped with a thermometer and a stirrer, 100ml of water, 100ml of tetrahydrofuran, 15g (43.94mmol) of biotin-N-hydroxysuccinimide ester and 3.96g (52.73mmol) of glycine were sequentially added under stirring, the pH was adjusted to 8.5 with a 30% sodium hydroxide solution, the pH was maintained at 8.5, after 1 hour of reaction at 25 ℃, a large amount of solid was precipitated by adjusting the pH to 2, the resulting mixture was filtered, and the filter cake was rinsed with 2ml of water and dried to obtain 12.7g of a white solid with a molar yield of 95.9%;
3) preparation of Biont-GHK (Boc) -OH:
in a 500ml three-necked flask equipped with a thermometer and a stirrer, 50ml of tetrahydrofuran and 16.59mmol of biotin-Gly-OH 5g were sequentially added under stirring, 2.82g (17.42mmol) of N, N' -carbonyldiimidazole was added under cooling in an ice bath, and when the reaction solution did not bubble any more, the solution was dropped into a tetrahydrofuran solution of His-lys (boc) -oh7.63g (19.91mmol) and 2.01g (19.91mmol) of triethylamine, and after completion of the reaction at 30 ℃ for 3 hours, the solution was concentrated under reduced pressure to obtain an oil, 20ml of water and 50ml of ethyl acetate were added, pH 2.3 was adjusted with 6M hydrochloric acid, and the organic phase was washed with 15ml of brine; adding 5g anhydrous magnesium sulfate, stirring for half an hour, filtering, and concentrating under reduced pressure to obtain 10.4 yellow oil with molar yield of 94.0%;
4) preparation of biotin tripeptide-1:
30ml of 4mol/L hydrochloric acid and 30ml of Biotin-GHK (Boc) -OH5g (7.50mmol) are sequentially added into a 500ml three-necked flask with a thermometer and a stirrer under stirring, the mixture reacts for 2 hours at 25 ℃, 30ml of acetonitrile is dropwise added into the feed liquid, the mixture stands for crystal growing at 7 ℃, the mixture is filtered after 24 hours, and the product with the purity of 98 percent is obtained by vacuum drying, wherein the molar yield is 92.9 percent.
Claims (5)
1. A liquid phase synthesis method of biotin tripeptide-1 is characterized by comprising the following steps:
1) preparation of biotin-N-hydroxysuccinimide ester: adding tetrahydrofuran, biotin, N-hydroxysuccinimide and 1, 3-dicyclohexylcarbodiimide in a reaction device in sequence under stirring, heating for reflux reaction, filtering, and concentrating the filtrate under reduced pressure to obtain a white solid;
2) preparation of Biotin-Gly-OH: adding water, tetrahydrofuran, the biotin-N-hydroxysuccinimide ester obtained in the step 1) and glycine into a reaction device in sequence under stirring, adjusting the pH value to 8.5 by using a 30% sodium hydroxide solution, reacting at room temperature, adjusting the pH value to 2 after the reaction is finished to separate out a large amount of solid, filtering, leaching a filter cake by using water, and drying in vacuum to obtain a white solid;
3) preparation of Biont-GHK (Boc) -OH: adding tetrahydrofuran and the Biontin-Gly-OH obtained in the step 2) in a reaction device in sequence under stirring, cooling in an ice bath, adding N, N' -carbonyldiimidazole, dripping the material into a tetrahydrofuran solution of His-Lys (Boc) -OH and triethylamine when the reaction solution does not bubble any more, reacting at room temperature after dripping, concentrating under reduced pressure after the reaction is finished to obtain an oily substance, adding water and ethyl acetate, adjusting the pH value to be 2.0-2.5 by using a hydrochloric acid solution, and washing an organic phase by using brine; drying with anhydrous magnesium sulfate, and concentrating under reduced pressure to obtain yellow oily substance;
4) preparation of biotin tripeptide-1: and (2) sequentially adding 4M hydrochloric acid and the Biotin-GHK (Boc) -OH obtained in the step 3) in a reaction device under stirring, reacting at room temperature, dropwise adding an organic solvent into the feed liquid after the reaction is finished, standing for growing crystals, filtering after 24 hours, and drying in vacuum to obtain the Biotin tripeptide-1.
2. The liquid-phase synthesis method of biotin tripeptide-1 according to claim 1, wherein the reflux reaction time in step 1) is controlled to be 1-3 hours.
3. The liquid-phase synthesis method of biotin tripeptide-1 according to claim 1, wherein the reaction temperature in step 2) is controlled to 25 ± 5 ℃ and the reaction time is 1-2 hours.
4. The liquid-phase synthesis method of biotin tripeptide-1 according to claim 1, wherein the reaction temperature in step 3) is controlled to be 30 ± 5 ℃ and the reaction time is 2-3 hours.
5. The liquid-phase synthesis method of biotin tripeptide-1 according to claim 1, characterized in that in step 4), the reaction temperature is controlled to 25 ± 5 ℃, the reaction time is 1-2 hours, the crystallization solvent is acetonitrile, and the crystallization temperature is controlled to 5 ± 3 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113896762A (en) * | 2021-12-06 | 2022-01-07 | 浙江湃肽生物有限公司深圳分公司 | Liquid phase synthesis method of biotin tripeptide-1 |
| CN117126230A (en) * | 2023-10-23 | 2023-11-28 | 广州同隽医药科技有限公司 | Synthesis method and application of tripeptide-1 and blue copper peptide |
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| US20110201562A1 (en) * | 2005-04-01 | 2011-08-18 | Sederma | Formulations and method for treating baldness |
| WO2012164488A2 (en) * | 2011-06-01 | 2012-12-06 | Sederma | New cosmetic or dermopharmaceutical topical use of a mixture of a ghk tripeptide and gqpr tetrapeptide |
| CN107778349A (en) * | 2017-11-29 | 2018-03-09 | 陕西慧康生物科技有限责任公司 | A kind of method of low cost synthesis GHK acetate |
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2019
- 2019-12-31 CN CN201911412735.4A patent/CN111004304B/en active Active
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| US20110201562A1 (en) * | 2005-04-01 | 2011-08-18 | Sederma | Formulations and method for treating baldness |
| WO2012164488A2 (en) * | 2011-06-01 | 2012-12-06 | Sederma | New cosmetic or dermopharmaceutical topical use of a mixture of a ghk tripeptide and gqpr tetrapeptide |
| CN107778349A (en) * | 2017-11-29 | 2018-03-09 | 陕西慧康生物科技有限责任公司 | A kind of method of low cost synthesis GHK acetate |
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| Title |
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| V. ARUL,ET AL.: ""Biotinylated GHK Peptide Incorporated Collagenous Matrix:A Novel Biomaterial for Dermal Wound Healing in Rats"", 《J BIOMED MATER RES B APPL BIOMATER》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113896762A (en) * | 2021-12-06 | 2022-01-07 | 浙江湃肽生物有限公司深圳分公司 | Liquid phase synthesis method of biotin tripeptide-1 |
| CN117126230A (en) * | 2023-10-23 | 2023-11-28 | 广州同隽医药科技有限公司 | Synthesis method and application of tripeptide-1 and blue copper peptide |
| CN117126230B (en) * | 2023-10-23 | 2024-02-13 | 广州同隽医药科技有限公司 | Synthesis method and application of tripeptide-1 and blue copper peptide |
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