CN106554407B - Synthetic method of ularitide - Google Patents
Synthetic method of ularitide Download PDFInfo
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- CN106554407B CN106554407B CN201510644646.8A CN201510644646A CN106554407B CN 106554407 B CN106554407 B CN 106554407B CN 201510644646 A CN201510644646 A CN 201510644646A CN 106554407 B CN106554407 B CN 106554407B
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- ularitide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
Abstract
The invention relates to the technical field of medicines, and discloses a synthetic method of ularitide. The synthetic method of the invention adds the crude peptide or the refined peptide of the ularitide linear peptide into the ionic liquid with the melting point below 20 ℃, and oxidizes the ionic liquid for 0.5 to 3 hours in the alkaline environment to obtain the crude product of the ularitide forming the disulfide bond. The method uses the room-temperature ionic liquid to replace a liquid-phase oxidation medium of an organic solvent and water in the traditional oxidation link, so that the purity of the crude peptide and the total yield of the refined peptide of the solid-phase synthesized ularitide are obviously improved, meanwhile, the oxidation reaction time can be greatly reduced, the operation is convenient, and the generation of organic waste liquid is avoided.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a synthetic method of ularitide.
Background
Urapide (Ularitide) is natriuretic peptide separated from human urine, and has an analytical structure similar to atrial natriuretic peptide, and is different only in that four amino acid residues are added at the N-terminal of the sequence. Relevant researches show that the ularitide has various effects of expanding blood vessels, expanding bronchus, inducing diuresis and the like, and has certain treatment effect on heart failure, renal failure, pulmonary hypertension and bronchial asthma clinically.
The structure of the ularitide is a polypeptide which is composed of 32 amino acids and contains a pair of intramolecular disulfide bonds, the polypeptide is generally prepared by chemical synthesis, and the sequence structure is as follows:
H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH(S-S)。
at present, there are two main methods for synthesizing ularitide. One is to complete the coupling of linear peptide resin in a solid phase and then form a corresponding disulfide bond by iodine solid phase oxidation; another method is to synthesize linear peptide precursors in solid phase and then oxidize in liquid phase in very low concentration organic solvent/water solution to form the corresponding disulfide bonds.
As can be seen from the existing method, the synthesis of the ularitide is mainly divided into two stages of amino acid coupling and oxidation reaction. In the former, the obtained linear peptide resin is added with solid iodine simple substance, oxidized to form disulfide bond, oxidized cyclized peptide resin is obtained, and the oxidized cyclized peptide resin is cracked to obtain a crude product. However, the oxidation of the peptide resin by iodine is difficult to completely elute in the whole process, so that the final pure product, namely the crude drug, contains a trace amount of iodine, and the crude drug is yellow. In addition, the peptide is oxidized by a strong oxidant, so that the secondary structure of the peptide sequence is easily damaged, and the bioactivity and the drug effect of the peptide are influenced finally.
And the latter purifies the crude peptide to obtain fine peptide. Dissolving linear peptide with methanol or acetonitrile, adding water to dilute to 10 deg.C-3And (3) oxidizing the mixture for 72 hours at the concentration of mg/ml under the air and alkaline conditions to obtain the oxidized crude cyclic peptide. And purifying the crude cyclic peptide by HPLC to obtain the refined peptide. Compared with the former, the method has the advantages that the spatial secondary structure of the final product can be completely reserved, and the bioactivity of the final refined peptide is better than that of the former. However, this scheme is not suitable for large-scale production because the dissolution of the ularitide is difficult, and the final oxidation needs to be carried out at a very dilute concentration, which makes the operation very difficult and the reaction time long. In addition, the method can generate a large amount of mixed waste liquid of the organic solvent and water, and is not beneficial to the requirement of green chemistry in the aspect of environmental protection.
Disclosure of Invention
In view of the above, the present invention aims to provide a synthetic method of ularitide, which can shorten the oxidation reaction time of ularitide linear peptide and avoid the generation of organic waste liquid.
Another object of the present invention is to provide a method for synthesizing ularitide, such that the method can improve the purity of the crude peptides of ularitide and the total yield of refined peptides.
In order to achieve the purpose, the invention provides the following technical scheme:
a synthetic method of ularitide is characterized in that ularitide linear peptide crude peptide or refined peptide is added into ionic liquid with the melting point below 20 ℃, and oxidation is carried out for 0.5-3h under the alkaline condition, so as to obtain the ularitide crude product with disulfide bonds.
Aiming at the defects of overlong oxidation reaction time, difficult operation, organic waste liquid generation and the like in the existing synthesis method, the ionic liquid with the melting point below 20 ℃ is used for replacing the existing organic solvent and water as a liquid phase oxidation medium, the system has very good solubility for the ularitide, can be oxidized in a higher concentration range, does not need to be diluted to a very low concentration, is convenient to operate, can greatly shorten the oxidation reaction time, avoids the generation of the organic waste liquid, increases the bioactivity of the ularitide, and can realize large-scale production.
Wherein the alkaline condition can be adjusted by referring to the alkaline condition of the existing liquid phase oxidation method, for example, adjusting the pH to be 7.0-7.5 or higher, and the oxidation reaction is stopped to adjust the pH to be acidic, and the oxidation time can be selected to be 0.5h, 1h, 2h or 3h in some embodiments of the invention.
Preferably, the cation of the ionic liquid is a 1-ethyl-3-methylimidazolium cation or a 1-butyl-3-methylimidazolium cation.
Preferably or further preferably, the ionic liquid is 1-ethyl-3-methylimidazolium acetate [ C2mim ]][OAc]1-Ethyl-3-methylimidazolium trifluoroacetate [ C2mim ]][TFA]1-ethyl-3-methylimidazolium hexafluorophosphate [ C2mim ]][PF6]1-butyl-3-methylimidazolyl acetate [ C4mim ]][OAc]1-butyl-3-methylimidazolium trifluoroacetate salt [ C4mim ]][TFA]Or 1-butyl-3-methylimidazolium hexafluorophosphate [ C4mim ]][PF6]。
Preferably, the concentration of the crude peptide or the refined peptide of the oxidized ularitide linear peptide is 20-200mg of linear peptide/mL of ionic liquid. In some embodiments of the invention, the concentration of the crude or refined peptide of the ularitide linear peptide can be selected from 20mg/mL, 50mg/mL, 100mg/mL or 200 mg/mL.
The crude peptide or the refined peptide of the ularitide linear peptide in the synthetic method can be prepared according to the conventional synthetic method in the field, and as a preferable scheme, the invention is prepared by a solid phase synthetic method, wherein the solid phase synthetic method comprises the step of synthesizing according to the amino acid sequence of the ularitide by a one-by-one synthetic method or a fragment method.
In the process of synthesizing one by one, the first amino acid Tyr at the C end of the ularitide is coupled with a resin carrier such as wang resin, and then subsequent coupling is carried out according to an amino acid sequence; in the synthesis by the fragment method, the peptide sequence of the ularitide can be divided into a plurality of fragments according to actual needs to be synthesized simultaneously, and finally, each fragment is coupled.
During the synthesis, the skilled person will generally perform the synthesis using protected amino acids. The protected amino acid or protected aa (aa refers to a specific amino acid) refers to an amino acid which is easy to generate an interference reaction group such as an amino group, a carboxyl group and the like on the main chain and the side chain of the amino acid by protecting groups. For amino acids whose side chain needs to be protected in the synthesis, the side chain structure is well known to those skilled in the art, and it is known to use commonly used protecting groups to protect amino groups, carboxyl groups, etc. on the side chain of amino acids.
The synthesis process is illustrated by a one-by-one synthesis method:
step 1, coupling the protected Tyr and Wang resin in a coupling system;
step 2, coupling the following remaining protected amino acids one by using a coupling system according to a peptide sequence from the C end to the N end of the ularitide:
Fmoc-Arg(Pbf)-OH，Fmoc-Phe-OH，Fmoc-Ser(tBu)-OH，Fmoc-Asn(Trt)-OH，Fmoc-Cys(Trt)-OH，Fmoc-Gly-OH，Fmoc-Leu-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Ala-OH，Fmoc-Gly-OH，Fmoc-Ile-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Met-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Gly-OH，Fmoc-Gly-OH，Fmoc-Phe-OH，Fmoc-Cys(Trt)-OH，Fmoc-Ser(tBu)-OH，Fmoc-Ser(tBu)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Leu-OH，Fmoc-Ser(tBu)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Pro-OH，Fmoc-Ala-OH，Fmoc-Thr(tBu)-OH；
and removing the Fmoc protecting group after the coupling is finished to obtain the crude peptide of the ularitide linear peptide, and purifying the obtained refined peptide.
The coupling system for solid-phase synthesis of the residual amino acid comprises DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
The cleavage was performed using TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2Cracking with 85-95:2-5:0-5:0-2:1-5 cracking reagent for 1.5-3.5 hr; more preferably, the volume ratio TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2Cleavage with 90:3:2:1:4 cleavage reagent for 2.5 h.
In the coupling process of the protected Try and the subsequent amino acid, because the N end is protected by protecting groups such as Fmoc and the like, each deprotection is required, which is a conventional step in solid phase synthesis, and a DMF solution containing 20% hexahydropyridine can be adopted, and other suitable reagents can be used for removing.
In addition, the invention also comprises a step of purifying the crude product of the ularitide, wherein the purification is further preferably HPLC purification.
More specific HPLC purification steps are as follows:
taking the oxidized crude product of the ularitide, adopting a Waters 2454RP-HPLC system, the wavelength is 220nm, a chromatographic column is a 100 x 500mm reversed phase C18 column, and the mobile phase is as follows: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting the target peak fraction at 6 ml/min, performing rotary evaporation and concentration, and performing freeze-drying to obtain the ularitide refined peptide.
The method for oxidizing the ularitide linear peptide can improve the purity of the ularitide crude peptide obtained by the reaction to 62.47-77.52%, while the purity of the crude peptide obtained by the existing liquid phase oxidation method is only 48.52%, the total yield of the ularitide can reach 58.3-74.7% after purification, and the total yield of the ularitide purified by the existing method is 36.3%, and the two have very obvious difference compared with each other.
Based on the beneficial effects brought by the ionic liquid in the oxidation reaction of the ularitide linear peptide, the invention provides the application of the ionic liquid in the formation of the ularitide through the oxidation of the ularitide linear peptide.
Among them, the ionic liquid is preferably an ionic liquid having a melting point of 20 ℃ or lower. More preferably, the cation of the ionic liquid is a 1-ethyl-3-methylimidazolium cation or a 1-butyl-3-methylimidazolium cation.
Preferably or further preferably, the ionic liquid is 1-ethyl-3-methylimidazolium acetate [ C2mim ]][OAc]1-Ethyl-3-methylimidazolium trifluoroacetate [ C2mim ]][TFA]1-ethyl-3-methylimidazolium hexafluorophosphate [ C2mim ]][PF6]1-butyl-3-methylimidazolyl acetate [ C4mim ]][OAc]1-butyl-3-methylimidazolium trifluoroacetate salt [ C4mim ]][TFA]Or 1-butyl-3-methylimidazolium hexafluorophosphate [ C4mim ]][PF6]。
The ionic liquid carrier used in the present invention can be synthesized according to a direct synthesis method or a two-step method which is common in the art after the cation and anion are defined, or can be obtained commercially.
According to the technical scheme, the room-temperature ionic liquid replaces a liquid-phase oxidation medium of an organic solvent and water in the traditional oxidation link, so that the purity of the crude peptide and the total yield of the refined peptide of the solid-phase synthesized ularitide are obviously improved, the oxidation reaction time can be greatly reduced, the operation is convenient, and the generation of organic waste liquid is avoided.
Detailed Description
The invention discloses a synthetic method of ularitide, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the synthesis of the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be practiced and applied by modifying or appropriately combining the compounds and processes described herein without departing from the spirit, scope and spirit of the invention.
Some abbreviations and key terms in the present invention are defined as in the following table, reagents and materials commonly used in the synthesis of protected amino acids and the like are available from gill biochemical company, tianjin nan kao and cheng company.
| Fmoc | 9-fluorenylmethoxycarbonyl group |
| Boc | Tert-butyloxycarbonyl radical |
| tBu | Tert-butyl radical |
| Trt | Trityl radical |
| Pbf | 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl |
| DMF | N, N-dimethylformamide |
| DCM | Methylene dichloride |
| DBLK | 20% piperidine/DMF solution |
| DIC | N, N-diisopropylCarbodiimides |
| DIPEA | N, N-diisopropylethylamine |
| PyBOP | Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphates |
| TBTU | O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate |
| HBTU | O-benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate |
| HOBT | 1-hydroxybenzotriazoles |
| HOAT | 1-hydroxy-7 azo-benzotriazoles |
| TFA | Trifluoroacetic acid |
| EDT | 1, 2-ethanedithiol |
| PhOH | Phenol and its preparation |
| TIS | Tri-isopropyl silane |
In a specific embodiment, unless otherwise specified, when the present invention is compared with the conventional oxidation method, the crude peptide or the refined peptide of the ularitide linear peptide used in the present invention is derived from the same solid phase synthesis to ensure comparability of the comparison test.
The invention is further illustrated by the following examples.
Example 1: preparation of crude wularitide linear peptide
1mol of the ularitide is synthesized by a solid phase synthesis mode one by one to obtain 3510g of crude peptide, the yield of the crude peptide is 100.1 percent, and the purity is 78.43 percent.
Example 2: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude peptide of the linear peptide obtained in example 1 was dissolved in 5.0L of [ C2mim ] [ OAc ] (oxidation concentration 20mg/mL), the pH of the solution was adjusted to 7.0 to 7.5 basic atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 2.0 hours, 0.5L of aqueous 0.1% TFA solution was added to adjust the pH of the solution to acidic, and oxidation was completed, whereby the crude purity of ularitide was 62.47%.
Example 3: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 5.0L of [ C2mim ] [ TFA ] (oxidation concentration: 20mg/mL), the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 2.0 hours, and 0.5L of a 0.1% aqueous solution of TFA was added to adjust the pH to acidic, whereby the oxidation was completed. The purity of the obtained crude product of the ularitide is 76.81%.
Example 4: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 2.0L of [ C2mim ] [ TFA ] (oxidation concentration: 50mg/mL), the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 1.0 hour, and 0.5L of a 0.1% aqueous solution of TFA was added to adjust the pH to acidic, whereby the oxidation was completed. The purity of the obtained crude product of the ularitide is 75.48%.
Example 5: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L of [ C2mim ] [ TFA ] (oxidation concentration: 100mg/mL), the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 1.0 hour, and 0.5L of a 0.1% aqueous solution of TFA was added to adjust the pH to acidic, thereby completing the oxidation. The purity of the obtained crude product of the ularitide is 76.01 percent.
Example 6: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 0.5L of [ C2mim ] [ TFA ] (oxidation concentration: 200mg/mL), the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 0.5 hour, and 0.5L of a 0.1% aqueous solution of TFA was added to adjust the pH to acidic, whereby the oxidation was completed. The purity of the obtained crude product of the ularitide is 73.05%.
Example 7: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L of [ C2mim ]][PF6]Adjusting the pH of the solution to 7.0-7.5 alkaline environment with ammonia water (oxidation concentration is 100mg/mL), slightly stirring at room temperature, carrying out oxidation reaction for 1.0h, adding 0.5L aqueous solution of TFA with concentration of 0.1% to adjust the pH to acidity, and completing the oxidation. The purity of the obtained crude product of the ularitide is 77.11%.
Example 8: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L of [ C4mim ] [ OAc ] (oxidation concentration: 100mg/mL), the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 1.0 hour, and 0.5L of 0.1% aqueous TFA solution was added to adjust the pH to acidity, whereby the oxidation was completed. The purity of the obtained crude product of the ularitide is 70.49%.
Example 9: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L of [ C4mim ] [ TFA ] (oxidation concentration: 100mg/mL), the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, the mixture was stirred slightly at room temperature, oxidation was carried out for 1.0 hour, and 0.5L of a 0.1% aqueous solution of TFA was added to adjust the pH to acidic, thereby completing the oxidation. The purity of the obtained crude product of the ularitide is 76.39%.
Example 10: oxidation reaction of crude peptide of wularitide linear peptide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L of [ C4mim ]][PF6]Adjusting the pH of the solution to 7.0-7.5 alkaline environment with ammonia water (oxidation concentration is 100mg/mL), slightly stirring at room temperature, carrying out oxidation reaction for 1.0h, adding 0.5L aqueous solution of TFA with concentration of 0.1% to adjust the pH to acidity, and completing the oxidation. The purity of the obtained crude product of the ularitide is 77.52%.
Example 11: preparation of ularitide fine peptide
Taking the oxidized crude peptide solution in example 2, adopting a Waters 2454RP-HPLC system, the wavelength is 220nm, the chromatographic column is a 100X 500mm reversed phase C18 column, and the mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: collecting the target peak fraction at 6 ml/min, rotary evaporating for concentration, and lyophilizing to obtain 58.32g of ularitide refined peptide, HPLC purity of 99.34%, and total yield of 58.3%.
Example 12: preparation of ularitide fine peptide
The crude peptide solution oxidized in example 3 was taken, and lyophilized by the purification method of example 11 to obtain 72.58g of urotropine refined peptide with HPLC purity of 99.15% and total yield of 72.6%.
Example 13: preparation of ularitide fine peptide
The oxidized crude peptide solution obtained in example 4 was lyophilized by the purification method of example 11 to obtain 71.94g of urotropine refined peptide with HPLC purity of 99.27% and total yield of 72.0%.
Example 14: preparation of ularitide fine peptide
The crude peptide solution oxidized in example 5 was subjected to lyophilization by the purification method of example 11 to obtain 73.08g of urotropine refined peptide, the HPLC purity was 99.85%, and the total yield was 73.1%.
Example 15: preparation of ularitide fine peptide
The oxidized crude peptide solution obtained in example 6 was lyophilized by the purification method of example 11 to obtain 67.44g of urotropine refined peptide with HPLC purity of 99.01% and total yield of 67.5%.
Example 16: preparation of ularitide fine peptide
The crude peptide solution oxidized in example 7 was subjected to lyophilization by the purification method of example 11 to obtain 74.13g of urotropine refined peptide, the HPLC purity was 99.30%, and the total yield was 74.2%.
Example 17: preparation of ularitide fine peptide
The crude peptide solution oxidized in example 8 was subjected to lyophilization by the purification method of example 11 to obtain 65.59g of urotropine refined peptide, HPLC purity was 99.20%, and total yield was 65.6%.
Example 18: preparation of ularitide fine peptide
The crude peptide solution oxidized in the example 9 was taken, and lyophilized by the purification method of the example 11 to obtain 73.25g of the urotropine refined peptide, the HPLC purity was 99.47%, and the total yield was 73.3%.
Example 19: preparation of ularitide fine peptide
The crude peptide solution oxidized in example 10 was taken, and lyophilized to obtain 74.67g of urotropine refined peptide with HPLC purity of 99.36% and total yield of 74.7% by the purification method of example 11.
Example 20: oxidation reaction of existing organic solvent/aqueous solution liquid phase ularitide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L DMF, and diluted to 5L with 4L of water, the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, and slightly stirred at room temperature, and oxidized for 24.0 hours, and then 0.5L of aqueous 0.1% TFA solution was added to adjust the pH to acidity, thereby completing the oxidation. The target product cannot be obtained, and the dimer forming intermolecular disulfide bond is obtained.
Example 21: oxidation reaction of existing organic solvent/aqueous solution liquid phase ularitide
100g of the crude linear peptide obtained in example 1 was dissolved in 1.0L DMF, and diluted to 100L with 99L of water, the solution was adjusted to pH 7.0 to 7.5 in an alkaline atmosphere with ammonia, and slightly stirred at room temperature, and oxidized for 48.0 hours (monitoring the progress of the reaction by HPLC), and then 0.5L of aqueous 0.1% TFA was added to adjust the pH to acidity, thereby completing the oxidation. The purity of the obtained crude product of the ularitide is 48.52 percent.
Example 22: preparation of oxidized refined peptide of existing organic solvent/aqueous solution liquid phase ularitide
The crude peptide solution oxidized in example 21 was concentrated to obtain 5L of a reaction solution, which was lyophilized in the same manner as in example 11 to obtain 36.58g of urotropine arginine peptide with HPLC purity of 99.31% and total yield of 36.6%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A synthetic method of ularitide is characterized by comprising the following steps:
adding the crude peptide or the refined peptide of the ularitide linear peptide into an ionic liquid with the melting point below 20 ℃, and oxidizing for 0.5-3.0h under the alkaline condition to obtain a crude product of the ularitide forming a disulfide bond; the ionic liquid is 1-ethyl-3-methylimidazolium acetate [ C2mim ]][OAc]1-Ethyl-3-methylimidazolium trifluoroacetate [ C2mim ]][TFA]1-ethyl-3-methylimidazolium hexafluorophosphate [ C2mim ]][PF6]1-butyl-3-methylimidazolyl acetate [ C4mim ]][OAc]1-butyl-3-methylimidazolium trifluoroacetate salt [ C4mim ]][TFA]Or 1-butyl-3-methylimidazolium hexafluorophosphate [ C4mim ]][PF6]。
2. The method for synthesizing the urotropine in the claim 1, wherein the concentration of the crude peptide or the refined peptide of the urotropine linear peptide during the oxidation is 20-200mg of the linear peptide/mL of the ionic liquid.
3. The method for synthesizing the ularitide linear peptide as claimed in claim 1, wherein the ularitide linear peptide crude peptide or refined peptide is prepared by a solid phase synthesis process.
4. The method of claim 1, further comprising purifying the crude ularitide.
5. The synthetic method of claim 4 wherein the purification is HPLC purification.
6. The synthetic method of claim 5 wherein the HPLC purification step is:
taking the oxidized crude product of the ularitide, adopting a Waters 2454RP-HPLC system, the wavelength is 220nm, a chromatographic column is a 100 x 500mm reversed phase C18 column, and the mobile phase is as follows: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting the target peak fraction at 6 ml/min, performing rotary evaporation and concentration, and performing freeze-drying to obtain the ularitide refined peptide.
7. The application of the ionic liquid in the oxidation of the ularitide linear peptide to form the ularitide; the ionic liquid is 1-ethyl-3-methylimidazolium acetate [ C2mim ]][OAc]1-Ethyl-3-methylimidazolium trifluoroacetate [ C2mim ]][TFA]1-ethyl-3-methylimidazolium hexafluorophosphate [ C2mim ]][PF6]1-butyl-3-methylimidazolyl acetate [ C4mim ]][OAc]1-butyl-3-methylimidazolium trifluoroacetate salt [ C4mim ]][TFA]Or 1-butyl-3-methylimidazolium hexafluorophosphate [ C4mim ]][PF6]。
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| CN103145827A (en) * | 2013-03-04 | 2013-06-12 | 吉尔生化（上海）有限公司 | Solid-phase synthesis method of ularitide |
| CN104371018A (en) * | 2014-01-22 | 2015-02-25 | 江苏汉邦科技有限公司 | Ularitide preparation method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103145827A (en) * | 2013-03-04 | 2013-06-12 | 吉尔生化（上海）有限公司 | Solid-phase synthesis method of ularitide |
| CN104371018A (en) * | 2014-01-22 | 2015-02-25 | 江苏汉邦科技有限公司 | Ularitide preparation method |
Non-Patent Citations (3)
| Title |
|---|
| Application of Room-Temperature Aprotic and Protic Ionic;Pascal Heimer;《CHEMBIOCHEM》;20141115;第15卷(第18期);第1-13页 * |
| Ionic Liquid Applications in Peptide Chemistry:;Alesia A. Tietze;《molecules》;20120405;第17卷(第4期);全文 * |
| Miriam Böhm.Ionic liquids as reaction media for oxidative folding and native chemical.《Journal of Molecular Liquids》.2014,第192卷第67-70. * |
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| CN106554407A (en) | 2017-04-05 |
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