CN110256532B - RGD cyclopeptide synthesis method - Google Patents
RGD cyclopeptide synthesis method Download PDFInfo
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- CN110256532B CN110256532B CN201910595934.7A CN201910595934A CN110256532B CN 110256532 B CN110256532 B CN 110256532B CN 201910595934 A CN201910595934 A CN 201910595934A CN 110256532 B CN110256532 B CN 110256532B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/52—Cyclic peptides containing at least one abnormal peptide link with only normal peptide links in the ring
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses an RGD cyclopeptide synthesis method, which comprises the following steps: s1, obtaining fully protected linear peptide 1) obtaining Mtt-Lys (Boc) -NHS; 2) Adding amino acids to Mtt-Lys (Boc) -NHS and forming a linkage; 3) Linking the N-terminus of the next amino acid to the C-terminus of the previous amino acid; 4) Taking H-D-Phe-OMe as a terminal raw material, and obtaining a fully-protected straight-chain peptide according to the step 3); s2, obtaining the fully-protected cyclic peptide, and dissolving the fully-protected cyclic peptide by using DCM (DCM) added with TFA to form an amide ring; s3, cutting and purifying; 5) Putting the fully-protected cyclic peptide into the reaction kettle for cutting for 2 hours; 6) Separating out with glacial ethyl ether, centrifuging to obtain crude polypeptide peptide, washing with glacial ethyl ether for 3 times, removing part of impurities, vacuum pumping, and purifying with HPLC to obtain pure product. The method has the advantages of simple operation, pure product, high yield and low racemization rate.
Description
Technical Field
The invention relates to a method for synthesizing cyclopeptide, in particular to a method for synthesizing RGD cyclopeptide.
Background
The RGD sequence is a universal recognition site of extracellular matrix and integrin, and can increase the biological stability of the peptide. The RGD sequence peptide has wide bioactivity, can be used for treating cardiovascular diseases, osteoporosis, inflammation and other diseases, and can also prevent and treat tumors caused by abnormal cell adhesion, especially the metastasis of developmental tumors; on the other hand, RGD sequence peptides can also serve as agonists, promoting regeneration of injured organs and tissues, healing of wounds, and the like, and RGD serves as a receptor for some integrins, the selectivity of which depends in part on the conformation of RGD and the amino acid residues around RGD. In the traditional liquid phase cyclization synthesis method, a condensation reagent is required to be added in the synthesis process, the condensation reagent can bring byproducts and impurities, and the product yield is low and the cost is high.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an RGD cyclopeptide synthesis method which is simple to operate, pure in product, high in yield and low in racemization rate.
In order to solve the technical problem, the invention provides an RGD cyclopeptide synthesis method, which comprises the following steps:
s1, obtaining a fully protected linear peptide
1) Forming an activated ester at the C-terminus of Mtt-Lys (Boc) -OH with an activating reagent starting with Mtt-Lys (Boc) -OH to obtain Mtt-Lys (Boc) -NHS;
2) Adding an amino acid into Mtt-Lys (Boc) -NHS under alkaline environment, and reacting the activated ester of Mtt-Lys (Boc) -NHS with the amino group at the N-terminal of the amino acid to form a connection;
3) Activating the carboxyl group of the amino acid to form an activated ester under the conditions of step S1, and connecting the N terminal of the next amino acid to the C terminal of the previous amino acid under the conditions of step S2;
4) Taking H-D-Phe-OMe as a terminal raw material, connecting the N end of the H-D-Phe-OMe to the C end of the last amino acid according to the step 3) to obtain a fully protected linear peptide;
s2, obtaining the fully-protected cyclic peptide
After the fully protected linear peptide is subjected to rotary evaporation and freeze-drying, DCM added with TFA is used for dissolving to form an amide ring, and the fully protected cyclic peptide is obtained, wherein the weight parts of DCM: TFA =20:1, then refluxing in oil bath at 50 ℃ overnight, and finally spin-drying the fully-protected cyclic peptide;
s3, cutting and purifying
5) In parts by weight, as TFA: thioanisole: EDT (electric discharge machining): anisole =90:5:3:2 preparing a cutting fluid, and placing the fully-protected cyclic peptide into the cutting fluid for cutting for 2 hours;
6) Separating out with glacial ethyl ether, centrifuging to obtain crude polypeptide peptide, washing with glacial ethyl ether for 3 times, removing part of impurities, vacuum drying, and purifying with HPLC to obtain pure product.
Preferably, in step S2, the method for obtaining the fully protected cyclic peptide comprises the following steps:
after the full-protection linear peptide is subjected to rotary evaporation and freeze-drying, DCM with TFA is added for dissolving, and the TFA can catalyze the reaction of C-terminal methyl ester and amino to form an amide ring while removing N-terminal Mtt nudine acid, so that the full-protection cyclic peptide is obtained, wherein the DCM comprises the following components in parts by weight: TFA =20:1, dissolving each mmol of the fully protected linear peptide in 0.5L of solvent, then refluxing the fully protected cyclic peptide in an oil bath overnight at 50 ℃, and finally spin-drying the fully protected cyclic peptide under reduced pressure.
Preferably, step 1) comprises the following steps:
mixing raw materials, an activating reagent HOSU and an activating reagent DCC in a ratio of 1:1.01:1.02 parts by weight in DCM overnight to form an activated ester at the C-terminus of the starting material, and then insoluble material was removed by suction filtration and the solution was spin-dried under reduced pressure.
Preferably, the step 2) comprises the following steps:
under the alkaline environment, taking DCM as a solvent, and mixing the product in the step 1) and a new raw material in a proportion of 1: dissolving 1, dropwise adding TEA with pH of 8, reacting for 4 hours, reacting the activated ester of the product in the previous step with the amino group of the new raw material to form a connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin-drying to obtain fragments, adding diethyl ether, stirring to obtain a turbid substance, performing suction filtration by using a Buchner funnel, washing with diethyl ether twice to remove impurities, and performing suction-drying.
Preferably, the method comprises the following steps:
s1, obtaining the fully-protected linear peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe
1) Starting with Mtt-Lys (Boc) -OH, the activation reagents HOSU and DCC were mixed at a ratio of 1:1.01:1.02 reacting in DCM overnight to form an activated ester with Mtt-Lys (Boc) -OH at C-terminus to give Mtt-Lys (Boc) -NHS, suction filtering off insoluble material, and spin drying the solution under reduced pressure to give pure Mtt-Lys (Boc) -NHS;
2) Mtt-Lys (Boc) -NHS was reacted with NH2-Arg (Pbf) -OH in a basic atmosphere in 1: dissolving 1, dropwise adding TEA with pH of 8 to react for 4 hours, enabling the activated ester of Mtt-Lys (Boc) -NHS to react with the amino group at the N end of the amino acid and form connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -OH, adding ether to the fragment, stirring the fragment into a turbid substance, performing suction filtration by using a Buchner funnel, washing the fragment with ether twice to remove impurities, and performing suction drying;
3) Mtt-Lys (Boc) -Arg (Pbf) -OH was reacted with activating reagent HOSU, DCC at 1:1.01:1.02 reacting in DCM overnight to form an activated ester at C with Mtt-Lys (Boc) -Arg (Pbf) -OH to obtain Mtt-Lys (Boc) -Arg (Pbf) -NHS, suction filtering off insoluble material, and spin-drying the solution under reduced pressure to obtain purified Mtt-Lys (Boc) -Arg (Pbf) -NHS;
Mtt-Lys (Boc) -Arg (Pbf) -NHS was reacted with NH2-Gly-OH in a basic atmosphere in the presence of DCM as solvent at a molar ratio of 1: dissolving 1, dropwise adding TEA with pH of 8 to react for 4 hours, enabling the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -NHS to react with the amino group at the N end of the amino acid and form connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin-drying to obtain a segment Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH, adding diethyl ether into the segment, stirring the segment into a turbid substance, performing suction filtration by using a Buchner funnel, washing the residue twice by using the diethyl ether to remove impurities, and performing suction drying;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH to form an activated ester at C-terminus to obtain Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS, followed by suction filtration to remove insoluble material and spin-drying the solution under reduced pressure to obtain pure Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS was reacted with NH2-Asp (Otbu) -OH in a basic atmosphere in DCM with 1: dissolving 1, dropwise adding TEA with pH of 8 for reaction for 4 hours, reacting the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS with the amino group at the N end of the amino acid to form a link, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin-drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH, adding diethyl ether, stirring to obtain a turbid substance, performing suction filtration by using a Buchner funnel, washing with diethyl ether twice to remove impurities, and performing suction drying;
4) Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 reacting in DCM overnight to form an activated ester at C-terminus for Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH to obtain Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS, suction filtering to remove insoluble material, and spin-drying the solution under reduced pressure to obtain pure Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS was reacted with H-D-Phe-OMe in a basic atmosphere in the presence of DCM as solvent at a ratio of 1: dissolving the peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, dropwise adding TEA with pH of 8, reacting for 4 hours to enable the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS to react with the amino group at the N end of the amino acid to form a connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain the fully protected linear peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, adding diethyl ether, stirring to obtain a turbid substance, performing suction filtration by using a Buchner funnel, washing twice by diethyl ether to remove impurities, and performing suction drying;
s2, obtaining the fully-protected cyclic peptide
After the fully protected linear peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe is subjected to rotary evaporation and freeze-drying, the fully protected linear peptide is dissolved by DCM added with TFA, and the TFA can catalyze the reaction of C-terminal methyl ester and amino to form an amide ring while removing N-terminal Mtt nudine to obtain the fully protected cyclic peptide, wherein the weight parts of DCM: TFA =20:1, dissolving each mmol of fully-protected linear peptide in 0.5L of solvent, then refluxing the solution in oil bath at 50 ℃ overnight, and finally, carrying out decompression and spin-drying on the fully-protected cyclic peptide;
s3, cutting and purifying
5) In parts by weight, as TFA: thioanisole: EDT (electro-thermal transfer coating): anisole =90:5:3:2 preparing a cutting fluid, and placing the fully-protected cyclic peptide into the cutting fluid for cutting for 2 hours;
6) Separating out the polypeptide crude peptide by using glacial ethyl ether, centrifuging to obtain the polypeptide crude peptide, then washing for 3 times by using the glacial ethyl ether, removing partial impurities, decompressing, draining, and finally purifying by using HPLC to obtain the pure RGD full peptide, wherein the structural formula is as follows: .
Compared with the prior art, the invention has the beneficial effects that:
the synthesis method disclosed by the invention is simple to operate, does not need a condensation reagent in a cyclization process, reduces byproducts and impurities brought by the condensation reagent, and is high in product yield and low in racemization rate.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments of the present invention will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a high performance liquid chromatography report;
FIG. 2 is a mass spectrometry report.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, are within the scope of protection of the present invention.
Examples
The invention discloses an RGD cyclopeptide synthesis method, which comprises the following steps:
s1, obtaining a fully protected linear peptide
1) Forming an activated ester at the C-terminus of Mtt-Lys (Boc) -OH with an activating reagent starting with Mtt-Lys (Boc) -OH to obtain Mtt-Lys (Boc) -NHS;
2) Adding an amino acid to Mtt-Lys (Boc) -NHS under alkaline conditions, reacting the activated ester of Mtt-Lys (Boc) -NHS with the amino group at the N-terminus of the amino acid and forming a linkage;
3) Activating the carboxyl group of the amino acid to form an activated ester under the conditions of step S1, and connecting the N terminal of the next amino acid to the C terminal of the previous amino acid under the conditions of step S2;
4) Taking H-D-Phe-OMe as a terminal raw material, connecting the N end to the C end of the last amino acid according to the step 3) to obtain a fully-protected linear peptide;
s2, obtaining the fully-protected cyclic peptide
After the fully protected linear peptide is subjected to rotary evaporation and freeze-drying, DCM added with TFA is used for dissolving to form an amide ring, and the fully protected cyclic peptide is obtained, wherein the weight parts of DCM: TFA =20:1, then refluxing in oil bath at 50 ℃ overnight, and finally spin-drying the fully-protected cyclic peptide;
s3, cutting and purifying
5) Measured as parts by weight, as TFA: thioether ether: EDT (electro-thermal transfer coating): anisole =90:5:3:2 preparing a cutting fluid, and placing the fully-protected cyclic peptide into the cutting fluid for cutting for 2 hours;
6) Separating out with glacial ethyl ether, centrifuging to obtain crude polypeptide peptide, washing with glacial ethyl ether for 3 times, removing part of impurities, vacuum drying, and purifying with HPLC to obtain pure product.
As a preferred implementation of the embodiment, the method comprises the following steps:
s1, obtaining the full protection straight-chain peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe
4) Starting with Mtt-Lys (Boc) -OH, the activation reagents HOSU and DCC were mixed at a ratio of 1:1.01:1.02 reacting in DCM overnight to form an activated ester with Mtt-Lys (Boc) -OH at C-terminus to give Mtt-Lys (Boc) -NHS, suction filtering off insoluble material, and spin drying the solution under reduced pressure to give pure Mtt-Lys (Boc) -NHS;
5) Mtt-Lys (Boc) -NHS was reacted with NH in a basic environment in DCM 2 -Arg (Pbf) -OH is present at 1: dissolving 1, dropwise adding TEA with pH of 8 to react for 4 hours, reacting the Mtt-Lys (Boc) -NHS activated ester with the amino group at the N end of the amino acid to form a connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -OH, adding diethyl ether, stirring to obtain a turbid substance, performing suction filtration by using a Buchner funnel, washing with diethyl ether twice to remove impurities, and performing suction drying;
6) Mtt-Lys (Boc) -Arg (Pbf) -OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -OH to form an activated ester at C-terminus to give Mtt-Lys (Boc) -Arg (Pbf) -NHS, which is then filtered off by suction and the solution is spun dry under reduced pressure to give pure Mtt-Lys (Boc) -Arg (Pbf) -NHS;
Mtt-Lys (Boc) -Arg (Pbf) -NHS is reacted with NH in a basic environment using DCM as a solvent 2 -Gly-OH is substituted with 1: dissolving 1, dropwise adding TEA with pH of 8 to react for 4 hours, reacting the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -NHS with the amino group at the N end of the amino acid to form a connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH, adding diethyl ether into the fragment, stirring the fragment into a turbid substance, performing suction filtration on the turbid substance by using a Buchner funnel, washing the fragment twice by using diethyl ether to remove impurities, and performing suction drying;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH to form an activated ester at C-terminus to obtain Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS, followed by suction filtration to remove insoluble material and spin-drying the solution under reduced pressure to obtain pure Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS;
under the alkaline environment, mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS and NH are mixed by taking DCM as a solvent 2 -Asp (Otbu) -OH is substituted with 1: dissolving 1, dropwise adding TEA with pH of 8 for reacting for 4 hours to enable the active ester of Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS to react with the amino group at the N end of the amino acid and form connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH, adding diethyl ether into the fragment, stirring the fragment into a turbid substance, performing suction filtration on the turbid substance by using a Buchner funnel, washing the fragment twice by using the diethyl ether to remove impurities, and performing suction drying;
4) Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH to form an activated ester at C-terminus to give Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS, which is then filtered to remove insoluble material and the solution is spun dry under reduced pressure to give pure Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS was reacted with H-D-Phe-OMe in a basic atmosphere in the presence of DCM as solvent at a ratio of 1: dissolving, dropwise adding TEA with pH of 8 to react for 4 hours, leading the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS to react with the amino group at the N end of the amino acid and form a connection, leading the HPLC liquid phase analysis to be a single main peak, carrying out reduced pressure spin-drying to obtain the fully protected straight-chain peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, then adding ether to the fully protected straight-chain peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, stirring the fully protected straight-chain peptide into a turbid substance, carrying out suction filtration by a Buchner funnel, washing twice by the ether to remove impurities, and carrying out suction drying;
s2, obtaining the fully-protected cyclic peptide
After rotary evaporation and freeze-drying of the fully protected linear peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, dissolving the fully protected linear peptide with DCM added with TFA, and removing the N-terminal Mtt nudic acid by the TFA, the TFA can catalyze the reaction of C-terminal methyl ester and amino to form an amide ring to obtain the fully protected cyclic peptide, wherein the weight portions of DCM: TFA =20:1, dissolving each mmol of fully-protected linear peptide in 0.5L of solvent, then refluxing the solution in oil bath at 50 ℃ overnight, and finally, carrying out decompression and spin-drying on the fully-protected cyclic peptide;
s3, cutting and purifying
5) Measured as parts by weight, as TFA: thioether ether: EDT (electro-thermal transfer coating): anisole =90:5:3:2 preparing a cutting fluid, and placing the fully-protected cyclic peptide into the cutting fluid for cutting for 2 hours;
6) Separating out the polypeptide crude peptide by using glacial ethyl ether, centrifuging to obtain the polypeptide crude peptide, then washing for 3 times by using the glacial ethyl ether, removing partial impurities, decompressing, draining, and finally purifying by using HPLC to obtain the pure RGD full peptide, wherein the structural formula is as follows:
it is to be noted that the proportions of the present invention are in parts by weight, and some common abbreviations have the following meanings:
mtt: 4-Methyltriphenylene
Lys: lysine
Boc: tert-butyloxycarbonyl radical
Arg: arginine
Gly: glycine
Asp: aspartic acid
Phe: phenylalanine (PHE)
H-D-Phe-OMe: d-phenylalanine methyl ester
HOSU: n-hydroxysuccinimide
DCC: dicyclohexylcarbodiimide
DCM: methylene dichloride
TFA: trifluoroacetic acid (trifluoroacetic acid)
EDT (electric discharge machining): 1, 2-ethanedithiol
Pbf:2, 4,6, 7-pentamethylbenzofuran-5-sulfonyl
And Otbu: tertiary butyl ester
Acetonitriles: acetonitrile (ACN)
Performance analysis
Referring to fig. 1, it is a report of HPLC analysis of RGD whole peptide, table 1 shows HPLC analysis conditions, and table 2 shows HPLC analysis results. It can be seen that the peak area ratio of the generated RGD full peptide is 98.27%, the peak area of impurities is only 1.724%, and compared with the traditional RGD full peptide synthesis method, the RGD full peptide synthesized by the synthesis method provided by the invention has extremely high purity.
Referring to FIG. 2, it is reported in MS analysis of RGD full peptide, and Table 3 is MS analysis conditions. It can be seen that the kind and the number of the ionic groups of the synthesized product are basically consistent with those of the ionic groups in the RGD full peptide, further indicating that the purity of the synthesized RGD full peptide is higher. TABLE 1 HPLC analysis conditions
TABLE 2 HPLC analysis results
TABLE 3 MS analysis conditions
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. A synthesis method of RGD cyclopeptide is characterized by comprising the following steps:
s1, obtaining a fully protected linear peptide
1) Forming an activated ester at the C-terminus of Mtt-Lys (Boc) -OH with an activating reagent starting with Mtt-Lys (Boc) -OH to obtain Mtt-Lys (Boc) -NHS;
2) Adding an amino acid to Mtt-Lys (Boc) -NHS under alkaline conditions, reacting the activated ester of Mtt-Lys (Boc) -NHS with the amino group at the N-terminus of the amino acid and forming a linkage;
3) Activating the carboxyl group of the amino acid to form an activated ester under the conditions of step S1, and connecting the N-terminal of the next amino acid to the C-terminal of the previous amino acid under the conditions of step S2;
4) Taking H-D-Phe-OMe as a terminal raw material, connecting the N end of the H-D-Phe-OMe to the C end of the last amino acid according to the step 3) to obtain a fully protected linear peptide;
s2, obtaining the fully-protected cyclic peptide
After the fully protected linear peptide is subjected to rotary evaporation and freeze-drying, DCM added with TFA is used for dissolving to form an amide ring, and the fully protected cyclic peptide is obtained, wherein the weight parts of DCM: TFA =20:1, then refluxing in an oil bath at 50 ℃ overnight, and finally spin-drying the fully-protected cyclic peptide;
s3, cutting and purifying
5) In parts by weight, as TFA: thioether ether: EDT (electro-thermal transfer coating): anisole =90:5:3:2 preparing a cutting fluid, and placing the fully-protected cyclic peptide into the cutting fluid for cutting for 2 hours;
6) Separating out with glacial ethyl ether, centrifuging to obtain crude polypeptide peptide, washing with glacial ethyl ether for 3 times, removing part of impurities, vacuum-pumping, and purifying with HPLC to obtain pure product; in the step S2, the method for obtaining the fully protected cyclic peptide includes the following steps:
after the full-protection linear peptide is subjected to rotary evaporation and freeze-drying, DCM with TFA is added for dissolving, and the TFA can catalyze the reaction of C-terminal methyl ester and amino to form an amide ring while removing N-terminal Mtt nudine acid, so that the full-protection cyclic peptide is obtained, wherein the DCM comprises the following components in parts by weight: TFA =20:1, dissolving each mmol of fully-protected linear peptide in 0.5L of solvent, then refluxing in oil bath at 50 ℃ overnight, and finally, carrying out decompression and spin-drying on the fully-protected cyclic peptide;
the step 1) comprises the following steps:
mixing raw materials, an activating reagent HOSU and an activating reagent DCC in a ratio of 1:1.01:1.02 parts by weight of the raw materials are reacted in DCM overnight, activated ester is formed at the C end of the raw materials, insoluble substances are removed by suction filtration, and the solution is decompressed and dried in a spinning mode;
the step 2) comprises the following steps:
under the alkaline environment, taking DCM as a solvent, and mixing the product in the step 1) and a new raw material in a proportion of 1: dissolving 1, dropwise adding TEA with pH of 8, reacting for 4 hours, reacting the activated ester of the product in the previous step with the amino group of the new raw material to form a connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin-drying to obtain fragments, adding diethyl ether, stirring to obtain a turbid substance, performing suction filtration by using a Buchner funnel, washing with diethyl ether twice to remove impurities, and performing suction-drying.
2. The method of synthesizing an RGD cyclic peptide according to claim 1, comprising the steps of:
s1, obtaining the fully-protected linear peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe
1) Starting with Mtt-Lys (Boc) -OH, mixed with activating reagents HOSU and DCC at a ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -OH to form an activated ester at C-terminus to give Mtt-Lys (Boc) -NHS, which is then filtered off by suction and the solution is dried under reduced pressure to give pure Mtt-Lys (Boc) -NHS;
2) Mtt-Lys (Boc) -NHS was reacted with NH2-Arg (Pbf) -OH in a basic reaction mixture of 1: dissolving 1, dropwise adding TEA with pH of 8 to react for 4 hours, enabling the activated ester of Mtt-Lys (Boc) -NHS to react with the amino group at the N end of the amino acid and form connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -OH, adding ether to the fragment, stirring the fragment into a turbid substance, performing suction filtration by using a Buchner funnel, washing the fragment with ether twice to remove impurities, and performing suction drying;
3) Mtt-Lys (Boc) -Arg (Pbf) -OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -OH to form an activated ester at C-terminus to give Mtt-Lys (Boc) -Arg (Pbf) -NHS, which is then filtered off by suction and the solution is spun dry under reduced pressure to give pure Mtt-Lys (Boc) -Arg (Pbf) -NHS;
Mtt-Lys (Boc) -Arg (Pbf) -NHS was reacted with NH2-Gly-OH in a basic atmosphere in the presence of DCM as solvent at a molar ratio of 1: dissolving 1, dropwise adding TEA with pH of 8 to react for 4 hours, enabling the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -NHS to react with the amino group at the N end of the amino acid and form connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin-drying to obtain a segment Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH, adding diethyl ether into the segment, stirring the segment into a turbid substance, performing suction filtration by using a Buchner funnel, washing the residue twice by using the diethyl ether to remove impurities, and performing suction drying;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -Gly-OH to form an activated ester at C-terminus to obtain Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS, followed by suction filtration to remove insoluble material and spin-drying the solution under reduced pressure to obtain pure Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS was reacted with NH2-Asp (Otbu) -OH in a basic atmosphere in DCM with a 1: dissolving 1, dropwise adding TEA with pH of 8 for reacting for 4 hours to enable the active ester of Mtt-Lys (Boc) -Arg (Pbf) -Gly-NHS to react with the amino group at the N end of the amino acid and form connection, performing HPLC liquid phase analysis to obtain a single main peak, performing reduced pressure spin drying to obtain a fragment Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH, adding diethyl ether into the fragment, stirring the fragment into a turbid substance, performing suction filtration on the turbid substance by using a Buchner funnel, washing the fragment twice by using the diethyl ether to remove impurities, and performing suction drying;
4) Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH was reacted with activating reagents HOSU, DCC at a molar ratio of 1:1.01:1.02 in DCM overnight to allow Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -OH to form an activated ester at C-terminus to give Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS, which is then filtered to remove insoluble material and the solution is spun dry under reduced pressure to give pure Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS;
Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS was reacted with H-DPhe-OMe in a basic atmosphere in DCM at 1: dissolving, dropwise adding TEA with pH of 8 to react for 4 hours, leading the activated ester of Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -NHS to react with the amino group at the N end of the amino acid and form a connection, leading the HPLC liquid phase analysis to be a single main peak, carrying out reduced pressure spin-drying to obtain the fully protected straight-chain peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, then adding ether to the fully protected straight-chain peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, stirring the fully protected straight-chain peptide into a turbid substance, carrying out suction filtration by a Buchner funnel, washing twice by the ether to remove impurities, and carrying out suction drying;
s2, obtaining the fully-protected cyclic peptide
After rotary evaporation and freeze-drying of the fully protected linear peptide Mtt-Lys (Boc) -Arg (Pbf) -Gly-Asp (Otbu) -D-Phe-OMe, dissolving the fully protected linear peptide with DCM added with TFA, and removing the N-terminal Mtt nudic acid by the TFA, the TFA can catalyze the reaction of C-terminal methyl ester and amino to form an amide ring to obtain the fully protected cyclic peptide, wherein the weight portions of DCM: TFA =20:1, dissolving each mmol of fully-protected linear peptide in 0.5L of solvent, then refluxing in oil bath at 50 ℃ overnight, and finally, carrying out decompression and spin-drying on the fully-protected cyclic peptide;
s3, cutting and purifying
5) In parts by weight, as TFA: thioether ether: EDT (electro-thermal transfer coating): anisole =90:5:3:2 preparing a cutting fluid, and placing the fully-protected cyclic peptide into the cutting fluid for cutting for 2 hours;
6) Separating with glacial ethyl ether, centrifuging to obtain crude polypeptide, washing with glacial ethyl ether for 3 times, removing part of impurities, and vacuum pumping
Drying, and purifying by HPLC to obtain pure RGD full peptide with the following structural formula:
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| CN109280080A (en) * | 2018-10-26 | 2019-01-29 | 马良会 | A kind of bicyclic RGD peptide synthetic method |
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| CN106518966A (en) * | 2016-10-24 | 2017-03-22 | 合肥国肽生物科技有限公司 | Synthetic method of RGD cyclopeptide |
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