CN110551178B - Method for synthesizing head-tail cyclic peptide containing proline - Google Patents
Method for synthesizing head-tail cyclic peptide containing proline Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
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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
- C07K7/64—Cyclic peptides containing only normal peptide links
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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 relates to a method for synthesizing a head-tail cyclic peptide containing proline, which comprises the following steps: 1) selecting solid-phase synthetic resin; 2) firstly coupling Fmoc-3-carboxyl-Pro-OAll, then obtaining NH- (3-carboxyl-solid phase synthetic resin) -Pro-OAll according to Fmoc solid phase synthetic strategy, and then sequentially coupling amino acid residues Fmoc-AA-OH in peptide sequence; 3) solid phase deprotection of the protecting group All to yield NH2-AAn- (3-carboxy-solid phase synthetic resin) -Pro-OH; 4) solid-phase head-to-tail cyclization: coupling the amino group on AAn with the carboxyl group on Pro; 5) cracking the solid phase synthetic resin to prepare intermediate cyclopeptide crude peptide; 6) removing the 3-position carboxyl of terminal Pro from the intermediate cyclopeptide crude peptide under the action of a decarboxylation agent to prepare the proline-containing cyclopeptide. The method is novel, mild in synthesis condition, simple in process and stable in process.
Description
Technical Field
The invention belongs to the field of drug synthesis, and particularly relates to a method for synthesizing proline-containing head-tail cyclic peptide.
Background
Many peptides with cyclic head-to-tail amide bonds are found in nature. The research of head-tail cyclic peptide drugs is also rapidly developed. With the progress of research, the synthesis of cyclic peptides has attracted increasing attention and certain efforts have been made in many studies.
The chemical synthesis of head-tail cyclic peptides can be largely divided into liquid phase method and solid phase method.
The liquid phase method is that the full protection peptide is prepared in a solid phase, then the liquid phase is cyclized, and finally the protecting group is removed. The liquid phase method is adopted to synthesize the peptide with the ring formed from the head and the tail, and the reaction needs to be carried out in a highly diluted solution (10)-3～10-4M) is carried out, the reaction is slow, and the activated carboxyl can avoid the decomposition reaction caused by single molecule or solvent induction effect. However, its successful synthesis requires linear precursors that rely on orthogonal protection, requires complex steps that undoubtedly affect the yield of the peptide, and requires coupling reagentsIs very high.
The solid phase method is to form a ring directly on a solid phase carrier. For peptide sequences containing Asp \ Glu or Asn \ Gln residues, the crude peptide synthesis is generally completed by adopting a method of adopting All protection for an Asp \ Glu main chain and connecting side chain carboxyl on a solid phase carrier, removing an All protection group after the coupling is completed, and completing amide bond cyclization on a solid phase.
Solid phase synthesis is much simpler than liquid phase synthesis, but the methods reported so far can be used only for amino acid residues with side chain groups in the peptide sequence, and the solid phase method is difficult if the peptide sequence does not have amino acid residues with side chains.
Disclosure of Invention
For head-tail cyclic peptide without containing amino acid with side chain and with Pro in peptide sequence, the invention provides a brand-new and high-efficiency synthesis method: firstly, connecting Fmoc-3-carboxyl-Pro-OAll on resin; then coupling other amino acid residues in peptide order; after the coupling is finished, removing All from the solid phase, and then forming a ring by the solid phase; finally, decarboxylation is carried out on the crude cyclic peptide to obtain the proline-containing head-tail cyclic peptide. The method is novel, mild in synthesis condition, simple in process and stable in process.
The synthetic route is as follows:
the invention relates to a method for synthesizing head-tail cyclic peptide containing alanine, which comprises the following steps: 1) selecting solid-phase synthetic resin;
2) firstly coupling Fmoc-3-carboxyl-Pro-OAll, then obtaining NH- (3-carboxyl-solid phase synthetic resin) -Pro-OAll according to Fmoc solid phase synthetic strategy, and then sequentially coupling amino acid residue Fmoc-AA-OH in peptide sequence to obtain NH2-AAn- (3-carboxy-solid phase synthetic resin) -Pro-OAll;
3) solid phase deprotection of the protecting group All to yield NH2-AAn- (3-carboxy-solid phase synthetic resin) -Pro-OH;
4) solid-phase head-to-tail cyclization: coupling the amino group on AAn with the carboxyl group on Pro;
5) cracking the solid phase synthetic resin to prepare intermediate cyclopeptide crude peptide;
6) removing the 3-position carboxyl of terminal Pro from the intermediate cyclopeptide crude peptide under the action of a decarboxylation agent to prepare the proline-containing cyclopeptide.
The solid phase synthetic resin in the step 1) is wang resin or 2-chloro resin, and the resin substitution degree is 0.1-3.0mmol/g, preferably 0.5-2.5mmol/g, and more preferably 1.0-1.5 mmol/g.
Step 2) is not particularly limited with respect to the Fmoc-AA-OH form of the amino acid, and a protecting group such as Trt, Boc, tBu, etc., which can be used, may be used or not according to the conventional knowledge in the art).
The Fmoc solid phase synthesis strategy coupling of amino acids in the step 2) comprises the following steps:
2.1) removing Fmoc, and then washing the resin by using a solvent until the Fmoc is completely removed by using a detection method;
2.2) dissolving and activating an appropriate amount of Fmoc-AA-OH to be coupled and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;
2.3) repeat 1) and 2) wherein the Fmoc removal reagent was a 20% piperidine/DMF solution (DB L K), i.e., a mixed solution of piperidine to DMF (by volume) at 1: 4.
The amide bond forming coupling agent in the step 2) of the invention is a combination of DIC and a compound A or a combination of DIA and a compound A and a compound B, wherein the compound A is HOBt or HOAt, the compound B is PyBOP, PyAOP, HATU, HBTU or TBTU, and the amide bond forming coupling agent is preferably a combination of DIC and the compound A. Further, the ratio of each component in the amide bond-forming coupling agent is DIC: a: 1.2:1.1 and DIA: a: B: 2.0:1.1:1.0 in terms of molar ratio.
The method for coupling Fmoc-3-carboxyl-Pro-OAll in the step 2) comprises the step of carrying out condensation coupling on the 3-position carboxyl of Fmoc-3-carboxyl-Pro-OAll and solid-phase synthetic resin by using an ester bond forming coupling agent, wherein the ester bond forming coupling agent is selected from one or more of HOBt and DMAP.
The reaction of step 2) is carried out in a solid phase reaction column. The solid-phase reaction column is not particularly limited, and may be any solid-phase reaction column capable of achieving the object. Further, the time for the coupling reaction of each amino acid is usually 1.5 to 4 hours, preferably 2 to 3 hours; the pressure is preferably normal pressure, and may be suitably increased or decreased; the temperature is preferably room temperature (i.e., 20. + -. 5 ℃ C.), and may be suitably elevated or reduced.
The reaction of step 2) is preferably carried out by swelling the resin prior to coupling, said washing and swelling steps being carried out in the art using any reagent which accomplishes this purpose, including DMF, NMP, dichloromethane, etc., preferably DMF.
The detection method employed in the reaction is any method known in the art for this purpose, such as chromatography or chemical calibration, preferably using a reagent that can determine the end of the reaction, preferably ninhydrin, which when used indicates a free amine in the polypeptide if the resin develops color, i.e., no protecting group on the amine.
In the step 2), Fmoc-AA-OH is natural amino acid or unnatural amino acid, and AAn is a peptide chain consisting of n amino acids, wherein n is more than or equal to 1 and is an integer.
Step 3), the catalyst used for solid phase removing the protecting group All is tetratriphenylphosphine palladium, the capturing agent is phenylsilane, and the solvent is DCM.
And 4) carrying out solid phase head-to-tail cyclization by using DIC + A or DIA + A + B, wherein A is HOBt or HOAt, and B is one or a combination of more of PyBOP, PyAOP, HATU, HBTU and TBTU.
The lysate in the step 5) is TFA and H2O, PhOMe, thioanisole. Preferred is TFA H2O pheme thioanisole 90:5:4:1(V: V).
Step 6), the decarboxylating agents are silver nitrate and sodium persulfate; the solvent is acetonitrile aqueous solution, and the reaction temperature is 0-100 ℃, preferably 30-70 ℃; the mass ratio of the crude cyclopeptide to silver nitrate and sodium persulfate substances is 1: 0.02: 2.
the present invention also includes an optional step 7) of purification. The purification step can be performed by reverse phase high pressure liquid chromatography. Further, the reversed-phase high-pressure liquid chromatography comprises: reversed-phase octadecylsilane is used as a stationary phase, 0.1% aqueous acetic acid solution/acetonitrile in volume ratio is used as a mobile phase, and the ratio of the 0.1% aqueous acetic acid solution/acetonitrile in volume ratio of the mobile phase is preferably 98:2 to 50:50, more preferably 80:20 to 60:40, and most preferably 70: 30. Collecting the target peak fraction, concentrating and freeze-drying.
Detailed Description
Synthesis of cyclic peptide [ Ala-Tyr-Ala-Phe-leu-Ile-Val-Pro ] cyclic octapeptide
EXAMPLE 1 preparation of Fmoc-3-carboxy-Pro-OAll-Wang resin
Weighing Wang resin 100g with substitution degree of 1.5mmol/g into a solid phase reaction column, adding DMF, bubbling nitrogen for swelling for 60 minutes, weighing Fmoc-3-carboxyl-Pro-OAll 42.1 g (100mmol), HOBt16.2 g (120mmol) and DMAP1.2 g (10mmol), dissolving with DMF, adding 20.3m L DIC at 0 ℃, activating for 5 minutes, adding into the reaction column, reacting for two hours, pumping out the solution, washing with DMF for six times, washing with DCM for three times, after methanol shrinkage, pumping out the resin to obtain 129 g of Fmoc-3-carboxyl-Pro-OAll-Wang resin, and detecting the substitution degree of 1.0 mmol/g.
EXAMPLE 2 preparation of peptide resin
Weighing 100g of Fmoc-3-carboxy-Pro-OAll-Wang resin with the substitution degree of 1.0mmol/g prepared in example 1, placing 100g of Fmoc-3-carboxy-Pro-OAll-Wang resin in a solid phase reaction column, adding 150m L DMF, carrying out nitrogen bubbling swelling for 60 minutes, then carrying out deprotection with 100m L DB L K for 6min +8min, and washing with 100m L DMF for 6 times, weighing 169.7g (500mmol) of Fmoc-Val-OH and 78.1g (500mmol) of HOBT, dissolving with 150m L DMF, adding 86.7m L (500mmol) DIC in an ice water bath for 3min, adding the mixture into the reaction column, reacting at room temperature for 2 hours, detecting the reaction end point with ninhydrin (if the resin is colorless and transparent), terminating the reaction if the resin is developed, extending the reaction for 1 hour), washing the resin with 150m L DMF for 3 times, adding 150m L DB L K6 min +8min, 150m L DMF, washing the resin for 6 times, washing the resin with trione, detecting the color of the resin, repeating the operations of Tyr-OH, withdrawing the Fmoc-Ala-OH, withdrawing the amino acid coupling procedure, and the amino acid coupling with 150 m-Phe-Ala 2, the amino acid coupling procedure, sequentially, and the amino acid coupling procedure of dry coupling to obtain dry amino acid coupling to obtain Fmoc-Ala-200 g of the amino acid coupling peptide coupling reagent (150 m.
EXAMPLE 3 solid phase removal of All
192.2g of the resin obtained in example 2 was added with 300m L DMF and swollen for 2 hours, the solvent was removed, DCM300m L was added, 50mmol of palladium tetratriphenylphosphine and 2000mmol of phenylsilane were sequentially added, the reaction was carried out at room temperature for 1 hour, after the reaction was completed, the solution was removed, washed three times with DCM (300m L× 3) and three times with DMF (300m L× 3), and an intermediate peptide resin was obtained.
Example 4 solid phase cyclization
DMF300ml, 78.1g (500mmol) HOBT and 86.7m L (210mmol) DIC were added to the intermediate peptide resin obtained in example 3 in this order and reacted at room temperature for 4 hours, after the reaction was completed, the solution was pumped off, DMF was washed six times (300m L× 6), dichloromethane was washed six times (300m L× 6), methanol was shrunk 2 times (150m L× 2), and vacuum drying was carried out to obtain 287.0g of intermediate cyclic peptide resin.
EXAMPLE 5 intermediate Cyclic peptide preparation
287.0g of intermediate cyclic peptide resin obtained in example 4 was put into a 5l three-necked flask, and added with TFA prepared in advance, H2O: PhOMe: benzylthioether ═ 90:5:4:1(V: V) 3L, reacted at room temperature for 2 hours, the resin was filtered under reduced pressure, the filtrate was collected, the resin was washed with a small amount of TFA, the filtrates were combined, the filtrate was slowly added to 30L glacial ethyl ether to precipitate, centrifuged, washed 5 times with glacial ethyl ether 5L, and dried under reduced pressure to obtain 81.2 g of intermediate cyclic crude peptide.
EXAMPLE 6 preparation of proline containing Cyclic peptides
Adding 81.2 g of intermediate cyclic crude peptide obtained in example 5 into a 500ml reaction bottle, adding 300ml of acetonitrile and 100M of L for dissolving, then adding 0.3g of silver nitrate and 47.6g of sodium persulfate, reacting at 60 ℃, directly loading the mixture onto a 10cm × 25cm preparation column for efficient liquid phase purification preparation after the reaction is finished, taking reverse octadecylsilane as a stationary phase and taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio is 70:30, performing equal gradient elution preparation, the flow rate is 70-80ml/min, the detection wavelength is 280nm, collecting target peak fractions, concentrating and freeze-drying to obtain 52.8g of a pure product, the purity is 99.5%, the yield is 60%, and the MS [ M + H ] + theoretical value is 876.1, and the experimental value is 876.1.
Synthesis of cyclic peptide [ Ala-Tyr-Ala-D-Phe-leu-Ile-Val-Pro ] cyclic octapeptide
EXAMPLE 7 preparation of peptide resin
Weighing 100g of Fmoc-3-carboxy-Pro-OAll-Wang resin with the substitution degree of 1.0mmol/g prepared in example 1, placing the resin in a solid phase reaction column, adding 150m L DMF, carrying out nitrogen bubbling for swelling for 60 minutes, then carrying out deprotection for 6min +8min and 100m L DMF with 100m L DB L K, washing for 6 times, weighing 169.7g (500mmol) of Fmoc-Val-OH and 78.1g (500mmol) HOBT, dissolving with 150m L DMF, adding 86.7m L (500mmol) DIC under ice water bath for activation for 3min, adding the mixture into the reaction column, reacting for 2 hours at room temperature, detecting the reaction end point with ninhydrin (if the resin is colorless and transparent), terminating the reaction for 1 hour if the resin is developed), washing the resin for 3 times with 150m L DMF, adding 150m L DB L K3876 min +8min, 150m L DMF, washing the resin for 6 times, detecting the resin with ninhydrin for 1 hour, repeating the operations of Tyr-OH, withdrawing the resin after the substitution degree of Fmoc-OH, and the procedures of Fmoc-OH, further carrying out coupling reaction for 24 g of Fmoc-OH, further, amino acid coupling for 24 g-Ala, amino acid coupling, dry coupling, 300 g-Ala-5-OH, and further carrying out the steps of Fmoc coupling.
EXAMPLE 8 solid phase removal of All
190.1g of the resin obtained in example 7 was added with 300m L DMF to swell for 2 hours, the solvent was removed, DCM300m L was added, 50mmol of palladium tetratriphenylphosphine and 2000mmol of phenylsilane were sequentially added to react at room temperature for 1 hour, after the reaction was completed, the solution was removed, washed three times with DCM (300m L× 3) and three times with DMF (300m L× 3) to obtain an intermediate peptide resin.
Example 9 solid phase cyclization
DMF300ml, 78.1g (500mmol) HOBT and 86.7m L (210mmol) DIC were added to the intermediate peptide resin obtained in example 8 in this order and reacted at room temperature for 4 hours, after the reaction was completed, the solution was pumped off, DMF was washed six times (300m L× 6), dichloromethane was washed six times (300m L× 6), methanol was contracted 2 times (150m L× 2), and vacuum drying was carried out to obtain 285.0g of intermediate cyclic peptide resin.
EXAMPLE 10 intermediate Cyclic peptide preparation
285.0g of the intermediate cyclic peptide resin obtained in example 9 was put into a 5l three-necked flask, and added with TFA prepared in advance: H2O: PhOMe: benzylthioether ═ 90:5:4:1(V: V) 3L to react at room temperature for 2 hours, the resin was filtered under reduced pressure, the filtrate was collected, the resin was washed with a small amount of TFA, the filtrates were combined, the filtrate was slowly added to 30L of glacial ethyl ether to precipitate, centrifuged, washed 5 times with glacial ethyl ether 5L, and dried under reduced pressure to obtain 80.2 g of intermediate cyclic crude peptide.
EXAMPLE 11 preparation of proline-containing cyclopeptide
Adding 80.2 g of intermediate cyclic crude peptide obtained in example 10 into a 500ml reaction bottle, adding 300ml of acetonitrile and 100M of L for dissolving, then adding 0.3g of silver nitrate and 47.6g of sodium persulfate, reacting at 60 ℃, directly loading 10cm × 25cm of prepared column for efficient liquid phase purification preparation after the reaction is finished, taking reverse octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio is 70:30, performing equal gradient elution preparation, the flow rate is 70-80ml/min, the detection wavelength is 280nm, collecting target peak fraction, concentrating and freeze-drying to obtain 51.6g of pure product, the purity is 99.5%, the yield is 58.6%, and the MS [ M + H ] + theoretical value is 876.1, and the experimental value is 876.1.
Synthesis of cyclic peptide [ Met-Ala-Tyr-Ala-Phe-leu-Ile-Val-Pro ] cyclic nonapeptide
EXAMPLE 12 preparation of peptide resin
Weighing 100g of Fmoc-3-carboxy-Pro-OAll-Wang resin with the substitution degree of 1.0mmol/g prepared in example 1, placing the resin in a solid phase reaction column, adding 150m L DMF, carrying out nitrogen bubbling for swelling for 60 minutes, then carrying out deprotection for 6min +8min and 100m L DMF with 100m L DB L K, washing for 6 times with 100m L DMF, weighing 169.7g (500mmol) of Fmoc-Val-OH and 78.1g (500mmol) of HOBT, dissolving with 150m L DMF, adding 86.7m L (500mmol) DIC under ice water bath for activation for 3min, adding the mixture into the reaction column, reacting for 2 hours at room temperature, detecting the reaction end point with ninhydrin (if the resin is colorless and transparent, stopping the reaction if the resin is developed, prolonging the reaction for 1 hour), washing the resin for 3 times with 150m L DMF, adding 150m L DB L K3876 min +8min, 150m L DMF, washing the resin for 6 times with triton, detecting the color of the resin, washing the resin with triton the resin, repeating the operations of Fmoc OH, Fmoc-OH, Fmoc coupling, Met-OH, Fmoc coupling for 240 g, Fmoc-Ala-200 g, and Met coupling to obtain dry coupling procedures, and Met coupling the following steps, Fmoc coupling to obtain dry amino acid coupling to obtain Fmoc-Phe-Ala-200 g.
EXAMPLE 13 solid phase removal of All
240.1g of the resin obtained in example 12 was added with 500m L DMF to swell for 2 hours, the solvent was removed, DCM300m L was added, 50mmol of palladium tetratriphenylphosphine and 2000mmol of phenylsilane were sequentially added to react at room temperature for 1 hour, after the reaction was completed, the solution was removed, washed three times with DCM (300m L× 3) and three times with DMF (300m L× 3) to obtain an intermediate peptide resin.
Example 14 solid phase cyclization
DMF500ml, 78.1g (500mmol) HOBT and 86.7m L (210mmol) DIC were added to the intermediate peptide resin obtained in example 13 in this order and reacted at room temperature for 4 hours, after completion of the reaction, the solution was pumped off, DMF was washed six times (300m L× 6), dichloromethane was washed six times (300m L× 6), methanol was contracted 2 times (150m L× 2), and vacuum-dried to obtain 325.0g of intermediate cyclic peptide resin.
EXAMPLE 15 intermediate Cyclic peptide preparation
325.0g of the intermediate cyclopeptide resin obtained in example 14 was charged into a 5l three-necked flask, and a pre-prepared TFA H was added2O PhOMe-benzylthion-e-90: 5:4:1(V: V) 4L, at room temperature for 2 hours, filtering the resin under reduced pressure, collecting the filtrate, washing the resin with a small amount of TFA, combining the filtrates, slowly adding the filtrate to 40L glacial ethyl ether for precipitation, centrifuging, washing 5 times with glacial ethyl ether 5L, and drying under reduced pressure to obtain 85.2 g of intermediate cyclic crude peptide.
EXAMPLE 16 preparation of proline containing Cyclic peptides
Adding 85.2 g of intermediate cyclic crude peptide obtained in example 15 into a 500ml reaction bottle, adding 300ml of acetonitrile and 100M of L for dissolving, then adding 0.35g of silver nitrate and 48.6g of sodium persulfate, reacting at 60 ℃, directly loading the mixture onto a 10cm × 25cm preparation column for efficient liquid phase purification preparation after the reaction is finished, taking reverse octadecylsilane as a stationary phase and taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio is 70:30, performing equal gradient elution preparation, the flow rate is 70-80ml/min, the detection wavelength is 280nm, collecting target peak fractions, concentrating and freeze-drying to obtain 60.6g of a pure product, the purity is 99.5%, the yield is 55.6%, and the MS [ M + H ] + theoretical value is 1007.3 and the experimental value is 1007.3.
Synthesis of Cyclo [ D-Met-Ala-Tyr-Ala-Phe-leu-Ile-Val-Pro ] cyclic nonapeptide
EXAMPLE 17 preparation of peptide resin
Weighing 100g of Fmoc-3-carboxy-Pro-OAll-Wang resin with the substitution degree of 1.0mmol/g prepared in example 1, placing 100g of Fmoc-3-carboxy-Pro-OAll-Wang resin in a solid phase reaction column, adding 150m L DMF, carrying out nitrogen bubbling swelling for 60 minutes, then carrying out deprotection with 100m L DB L K for 6min +8min, and washing with 100m L DMF for 6 times, weighing 169.7g (500mmol) of Fmoc-Val-OH and 78.1g (500mmol) of HOBT, dissolving with 150m L DMF, adding 86.7m L (500mmol) DIC in an ice water bath, activating for 3min, adding the mixture into the reaction column, reacting at room temperature for 2 hours, detecting the reaction end point with ninhydrin (if the resin is colorless and transparent), terminating the reaction if the resin is developed, extending the reaction for 1 hour), washing the resin with 150m L DMF for 3 times, adding 150m L DB L K +8min, 150m L DMF, washing the resin for 6 times, detecting the resin with trione, repeating the operations of Tyr, detecting the color of peptide coupling with Fmoc-OH, and peptide coupling with Met-Ala-24 g of Fmoc-P, and Met-L K-P, sequentially, and coupling the amino acid coupling to obtain dry peptide D-Ala, and Met-L g of the amino acid coupling to obtain dry peptide coupling reagents.
EXAMPLE 18 solid phase removal of All
245.2g of the resin obtained in example 17 was added with 500m L of DMF to swell for 2 hours, the solvent was removed, DCM300m L was added, 50mmol of palladium tetratriphenylphosphine and 2000mmol of phenylsilane were sequentially added to react at room temperature for 1 hour, after the reaction was completed, the solution was removed, washed three times with DCM (300m L× 3) and three times with DMF (300m L× 3) to obtain an intermediate peptide resin.
Example 19 solid phase cyclization
DMF500ml, 78.1g (500mmol) HOBT and 86.7m L (210mmol) DIC were added to the intermediate peptide resin obtained in example 18 in this order and reacted at room temperature for 4 hours, after the reaction was completed, the solution was pumped off, DMF was washed six times (300m L× 6), dichloromethane was washed six times (300m L× 6), methanol was shrunk 2 times (150m L× 2), and vacuum drying was carried out to obtain 329.0g of intermediate cyclic peptide resin.
EXAMPLE 20 intermediate Cyclic peptide preparation
329.0g of intermediate cyclic peptide resin obtained in example 19 was put into a 5l three-necked flask, and added with TFA prepared in advance, H2O: PhOMe: benzylthioether ═ 90:5:4:1(V: V) 4L, reacted at room temperature for 2 hours, the resin was filtered under reduced pressure, the filtrate was collected, the resin was washed with a small amount of TFA, the filtrates were combined, the filtrate was slowly added to 40L glacial ethyl ether to precipitate, centrifuged, washed 5 times with glacial ethyl ether 5L, and dried under reduced pressure to obtain 86.2 g of intermediate cyclic crude peptide.
EXAMPLE 21 preparation of proline-containing cyclopeptide
86.2 g of intermediate cyclic crude peptide obtained in example 20 is added into a 500ml reaction bottle, 300ml of acetonitrile and 100M of L are added for dissolving, then 0.35g of silver nitrate and 48.6g of sodium persulfate are added for reaction at 60 ℃, after the reaction is finished, the intermediate cyclic crude peptide is directly loaded onto a 10cm × 25cm preparation column for high-efficiency liquid phase purification preparation, reverse octadecylsilane is used as a stationary phase, 0.1% acetic acid aqueous solution/acetonitrile in volume ratio is used as a mobile phase, the ratio is 70:30, the preparation is carried out by equal gradient elution, the flow rate is 70-80ml/min, the detection wavelength is 280nm, a target peak fraction is collected, concentrated and freeze-dried, and a pure product 61.6g is obtained, the purity is 99.5%, the yield is 56.5%, the MS [ M + H ] + theoretical value is 1007.3, and the experimental value is 1007.3.
For head-tail cyclic peptide without containing amino acid with side chain and with Pro in peptide sequence, the invention provides a brand-new and high-efficiency synthesis method: firstly, connecting Fmoc-3-carboxyl-Pro-OAll on resin; then coupling other amino acid residues in peptide order; after the coupling is finished, removing All from the solid phase, and then forming a ring by the solid phase; finally, decarboxylation is carried out on the crude cyclic peptide by using silver nitrate and sodium persulfate to obtain the proline-containing head and tail cyclic peptide. The method is novel, mild in synthesis condition, simple in process and stable in process.
The synthesis method is also a protection point.
The invention provides a brand new method for preparing proline-containing head-tail cyclic peptide. The method has the advantages of simple operation, simplified process, environmental protection, high economic benefit, large-scale production and the like.
1)Contemporary strategies for peptide Macrocyclization.《NatureChemistry》，2011，3(7)：509-524
2) Persulfate/Silver Ion De carboxylation of carboxy acid of preparation of Alkanes, Alkenes, and Alcohols.J.org.chem.,1983,40(20), 3575-3577.
Claims (15)
1. A method for synthesizing a head-to-tail cyclic peptide containing proline, which comprises the following steps: 1) selecting solid-phase synthetic resin;
2) firstly coupling Fmoc-3-carboxyl-Pro-OAll, then obtaining NH- (3-carboxyl-solid phase synthetic resin) -Pro-OAll according to Fmoc solid phase synthetic strategy, and then sequentially coupling amino acid residue Fmoc-AA-OH in peptide sequence to obtain NH2-AAn- (3-carboxy-solid phase synthetic resin) -Pro-OAll;
3) solid phase deprotection of the protecting group All to yield NH2-AAn- (3-carboxy-solid phase synthetic resin) -Pro-OH;
4) solid-phase head-to-tail cyclization: coupling the amino group on AAn with the carboxyl group on Pro;
5) cracking the solid phase synthetic resin to prepare intermediate cyclopeptide crude peptide;
6) removing the 3-position carboxyl of terminal Pro from the intermediate cyclopeptide crude peptide under the action of a decarboxylation agent to prepare the proline-containing cyclopeptide.
2. The method for synthesizing a head-to-tail cyclic peptide containing proline according to claim 1, wherein the decarboxylating agent in step 6) is a mixture of silver nitrate and sodium persulfate.
3. The method for synthesizing the proline-containing head-tail cyclic peptide according to claim 2, wherein the solvent used in the step 6) is acetonitrile water solution, the reaction temperature is 0-100 ℃, and the mass ratio of the intermediate cyclic peptide crude peptide to the substances of silver nitrate and sodium persulfate is 1: 0.02: 2.
4. the method for synthesizing the proline-containing head-tail cyclic peptide according to claim 3, wherein the reaction temperature in step 6) is 30-70 ℃.
5. The method for synthesizing the proline-containing head-to-tail cyclic peptide in claim 1, wherein the Fmoc-3-carboxy-Pro-OAll is coupled in step 2) by condensing and coupling the carboxy group at position 3 of the Fmoc-3-carboxy-Pro-OAll with a solid-phase synthetic resin by using an ester bond forming coupling agent, wherein the ester bond forming coupling agent is selected from one or more of HOBt and DMAP.
6. The method for synthesizing a head-to-tail cyclic peptide containing proline according to claim 1, wherein the coupling of amino acids by Fmoc solid phase synthesis strategy in step 2) comprises the following steps:
2.1) removing Fmoc, and then washing the resin by using a solvent until the Fmoc is completely removed by using a detection method;
2.2) dissolving and activating an appropriate amount of Fmoc-AA-OH to be coupled and an amide bond forming coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction termination is detected by a detection method;
2.3) repeat 2.1) and 2.2) with the Fmoc removal reagent being 20% piperidine/DMF solution.
7. The process for the synthesis of a proline-containing head-to-tail cyclic peptide according to claim 6, wherein the amide bond-forming coupling reagent in step 2) is a combination of DIC and Compound A or a combination of DIA and Compound A and Compound B, wherein Compound A is HOBt or HOAt and Compound B is PyBOP, PyAOP, HATU, HBTU or TBTU.
8. The method for synthesizing a head-to-tail cyclic peptide containing proline according to claim 7, wherein the amide bond-forming coupling agent is a combination of DIC and Compound A.
9. The method for synthesizing proline-containing head-to-tail cyclic peptide according to claim 1, wherein the reagent used in the solid phase head-to-tail cyclization in step 4) is DIC + A or DIA + A + B, wherein A is HOBt or HOAt, and B is one or a combination of PyBOP, PyAOP, HATU, HBTU and TBTU.
10. The method for synthesizing proline-containing head-tail cyclic peptide according to claim 1, wherein the cleavage liquid used in step 5) is TFA、H2O, PhOMe, thioanisole.
11. The method for synthesizing proline-containing head-to-tail cyclic peptide according to claim 10, wherein the cleavage liquid used in the step 5) is TFA H2O PhOMe thioanisole 90:5:4: 1.
12. The proline containing head-to-tail cyclic peptide synthesis process of claim 1, further comprising an optional step 7) of purification, which may employ reverse phase high pressure liquid chromatography.
13. The method for proline-containing head-to-tail cyclic peptide synthesis of claim 12, the reverse phase high pressure liquid chromatography comprising: taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, taking the ratio of the 0.1% acetic acid aqueous solution to the acetonitrile in the mobile phase as 98:2 to 50:50, collecting target peak fractions, concentrating and freeze-drying.
14. The method for synthesizing a proline-containing head-to-tail cyclic peptide according to claim 13, wherein the ratio of the 0.1% aqueous acetic acid solution to the acetonitrile in the mobile phase is 80:20 to 60: 40.
15. The method for synthesizing a cyclic peptide containing proline according to claim 13, wherein the ratio of the 0.1% aqueous acetic acid solution to acetonitrile in the mobile phase is 70: 30.
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| CN201810554787.4A CN110551178B (en) | 2018-06-01 | 2018-06-01 | Method for synthesizing head-tail cyclic peptide containing proline |
| PCT/CN2018/095427 WO2019227612A1 (en) | 2018-06-01 | 2018-07-12 | Method for synthesizing head-to-tail cyclic peptide containing proline |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6008058A (en) * | 1993-06-18 | 1999-12-28 | University Of Louisville | Cyclic peptide mixtures via side chain or backbone attachment and solid phase synthesis |
| CN1323313A (en) * | 1998-04-15 | 2001-11-21 | 阿温蒂斯药物制品公司 | Process for the preparation of resin-bound cyclic peptides |
| CN104861045A (en) * | 2014-02-20 | 2015-08-26 | 复旦大学 | Cyclopeptide compound GG6F and preparation method thereof |
| WO2017079821A1 (en) * | 2015-11-11 | 2017-05-18 | Encycle Therapeutics, Inc. | Fragment synthesis of cyclic peptides |
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| US8569452B2 (en) * | 2011-02-17 | 2013-10-29 | American Peptide Company, Inc. | Preparation of phalloidin and its derivatives |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6008058A (en) * | 1993-06-18 | 1999-12-28 | University Of Louisville | Cyclic peptide mixtures via side chain or backbone attachment and solid phase synthesis |
| CN1323313A (en) * | 1998-04-15 | 2001-11-21 | 阿温蒂斯药物制品公司 | Process for the preparation of resin-bound cyclic peptides |
| CN104861045A (en) * | 2014-02-20 | 2015-08-26 | 复旦大学 | Cyclopeptide compound GG6F and preparation method thereof |
| WO2017079821A1 (en) * | 2015-11-11 | 2017-05-18 | Encycle Therapeutics, Inc. | Fragment synthesis of cyclic peptides |
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