CN114230653A - Preparation method of chlorotoxin - Google Patents
Preparation method of chlorotoxin Download PDFInfo
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- CN114230653A CN114230653A CN202210098956.4A CN202210098956A CN114230653A CN 114230653 A CN114230653 A CN 114230653A CN 202210098956 A CN202210098956 A CN 202210098956A CN 114230653 A CN114230653 A CN 114230653A
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Abstract
The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of chlorotoxin. According to the method, chlorotoxin is prepared through a process combining solid-phase fragment synthesis and liquid-phase synthesis, and the chlorotoxin is divided into four fragments of 1-5, 6-9, 10-13 and 14-36 to be subjected to segmented coupling. The method is simple and convenient to operate, and the synthesis period of the process is greatly shortened. The solid-liquid combined synthesis method improves the yield and purity of the chlorotoxin, reduces the occurrence of side reactions and the types of byproducts, and ensures that the total yield can reach more than 25 percent and the purity can reach more than 99 percent.
Description
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of chlorotoxin.
Background
Chlorotoxin is a peptide component derived from the venom of the scorpion (leiuriius quinquetiatus), and has the amino acid sequence:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2(Disulfide bridge:Cys2-Cys19,Cys5-Cys28,Cys16-Cys33,Cys20-Cys35)。
the molecular structural formula is as follows:
studies have shown that chlorotoxin can specifically bind to tumor cells, and thus, chlorotoxin can be used as a targeting agent to deliver cytotoxic and/or imaging agents to a variety of tumors, including metastatic tumors and brain tumors such as glioblastomas. For example, chlorotoxin has been conjugated to the radioisotope iodine-131, and such chlorotoxin conjugates have been shown to be effective anti-tumor therapeutics. Other chlorotoxin conjugates, including fusion proteins such as chlorotoxin-GST fusion protein linked to saporin, have also been shown to significantly and selectively kill tumor cells. At present, the mass production of chlorotoxin is generally carried out by a biological fermentation method, and the low-cost mass industrial production of chlorotoxin is not realized yet.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing chlorotoxin.
The preparation method of chlorotoxin provided by the invention comprises the following steps:
sequentially coupling a peptide segment B, a peptide segment C and a peptide segment D on the resin peptide connected with the peptide segment A, then removing a protecting group, cracking from the resin and cyclizing to obtain chlorotoxin;
the peptide segment A comprises 14 th to 36 th amino acid residues of chlorotoxin;
the peptide segment B comprises 10 th to 13 th amino acid residues of chlorotoxin;
the peptide segment C comprises 6 th to 9 th amino acid residues of chlorotoxin;
the peptide segment D comprises amino acid residues at 1-5 th positions of chlorotoxin.
Experiments of the invention show that the problem that a plurality of amino acids are difficult to condense exists in the process of preparing chlorotoxin by coupling the amino acids one by one according to the peptide sequence of the chlorotoxin, and meanwhile, the method influences the synthesis yield of polypeptide products in purification, so that the purity of products with unsatisfactory yield is required to be improved. Through the adoption of a plurality of sectional synthesis modes, the results show that compared with other sections, 36 amino acids in the chlorotoxin peptide sequence are coupled in sections according to the form of four segments of 1-5, 6-9, 10-13 and 14-36, and higher yield and purity can be obtained. In addition, the side chain protecting groups of the amino acid residues in each peptide segment are reasonably selected, and the yield and the purity of the product are improved.
In the invention, the resin for connecting the peptide segment A is as follows:
Fmoc-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-Resin；
wherein the resin is selected from AMResin, MBHAResin, Rinkamide AMResin or Rinkamide MBHAResin. In the embodiment of the invention, the resin is AMResin. In some embodiments, the resin is AMResin with a degree of substitution of 0.316 mmol/g.
In the invention, the preparation method of the resin peptide of the connecting peptide segment A adopts a solid phase stepwise synthesis method. The method specifically comprises the following steps:
coupling Fmoc-Arg (Pbf) -OH with a resin to produce Fmoc-Arg (Pbf) -resin;
Fmoc-Arg (Pbf) -resin Fmoc protecting group removal, coupling of Fmoc-Cys (Trt) -OH, Fmoc-Leu-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (Trt) -OH, Fmoc-Cys (Trt-OH, Fmoc-Asp (Trt) -OH, Fmoc-Cys (Trt-OH, Fmoc-Asp (Trt-OH, Fmoc-Asp (Trt-OH, Fmoc-Asp (Tab-OH, Fmoc) -OH, Fmoc-Asp (Tab-OH, Fmoc) -OH, Fmoc-Asp (Tab-OH, Fmoc-Asp (Tab-OH, Fmoc-Cys-OH, Fmoc-Asp (Tab-Cys-OH, Fmoc-Cys-C-OH, Fmoc-Asp (Tab-OH, Fmoc-Asp (Tab-Cys-Asp (Tab-OH, and the like, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH.
In the invention, the peptide fragment B is: Y-His (X1)10-Gln(X2)11-Met12-Ala13-OH。
In the invention, Y in the peptide segment B is Fmoc, Boc, Alloc, Trt, Mmt or Mtt; in some embodiments, Y is Fmoc.
In the invention, the X1 is Boc, Cbz, Alloc, Mmt, Mtt or Trt. In some embodiments, the X1 is Boc.
In the invention, X2 is H, Boc, Cbz, Alloc, Mmt, Mtt, Trt or tBu. In some embodiments, the X2 is Trt.
In the invention, the preparation method of the peptide fragment B adopts a solid phase stepwise synthesis method. The method specifically comprises the steps of coupling Fmoc-Ala-OH with resin, sequentially coupling Fmoc-Met-OH, Fmoc-Gln (Trt) -OH and Fmoc-His (Boc) -OH, and then cracking from the resin to obtain a peptide segment B.
In the invention, the peptide fragment C is: Y-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-OH；
In the invention, Y in the peptide segment C is Fmoc, Boc, Alloc, Trt, Mmt or Mtt; in some embodiments, Y is Fmoc.
In the invention, the X3 is H, tBu, Mmt, Mtt or Trt; in some embodiments, the X3 is tBu.
In the invention, the X4 is All, tBu, Mmt, Mtt or Trt. In some embodiments, the X4 is tBu.
In the invention, the preparation method of the peptide fragment C adopts a solid phase stepwise synthesis method. The method specifically comprises the steps of coupling Fmoc-Asp (tBu) -OH with resin, sequentially coupling Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -OH and Fmoc-Phe-OH, and then cleaving from the resin to obtain a peptide fragment C.
In the invention, the peptide segment D is: Y-Met1-Cys(X5)2-Met3-Pro4-Cys(X5)5-OH。
In the invention, Y in the peptide segment D is Fmoc, Boc, Alloc, Trt, Mmt or Mtt; in some embodiments, Y is Fmoc.
In the present invention, the X5 is Acm, tBu, Mmt, Mtt or Trt, and in some embodiments, the X5 is Trt.
In the invention, the preparation method of the peptide fragment D adopts a solid phase stepwise synthesis method. The method specifically comprises the steps of coupling Fmoc-Cys (Trt) -OH with resin, then coupling Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Cys (Trt) -OH and Fmoc-Met-OH in sequence, and then cracking from the resin to obtain a peptide segment D.
In embodiments of the invention, the coupling agent is selected from one or more of HBTU, HATU, PyBop, DIC, DCC, TBTU, HBOT, HOAT and Cl-HOBT. In some embodiments, the coupling reagent is HOBt and DIC and the solvent for the coupling reaction is DMF. In some embodiments, the coupling agent is used in an amount of 1.0eq to 5.0 eq. The coupling reaction time is preferably 0.5-10 h.
In the embodiment of the invention, the removing agent for removing the protecting group is a DMF solution of piperidine or piperazine.
In some embodiments, the deprotecting agent to remove the Fmoc protecting group is 20% piperidine/DMF. The dosage of the Fmoc protecting group removing agent is 5-30 ml per gram of resin.
If there is an Alloc side chain protecting group, it is necessary to first use Pd (PPh) as a deprotecting agent3)4Or Pd (PPh)3)2Cl2The protective group is removed by combining a palladium catalytic agent (equivalent weight is 0.02-1.0 eq) with a silane reagent (equivalent weight is 2-10 eq) in an equivalent catalytic amount, and the solvent is composed of DMF, THF and DCM in any proportion. Then the next step of cleavage is carried out.
In the embodiment of the inventionThe cracking agent comprises at least one of TFA, PhSMe, EDT, TIS and TFE and H2And O. In the present invention, said TFA, PhSMe, EDT, TIS, TFE and H2The volume ratio of O is 100 (0-20): 0-20: (0-20). In some embodiments, the cleavage agent comprises TFA, EDT, TIS, and H2And O. Wherein TFA, EDT, TIS and H2The volume ratio of O is 90:5:2.5: 2.5.
In the embodiment of the invention, the cyclizing reagent is selected from one or more of elementary iodine, hydrogen peroxide, DMSO, reduced glutathione, oxidized glutathione, air, oxygen, ozone, sodium sulfite, ammonia water, phosphoric acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid and sodium chloride.
In the embodiment of the invention, the cyclized solvent is one or more of water, methanol, ethanol, acetonitrile, DMF and DMSO.
In an embodiment of the present invention, the cyclization comprises: dissolving the straight-chain chlorotoxin cleaved from the branches, and performing an aeration reaction for 24 hours. The dissolving reagent comprises water and 0.01-0.5M Tris-HCl, 0.1-0.3M NaCl, 1-10 mM reduced glutathione and 0.1-1 mM oxidized glutathione, and the pH value is 7.5-8.0. In some embodiments, the solubilized reagents comprise water and 0.1M Tris-HCl,0.2M NaCl,5mM reduced glutathione and 0.5mM oxidized glutathione, pH 7.8.
In the invention, the cyclization temperature is-10-40 ℃, and the cyclization time is 30 min-7 d.
In the embodiment of the invention, the method further comprises a step of purification by Prep-HPLC after the chlorotoxin is obtained, wherein a chromatographic column of the Prep-HPLC is a reversed phase C18 column, a gradient elution mode is adopted, a mobile phase A is 0.2 wt% acetic acid aqueous solution, and a mobile phase B is acetonitrile solution. The gradient elution mode is that the mobile phase B maintains 5 percent and the mobile phase A maintains 95 percent in 0-5 min, the mobile phase B increases from 15 percent to 80 percent in 5.01-50 min, and the mobile phase B maintains 95 percent in 50.1-60 min. The size of the column was 50X 250mm, and the sample loading solution was 20% acetic acid solution.
According to the invention, chlorotoxin is prepared by a process combining solid-phase fragment synthesis and liquid-phase synthesis, and is divided into four fragments of 1-5, 6-9, 10-13 and 14-36 for segmented coupling. The method is simple and convenient to operate, and the synthesis period of the process is greatly shortened. The solid-liquid combined synthesis method improves the yield and purity of the chlorotoxin, reduces the occurrence of side reactions and the types of byproducts, and ensures that the total yield can reach more than 25 percent and the purity can reach more than 99 percent.
Drawings
FIG. 1 is a UPLC chromatogram of a comparative example 1 product of the present invention;
FIG. 2 is a UPLC chromatogram of a comparative example 2 product of the present invention;
FIG. 3 is a UPLC chromatogram of the product of example 1 of the present invention.
Detailed Description
The invention provides a method for preparing chlorotoxin, and a person skilled in the art can use the contents to reference the contents and appropriately improve process parameters to realize the preparation. 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 methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The preparation method of chlorotoxin provided by the invention comprises the following steps:
step 1: using Fmoc-AA-OH as raw material, solid phase stepwise synthesis to obtain resin peptide Fmoc-Arg (Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-Resin；
Step 2: using fragment Fmoc-His (X1)10-Gln(X2)11-Met12-Ala13-OH、Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9And Y-Met1-Cys(X5)2-Met3-Pro4-Cys(X5)5the-OH and the resin obtained in the step one are subjected to solid phase stepwise coupling to obtain the resin peptide Y-Met1-Cys(X5)2-Met3-Pro4-Cys(X5)5-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-His(X1)10-Gln(X2)11-Met12-Ala13-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-Resin；
And step 3: and (3) removing a protecting group from the resin peptide obtained in the step two, and cracking the resin peptide from the resin to obtain a chlorotoxin linear chain crude product:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2；
and 4, step 4: and (3) carrying out liquid-phase oxidative cyclization on the straight-chain crude chlorotoxin to obtain a crude chlorotoxin solution, and finally purifying and freeze-drying to obtain a pure chlorotoxin product.
In the method for preparing chlorotoxin provided by the invention, in step 1, Fmoc-Arg (Pbf) -resin is firstly synthesized in a solid phase manner.
The preferable concrete is as follows: coupling Fmoc-Arg (Pbf) -OH to resin, Fmoc-Arg (Pbf) -resin.
Adding the resin into a solid phase reaction column, swelling the resin by using a solvent, dissolving an Fmoc-Arg (Pbf) -OH coupling agent in the solvent, activating in an ice bath, adding the mixture into the solid phase reaction column, reacting, washing by using the solvent, draining, and carrying out the next reaction.
Such solvents include, but are not limited to, DMF.
The coupling agent is preferably selected from one or more of HBTU, HATU, PyBop, DIC, DCC, TBTU, HBOT, HOAT, HOBT, Oxyma and Cl-HOBT. The coupling agent and Fmoc-Arg (Pbf) -OH are in a molar ratio of 1: 1-1: 5.
the Resin is selected from AM Resin, MBHA Resin, Rink Amide AM Resin or Rink Amide MBHA Resin, and the substitution degree is 0.1 mmol/g-0.8 mmol/g. The present invention is not limited in its source, and may be commercially available.
Wherein the swelling time is preferably 20-30 min; the activation time is preferably 10-15 min; the reaction time is preferably 0.5-10 h.
Coupling amino acids on said Fmoc-Arg (Pbf) -resin according to the peptidic sequence of chlorotoxin to give Y-Met1-Cys(X5)2-Met3-Pro4-Cys(X5)5-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-His(X1)10-Gln(X2)11-Met12-Ala13-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-Resin。
The amino acid is selected from Y-Met1-Cys (X5)2-Met3-Pro4-Cys(X5)5-OH、Fmoc-Cys(X5)5-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-OH、Fmoc-His(X1)10-Gln(X2)11-Met12-Ala13-OH, Fmoc-Met-OH, Fmoc-Cys (X5) -OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr (X3) -OH, Fmoc-Asp (X4) -OH, Fmoc-His (X1) -OH, Fmoc-Gln (X2) -OH, Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH and the like.
Firstly, before coupling amino acid in each step, removing Fmoc protecting group, washing with solvent, dissolving Fmoc-AA-OH and coupling agent in the solvent, activating in ice bath, adding into a solid phase reaction column, reacting, washing with solvent, and pumping to dry to directly couple next amino acid.
The removing time of the Fmoc protecting group is 10-15 min. The swelling time is preferably 20-30 min; the activation time is preferably 10-15 min; the coupling reaction time is preferably 0.5-10 h. The coupling agent is preferably selected from one or more of HBTU, HATU, PyBop, DIC, DCC, TBTU, HBOT, HOAT, HOBT, Oxyma and Cl-HOBT. The dosage of the coupling agent is 1.0 eq-5.0 eq
The amino acids were then coupled in sequence as described above, finally shrunk more than twice with MeOH and dried.
According to the invention, the removing agent of the Fmoc protecting group is preferably a DMF solution of piperidine or piperazine with the volume concentration of 10-50%; more preferably from 20% to 40% by volume of piperidine or piperazine in DMF. The dosage of the Fmoc protecting group removing agent is between 5 and 30ml per gram of resin.
The coupling of the invention is specifically coupling in sequence:
Y-Met1-Cys(X5)2-Met3-Pro4-Cys(X5)5-OH、Fmoc-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-OH、Fmoc-His(X1)10-Gln(X2)11-Met12-Ala13-OH、Fmoc-Arg(Pbf)14-OH、Fmoc-Lys(Boc)15-OH、Fmoc-Cys(Trt)16-OH、Fmoc-Asp(tBu)17-OH、Fmoc-Asp(tBu)18-OH、Fmoc-Cys(Trt)19-OH、Fmoc-Cys(Trt)20-OH、Fmoc-Gly21-OH、Fmoc-Gly22-OH、Fmoc-Lys(Boc)23-OH、Fmoc-Gly24-OH、Fmoc-Arg(Pbf)25-OH、Fmoc-Gly26-OH、Fmoc-Lys(Boc)27-OH、Fmoc-Cys(Trt)28-OH、Fmoc-Tyr(tBu)29-OH、Fmoc-Gly30-OH、Fmoc-Pro31-OH、Fmoc-Gln(Trt)32-OH、Fmoc-Cys(Trt)33-OH、Fmoc-Leu34-OH、Fmoc-Cys(Trt)35-OH、Fmoc-Arg(Pbf)36-OH。
subjecting said Y-Met to1-Cys(X5)2-Met3-Pro4-Cys(X5)5-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-His(X1)10-Gln(X2)11-Met12-Ala13-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-Resin cleavage to give crude chlorotoxin linear chain:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys)33-Leu34-Cys35-Arg36-NH2；
the X1, X2, X3, X4 and X5 are H, Boc, Cbz, Alloc, Mmt, Mtt, Trt, tBu and the like. And Y is Fmoc, Boc, Alloc, Trt, Mmt, Mtt and the like.
The lysis solution adopted by the cracking method comprises a mixture of TFA, PhSMe, EDT, TIS, TFE and H2O.
The volume ratio of TFA, PhSMe, EDT, TIS, TFE and H2O is 100 (0-20): 0-20 (: 0-20).
The dosage of the lysis solution is between 5ml and 30ml per gram of resin.
The cracking time is 30-240 min. The cracking frequency is preferably 1-3 times, and the cracking solution is combined, concentrated, settled, centrifuged, washed and dried to obtain the chlorotoxin straight-chain crude product.
The temperature of the cracking reaction is-10-35 ℃, the reaction time is 30-240 min, and the solvent comprises but is not limited to methyl tert-butyl ether. The temperature of the sedimentation is-5 ℃ to 5 ℃; the number of washing is preferably 2 to 3. The drying is preferably vacuum drying, and the specific drying parameters are not limited in the present invention and are well known to those skilled in the art.
Liquid phase cyclization is carried out on a chlorotoxin straight-chain crude product, the concentration of chlorotoxin can be selected from 0.5mg/ml to 100mg/ml, a cyclization reagent can be one or more of iodine simple substance, hydrogen peroxide, DMSO, reduced glutathione, oxidized glutathione, air, oxygen, ozone, sodium sulfite, ammonia water, phosphoric acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid, sodium chloride and the like, one or more of solvent water, methanol, ethanol, acetonitrile, DMF, DMSO and the like are mixed, the cyclization temperature is-10 ℃ to 40 ℃, and the cyclization time is 30min to 7 d.
Then, the mixture is purified by Prep-HPLC and is freeze-dried to obtain chlorotoxin.
The Prep-HPLC purification method adopted in the embodiment of the invention is as follows: dissolving the crude product with 20% acetic acid solution, purifying with Waters RP-HPLC system at wavelength of 220nm, using 50 × 250mm reversed phase C18 column as chromatographic column, and gradient eluting with mobile phase A of 0.2% acetic acid solution and phase B of acetonitrile.
During the coupling condensation by the step-by-step method, a plurality of amino acids are difficult to condense, and the synthesis yield of a polypeptide product is influenced during purification. The influence of difficult peptide impurities, especially deletion peptide impurities on the product quality and yield is avoided by the fragment method synthesis. The total yield is improved to more than 25 percent from 8 percent in the stepwise synthesis method, and the purity is improved to more than 99 percent from 95 percent.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The amino acids and reagents used in the preparation method of the polypeptide provided by the invention can be purchased from the market.
Abbreviations and english means include: fmoc 9-fluorenylmethoxycarbonyl, HOBT 1-hydroxybenzotriazole, Cl-HOBT 6-chloro-1-hydroxybenzotriazole, HOAT 1-hydroxy-7-azabenzotriazole, DMF N, N-dimethylformamide, DCM dichloromethane, TFA trifluoroacetic acid, PhSMe phenylmethyl sulfide, TIS triisopropylsilane, EDT ethanedithiol, DIPEAN, N-diisopropylethylamine. HBTU benzotriazole-1-tetramethylhexafluorophosphate, HATU 2- (7-azobenzotriazol) -N, N, N ', N ' -tetramethyluronium hexafluorophosphate, TBTUO-benzotriazol-N, N, N ', N ' -tetramethyluronium tetrafluoroborate, PyBop benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, DIC N, N ' -diisopropylcarbodiimide, DCC dicyclohexylcarbodiimide, NMM N-methylmorpholine, DIEA N, N-diisopropylethylamine, TEA triethylamine, Mmt p-methoxytrityl, Mtt p-methyltriphenyl, Trt trityl, t-Bu t-butyl, All allyl, Alloc allyloxycarbonyl, Acm acetamidomethyl.
The invention is further illustrated by the following examples:
example 1: fragment coupling method
Step 1:
Fmoc-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36synthesis of AM Resin:
removing Fmoc protecting groups: AM Resin (31.646g, 10.0mmol) with a degree of substitution of 0.316mmol/g was weighed into a solid phase reaction column, the Resin was swollen with DMF (310ml) for 30min, and the solvent was drained. The Fmoc protection was removed with DBLK (20% piperidine/DMF, 200ml) for 10min, twice protected and then washed 6 times with DMF (300ml) (a little resin was added to 0.3ml of 5% ninhydrin ethanol solution and heated at 100 ℃ for 5-10 min, the resin did not turn blue).
Fmoc-Arg (Pbf) -OH coupling: 12.981g of Fmoc-Arg (Pbf) -OH (20.0mmol) is weighed and dissolved in DMF (200ml), 2.709g of HOBt (20.0mmol) and DIC (3.1ml) are added under ice bath for activation for 10min and then added into a solid phase reaction column for reaction for 2 h at room temperature (0.3 ml of 5% ninhydrin ethanol solution is added into a little resin, and the resin is heated for 5-10 min at 100 ℃ without discoloration). Washed 6 times with DMF (300ml) and taken up directly in the next reaction step.
Fmoc-Cys (Trt) coupled one by one as described above35-OH、Fmoc-Leu34-OH、Fmoc-Cys(Trt)33-OH、Fmoc-Gln(Trt)32-OH、Fmoc-Pro31-OH、Fmoc-Gly30-OH、Fmoc-Tyr(tBu)29-OH、Fmoc-Cys(Trt)28-OH、Fmoc-Lys(Boc)27-OH、Fmoc-Gly26-OH、Fmoc-Arg(Pbf)25-OH、Fmoc-Gly24-OH、Fmoc-Lys(Boc)23-OH、Fmoc-Gly22-OH、Fmoc-Gly21-OH、Fmoc-Cys(Trt)20-OH、Fmoc-Cys(Trt)19-OH、Fmoc-Asp(tBu)18-OH、Fmoc-Asp(tBu)17-OH、Fmoc-Cys(Trt)16-OH、Fmoc-Lys(Boc)15-OH、Fmoc-Arg(Pbf)14-OH, wherein the Fmoc protecting group is removedThe operations of the groups are the same, and the condensation reaction time of the amino acid is between 2 and 5 hours (based on ninhydrin color development).
The above amino acid coupling was completed to give a resin:
Fmoc-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-AM Resin
step 2:
Boc-Met1-Cys(Trt)2-Met3-Pro4-Cys(Trt)5-Phe6-Thr(tBu)7-Thr(tBu)8-Asp(tBu)9-His(Boc)10-Gln(Trt)11-Met12-Ala13-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36synthesis of-Resin
Removing Fmoc protecting groups: the Fmoc protection was removed with DBLK (20% piperidine/DMF, 200ml) for 10min, twice protected and then washed 6 times with DMF (300ml) (a little resin was added to 0.3ml of 5% ninhydrin ethanol solution and heated at 100 ℃ for 5-10 min, the resin did not turn blue).
Fmoc-His(Boc)10-Gln(Trt)11-Met12-Ala13-OH coupling: 20.107g of Fmoc-His (Boc) were weighed10-Gln(Trt)11-Met12-Ala13-OH in DMF (250ml) solutionAdding 2.711g of HOBt (20.0mmol) and DIC (3.1ml) under ice bath for activation for 10min, adding into a solid phase reaction column, and reacting at room temperature for 3h (adding a little resin into 0.3ml of 5% ninhydrin ethanol solution, heating at 100 ℃ for 5-10 min, and the resin is not discolored). Washed 6 times with DMF (300ml) and taken up directly in the next reaction step.
Fmoc-Phe coupling was performed as described above one by one6-Thr(tBu)7-Thr(tBu)8-Asp(tBu)9-OH and Boc-Met1-Cys(Trt)2-Met3-Pro4-Cys(Trt)5And (4) OH, wherein the operations for removing Fmoc protecting groups are the same, and the time for amino acid condensation reaction is between 2 and 5 hours (based on ninhydrin color development).
The above amino acid coupling was accomplished to give a resin which was washed twice with DCM (300ml) and MeOH (300ml) alternately, then three times with MeOH (300ml) and finally dried in vacuo to give 83.766g of chlorotoxin resinoid.
And step 3:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2synthesis of (2)
81.789g of chlorotoxin resin peptide was put into a 1L bottle of eggplant form, and 500ml of TFA, EDT, TIS, H was added2O (volume ratio 90:5:2.5:2.5), reacted at room temperature for 3h, filtered, and washed twice with 200ml TFA. And (3) combining the organic phases, concentrating the organic phases to about 200ml at the temperature of 30-35 ℃, dropwise adding the concentrated solution into 4L of methyl tert-butyl ether (-5 ℃) for settling, centrifuging and drying. Finally, 34.336g of crude chlorotoxin linear peptide are obtained.
And 4, step 4:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2(Disulfide bridge:Cys2-Cys19,Cys5-Cys28,Cys16-Cys33,Cys20-Cys35) Synthesis of (2)
34.107g of crude chlorotoxin are dissolved in 17L of mixed solution (the mixed solution is a mixed aqueous solution of 0.1M Tris-HCl,0.2M NaCl,5mM reduced glutathione and 0.5mM oxidized glutathione, and the pH value is 7.8), and after the dissolution is finished, the pH value of the mixed solution is adjusted to 7.5-8.0 by using 1M acetic acid and 1M ammonia water. Blowing air to react for 24h at room temperature, directly purifying the reaction liquid by reverse phase liquid preparative chromatography, combining and concentrating the purified liquid with the purity of more than 99% by HPLC (high performance liquid chromatography) to 1/3-1/2 of the volume before concentration, and freeze-drying to obtain the chlorotoxin 10.266g with the purity of 99.17%, wherein the total yield is 25.67%, and the UPLC spectrogram is shown in figure 3.
Comparative example 1: stepwise coupling process
Step 1:
Boc-Met1-Cys(Trt)2-Met3-Pro4-Cys(Trt)5-Phe6-Thr(tBu)7-Thr(tBu)8-Asp(tBu)9-His(Boc)10-Gln(Trt)11-Met12-Ala13-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36synthesis of-Resin
Removing Fmoc protecting groups: AM Resin (15.846g, 5.0mmol) with a degree of substitution of 0.316mmol/g was weighed into a solid phase reaction column, the Resin was swollen with DMF (150ml) for 30min, and the solvent was drained. The Fmoc protection was removed with DBLK (20% piperidine/DMF, 100ml) for 10min, twice protected and then washed 6 times with DMF (150ml) (a little resin was added to 0.3ml of 5% ninhydrin ethanol solution and heated at 100 ℃ for 5-10 min, the resin did not turn blue).
Fmoc-Arg (Pbf) -OH coupling: 6.581g of Fmoc-Arg (Pbf) -OH (10.0mmol) is weighed and dissolved in DMF (200ml), 1.365g of HOBt (10.0mmol) and DIC (1.6ml) are added under ice bath for activation for 10min and then added into a solid phase reaction column for reaction for 2 h at room temperature (0.3 ml of 5% ninhydrin ethanol solution is added into a little resin, and the resin is heated for 5-10 min at 100 ℃ without discoloration). Washed 6 times with DMF (150ml) and taken up directly in the next reaction step.
Fmoc-Cys (Trt) coupled one by one as described above35-OH、Fmoc-Leu34-OH、Fmoc-Cys(Trt)33-OH、Fmoc-Gln(Trt)32-OH、Fmoc-Pro31-OH、Fmoc-Gly30-OH、Fmoc-Tyr(tBu)29-OH、Fmoc-Cys(Trt)28-OH、Fmoc-Lys(Boc)27-OH、Fmoc-Gly26-OH、Fmoc-Arg(Pbf)25-OH、Fmoc-Gly24-OH、Fmoc-Lys(Boc)23-OH、Fmoc-Gly22-OH、Fmoc-Gly21-OH、Fmoc-Cys(Trt)20-OH、Fmoc-Cys(Trt)19-OH、Fmoc-Asp(tBu)18-OH、Fmoc-Asp(tBu)17-OH、Fmoc-Cys(Trt)16-OH、Fmoc-Lys(Boc)15-OH、Fmoc-Arg(Pbf)14-OH、Fmoc-Ala13-OH、Fmoc-Met12-OH、Fmoc-Gln(Trt)11-OH、Fmoc-His(Boc)10-OH、Fmoc-Asp(tBu)9-OH、Fmoc-Thr(tBu)8-OH、Fmoc-Thr(tBu)7-OH、Fmoc-Phe6-OH、Fmoc-Cys(Trt)5-OH、Fmoc-Pro4-OH、Fmoc-Met3-OH、Fmoc-Cys(Trt)2-OH、Boc-Met1And (4) removing Fmoc protecting groups from-OH, wherein the operations are the same, and the condensation reaction time of amino acid is 2-5 h (based on ninhydrin color development).
The above amino acid coupling was completed to give a resin which was washed twice with DCM (150ml) and MeOH (150ml) alternately, then three times with MeOH (150ml) and finally dried in vacuo to give 33.465g of chlorotoxin resinoid.
Step 2:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2synthesis of (2)
30.257g of chlorotoxin resin peptide was put into a 1L bottle of eggplant form, and 150ml of TFA, EDT, TIS, H was added2O (volume ratio 90:5:2.5:2.5), reacted at room temperature for 3h, filtered, and washed twice with 100ml TFA. And (3) combining the organic phases, concentrating the organic phases to about 70ml at the temperature of 30-35 ℃, dropwise adding the concentrated solution into 1.5L of methyl tert-butyl ether (-5 ℃) for settling, centrifuging and drying. Finally, 8.896g of crude chlorotoxin linear peptide are obtained.
And step 3:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2(Disulfide bridge:Cys2-Cys19,Cys5-Cys28,Cys16-Cys33,Cys20-Cys35) Synthesis of (2)
8.533g of crude chlorotoxin are dissolved in 4.3L of mixed solution (the mixed solution is a mixed aqueous solution of 0.1M Tris-HCl,0.2M NaCl,5mM reduced glutathione and 0.5mM oxidized glutathione, and the pH value is 7.8), and after the dissolution is finished, the pH value of the mixed solution is adjusted to 7.5-8.0 by using 1M acetic acid and 1M ammonia water. Blowing air to react for 24h at room temperature, directly preparing and purifying the reaction solution under high pressure, concentrating and freeze-drying. The chlorotoxin with the purity of 92.71 percent is obtained in 1.623g and the yield is 8.1 percent, and the UPLC spectrum is shown in figure 1.
Comparative example 2: fragment coupling method
Step 1:
Fmoc-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36synthesis of-AM Resin
Removing Fmoc protecting groups: AM Resin (15.887g, 5.0mmol) with a degree of substitution of 0.316mmol/g was weighed into a solid phase reaction column, the Resin was swollen with DMF (150ml) for 30min, and the solvent was drained. The Fmoc protection was removed with DBLK (20% piperidine/DMF, 100ml) for 10min, twice protected and then washed 6 times with DMF (150ml) (a little resin was added to 0.3ml of 5% ninhydrin ethanol solution and heated at 100 ℃ for 5-10 min, the resin did not turn blue).
Fmoc-Arg (Pbf) -OH coupling: 6.541g of Fmoc-Arg (Pbf) -OH (10.0mmol) is weighed and dissolved in DMF (100ml), 1.361g of HOBt (10.0mmol) and DIC (1.6ml) are added under ice bath for activation for 10min and then added into a solid phase reaction column for reaction for 2 h at room temperature (0.3 ml of 5% ninhydrin ethanol solution is added into a little resin, and the resin is heated for 5-10 min at 100 ℃ without discoloration). Washed 6 times with DMF (300ml) and taken up directly in the next reaction step.
Fmoc-Cys (Trt) coupled one by one as described above35-OH、Fmoc-Leu34-OH、Fmoc-Cys(Trt)33-OH、Fmoc-Gln(Trt)32-OH、Fmoc-Pro31-OH、Fmoc-Gly30-OH、Fmoc-Tyr(tBu)29-OH、Fmoc-Cys(Trt)28-OH、Fmoc-Lys(Boc)27-OH、Fmoc-Gly26-OH、Fmoc-Arg(Pbf)25-OH、Fmoc-Gly24-OH、Fmoc-Lys(Boc)23-OH、Fmoc-Gly22-OH、Fmoc-Gly21-OH、Fmoc-Cys(Trt)20-OH、Fmoc-Cys(Trt)19-OH、Fmoc-Asp(tBu)18-OH、Fmoc-Asp(tBu)17-OH、Fmoc-Cys(Trt)16-OH、Fmoc-Lys(Boc)15-OH、Fmoc-Arg(Pbf)14And (4) OH, wherein the operations for removing Fmoc protecting groups are the same, and the time for amino acid condensation reaction is between 2 and 5 hours (based on ninhydrin color development).
The above amino acid coupling was completed to give a resin:
Fmoc-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-AM Resin
step 2:
Boc-Met1-Cys(Trt)2-Met3-Pro4-Cys(Trt)5-Phe6-Thr(tBu)7-Thr(tBu)8-Asp(tBu)9-His(Boc)10-Gln(Trt)11-Met12-Ala13-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36synthesis of-Resin
Removing Fmoc protecting groups: the Fmoc protection was removed with DBLK (20% piperidine/DMF, 200ml) for 10min, twice protected and then washed 6 times with DMF (300ml) (a little resin was added to 0.3ml of 5% ninhydrin ethanol solution and heated at 100 ℃ for 5-10 min, the resin did not turn blue).
Fmoc-His(Boc)10-Gln(Trt)11-Met12-Ala13-OH coupling: 10.057g of Fmoc-His (Boc) were weighed10-Gln(Trt)11-Met12-Ala13dissolving-OH in a DMF (1250ml) solution, adding 1.355g of HOBt (10.0mmol) and DIC (1.6ml) under ice bath for activation for 10min, adding into a solid phase reaction column, and reacting at room temperature for 3h (adding a little resin into 0.3ml of 5% ninhydrin ethanol solution, heating at 100 ℃ for 5-10 min, and the resin does not change color). Washed 6 times with DMF (150ml) and taken up directly in the next reaction step.
Fmoc-Phe coupling was performed as described above one by one6-Thr(tBu)7-Thr(tBu)8-Asp(tBu)9-OH、Fmoc-Cys(Trt)5-OH、Fmoc-Pro4-OH、Fmoc-Met3-OH、Fmoc-Cys(Trt)2-OH and Boc-Met1And (4) removing Fmoc protecting groups from-OH, wherein the operations are the same, and the condensation reaction time of amino acid is 2-5 h (based on ninhydrin color development).
The above amino acid coupling was completed to give a resin which was washed twice with DCM (150ml) and MeOH (150ml) alternately, then three times with MeOH (150ml) and finally dried in vacuo to give 39.568g of chlorotoxin resinoid.
And step 3:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2synthesis of (2)
39.568g of chlorotoxin resin peptide was put into a 1L bottle of eggplant shape, and 300ml of TFA, EDT, TIS, H was added2O (volume ratio 90:5:2.5:2.5), reacted at room temperature for 3h, filtered, and washed twice with 100ml TFA. And (3) combining the organic phases, concentrating the organic phases to about 100ml at the temperature of 30-35 ℃, dropwise adding the concentrated solution into 2L of methyl tert-butyl ether (-5 ℃) for settling, centrifuging and drying. Finally, 14.778g of crude chlorotoxin linear peptide are obtained.
And 4, step 4:
Met1-Cys2-Met3-Pro4-Cys5-Phe6-Thr7-Thr8-Asp9-His10-Gln11-Met12-Ala13-Arg14-Lys15-Cys16-Asp17-Asp18-Cys19-Cys20-Gly21-Gly22-Lys23-Gly24-Arg25-Gly26-Lys27-Cys28-Tyr29-Gly30-Pro31-Gln32-Cys33-Leu34-Cys35-Arg36-NH2(Disulfide bridge:Cys2-Cys19,Cys5-Cys28,Cys16-Cys33,Cys20-Cys35) Synthesis of (2)
14.458g of crude chlorotoxin are dissolved in 7.3L of mixed solution (the mixed solution is a mixed aqueous solution of 0.1M Tris-HCl,0.2M NaCl,5mM reduced glutathione and 0.5mM oxidized glutathione, and the pH value is 7.8), and after the dissolution is finished, the pH value of the mixed solution is adjusted to 7.5-8.0 by using 1M acetic acid and 1M ammonia water. Blowing air to react for 24h at room temperature, directly preparing and purifying the reaction solution under high pressure, concentrating and freeze-drying. The chlorotoxin with the purity of 97.94% 3.237g is obtained, the yield is 16.17%, and the UPLC spectrum is shown in figure 2.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. A method of producing chlorotoxin, comprising:
sequentially coupling a peptide segment B, a peptide segment C and a peptide segment D on the resin peptide connected with the peptide segment A, then removing a protecting group, cracking from the resin and cyclizing to obtain chlorotoxin;
the peptide segment A comprises 14 th to 36 th amino acid residues of chlorotoxin;
the peptide segment B comprises 10 th to 13 th amino acid residues of chlorotoxin;
the peptide segment C comprises 6 th to 9 th amino acid residues of chlorotoxin;
the peptide segment D comprises amino acid residues at 1-5 th positions of chlorotoxin.
2. The method according to claim 1, wherein the resin for connecting peptide fragment A is:
Fmoc-Arg(Pbf)14-Lys(Boc)15-Cys(Trt)16-Asp(tBu)17-Asp(tBu)18-Cys(Trt)19-Cys(Trt)20-Gly21-Gly22-Lys(Boc)23-Gly24-Arg(Pbf)25-Gly26-Lys(Boc)27-Cys(Trt)28-Tyr(tBu)29-Gly30-Pro31-Gln(Trt)32-Cys(Trt)33-Leu34-Cys(Trt)35-Arg(Pbf)36-Resin；
wherein the Resin is selected from AM Resin, MBHA Resin, Rink Amide AM Resin or Rink Amide MBHA Resin.
3. The production method according to claim 1,
the peptide fragment B is as follows: Y-His (X1)10-Gln(X2)11-Met12-Ala13-OH；
Y is Fmoc, Boc, Alloc, Trt, Mmt or Mtt;
the X1 is Boc, Cbz, Alloc, Mmt, Mtt or Trt;
and the X2 is H, Boc, Cbz, Alloc, Mmt, Mtt, Trt or tBu.
4. The method according to claim 1, wherein the peptide fragment C is: Y-Phe6-Thr(X3)7-Thr(X3)8-Asp(X4)9-OH；
Y is Fmoc, Boc, Alloc, Trt, Mmt or Mtt;
the X3 is H, tBu, Mmt, Mtt or Trt;
and the X4 is All, tBu, Mmt, Mtt or Trt.
5. The method according to claim 1, wherein the peptide fragment D is: Y-Met1-Cys(X5)2-Met3-Pro4-Cys(X5)5-OH；
Y is Fmoc, Boc, Alloc, Trt, Mmt or Mtt;
and the X5 is Acm, tBu, Mmt, Mtt or Trt.
6. The process according to any one of claims 1 to 5, wherein the coupling agent is one or more selected from HBTU, HATU, PyBop, DIC, DCC, TBTU, HBOT, HOAT and Cl-HOBT.
7. The process according to any one of claims 1 to 5, wherein the deprotecting agent is a solution of piperidine or piperazine in DMF.
8. The method of any one of claims 1-5, wherein the cracking agent comprises TFA, PhSMe, EDT, TIS, TFE, and H2O。
9. The production method according to any one of claims 1 to 5, wherein the cyclizing reagent is one or more selected from the group consisting of elemental iodine, hydrogen peroxide, DMSO, reduced glutathione, oxidized glutathione, air, oxygen, ozone, sodium sulfite, aqueous ammonia, phosphoric acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid, and sodium chloride;
the cyclized solvent is one or more of water, methanol, ethanol, acetonitrile, DMF and DMSO.
10. The preparation method according to any one of claims 1 to 9, further comprising a purification step by Prep-HPLC after the chlorotoxin is obtained, wherein the chromatography column of Prep-HPLC is a reversed phase C18 column, and the mobile phase is a mixture of 0.2 wt% acetic acid aqueous solution and acetonitrile.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115806607A (en) * | 2022-12-27 | 2023-03-17 | 哈尔滨吉象隆生物技术有限公司 | Site-directed cyclization method of chlorotoxin polypeptide |
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| CN105801706A (en) * | 2014-12-31 | 2016-07-27 | 深圳先进技术研究院 | Scorpion chlorotoxin polypeptide-ferritin heavy chain fused protein, self-assembled protein nanocage, preparation method therefor and application |
| US9518099B1 (en) * | 2015-09-13 | 2016-12-13 | Wenzhou Institute Of Biomaterials And Engineering | Refolded chlorotoxin, chlorotoxin variant, refolded chlorotoxin variant, and preparation technology thereof |
| CN109456401A (en) * | 2018-12-03 | 2019-03-12 | 成都诺和晟泰生物科技有限公司 | A kind of synthetic method of Suo Malu peptide |
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| CN105801706A (en) * | 2014-12-31 | 2016-07-27 | 深圳先进技术研究院 | Scorpion chlorotoxin polypeptide-ferritin heavy chain fused protein, self-assembled protein nanocage, preparation method therefor and application |
| US9518099B1 (en) * | 2015-09-13 | 2016-12-13 | Wenzhou Institute Of Biomaterials And Engineering | Refolded chlorotoxin, chlorotoxin variant, refolded chlorotoxin variant, and preparation technology thereof |
| CN109456401A (en) * | 2018-12-03 | 2019-03-12 | 成都诺和晟泰生物科技有限公司 | A kind of synthetic method of Suo Malu peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115806607A (en) * | 2022-12-27 | 2023-03-17 | 哈尔滨吉象隆生物技术有限公司 | Site-directed cyclization method of chlorotoxin polypeptide |
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