CN115806607A - Site-directed cyclization method of chlorotoxin polypeptide - Google Patents
Site-directed cyclization method of chlorotoxin polypeptide Download PDFInfo
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- CN115806607A CN115806607A CN202211684309.8A CN202211684309A CN115806607A CN 115806607 A CN115806607 A CN 115806607A CN 202211684309 A CN202211684309 A CN 202211684309A CN 115806607 A CN115806607 A CN 115806607A
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Abstract
A fixed-point cyclization method of chlorotoxin polypeptide belongs to the technical field of polypeptide. In order to solve the problem that the purity and yield of the product are low due to isomer impurities generated by mismatching disulfide bonds in the existing chlorotoxin synthesis method, the invention provides a method for synthesizing chlorotoxin polypeptide by forming four pairs of disulfide bonds through site-specific cyclization, which comprises the steps of synthesizing chlorotoxin polypeptide precursor resin through a solid phase; solid phase oxidation to form a first pair of disulfide bonds; solid phase oxidation to form a second pair of disulfide bonds; solid phase oxidation to form a third pair of disulfide bonds; liquid phase oxidation forms a fourth pair of disulfide bonds. The method adopts a specific amino acid side chain protecting group, forms a disulfide bond at a fixed point in the cyclization process, reduces the generation of mismatched byproducts, greatly improves the purity of the crude product of the chlorotoxin polypeptide, improves the yield of the crude product, reduces the production cost, and is suitable for large-scale production.
Description
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a site-specific cyclization method of chlorotoxin polypeptide.
Background
Chlorotoxin (chlorotoxin) is a scorpion toxin polypeptide, is separated from the Israeli scorpio (leiurus quinquequestraq chemical hookum), and has huge application prospects as a selective targeting new drug for human glioma and a specific marker for diagnosis (including grade judgment).
It has been found that human brain astrocytomas produce a unique voltage-dependent chloride current (referred to as GCC current, the chloride channel of which is referred to as GCC channel) that is not produced in human normal cells (including normal glial cells) as well as human tumor cells of non-glial origin, and that this chloride current can be driven by Cl - The channel inhibitor chlorotoxin effectively inhibits and causes a change in the proliferation rate of brain tumor cells, presumably Cl - The channels are involved in the regulation of astrocytoma growth. GCC currents sensitive to chlorotoxin persist in implanted glioma cells but not in neighboring normal host glial cells. This unique chloride channel was confirmed to be unique to glioma. The selective binding of chlorotoxin to this chloride channel provides a powerful new approach to identify and treat gliomas.Chlorotoxin selectively binds to glioma cells, while normal human tissues including brain, kidney, intestine, etc. exhibit a negative immunostaining with chlorotoxin.
At present, the mass production of the chlorotoxin is generally carried out by a biological fermentation method, and the low-cost mass industrial production of the chlorotoxin is not realized. Chinese patent CN114230653A describes a method of forming four pairs of disulfide bonds by one-step oxidation: the method comprises the steps of firstly preparing chlorotoxin linear peptide resin by a process combining solid-phase fragment synthesis and liquid-phase synthesis, then cracking the chlorotoxin resin to remove all protecting groups and resin solid-phase carriers to obtain chlorotoxin linear crude peptide, and finally performing one-step oxidation reaction on the linear peptide by adopting an oxidation system to obtain the chlorotoxin. Although the target structure can be obtained by the one-step oxidation method, generation of disulfide bond mismatching isomer impurities cannot be avoided, and the obtained crude product has low purity and low overall yield.
Therefore, the method for synthesizing chlorotoxin, which has the advantages of mild reaction conditions, low cost, high yield, high product purity, simple process, stable process and suitability for large-scale production, needs to be explored urgently.
Disclosure of Invention
The invention provides a fixed-point cyclization method for synthesizing chlorotoxin, aiming at the problem that isomer impurities are generated due to mismatching of disulfide bonds in the chlorotoxin synthesis method, so that four pairs of full-crossed disulfide bonds are directionally and efficiently synthesized.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the first object of the present invention is to provide a site-directed cyclization method of chlorotoxin polypeptide, which comprises the following steps:
1) Preparing chlorotoxin polypeptide precursor resin: fmoc-Arg (pbf) -OH reacts with carrier resin to obtain Fmoc-Arg (pbf) -resin, the Fmoc-Arg (pbf) -resin is taken as a solid phase carrier, fmoc-AA-OH is coupled in a one-by-one coupling mode according to the sequence from C end to N end in the presence of an activation system to obtain chlorotoxin polypeptide precursor resin, wherein Cys side chains corresponding to the 2 nd and 19 th sites of chlorotoxin polypeptide are protected by Mtt, cys side chains corresponding to the 5 th and 28 th sites of chlorotoxin polypeptide are protected by Trt, cys side chains corresponding to the 16 th and 33 th sites of chlorotoxin polypeptide are protected by Acm, and Cys side chains corresponding to the 20 th and 35 th sites of chlorotoxin polypeptide are protected by Dpm;
2) Removing the Mtt protecting group of the chlorotoxin polypeptide precursor resin obtained in the step 1) by using a deprotection agent;
3) Oxidizing the chlorotoxin polypeptide precursor resin obtained in the step 2) by using an oxidizing agent to form a first pair of disulfide bonds, so as to obtain chlorotoxin polypeptide precursor resin containing a disulfide bond;
4) Removing the Trt protecting group of the chlorotoxin polypeptide precursor resin containing the dithio-rings obtained in the step 3) by using a deprotection agent;
5) Oxidizing the mono-disulfide cyclopeptide obtained in the step 4) by using an oxidizing agent to form a second pair of disulfide bonds to obtain a di-disulfide cyclopeptide;
6) Removing the Acm protecting group of Cys in the double-disulfide cyclic peptide obtained in the step 5) by using a deprotection agent, and oxidizing to form a third pair of disulfide bonds;
7) Cutting the chlorotoxin polypeptide precursor resin containing three pairs of disulfide rings obtained in the step 6) by using a lysis solution, and simultaneously removing a Dpm protective group;
8) Oxidizing the three pairs of disulfide cyclic peptides obtained in the step 7) with an oxidizing agent to form a fourth pair of disulfide bonds, so as to obtain chlorotoxin polypeptides containing the four pairs of disulfide cyclic peptides;
in one embodiment of the invention, the carrier resin in step 1) is rinkAmideAMResin or rinkAmidemBHAResin, and the resin substitution degree is 0.1-1.0 mmol/g.
Preferably, the resin substitution is from 0.2 to 0.5mmol/g.
In one embodiment of the invention, the activation system in the step 1) is any one of HOBt/DIC, HBTU/HOBt/DIPEA and TBTU/HOBt/DIPEA, and the feeding multiple is 1-10eq.
Preferably, the activation system in the step 1) is HOBt/DIC, and the feeding multiple is 3-5eq.
In one embodiment of the invention, the coupling of Fmoc-AA-OH in the order C-to N-terminal as described in step 1) is Fmoc-Arg (Pbf) 36 -OH、Fmoc-Cys(Dpm) 35 -OH、Fmoc-Leu 34 -OH、Fmoc-Cys(Acm) 33 -OH、Fmoc-Gln(Trt) 32 -OH、Fmoc-Pro 31 -OH、Fmoc-Gly 30 -OH、Fmoc-Tyr(tBu) 29 -OH、Fmoc-Cys(Trt) 28 -OH、Fmoc-Lys(Boc) 27 -OH、Fmoc-Gly 26 -OH、Fmoc-Arg(Pbf) 25 -OH、Fmoc-Gly 24 -OH、Fmoc-Lys(Boc) 23 -OH、Fmoc-Gly 22 -OH、Fmoc-Gly 21 -OH、Fmoc-Cys(Dpm) 20 -OH、Fmoc-Cys(Mtt) 19 -OH、Fmoc-Asp(tBu) 18 -OH、Fmoc-Asp(tBu) 17 -OH、Fmoc-Cys(Acm) 16 -OH、Fmoc-Lys(Boc) 15 -OH、Fmoc-Arg(Pbf) 14 -OH、Fmoc-Ala 13 -OH、Fmoc-Met 12 -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-Phe 6 -OH、Fmoc-Cys(Trt) 5 -OH、Fmoc-Pro 4 -OH、Fmoc-Met 3 -OH、Fmoc-Cys(Mtt) 2 -OH、Boc-Met 1 -OH。
The amino acid sequence of the polypeptide containing four pairs of dithiocyclopeptide chlorotoxin is shown in SEQ ID NO.1, and specifically comprises the following steps:
MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR；
Disulfidebridge：Cys 2 -Cys 19 ,Cys 5 -Cys 28 ,Cys 16 -Cys 33 ,Cys 20 -Cys 35 ；
disulfide bonds are located in Cys 2-19 、Cys 5-28 、Cys 16-33 And Cys 20-35 。
In an embodiment of the present invention, the deprotection agent in step 2) is a mixed solution of TFA and DCM, and the volume ratio of TFA to DCM is (1-5): (95-99).
Preferably, the volume ratio of TFA to DCM is (1-3): (97 to 99).
In one embodiment of the present invention, the solvent used in the washing step may be any reagent in the art that can achieve this purpose, such as DMF, NMP, dichloromethane, and the like, preferably DMF.
In one embodiment of the present invention, the deprotection time in step 2) is 5-50min.
Preferably, the deprotection time in step 2) is 10-30min.
In one embodiment of the present invention, the oxidizing agent in step 3) is DMSO.
In one embodiment of the present invention, the oxidation time in step 3) is 1 to 20 hours.
Preferably, the oxidation time in step 3) is 3-8h.
In an embodiment of the present invention, the deprotection agent in step 4) is a mixed solution of TFA and DCM, and the volume ratio of TFA to DCM (10-20): (80-90).
Preferably, the volume ratio of TFA to DCM is (10-15): (85-90).
In one embodiment of the present invention, the deprotection time in step 4) is 10 to 60min.
Preferably, the deprotection time in step 4) is 10-30min.
In one embodiment of the present invention, the oxidizing agent in step 5) is DMSO.
In one embodiment of the present invention, the oxidation time in step 5) is 1 to 20 hours.
Preferably, the oxidation time in step 5) is 3-8h.
In one embodiment of the present invention, the deprotection agent in step 6) comprises I 2 DMF solution of (1), I 2 The mass fraction in DMF is 1-10 wt%.
Preferably, I 2 The mass fraction in DMF is 5-10 wt%.
In one embodiment of the present invention, the deprotection and oxidation time in step 6) is 1 to 10 hours.
Preferably, the deprotection and oxidation time of step 6) is 2-5 h.
In one embodiment of the present invention, the lysis solution in step 7) is TFA, a mixed solution of EDT and water, and the volume ratio of the EDT to the water is (90-95): (2-5): (2-5).
In one embodiment of the present invention, the cracking time in step 7) is 1 to 5 hours.
Preferably, the cracking time in step 7) is 2-3 h.
In one embodiment of the present invention, the oxidizing agent in step 8) is a 3% hydrogen peroxide solution.
In one embodiment of the present invention, the feed-to-solution ratio of the three pairs of dithiocyclic peptides obtained in step 7) in step 8) to the hydrogen peroxide solution is 1g: (0.5-5) mL.
Preferably, the feed-to-liquid ratio of the three pairs of dithiocyclic peptides obtained in step 7) in step 8) to the hydrogen peroxide solution is 1g: (1-3) mL.
In one embodiment of the present invention, the oxidation time in step 8) is 1 to 10 hours.
Preferably, the oxidation time in step 8) is 3 to 5 hours.
In one embodiment of the present invention, the site-directed cyclization method further comprises a step of purifying the obtained chlorotoxin polypeptide containing four pairs of dithiocyclopeptides, specifically, a reversed-phase high-pressure liquid chromatography method is adopted for purification.
In one embodiment of the invention, the reversed phase high pressure liquid chromatography comprises: taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% trifluoroacetic acid water solution/acetonitrile as a mobile phase, carrying out elution at a gradient change rate of every 10 minutes after the chlorotoxin crude peptide is loaded, and collecting a target product, wherein the wavelength of the chlorotoxin crude peptide is 220 nm. And (3) carrying out secondary purification on the target product with the purity of more than 90%, wherein the mobile phases are 0.1% acetic acid aqueous solution and acetonitrile, the wavelength is 220nm, loading a sample after the primary purification of the chlorotoxin, eluting at a gradient change rate of one gradient every 12 minutes, collecting the target product, concentrating and freeze-drying to obtain a final finished product.
The invention has the beneficial effects that:
compared with the prior art, the invention adopts the method for synthesizing the chlorotoxin polypeptide by completely selectively forming four pairs of disulfide bonds: and (3) solid-phase synthesis of chlorotoxin polypeptide precursor resin, wherein a first pair of disulfide bonds are formed by solid-phase oxidation, a second pair of disulfide bonds are formed by solid-phase oxidation, a third pair of disulfide bonds are formed by solid-phase oxidation, and a fourth pair of disulfide bonds are formed by liquid-phase oxidation after a cleavage reaction is carried out to obtain chlorotoxin crude peptide.
The method avoids the generation of disulfide bond mismatching isomers by forming four pairs of disulfide bonds completely and selectively, can obtain crude peptide with higher purity, improves the total yield, and reduces the production cost; meanwhile, before the chlorotoxin precursor resin undergoes a cracking reaction, three pairs of disulfide bonds are formed by solid-phase oxidation, so that the difficulty in forming the disulfide bonds is reduced.
The fixed-point cyclization method provided by the invention has the advantages of high product purity, high yield, simple and easily-obtained raw materials, low cost, simple and stable process, suitability for large-scale production and the like.
Drawings
FIG. 1 is an HPLC chart of chlorotoxin depsipeptide obtained in example 1;
FIG. 2 is an HPLC chart of chlorotoxin depsipeptide obtained in example 2;
FIG. 3 is an HPLC chart of chlorotoxin depsipeptide obtained in example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to specific embodiments and the accompanying drawings. The experimental procedures used in the examples below are conventional, unless otherwise specified, and the materials, reagents, methods and apparatus used are conventional in the art, and those skilled in the art are commercially available.
The abbreviations used in the present invention have the meanings given in Table 1.
TABLE 1 meanings of abbreviations used in the present invention
Example 1
The method comprises the following steps: preparation of chlorotoxin polypeptide precursor resin
Removing Fmoc protection from resin: rinkAmideAMResin (333.3 g, 100mmol) with a degree of substitution of 0.30mmol/g was weighed into a reaction vessel, the resin was swollen with DMF (1.8L) for 30 minutes, and the solvent was drained. The resin was charged with 25% piperidine/DMF1.8L, reacted for 15 minutes, the solvent was drained, and the resin was washed 6 times with 1.8LDMF each time to remove Fmoc protection. Ninhydrin test resin is positive.
Fmoc-Arg(Pbf) 36 -OH coupling: 194.63g of Fmoc-Arg (Pbf) was weighed 36 -OH (300 mmol) is dissolved in 1.5LDMF solution, 40.53g HOBt (300 mmol) and DIC (46.5 mL) are added into the amino acid solution for activation for 3min, the mixture is added into a reaction kettle and reacted for 2h at room temperature, a small amount of resin is subjected to indene detection, and ninhydrin detection shows that the resin is negative. The reaction solution was drained and the resin was washed 2 times with 1.8LDMF each time by adding DMF. The resin was charged with 25% piperidine/DMF1.8L for 15 minutes, the solvent was drained and the resin was washed 6 times with DMF, 1.8LDMF each time, and the drainage was used directly in the next step.
Coupling Fmoc-AA-OH successively as Fmoc-Cys (Dpm) according to the above procedure 35 -OH、Fmoc-Leu 34 -OH、Fmoc-Cys(Acm) 33 -OH、Fmoc-Gln(Trt) 32 -OH、Fmoc-Pro 31 -OH、Fmoc-Gly 30 -OH、Fmoc-Tyr(tBu) 29 -OH、Fmoc-Cys(Trt) 28 -OH、Fmoc-Lys(Boc) 27 -OH、Fmoc-Gly 26 -OH、Fmoc-Arg(Pbf) 25 -OH、Fmoc-Gly 24 -OH、Fmoc-Lys(Boc) 23 -OH、Fmoc-Gly 22 -OH、Fmoc-Gly 21 -OH、Fmoc-Cys(Dpm) 20 -OH、Fmoc-Cys(Mtt) 19 -OH、Fmoc-Asp(tBu) 18 -OH、Fmoc-Asp(tBu) 17 -OH、Fmoc-Cys(Acm) 16 -OH、Fmoc-Lys(Boc) 15 -OH、Fmoc-Arg(Pbf) 14 -OH、Fmoc-Ala 13 -OH、Fmoc-Met 12 -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-Phe 6 -OH、Fmoc-Cys(Trt) 5 -OH、Fmoc-Pro 4 -OH、Fmoc-Met 3 -OH、Fmoc-Cys(Mtt) 2 -OH、Boc-Met 1 -OH. After the last amino acid coupling is finished, adding DCM to wash the resin for 6 times,the chlorotoxin polypeptide precursor resin was obtained by suction drying with 1.8LDCM each time.
Step two: removal of the protecting group Mtt
2L of 2% TFA/DCM (v/v) solution was prepared, the chlorotoxin polypeptide precursor resin obtained in step one was added, reaction was performed for 20min, the reaction solution was drained, 2L of 2% TFA/DCM (v/v) solution was added again, reaction was performed for 20min, the reaction solution was drained, and DMF was added and washed 6 times (2L/time) and drained.
Step three: preparation of Monodithiocyclopeptides
And (3) adding 2LDMSO into the Mtt-protecting-group-removed resin obtained in the step two, reacting for 5h, draining the reaction solution, adding DCM, washing for 6 times (2L/time), and draining.
Step four: removal of protecting group Trt
Preparing 2L of 15% TFA/DCM (v/v) solution, adding the chlorotoxin polypeptide precursor resin obtained in step three, reacting for 30min, draining the reaction solution, adding 2L of 15% TFA/DCM (v/v) solution again, reacting for 30min, draining the reaction solution, adding DMF, washing 6 times (2L/times), and draining.
Step five: preparation of dithiocyclopeptides
And (3) adding 2LDMSO into the resin which is obtained in the step four and is subjected to the removal of the protecting group Trt, reacting for 5 hours, draining the reaction solution, adding DMF, washing for 6 times (2L/time), and draining.
Step six: preparation of three pairs of disulfide bond cyclopeptides
Preparation of 80g/L of I 2 Adding 3L of DMF solution into the resin obtained in the fifth step, reacting for 3h at room temperature, draining the reaction solution, adding DMF, washing for 6 times (2L/time), draining, adding methanol (3L/time) for shrinking for 3 times (10 min each time), draining the methanol, and vacuum drying to obtain 920.57g of resin.
Step seven: removal of protecting group Dpm and cleavage resin
Preparing 9.2L of lysate TFA/EDT/water =95/2.5/2.5 (v/v), respectively measuring 8740mL of LTFA, 230mL of LEDT and 230mL of water, adding the obtained mixture into a reaction kettle, uniformly stirring, adding 920.57g of the resin obtained in the sixth step into the reaction kettle, reacting for 2 hours at room temperature, filtering the resin under reduced pressure, and collecting filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was added slowly to 50L of iced ether for precipitation, centrifuged, washed 5 times with iced ether and dried under vacuum to give 397.23g of crude peptide.
Step eight: preparation of four pairs of disulfide cyclic peptides
And (3) adding 397g of the crude peptide containing the three pairs of disulfide bonds obtained in the step seven into 397L of water for dissolving, after dissolving and clarification, adjusting the pH value to 6.8-7.2 by using 10% ammonia water, measuring 794mL of hydrogen peroxide solution with the mass fraction of 3% and adding the hydrogen peroxide solution into the crude peptide solution, stirring and reacting for 4 hours, adjusting the pH value to 4.0-5.0 by using acetic acid, and obtaining the chlorotoxin polypeptide containing the four pairs of disulfide bonds.
Step nine: chlorotoxin polypeptide purification
And (5) filtering the chlorotoxin polypeptide solution obtained in the step eight by using a 0.45-micron filter membrane, and purifying by adopting a reversed-phase high-pressure liquid chromatography. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% trifluoroacetic acid water solution/acetonitrile as a mobile phase, carrying out elution at a gradient change rate of every 10 minutes after the chlorotoxin crude peptide is loaded, and collecting a target product, wherein the wavelength of the chlorotoxin crude peptide is 220 nm. And (2) performing secondary purification on the target product with the purity of more than 90%, wherein the mobile phases are 0.1% acetic acid aqueous solution and acetonitrile, the wavelength is 220nm, sampling the sample after primary purification of the chlorotoxin, eluting at a gradient change rate of one gradient every 12 minutes, collecting the target product, concentrating and freeze-drying to obtain 118.19g of finished chlorotoxin product with the purity of 99.64% and the total yield of 29.58%. The HPLC chart of the final chlorotoxin product is shown in FIG. 1.
Total yield (%) = weight of finished product ÷ theoretical yield × 100%
Theoretical yield = synthesis scale (100 mmol) × molecular weight (3995.7 g/mol)/1000 =399.57g
Total yield (%) = finished product weight (118.19 g) ÷ theoretical yield (399.57 g) × 100% =29.58%
Example 2
The method comprises the following steps: preparation of chlorotoxin polypeptide precursor resin
Removing Fmoc protection from resin: rinkAmideAMResin (100.0 g, 100mmol) was weighed out at a substitution rate of 1.00mmol/g, charged into a reaction vessel, the resin was swollen with DMF (0.6L) for 30 minutes, and the solvent was drained. The resin was added with 25% piperidine/DMF0.6L, reacted for 15 minutes, the solvent was drained and DMF was added to wash the resin 6 times with 0.6LDMF each time to remove Fmoc protection. Ninhydrin test resin positive.
Fmoc-Arg(Pbf) 36 -OH coupling: 648.8g of Fmoc-Arg (Pbf) was weighed 36 dissolving-OH (1000 mmol) in 1.2LDMF solution, adding 135.1g HOBt (1000 mmol) and DIC (155 ml) into the amino acid solution for activation for 3min, adding into the reaction kettle, reacting at room temperature for 2 hr, performing indene detection on a small amount of resin, and detecting that the resin is negative by ninhydrin detection. The reaction solution was drained and DMF was added to wash the resin 2 times with 0.8LDMF each time. The resin was charged with 25% piperidine/DMF0.8L for 15 minutes, the solvent was drained and the resin was washed 6 times with DMF, 0.8LDMF each time, and the drainage was used directly in the next step.
Coupling Fmoc-AA-OH one by one according to the above operation to obtain Fmoc-Cys (Dpm) 35 -OH、Fmoc-Leu 34 -OH、Fmoc-Cys(Acm) 33 -OH、Fmoc-Gln(Trt) 32 -OH、Fmoc-Pro 31 -OH、Fmoc-Gly 30 -OH、Fmoc-Tyr(tBu) 29 -OH、Fmoc-Cys(Trt) 28 -OH、Fmoc-Lys(Boc) 27 -OH、Fmoc-Gly 26 -OH、Fmoc-Arg(Pbf) 25 -OH、Fmoc-Gly 24 -OH、Fmoc-Lys(Boc) 23 -OH、Fmoc-Gly 22 -OH、Fmoc-Gly 21 -OH、Fmoc-Cys(Dpm) 20 -OH、Fmoc-Cys(Mtt) 19 -OH、Fmoc-Asp(tBu) 18 -OH、Fmoc-Asp(tBu) 17 -OH、Fmoc-Cys(Acm) 16 -OH、Fmoc-Lys(Boc) 15 -OH、Fmoc-Arg(Pbf) 14 -OH、Fmoc-Ala 13 -OH、Fmoc-Met 12 -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-Phe 6 -OH、Fmoc-Cys(Trt) 5 -OH、Fmoc-Pro 4 -OH、Fmoc-Met 3 -OH、Fmoc-Cys(Mtt) 2 -OH、Boc-Met 1 -OH. After the last amino acid coupling, adding DCM to wash the resin for 6 times, each time using 0.8LDCM, suction drying to obtain chlorotoxin polypeptide precursor resin.
Step two: removal of the protecting group Mtt
Preparation of 1.0L of 5% TFA/DCM (v/v) solution, addition of the chlorotoxin polypeptide precursor resin obtained in step one, reaction for 10min, draining the reaction solution, addition of 1.0L of 5% TFA/DCM (v/v) solution again, reaction for 10min, draining the reaction solution, washing 6 times (1L/time) with DMF, and draining.
Step three: preparation of Monodithiocyclopeptides
And (3) adding 1.0LDMSO into the Mtt-protecting group-removed resin obtained in the step two, reacting for 20 hours, draining the reaction solution, adding DCM (1.0L/time) for washing for 6 times, and draining.
Step four: removal of protecting group Trt
Preparing 20% (v/v) solution 1L of TFA/DCM (v/v), adding the chlorotoxin polypeptide precursor resin obtained in step three, reacting for 10min, draining the reaction solution, adding 20% (v/v) solution 1L of TFA/DCM (v/v) again, reacting for 10min, draining the reaction solution, adding DMF, washing 6 times (1L/time), and draining.
Step five: preparation of Didithiocyclopeptides
And (3) adding 1LDMSO into the resin which is obtained in the step four and is subjected to the removal of the protecting group Trt, reacting for 20 hours, draining the reaction solution, adding DMF (dimethyl formamide) to wash for 6 times (1L/time), and draining.
Step six: preparation of three pairs of disulfide bond cyclopeptides
Preparation of 50g/L of I 2 Adding 3L of DMF solution into the resin obtained in the step five, reacting for 2h at room temperature, draining the reaction solution, adding DMF, washing for 6 times (1L/time), draining, adding methanol (1L/time) for shrinking for 3 times (10 min each time), draining the methanol, and drying in vacuum to obtain 718.99g of resin.
Step seven: protecting group removal Dpm and cracking resin
Preparing lysate TFA/EDT/water =90/5/5 (v/v) 7.2L, respectively measuring 6840ml TFA, 180ml EDT and 180ml water, adding into a reaction kettle, uniformly stirring, adding 718.99g of the resin obtained in the sixth step into the reaction kettle, reacting at room temperature for 1h, filtering the resin under reduced pressure, and collecting filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was added slowly to 40L of iced ether for precipitation, centrifuged, washed 5 times with iced ether and dried in vacuo to give 317.16g of crude peptide.
Step eight: preparation of four pairs of disulfide cyclic peptides
And (3) adding 317g of the crude peptide containing the three pairs of disulfide bonds obtained in the step seven into 317L of water for dissolving, after the crude peptide is dissolved and clarified, adjusting the pH value to 6.8-7.2 by using 10% ammonia water, measuring 794ml of hydrogen peroxide solution with the mass fraction of 3% and adding the hydrogen peroxide solution into the crude peptide solution, stirring and reacting for 3 hours, and adjusting the pH value to 4.0-5.0 by using acetic acid to obtain the chlorotoxin polypeptide containing the four pairs of disulfide bonds.
Step nine: chlorotoxin polypeptide purification
And (5) filtering the chlorotoxin polypeptide solution obtained in the step eight by using a 0.45-micron filter membrane, and purifying by adopting a reversed-phase high-pressure liquid chromatography. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% trifluoroacetic acid water solution/acetonitrile as a mobile phase, carrying out elution at a gradient change rate of every 10 minutes after the chlorotoxin crude peptide is loaded, and collecting a target product, wherein the wavelength of the chlorotoxin crude peptide is 220 nm. And (2) carrying out secondary purification on the target product with the purity of more than 90%, wherein the mobile phases are 0.1% acetic acid aqueous solution and acetonitrile, the wavelength is 220nm, loading a sample after primary purification of the chlorotoxin, eluting at a gradient change rate of one gradient every 12 minutes, collecting the target product, concentrating and freeze-drying to obtain 97.22g of finished chlorotoxin product with the purity of 98.05% and the total yield of 24.33%. The HPLC profile of the final chlorotoxin product is shown in FIG. 2.
Total yield (%) = finished product weight ÷ theoretical yield × 100%
Theoretical yield = synthesis scale (100 mmol) × molecular weight (3995.7 g/mol)/1000 =399.57g
Total yield (%) = finished product weight (97.22 g) ÷ theoretical yield (399.57 g) × 100% =24.33%
Example 3
The method comprises the following steps: preparation of chlorotoxin polypeptide precursor resin
Removing Fmoc protection from resin: rinkAmideAMresin (1000g, 100mmol) with substitution of 0.10mmol/g was weighed into a reaction kettle, the resin was swollen with DMF (5.5L) for 30 minutes, and the solvent was drained. The resin was added with 25% piperidine/DMF5.5L, reacted for 15 minutes, the solvent was drained and DMF was added to wash the resin 6 times with 5.5LDMF each time to remove Fmoc protection. Ninhydrin test resin is positive.
Fmoc-Arg(Pbf) 36 -OH coupling: 129.75g of Fmoc-Arg (Pbf) were weighed 36 dissolving-OH (200 mmol) in 5.5LDMF solution, adding 27.02g HOBt (200 mmol) and DIC (31 ml) into amino acid solution for activation for 3min, adding into reaction kettle, reacting at room temperature for 2 hr, collecting small amount of resinThe resin is negative in ninhydrin test and indene test. The reaction was drained and the resin was washed 2 times with 5.5LDMF each time by adding DMF. The resin was added with 25% piperidine/DMF5.5L, reacted for 15 minutes, the solvent was drained, the resin was washed 6 times with DMF, 5.5LDMF each time, and the drain was used directly in the next reaction.
Coupling Fmoc-AA-OH one by one according to the above operation to obtain Fmoc-Cys (Dpm) 35 -OH、Fmoc-Leu 34 -OH、Fmoc-Cys(Acm) 33 -OH、Fmoc-Gln(Trt) 32 -OH、Fmoc-Pro 31 -OH、Fmoc-Gly 30 -OH、Fmoc-Tyr(tBu) 29 -OH、Fmoc-Cys(Trt) 28 -OH、Fmoc-Lys(Boc) 27 -OH、Fmoc-Gly 26 -OH、Fmoc-Arg(Pbf) 25 -OH、Fmoc-Gly 24 -OH、Fmoc-Lys(Boc) 23 -OH、Fmoc-Gly 22 -OH、Fmoc-Gly 21 -OH、Fmoc-Cys(Dpm) 20 -OH、Fmoc-Cys(Mtt) 19 -OH、Fmoc-Asp(tBu) 18 -OH、Fmoc-Asp(tBu) 17 -OH、Fmoc-Cys(Acm) 16 -OH、Fmoc-Lys(Boc) 15 -OH、Fmoc-Arg(Pbf) 14 -OH、Fmoc-Ala 13 -OH、Fmoc-Met 12 -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-Phe 6 -OH、Fmoc-Cys(Trt) 5 -OH、Fmoc-Pro 4 -OH、Fmoc-Met 3 -OH、Fmoc-Cys(Mtt) 2 -OH、Boc-Met 1 -OH. After the last amino acid coupling, adding DCM to wash the resin for 6 times, using 5.5LDCM each time, and pumping to dry to obtain chlorotoxin polypeptide precursor resin.
Step two: removal of the protecting group Mtt
Preparation of 6L% TFA/DCM (v/v) solution, addition of the chlorotoxin polypeptide precursor resin obtained in step one, reaction for 30min, draining the reaction solution, addition of 6L solution 4% TFA/DCM (v/v) again, reaction for 30min, draining the reaction solution, washing 6 times (6L/times) with DMF, and draining.
Step three: preparation of Monodithiocyclopeptides
And (3) adding 6LDMSO into the Mtt-protecting-group-removed resin obtained in the step two, reacting for 1h, draining the reaction solution, adding DCM, washing for 6 times (6L/time), and draining.
Step four: removal of protecting group Trt
Preparing 6L of 10% TFA/DCM (v/v) solution, adding the chlorotoxin polypeptide precursor resin obtained in step three, reacting for 20min, draining the reaction solution, adding again 20% TFA/DCM (v/v) solution 6L, reacting for 20min, draining the reaction solution, washing 6 times (6L/times) with DMF, and draining.
Step five: preparation of dithiocyclopeptides
And (3) adding 6LDMSO into the resin which is obtained in the step four and is subjected to the removal of the protecting group Trt, reacting for 1h, draining the reaction solution, adding DMF, washing for 6 times (6L/time), and draining.
Step six: preparation of three pairs of disulfide bond cyclopeptides
Preparation of 100g/L of I 2 Adding 6L of DMF solution into the resin obtained in the fifth step, reacting for 5h at room temperature, draining the reaction solution, adding DMF, washing for 6 times (6L/time), draining, adding methanol (6L/time) for shrinking for 3 times (10 min each time), draining the methanol, and drying in vacuum to obtain 1480.02g of resin.
Step seven: protecting group removal Dpm and cracking resin
Preparing lysate TFA/EDT/water =92/4/4 (v/v) 14.8L, respectively measuring 14060ml TFA, 370ml EDT and 370ml water, adding into a reaction kettle, uniformly stirring, adding 1480.02g of the resin obtained in the step six into the reaction kettle, reacting at room temperature for 5h, filtering the resin under reduced pressure, and collecting filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was added slowly to 80L of iced ether for precipitation, centrifuged, washed 5 times with iced ether and dried in vacuo to give 295.22g of crude peptide.
Step eight: preparation of four pairs of disulfide cyclic peptides
And (4) adding 295g of the crude peptide containing the three pairs of disulfide bonds obtained in the seventh step into 295L of water for dissolving, after the crude peptide is dissolved and clarified, adjusting the pH to 6.8-7.2 by using 10% ammonia water, measuring 794ml of hydrogen peroxide solution with the mass fraction of 3% and adding the hydrogen peroxide solution into the crude peptide solution, stirring and reacting for 5 hours, and adjusting the pH to 4.0-5.0 by using acetic acid to obtain the chlorotoxin polypeptide containing the four pairs of disulfide bonds.
Step nine: chlorotoxin polypeptide purification
And (5) filtering the chlorotoxin polypeptide solution obtained in the step eight by using a 0.45-micron filter membrane, and purifying by adopting a reversed-phase high-pressure liquid chromatography. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% trifluoroacetic acid water solution/acetonitrile as a mobile phase, carrying out elution at a gradient change rate of every 10 minutes after the chlorotoxin crude peptide is loaded, and collecting a target product, wherein the wavelength of the chlorotoxin crude peptide is 220 nm. And (2) carrying out secondary purification on a target product with the purity of more than 90%, wherein the mobile phase is 0.1% acetic acid water solution and acetonitrile, the wavelength is 220nm, loading a sample after primary purification of the chlorotoxin, eluting at a gradient change rate of one gradient every 12 minutes, collecting the target product, concentrating and freeze-drying to obtain 77.21g of finished chlorotoxin product with the purity of 99.10% and the total yield of 19.32%. The HPLC profile of the final chlorotoxin product is shown in FIG. 3.
Total yield (%) = finished product weight ÷ theoretical yield × 100%
Theoretical yield = synthesis scale (100 mmol) × molecular weight (3995.7 g/mol)/1000 =399.57g
Total yield (%) = finished product weight (77.21 g) ÷ theoretical yield (399.57 g) × 100% =19.32%
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A site-directed cyclization method of chlorotoxin polypeptide, wherein the site-directed cyclization method comprises the following steps:
1) Preparing chlorotoxin polypeptide precursor resin: fmoc-Arg (pbf) -OH reacts with carrier resin to obtain Fmoc-Arg (pbf) -resin, the Fmoc-Arg (pbf) -resin is taken as a solid phase carrier, fmoc-AA-OH is coupled in a one-by-one coupling mode according to the sequence from C end to N end in the presence of an activation system to obtain chlorotoxin polypeptide precursor resin, wherein Cys side chains corresponding to the 2 nd and 19 th sites of chlorotoxin polypeptide are protected by Mtt, cys side chains corresponding to the 5 th and 28 th sites of chlorotoxin polypeptide are protected by Trt, cys side chains corresponding to the 16 th and 33 th sites of chlorotoxin polypeptide are protected by Acm, and Cys side chains corresponding to the 20 th and 35 th sites of chlorotoxin polypeptide are protected by Dpm;
2) Removing the Mtt protecting group of the chlorotoxin polypeptide precursor resin obtained in the step 1) by using a deprotection agent;
3) Oxidizing the chlorotoxin polypeptide precursor resin obtained in the step 2) by using an oxidizing agent to form a first pair of disulfide bonds, so as to obtain chlorotoxin polypeptide precursor resin containing a single disulfide bond;
4) Removing the Trt protecting group of the chlorotoxin polypeptide precursor resin containing the dithio-rings obtained in the step 3) by using a deprotection agent;
5) Oxidizing the mono-disulfide cyclopeptide obtained in the step 4) by using an oxidizing agent to form a second pair of disulfide bonds to obtain a di-disulfide cyclopeptide;
6) Removing the Acm protecting group of Cys in the double-disulfide cyclic peptide obtained in the step 5) by using a deprotection agent, and oxidizing to form a third pair of disulfide bonds;
7) Cutting the chlorotoxin polypeptide precursor resin containing three pairs of disulfide rings obtained in the step 6) by using a lysis solution, and simultaneously removing a Dpm protective group;
8) Oxidizing the three pairs of disulfide cyclic peptides obtained in the step 7) by using an oxidizing agent to form a fourth pair of disulfide bonds, so as to obtain chlorotoxin polypeptides containing the four pairs of disulfide cyclic peptides;
the amino acid sequence of the polypeptide containing the four pairs of disulfide cyclopeptide chlorotoxin is shown in SEQ ID NO. 1;
disulfide bonds in Cys 2-19 、Cys 5-28 、Cys 16-33 And Cys 20-35 ；
Cys as a base 2 And Cys 19 The monomer of (1) is Fmoc-Cys (Mtt) -OH;
cys as mentioned 5 And Cys 28 The monomer of (A) is Fmoc-Cys (Trt) -OH;
cys as a base 16 And Cys 33 The monomer of (A) is Fmoc-Cys (Acm) -OH;
cys as a base 20 And Cys 35 The monomer of (a) is Fmoc-Cys (Dpm) -OH.
2. The site-directed cyclization method of claim 1, wherein said carrier resin of step 1) is Rink amidemamrein or Rink amidebbharesin.
3. The site-directed cyclization method of claim 1, wherein the activation system in step 1) is any one of HOBt/DIC, HBTU/HOBt/DIPEA and TBTU/HOBt/DIPEA.
4. The site-directed cyclization method of claim 1, wherein the deprotecting agent in step 2) is a mixed solution of TFA and DCM, and the volume ratio of TFA to DCM is (1-5): (95-99).
5. The site-directed cyclization method of claim 1, wherein the deprotection agent in step 4) is a mixed solution of TFA and DCM, and the volume ratio of TFA to DCM (10-20): (80 to 90).
6. The site-directed cyclization method according to claim 1, wherein the oxidizing agent in step 3) and step 5) is DMSO.
7. The site-directed cyclization method according to claim 1, wherein said deprotection agent in step 6) comprises I 2 DMF solution of (1), I 2 The mass fraction in DMF is 1-10 wt%.
8. The method of claim 1, wherein the lysis solution in step 7) is TFA, a mixture of EDT and water, and the volume ratio of EDT to water is (90-95): (2-5): (2-5).
9. The site-directed cyclization method according to claim 1, wherein the oxidizing agent in step 8) is a hydrogen peroxide solution with a mass fraction of 3%, and the feed-to-solution ratio of the trisdithiocyclic peptide obtained in step 7) to the hydrogen peroxide solution is 1g: (0.5-5) mL.
10. A chlorotoxin polypeptide produced by the site-directed cyclization method of any of claims 1-9.
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