A large-scale preparation method of conopeptide

Abstract

The invention discloses a large-scale preparation method of cono peptide. The large-scale preparation method of cono peptide of the invention comprises the preparation of linear peptide amino resin, cracking, linear peptide folding, crude peptide purification, using amino resin with a substitution degree of 0.4-0.8 mmol / g, sequentially coupling amino acid synthesis protected twenty-two peptide amino resin through solid phase synthesis, cracking; oxidizing one-step folding to form three pairs of disulfide bonds through cysteine ​​hydrochloride / DMSO buffer solution oxidation system, simple operation, low cost, suitable for industrial mass production; crude peptide is prepared and purified by HPLC to obtain cono peptide with a purity greater than 98%.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide drugs, and particularly relates to a large-scale synthesis and purification method of cono peptide. Background Art

[0002] Conotoxin CTX, also known as conotoxin peptide, is a type of small molecule polypeptide, belonging to neurotoxin. Conotoxin CTX is a component of the venom secreted by carnivorous marine molluscs of the family Conidae, Gastropoda. It is a weapon used by cone snails to paralyze prey and defend against external enemies. Each cone snail venom may contain 50 to 200 small molecule polypeptides.

[0003] Cono peptides are small molecule polypeptides expressed through signal peptide mediation. They are the smallest discovered nucleic acid-encoded animal neurotoxin peptides. Most of them are composed of 12 to 46 amino acid residues and contain 2 or 3 pairs of disulfide bonds. Their high-density disulfide bond structure greatly improves their stability, allowing cono peptides to maintain their activity in various physical and chemical environments.

[0004] Conotoxin, also known as Mu-conotoxin in English, has the following peptide sequence: pGlu 1 -Gly 2 -Cys 3 -Cys 4 -Asn 5 -Gly 6 -Pro 7 -Lys 8 -Gly 9 -Cys 10 -Ser 11 -Lys 12 -Ser 13 -Trp 14 -Cys 15 -Arg 16 -Asp 17 -His 18 -Ala 19 -Arg 20 -Cys 21 -Cys 22 -NH2, disulfide bond located at Cys 3 -Cys 15 、Cys 4 -Cys 21 and Cys 10 -Cys 22 Location.

[0005] At present, there are three main methods for preparing conotoxins, namely biological tissue extraction, chemical synthesis, and genetic engineering preparation. Among them, the yield of biological tissue extraction is limited, and the process of directly separating and extracting conotoxins from conus is complicated, difficult to extract, and costly; the genetic engineering preparation process is not mature, and both are difficult to achieve the industrialization of conotoxins and cannot meet market demand. Therefore, it is necessary to synthesize them in large quantities through solid-phase peptide synthesis to reduce costs in order to meet scientific research purposes and the growing market demand.

[0006] Prior art, such as publication number CN110894225A, discloses a large-scale preparation method of cono peptide, which sequentially couples peptide resins according to the amino acid sequence from the C-terminus to the N-terminus of the main chain of cono peptide, and then uses air oxidation to synthesize three pairs of disulfide bonds after cleavage, and finally purifies to prepare the product. This method uses air oxidation, the reaction time is too long, and the final yield is only 10%.

[0007] Patent CN116655765A discloses a liquid phase synthesis method of conotoxin, which uses 2,6-dioctadecyloxybenzyl alcohol as a carrier, including using Fmoc-Cys(MeBzl)-OH as a starting amino acid, which is activated and then esterified and condensed with the carrier to form Fmoc-Cys(MeBzl)-shs; after removing the Fmoc protecting group, it is condensed with the activated Fmoc-Cys(Acm)-OH, and after further removing the Fmoc protecting group, it is condensed with other Fmoc-amino acids to obtain a protected shs-conotoxin linear peptide, wherein Cys 3 、Cys 15 Protected with Trt group, Cys 4 Protected with Acm group, and Cys 10 The protected shs-conotoxin linear peptide is cleaved, and the protecting groups Trt, Acm and MeBzl of Cys are removed in sequence, and an oxidation reaction is performed after each removal of the protecting groups to prepare the conotoxin. The process of this method is relatively cumbersome and not suitable for large-scale production. Summary of the invention

[0008] In view of the above-mentioned defects in the prior art, the present invention provides a novel method for preparing conopeptide which has low cost, simple operation, mild reaction conditions and is conducive to industrialization.

[0009] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0010] A large-scale preparation method of conopeptide comprises: preparing conopeptide resin by solid phase synthesis method, cracking the conopeptide resin to obtain crude conopeptide line peptide, cyclizing and purifying the crude conopeptide line peptide to obtain conopeptide; in the solid phase synthesis method, coupling an amino acid reagent to an amino resin by a coupling agent;

[0011] The coupling agent comprises a combination of DIC and compound A, or the coupling agent comprises a combination of a base, compound A and compound B;

[0012] Compound A is at least one of Oxyma, a pyridine heterocyclic compound, HOBt and HOAt, and the pyridine heterocyclic compound has a pyridine group and an N heterocyclic group; wherein, when the pyridine heterocyclic compound is used as compound A to participate in the preparation of cono peptide, the yield of cono peptide is increased to 4.43 g, and the yield of cono peptide is increased to 31.26%. Oxyma is a new coupling agent, and the cono peptide resin generated in the reaction has high reactivity, which makes it show excellent effect in polypeptide synthesis. In the present invention, the yield and yield of cono peptide are significantly improved by replacing Oxyma with a pyridine heterocyclic compound.

[0013] Compound B is at least one of DIC, PyBOP, PyAOP, HATU, HBTU and TBTU, and the base is at least one of DIPEA, TEA, 2,4,6-TMP and NMM.

[0014] Preferably, the coupling agent is DIC and a pyridine heterocyclic compound; or, the coupling agent is DIC and a pyridine heterocyclic compound and a base.

[0015] Preferably, in the preparation of the pyridine heterocyclic compound, 3-aminopyridine-2-carboxylic acid is first reacted with benzyloxyamine hydrochloride to generate intermediate 1, and then intermediate 1 is reacted with acetaldehyde to generate intermediate 2; finally, benzyloxyamine is removed by catalytic hydrogenation to obtain the pyridine heterocyclic compound.

[0016] More preferably, 3-aminopyridine-2-carboxylic acid and benzyloxyamine hydrochloride are reacted in DCM containing TEA and EDCI, the amount of TEA is 0.5 to 2 molar parts, the amount of EDCI is 0.05 to 1 molar parts, and the amount of DCM is 2 to 8 molar parts.

[0017] More preferably, the intermediate 1 reacts with acetaldehyde in acetic acid, the amount of acetaldehyde used is 0.5 to 2 parts by mole, and the amount of acetic acid used is 0.5 to 2 parts by mole.

[0018] Preferably, the amount of compound A is 10-70 mole parts; or, the amount of compound B is 50-70 mole parts; or, the amount of base is 50-120 mole parts, and the degree of substitution on the amino resin is 0.4-0.8 mmol / g.

[0019] Preferably, the amino resin is AM Resin or MBHA Resin.

[0020] Preferably, a lysis solution is used in the lysis treatment, the lysis solution consists of TFA, EDT and Tis, and the volume ratio of TFA, EDT and Tis in the lysis solution is 80-90:5-10:5-10.

[0021] Preferably, in the preparation of the crude conoline peptide, isopropyl ether is used for precipitation.

[0022] Preferably, a buffer solution containing cysteine ​​hydrochloride is used in the cyclization treatment of the crude conoline peptide, wherein the buffer solution contains cysteine ​​hydrochloride, DMSO and water, the molar concentration of cysteine ​​hydrochloride is 0.1-5 mmol / L, and the volume percentage concentration of DMSO is 5-25%.

[0023] The preparation method of the present invention is to couple other amino acids of Fmoc protective groups through amino resin in a one-to-one coupling mode to obtain conopeptide peptide resin. The conopeptide peptide resin is subjected to a cleavage reaction to obtain a crude conopeptide line peptide. The three disulfide bonds in the crude conopeptide line peptide are cyclized, and the conopeptide is obtained through purification, freeze-drying and other steps under HPLC. Not only the yield of conopeptide is effectively improved, but also the method is simple and can realize the large-scale production of conopeptide.

[0024] Preferably, the preparation method of conopeptide is specifically as follows:

[0025] The crude peptide of cono peptide line is taken, dissolved and diluted with a buffer solution, the pH is adjusted with ammonia water, an oxidative folding reaction is carried out, the reaction end point is monitored by HPLC, and the cono peptide is obtained through purification and freeze-drying.

[0026] Preferably, in the method for preparing conopeptide, the mass of the crude conopeptide peptide is 100-200 parts by weight.

[0027] Preferably, in the method for preparing conopeptide, the molar concentration of cysteine ​​hydrochloride is 0.1-5 mmol / L.

[0028] Preferably, in the method for preparing conopeptide, the volume percentage concentration of DMSO is 5-25%.

[0029] Preferably, in the preparation method of conopeptide, the crude conopeptide peptide is dissolved and diluted to 1.0-5.0 mg / ml.

[0030] Preferably, in the method for preparing conopeptide, the pH is 7.5-9.0.

[0031] Preferably, the method for preparing the crude peptide of cono-spi peptide line is specifically as follows:

[0032] Take cono peptide resin, add a lysis solution mixed with TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at a certain temperature, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake with TFA, combine the filtrate, add the filtrate to isopropyl ether for precipitation, wash with isopropyl ether, and dry under vacuum to constant weight to obtain a crude cono peptide line peptide.

[0033] Preferably, in the method for preparing the crude cono-spigot peptide, the volume percentages of the components in the lysate are: TFA: 85-95%; Tis: 3-8%; EDT: 3-8%.

[0034] Preferably, in the method for preparing crude conoline peptide, the amount of lysate used is 6-10 mL / g.

[0035] Preferably, in the method for preparing crude cono-spiky peptide, the volume ratio of TFA, EDT and Tis in the lysate is 80-90:5-10:5-10.

[0036] Preferably, in the method for preparing crude conoline peptide, the reaction temperature is 20-25° C. and the reaction time is 2.0-4.0 h.

[0037] Preferably, in the method for preparing crude cono-peptide peptide, the temperature of the isopropyl ether used for precipitation is -10~0°C.

[0038] More preferably, the preparation method of conopeptide resin is specifically as follows:

[0039] The resin is added to swell, and the Fmoc protecting group is removed by reaction under the Fmoc deprotection agent, the resin is washed with a solvent, and the activated amino acid is added. Under the action of the coupling system, the amino acids are connected in sequence from the carboxyl end to the amino end in the coupling solvent according to the peptide sequence for coupling. After the reaction, the resin is washed and dried to obtain a twenty-two peptide amino resin. Specifically, according to the sequence of the cono peptide, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH are added in sequence. , Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Boc-Pyr-OH, until all amino acids are connected, shrink the resin with DCM and methanol, and dry it to obtain cono peptide resin.

[0040] Preferably, in the preparation method of conopeptide resin, the amino resin is AM Resin, MBHA Resin or other resins with equivalent functions.

[0041] Preferably, in the method for preparing conopeptide resin, the amount of amino resin used is 20 to 50 parts by weight.

[0042] Preferably, in the preparation method of conopeptide resin, the substitution degree of amino resin is 0.4-0.8 mmol / g.

[0043] Preferably, in the preparation method of conopeptide resin, DMF is used in swelling in an amount of 120 to 300 parts by volume, and the amount of amino resin and DMF used is in a ratio of 50 g:250 mL.

[0044] Preferably, in the preparation method of conopeptide resin, DMF is used in washing in an amount of 120 to 300 parts by volume.

[0045] Preferably, in the preparation method of the cono peptide resin, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Bo The amount of c-Pyr-OH used is 50 to 70 parts by mole, and the amount of Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc -The usage amount relationship of Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Boc-Pyr-OH and DMF is 60mmol:250mL.

[0046] Preferably, in the preparation method of cono peptide resin, the coupling agent is a composition of DIC and compound A. Compound A is Oxyma, a pyridine heterocyclic compound, salicylic acid, HOBt or HOAt. When salicylic acid is involved in the preparation of cono peptide, the yield and yield of cono peptide are significantly improved. This is mainly attributed to the fact that salicylic acid participates in the condensation reaction as an intermediate, and because of the presence of hydroxyl and oxime functional groups, it can interact with other functional groups to promote the esterification reaction, which is beneficial to the preparation of cono peptide resin, so that the yield and yield of cono peptide are significantly improved.

[0047] Preferably, in the preparation method of cono peptide resin, the coupling agent is a composition of a base and compound A and compound B, wherein compound A is Oxyma, a pyridine heterocyclic compound, salicylic hydroxamic acid, HOBt or HOAt, and compound B is DIC, PyBOP, PyAOP, HATU, HBTU or TBTU.

[0048] Preferably, in the method for preparing conopeptide resin, the base is at least one of DIPEA, TEA, 2,4,6-TMP and NMM.

[0049] Preferably, in the method for preparing conopeptide resin, the amount of compound A used is 10 to 70 parts by mole.

[0050] Preferably, in the method for preparing conopeptide resin, the amount of compound B used is 50 to 70 parts by mole.

[0051] Preferably, in the method for preparing conopeptide resin, the amount of base used is 50 to 120 parts by mole.

[0052] Preferably, in the method for preparing cono peptide resin, the coupling temperature is 20-35° C. and the reaction time is 90-120 min.

[0053] Preferably, in the preparation method of cono peptide resin, the amount of DCM used in the shrinkage is 50-350 parts by volume, and the amount of methanol used in the shrinkage is 50-350 parts by volume.

[0054] More preferably, the preparation method of the pyridine heterocyclic compound is specifically as follows:

[0055] Dissolve 3-aminopyridine-2-carboxylic acid in DCM, add TEA and EDCI, stir at room temperature and add benzyloxyamine hydrochloride overnight, and monitor by TLC. After the reaction is complete, wash with water and saturated brine, dry over anhydrous sodium sulfate, spin dry the solvent and pass through a column to obtain intermediate 1. Then add acetaldehyde and acetic acid and react overnight at a certain temperature until the reaction is complete, monitor by TLC, and spin dry the system to obtain intermediate 2. After dissolving the intermediate in methanol, add Pd / C, connect a hydrogen bag and heat the reaction at a certain temperature overnight, and detect the reaction by TLC. Filter, spin dry the solvent and pass through a column to obtain a pyridine heterocyclic compound.

[0056] Preferably, in the method for preparing the pyridine heterocyclic compound, the amount of 3-aminopyridine-2-carboxylic acid used is 0.5 to 2 molar parts.

[0057] Preferably, in the method for preparing the pyridine heterocyclic compound, the amount of DCM used is 2 to 8 parts by mole.

[0058] Preferably, in the method for preparing the pyridine heterocyclic compound, the amount of TEA used is 0.5 to 2 parts by mole.

[0059] Preferably, in the method for preparing the pyridine heterocyclic compound, the amount of EDCI used is 0.05 to 1 molar part.

[0060] Preferably, in the method for preparing the pyridine heterocyclic compound, the amount of acetaldehyde used is 0.5 to 2 parts by mole.

[0061] Preferably, in the method for preparing the pyridine heterocyclic compound, the amount of acetic acid used is 0.5 to 2 molar parts.

[0062] When PE and EA are mixed in a column system as eluents for column chromatography separation, the volume ratio of EA and PE in the column system changes gradually, and the volume ratio is 1:2~50.

[0063] Preferably, in the method for preparing the pyridine heterocyclic compound, the reaction temperature is 40-60°C.

[0064] The present invention adopts a one-by-one coupling mode to couple other amino acids of the Fmoc protective group to obtain a cono peptide resin, and then obtains the cono peptide through steps such as cyclization, purification and freeze drying, so it has the following beneficial effects: the cono peptide of the present invention has low cost, relatively simple operation steps, relatively mild reaction conditions and is conducive to the industrialization of the cono peptide. Therefore, the present invention is a novel cono peptide preparation method with low cost, simple operation, mild reaction conditions and conducive to the industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The target compound is conopeptide (molecular formula C 92 H 139 N 35 O 28 S6) Schematic diagram of mass spectrometry. DETAILED DESCRIPTION

[0066] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0068] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0069] The materials and reagents used in the following examples represent the following meanings:

[0070]

[0071] Embodiment 1:

[0072] The invention discloses a preparation method of conopeptide peptide resin.

[0073] First, Rink-AmideAM Resin was swollen with 250ml DMF for 60 min. Then, it was deprotected with 250ml 20% Pip / DMF solution for 30 min, and the resin was washed with DMF 6 times, with the amount of DMF used each time being 250ml; Fmoc-Cys(Trt)-OH and Oxyma were dissolved with 250ml DMF and cooled to 0℃, and DIC was taken and slowly added under stirring, and continued to stir for 10min to obtain the activation solution. Finally, the activation solution was added to the deprotected resin and reacted at 25℃ for 100min. The end point of the reaction was determined by ninhydrin detection. After the reaction, the resin was washed with DMF three times, with the amount of DMF used each time being 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 47.62 g, 20 mmol, the degree of substitution is 0.42 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of Oxyma used is 60 mmol, and the amount of DIC used is 60 mmol.

[0074] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 152.61 g of cono peptide resin.

[0075] Table 1 Cono peptide amino acid coupling sequence

[0076]

[0077] Embodiment 2:

[0078] The invention discloses a preparation method of conopeptide peptide resin.

[0079] First, Rink-AmideAM Resin was swollen with 250ml DMF for 60 min. Then, it was deprotected with 250ml 20% Pip / DMF solution for 30 min, and the resin was washed with DMF 6 times, with the amount of DMF used each time being 250ml; Fmoc-Cys(Trt)-OH and Oxyma were dissolved with 250ml DMF and cooled to 0℃, and DIC was taken and slowly added under stirring, and continued to stir for 10min to obtain the activation solution. Finally, the activation solution was added to the deprotected resin and reacted at 25℃ for 100min. The end point of the reaction was determined by ninhydrin detection. After the reaction, the resin was washed with DMF three times, with the amount of DMF used each time being 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 34.48 g, 20 mmol, the degree of substitution is 0.58 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of Oxyma used is 60 mmol, and the amount of DIC used is 60 mmol.

[0080] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 140.47 g of cono peptide resin.

[0081] Embodiment 3:

[0082] The invention discloses a preparation method of conopeptide peptide resin.

[0083] First, Rink-AmideAM Resin was swollen with 250ml DMF for 60 min. Then, it was deprotected with 250ml 20% Pip / DMF solution for 30 min, and the resin was washed with DMF 6 times, with the amount of DMF used each time being 250ml; Fmoc-Cys(Trt)-OH and Oxyma were dissolved with 250ml DMF and cooled to 0℃, and DIC was taken and slowly added under stirring, and continued to stir for 10min to obtain the activation solution. Finally, the activation solution was added to the deprotected resin and reacted at 25℃ for 100min. The end point of the reaction was determined by ninhydrin detection. After the reaction, the resin was washed with DMF three times, with the amount of DMF used each time being 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 25.32 g, 20 mmol, the degree of substitution is 0.79 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of Oxyma used is 60 mmol, and the amount of DIC used is 60 mmol.

[0084] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 130.31 g of cono peptide resin.

[0085] Embodiment 4:

[0086] The invention discloses a preparation method of conopeptide peptide resin.

[0087] First, Rink-AmideAM Resin was swollen with 250ml DMF for 60 min. Then, it was deprotected with 250ml 20% Pip / DMF solution for 30 min, and the resin was washed with DMF 6 times, with the amount of DMF used each time being 250ml; Fmoc-Cys(Trt)-OH and HOBt were dissolved with 250ml DMF and cooled to 0℃, and DIC was taken and slowly added under stirring, and continued to stir for 10min to obtain the activation solution. Finally, the activation solution was added to the deprotected resin and reacted at 25℃ for 100min. The end point of the reaction was determined by ninhydrin detection. After the reaction, the resin was washed with DMF three times, with the amount of DMF used each time being 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 34.49 g, 20 mmol, the degree of substitution is 0.58 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of HOBt used is 60 mmol, and the amount of DIC used is 60 mmol.

[0088] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 139.22 g of cono peptide resin.

[0089] Embodiment 5:

[0090] A method for preparing conopeptide resin.

[0091] First, Rink-AmideAM Resin was swollen with 250ml DMF for 60 min. Then, it was deprotected with 250ml 20% Pip / DMF solution for 30 min, and the resin was washed with DMF 6 times, with the amount of DMF used each time being 250ml; Fmoc-Cys(Trt)-OH, HOBt and DIPEA were dissolved with 250ml DMF and cooled to 0℃, and HBTU was taken and slowly added under stirring, and continued to stir for 10min to obtain the activation solution. Finally, the activation solution was added to the deprotected resin and reacted at 25℃ for 100min. The end point of the reaction was determined by ninhydrin detection. After the reaction, the resin was washed with DMF three times, with the amount of DMF used each time being 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 34.50 g, 20 mmol, the degree of substitution is 0.58 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of HOBt used is 60 mmol, the amount of HBTU used is 60 mmol, and the amount of DIPEA used is 120 mmol.

[0092] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 138.95 g of cono peptide resin.

[0093] Embodiment 6:

[0094] A method for preparing conopeptide resin.

[0095] First, Rink-AmideAM Resin was swollen with 250ml DMF for 60 min. Then, it was deprotected with 250ml 20% Pip / DMF solution for 30 min, and the resin was washed with DMF 6 times, with the amount of DMF used each time being 250ml; Fmoc-Cys(Trt)-OH, HOBt and NMM were dissolved with 250ml DMF and cooled to 0℃, and HBTU was taken and slowly added under stirring, and continued to stir for 10min to obtain the activation solution. Finally, the activation solution was added to the deprotected resin at 25℃ for 100min. The end point of the reaction was determined by ninhydrin detection. After the reaction, the resin was washed with DMF three times, with the amount of DMF used each time being 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 34.48 g, 20 mmol, the degree of substitution is 0.58 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of HOBt used is 60 mmol, the amount of HBTU used is 60 mmol, and the amount of NMM used is 120 mmol.

[0096] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 139.76 g of cono peptide resin.

[0097] Embodiment 7:

[0098] A method for preparing conopeptide resin.

[0099] First, 3-aminopyridine-2-carboxylic acid was dissolved in DCM, TEA and EDCI were added, and benzyloxyamine hydrochloride was added after stirring at room temperature for 10 min. The reaction was allowed to proceed overnight at room temperature and monitored by TLC. After the reaction was complete, the organic phase was washed with water and saturated brine, and then the organic phase was dried with anhydrous sodium sulfate, and the solvent was dried by spin column. The column system was polar PE:EA from 5:1 to 2:1 to obtain a white solid intermediate 1; then, acetaldehyde and acetic acid were added to the intermediate 1 and reacted at 55°C overnight until the reaction was complete, and monitored by TLC. After the reaction was completed, the system was dried by spin column and passed through the column to obtain a white solid intermediate 2. The column system was polar PE:EA from 50:1 to 20:1. Finally, the intermediate 2 was dissolved in methanol, Pd / C was added, and a hydrogen bag was connected to heat the reaction at 50°C overnight, and the reaction was detected by TLC. After the reaction was completed, the mixture was filtered, the solvent was dried by spin column, and the pyridine heterocyclic compound was obtained. The column system was polar PE:EA from 5:1 to 2:1.

[0100] The amount of 3-aminopyridine-2-carboxylic acid used is 1 mol, the amount of DCM used is 5 mol, the amount of TEA used is 1 mol, the amount of EDCI used is 0.1 mol, the amount of acetaldehyde used is 1 mol, and the amount of acetic acid used is 1 mol. When PE and EA are used as eluents for column chromatography separation in the column system, the ratio of the eluent gradually changes from 5:1 to 2:1.

[0101] The preparation of cono peptide resin is the same as in Example 1, except that oxyma is replaced by the pyridine heterocyclic compound of this example. First, Rink-AmideAM Resin is swollen with 250ml DMF for 60 min. Then, deprotect with 250ml 20% Pip / DMF solution for 30 min, wash the resin with DMF 6 times, and the amount of DMF used each time is 250ml; take Fmoc-Cys(Trt)-OH and pyridine heterocyclic compound, dissolve with 250ml DMF and cool to 0°C, take DIC, add slowly under stirring, and continue stirring for 10min to obtain activation solution. Finally, add the activation solution to the deprotected resin at 25°C for 100min. The end point of the reaction is based on the ninhydrin test. After the reaction, wash the resin with DMF three times, and the amount of DMF used each time is 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 47.62 g, 20 mmol, the degree of substitution is 0.42 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of DIC used is 60 mmol, and the amount of pyridine heterocyclic compound used is 20 mmol.

[0102] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 159.35 g of cono peptide resin.

[0103] Embodiment 8:

[0104] A method for preparing conopeptide resin.

[0105] The preparation of the pyridine heterocyclic compound is the same as in Example 7.

[0106] The preparation of cono peptide resin is the same as in Example 1, except that oxyma is replaced by the pyridine heterocyclic compound of this example. First, Rink-AmideAM Resin is swollen with 250ml DMF for 60 min. Then, deprotect with 250ml 20% Pip / DMF solution for 30 min, wash the resin with DMF 6 times, and the amount of DMF used each time is 250ml; take Fmoc-Cys(Trt)-OH and pyridine heterocyclic compound, dissolve with 250ml DMF and cool to 0°C, take DIC, add slowly under stirring, and continue stirring for 10min to obtain activation solution. Finally, add the activation solution to the deprotected resin at 25°C for 100min. The end point of the reaction is based on the ninhydrin test. After the reaction, wash the resin with DMF three times, and the amount of DMF used each time is 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 47.62 g, 20 mmol, the degree of substitution is 0.42 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of DIC used is 60 mmol, and the amount of pyridine heterocyclic compound used is 60 mmol.

[0107] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 164.15 g of cono peptide resin.

[0108] Embodiment 9:

[0109] A method for preparing conopeptide resin.

[0110] The preparation of the pyridine heterocyclic compound is the same as in Example 7.

[0111] The preparation of cono peptide resin is the same as in Example 1, except that oxyma is replaced with the pyridine heterocyclic compound and salicylic acid of this example. First, Rink-AmideAM Resin is swollen with 250ml DMF for 60 min. Then, deprotect with 250ml 20% Pip / DMF solution for 30 min, wash the resin with DMF 6 times, and the amount of DMF used each time is 250ml; take Fmoc-Cys(Trt)-OH, pyridine heterocyclic compound and salicylic acid, dissolve with 250ml DMF and cool to 0°C, take DIC, add slowly under stirring, and continue stirring for 10min to obtain an activation solution. Finally, the activation solution is added to the deprotected resin at 25°C for 100min. The end point of the reaction is based on the ninhydrin test. After the reaction, the resin is washed with DMF three times, and the amount of DMF used each time is 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 47.62 g, 20 mmol, the degree of substitution is 0.42 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of pyridine heterocyclic compound used is 60 mmol, the amount of DIC used is 60 mmol, and the amount of salicylic hydroxamic acid used is 20 mmol.

[0112] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 171.21 g of cono peptide resin.

[0113] Embodiment 10:

[0114] A method for preparing conopeptide resin.

[0115] The preparation of the pyridine heterocyclic compound is the same as in Example 7.

[0116] The preparation of cono peptide resin is the same as in Example 1, except that oxyma is replaced with the pyridine heterocyclic compound and salicylic acid of this example. First, Rink-AmideAM Resin is swollen with 250ml DMF for 60 min. Then, deprotect with 250ml 20% Pip / DMF solution for 30 min, wash the resin with DMF 6 times, and the amount of DMF used each time is 250ml; take Fmoc-Cys(Trt)-OH, pyridine heterocyclic compound and salicylic acid, dissolve with 250ml DMF and cool to 0°C, take DIC, add slowly under stirring, and continue stirring for 10min to obtain an activation solution. Finally, the activation solution is added to the deprotected resin at 25°C for 100min. The end point of the reaction is based on the ninhydrin test. After the reaction, the resin is washed with DMF three times, and the amount of DMF used each time is 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 47.62 g, 20 mmol, the degree of substitution is 0.42 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of pyridine heterocyclic compound used is 60 mmol, the amount of DIC used is 60 mmol, and the amount of salicylic hydroxamic acid used is 60 mmol.

[0117] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 179.09 g of cono peptide resin.

[0118] Comparative Example 1:

[0119] A method for preparing conopeptide resin.

[0120] The preparation of the pyridine heterocyclic compound is the same as in Example 7.

[0121] The preparation of cono peptide resin is the same as in Example 1, except that oxyma is replaced by the pyridine heterocyclic compound of this example. First, Rink-AmideAM Resin is swollen with 250ml DMF for 60 min. Then, deprotect with 250ml 20% Pip / DMF solution for 30 min, wash the resin with DMF 6 times, and the amount of DMF used each time is 250ml; take Fmoc-Cys(Trt)-OH and pyridine heterocyclic compound, dissolve with 250ml DMF and cool to 0°C, take DIC, add slowly under stirring, and continue stirring for 10min to obtain activation solution. Finally, add the activation solution to the deprotected resin at 25°C for 100min. The end point of the reaction is based on the ninhydrin test. After the reaction, wash the resin with DMF three times, and the amount of DMF used each time is 250ml, thereby obtaining Fmoc-Cys(Trt)-Rink-Amide AM Resin. The amount of Rink-AmideAM Resin used is 47.62 g, 20 mmol, the degree of substitution is 0.42 mmol / g, the amount of Fmoc-Cys(Trt)-OH used is 60 mmol, the amount of DIC used is 60 mmol, and the amount of pyridine heterocyclic compound used is 2 mmol.

[0122] The protected amino acids were connected in the order of Table 1 in the same manner as above. After the last amino acid was connected, the resin was washed with methanol 4 times, 300 ml each time, and dried to obtain 101.64 g of cono peptide resin.

[0123] Embodiment 11:

[0124] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0125] Take the cono peptide resin obtained in Example 1, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrates, add the filtrate to 7500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0126] The amount of cono peptide resin was 152.61 g, the amount of mixed lysate was 1200 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 62.8% and the weight was 35.4 g.

[0127] Embodiment 12:

[0128] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0129] Take the cono peptide resin obtained in Example 2, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7000 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0130] The amount of cono peptide resin was 140.47 g, the amount of mixed lysate was 1100 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 63.7% and the weight was 36.5 g.

[0131] Embodiment 13:

[0132] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0133] Take the cono peptide resin obtained in Example 3, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 6500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0134] The amount of cono-peptide resin was 130.31 g, the amount of mixed lysate was 1050 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono-peptide crude peptide was 58.2% and the weight was 33.2 g.

[0135] Embodiment 14:

[0136] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0137] Take the cono peptide resin obtained in Example 4, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7000 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0138] The amount of cono peptide resin was 139.22 g, the amount of mixed lysate was 1100 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 62.2% and the weight was 35.5 g.

[0139] Embodiment 15:

[0140] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0141] Take the cono peptide resin obtained in Example 5, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7000 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide line peptide.

[0142] The amount of cono peptide resin was 138.95 g, the amount of mixed lysate was 1100 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 53.9% and the weight was 33.5 g.

[0143] Embodiment 16:

[0144] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0145] Take the cono peptide resin obtained in Example 6, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7000 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0146] The amount of cono peptide resin was 139.76 g, the amount of mixed lysate was 1100 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 50.8% and the weight was 32.1 g.

[0147] Embodiment 17:

[0148] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0149] Take the cono peptide resin obtained in Example 7, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0150] The amount of cono peptide resin was 159.35 g, the amount of mixed lysate was 1200 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 64.9% and the weight was 35.2 g.

[0151] Embodiment 18:

[0152] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0153] Take the cono peptide resin obtained in Example 8, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0154] The amount of cono peptide resin was 164.15 g, the amount of mixed lysate was 1200 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 65.8% and the weight was 37.5 g.

[0155] Embodiment 19:

[0156] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0157] Take the cono peptide resin obtained in Example 9, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0158] The amount of cono peptide resin was 171.21 g, the amount of mixed lysate was 1200 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 66.6% and the weight was 37.6 g.

[0159] Embodiment 20:

[0160] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0161] Take the cono peptide resin obtained in Example 10, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0162] The amount of cono peptide resin was 179.09 g, the amount of mixed lysate was 1200 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 68.9% and the weight was 39.2 g.

[0163] Comparative Example 2:

[0164] The invention discloses a preparation method of a crude peptide of cono-spi peptide.

[0165] Take the cono peptide resin obtained in Comparative Example 1, add a mixed lysis solution of TFA, EDT and Tis to submerge the cono peptide resin, stir evenly, react at 25°C, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the filter cake twice with 200 ml TFA, combine the filtrate, add the filtrate to 7500 ml -10°C isopropyl ether for precipitation, wash with 2000 ml isopropyl ether 4 times, and dry under vacuum to constant weight to obtain a crude cono peptide peptide.

[0166] The amount of cono peptide resin was 101.64 g, the amount of mixed lysate was 1200 ml, the volume ratio of TFA:EDT:Tis was 90:5:5, the reaction time was 2.5 h, and the purity of the obtained cono peptide crude peptide was 60.9% and the weight was 34.3 g.

[0167] Embodiment 21:

[0168] A preparation method of conopeptide.

[0169] The crude cono-spi peptide obtained in Example 11 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with ammonia water. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0170] The mass of the crude cono peptide was 10 g, and the buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 5%, the pH was 8.0, the stirring time was 5.5 h, and the purity of the crude cono peptide was 71.2%.

[0171] Purification was performed using the method described in Example 36 to obtain 3.31 g of cono peptide with a purity of 98.87% and a yield of approximately 24.68%.

[0172] Embodiment 22:

[0173] A preparation method of conopeptide.

[0174] The crude cono-spi peptide obtained in Example 11 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with ammonia water. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0175] The mass of the crude cono peptide was 10 g, and the buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 72.2%.

[0176] Purification was performed using the method described in Example 36 to obtain 3.44 g of cono peptide with a purity of 99.10% and a yield of approximately 25.65%.

[0177] Embodiment 23:

[0178] A preparation method of conopeptide.

[0179] The crude cono-spi peptide obtained in Example 11 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with ammonia water. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0180] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 25%, the pH was 8.0, the stirring time was 2.5 h, and the purity of the crude cono peptide was 58.60%.

[0181] Purification was performed using the method described in Example 36 to obtain 2.82 g of cono peptide with a purity of 98.30% and a yield of approximately 21.03%.

[0182] Embodiment 24:

[0183] A preparation method of conopeptide.

[0184] The crude cono-spi peptide obtained in Example 12 was dissolved and diluted to 1.0 mg / ml with a buffer solution, the pH was adjusted with aqueous ammonia, and the reaction was stirred for a period of time. HPLC detection showed that the cyclization was complete.

[0185] The mass of the crude cono peptide was 10 g, and the buffer solution was prepared by cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 5.0 h, and the purity of the crude cono peptide was 68.88%.

[0186] Purification was performed using the method described in Example 36 to obtain 3.25 g of cono peptide with a purity of 98.90% and a yield of approximately 24.98%.

[0187] Embodiment 25:

[0188] A preparation method of conopeptide.

[0189] The crude cono-spi peptide obtained in Example 12 was dissolved and diluted to 2.0 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0190] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 51.11%.

[0191] Purification was performed using the method described in Example 36 to obtain 2.91 g of cono peptide with a purity of 98.11% and a yield of approximately 22.37%.

[0192] Embodiment 26:

[0193] A preparation method of conopeptide.

[0194] The crude cono-spi peptide obtained in Example 12 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with ammonia water. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0195] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 7.5, the stirring time was 6.5 h, and the purity of the crude cono peptide was 71.44%.

[0196] Purification was performed using the method described in Example 36 to obtain 3.22 g of cono peptide with a purity of 98.76% and a yield of approximately 24.75%.

[0197] Embodiment 27:

[0198] A preparation method of conopeptide.

[0199] The crude cono-spi peptide obtained in Example 14 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0200] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 9.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 66.74%.

[0201] Purification was performed using the method described in Example 36 to obtain 2.81 g of cono peptide with a purity of 98.50% and a yield of approximately 21.02%.

[0202] Embodiment 28:

[0203] A preparation method of conopeptide.

[0204] The crude cono-spi peptide obtained in Example 13 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0205] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 58.82%.

[0206] Purification was performed using the method described in Example 36 to obtain 2.21 g of cono peptide with a purity of 98.30% and a yield of approximately 15.47%.

[0207] Embodiment 29:

[0208] A preparation method of conopeptide.

[0209] The crude cono-spi peptide obtained in Example 14 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0210] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 69.31%.

[0211] Purification was performed using the method described in Example 36 to obtain 3.21 g of cono peptide with a purity of 99.01% and a yield of approximately 24.01%.

[0212] Embodiment 30:

[0213] A preparation method of conopeptide.

[0214] The crude cono-spi peptide obtained in Example 15 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0215] The mass of the crude cono peptide was 10 g, and the buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 55.01%.

[0216] Purification was performed using the method described in Example 36 to obtain 2.11 g of cono peptide with a purity of 98.05% and a yield of approximately 14.89%.

[0217] Embodiment 31:

[0218] A preparation method of conopeptide.

[0219] The crude cono-spi peptide obtained in Example 16 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0220] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 51.33%.

[0221] Purification was performed using the method described in Example 36 to obtain 1.99 g of cono peptide with a purity of 98.09% and a yield of approximately 13.46%.

[0222] Embodiment 32:

[0223] A preparation method of conopeptide.

[0224] The crude cono-spi peptide obtained in Example 17 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0225] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 72.3%.

[0226] Purification was performed using the method described in Example 36 to obtain 3.80 g of cono peptide with a purity of 99.12% and a yield of approximately 28.58%.

[0227] Embodiment 33:

[0228] A preparation method of conopeptide.

[0229] The crude cono-spi peptide obtained in Example 18 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0230] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 75.8%.

[0231] Purification was performed using the method described in Example 36 to obtain 4.43 g of cono peptide with a purity of 99.23% and a yield of approximately 31.26%.

[0232] Embodiment 34:

[0233] A preparation method of conopeptide.

[0234] The crude cono-spi peptide obtained in Example 19 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with aqueous ammonia. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0235] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 76.4%.

[0236] Purification was performed using the method described in Example 36 to obtain 4.82 g of cono peptide with a purity of 99.31% and a yield of approximately 33.87%.

[0237] Embodiment 35:

[0238] A preparation method of conopeptide.

[0239] The crude cono-spi peptide obtained in Example 20 was dissolved and diluted to 1.5 mg / ml with a buffer solution, and the pH was adjusted with ammonia water. The reaction was stirred for a period of time, and HPLC detection showed that the cyclization was complete.

[0240] The mass of the crude cono peptide was 10 g. The buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 77.2%.

[0241] Purification was performed using the method described in Example 36 to obtain 5.52 g of cono peptide with a purity of 99.35% and a yield of approximately 37.24%.

[0242] Comparative Example 3:

[0243] A preparation method of conopeptide.

[0244] The crude cono-spi peptide obtained in Comparative Example 2 was dissolved and diluted to 1.5 mg / ml with a buffer solution, the pH was adjusted with aqueous ammonia, and the reaction was stirred for a period of time. HPLC detection showed that the cyclization was complete.

[0245] The mass of the crude cono peptide was 10 g, and the buffer solution was prepared from cysteine ​​hydrochloride, DMSO and water, wherein the molar concentration of cysteine ​​hydrochloride was 1 mmol / L, the volume percentage concentration of DMSO was 15%, the pH was 8.0, the stirring time was 3.5 h, and the purity of the crude cono peptide was 71.3%.

[0246] Purification was performed using the method described in Example 36 to obtain 2.98 g of cono peptide with a purity of 97.11% and a yield of approximately 22.97%.

[0247] Table 2 Yield of conopeptide resin

[0248]

[0249] As can be seen from Table 2, the net increase of the cono peptide resin prepared in Example 2 is higher when the results of Example 1, Example 2 and Example 3 are compared, which shows that when the degree of substitution of Rink-AmideAM Resin is 0.58 mmol / g, it is beneficial to the preparation of the cono peptide resin; the four groups of results of Example 2, Example 4, Example 5 and Example 6 are compared, and the yield of the cono peptide resin prepared in Example 2 is higher, which shows that when compound A is Oxyma, it is beneficial to the preparation of the cono peptide resin; the five groups of results of Example 7, Example 8, Example 9, Example 10 and Example 1 are compared, and the yield of the cono peptide resin prepared in Example 1 is lower, indicating that the use of pyridine heterocyclic compounds and salicylic hydroxamic acid is beneficial to the preparation of the cono peptide resin; the three groups of results of Example 7, Example 8 and Comparative Example 1 are compared, and the yield of the cono peptide resin prepared in Comparative Example 1 is lower, indicating that when the amount of pyridine heterocyclic compounds is less, it is not conducive to the preparation of the cono peptide resin.

[0250] Table 3 Purity of crude conopeptide peptide

[0251]

[0252] As can be seen from Table 3, the results of Example 11, Example 17, Example 18 and Comparative Example 2 are compared. The purity of the crude peptides of the cono-peptide line peptide prepared in Example 17 and Example 18 is higher, while the purity of the crude peptides of the cono-peptide line peptide prepared in Comparative Example 2 is slightly lower than that in Example 11, which indicates that when the amount of the pyridine heterocyclic compound is small, it is not conducive to the preparation of the crude peptides of the cono-peptide line peptide, and when the amount of the pyridine heterocyclic compound is increased, it has a significant promoting effect on the preparation of the crude peptides of the cono-peptide line peptide; the results of Example 11, Example 12 and Example 13 are compared. The crude peptides of the cono-peptide line peptide prepared in Example 12 have a higher purity, which indicates that when the degree of substitution of Rink-AmideAM Resin is 0.58, the purity of the crude peptides of the cono-peptide line peptide prepared in Example 12 is higher. mmol / g, it is beneficial to the preparation of crude conopeptide peptide; comparing the results of Example 12, Example 14, Example 15 and Example 16, the crude conopeptide peptide prepared in Example 12 has a higher purity, which indicates that when compound A is Oxyma and compound B is DIC, it is beneficial to the preparation of crude conopeptide peptide; comparing the results of Example 18, Example 19 and Example 20, the crude conopeptide peptide prepared in Examples 19 and 20 has a higher purity, which indicates that the use of salicylic hydroxamic acid is beneficial to the preparation of crude conopeptide peptide.

[0253] Table 4 Quality and yield of conopeptides

[0254]

[0255] As can be seen from Table 4, compared with the results of Example 21, Example 22 and Example 23, the yield of the cono peptide prepared in Example 22 is higher, which indicates that when the buffer solution is 1mmol / L cysteine ​​hydrochloride and 15% DMSO aqueous solution, it is beneficial to the preparation of cono peptide; compared with the results of Example 22, Example 24 and Example 25, the yield of the cono peptide prepared in Example 22 is higher, which indicates that when the buffer solution is diluted to 1.5 mg / mL, it is beneficial to the preparation of cono peptide; compared with the results of Example 27, Example 28, Example 29, Example 30 and Example 31, the yield of the cono peptide prepared in Example 29 is higher, which indicates that the degree of substitution of Rink-AmideAM Resin is 0.58 mmol / g, when the pH is 8.0, it is more conducive to the preparation of cono peptide; compared with the results of Comparative Example 3, Example 21, Example 32 and Example 33, the yield of cono peptide prepared in Comparative Example 3 is lower than the yield of cono peptide prepared in Example 32 and Example 33, which shows that the use of pyridine heterocyclic compounds is beneficial to improving the yield of cono peptide. At the same time, when the amount of pyridine heterocyclic compounds is small, it is not conducive to improving the yield of cono peptide; compared with the results of Example 21, Example 34 and Example 35, the yield of cono peptide prepared in Example 34 and Example 35 is higher, which shows that the use of salicylic hydroxamic acid can effectively improve the yield of cono peptide.

[0256] Embodiment 36:

[0257] The steps for purifying the crude conopeptide are as follows.

[0258] 4.1 Preprocessing

[0259] The crude solution of cono peptide was filtered using a filter membrane with a pore size of 0.45 μm.

[0260] 4.2 Purification and preparation

[0261] The preparation and purification were carried out by high performance liquid chromatography.

[0262] Pure: Mobile phase A: 0.1% TFA in pure water; Mobile phase B: 0.1% TFA in acetonitrile.

[0263] Chromatographic column and filler information: Hanbang DAC-150; Nano-C18 unisil 10-120.

[0264] The wavelength is 214nm; the flow rate is 400ml / min; the sample load is 8~12g.

[0265] Prepare the gradient:

[0266]

[0267] Collect the components with purity ≥90%, perform purification multiple times, and combine the qualified components.

[0268] Second pure: Mobile phase A: 1% TFA in glacial acetic acid in water; Mobile phase B: 1% TFA in glacial acetic acid in acetonitrile.

[0269] Chromatographic column and filler information: Hanbang DAC-150; Nano-C18 unisil 10-120.

[0270] The wavelength is 214nm; the flow rate is 400ml / min; the sample load is 6~10g.

[0271] Prepare the gradient:

[0272]

[0273] Collect the components with purity ≥98%, perform multiple secondary purifications, and combine the qualified components.

[0274] 4.3 Pure concentrated freeze-dried

[0275] The pure product solution was added to a 20L rotary evaporator, set at 30°C and 60r / min, and concentrated by rotary evaporation to remove the organic solvent and water in the solution. After concentrating to a volume of ≤3L, it was transferred to a freeze-drying tray with a liquid surface thickness of about 8mm. After freeze-drying, a white powdery solid was obtained, which was the pure cono peptide.

[0276] Figure 1 The target compound conopeptide (molecular formula C 92 H 139 N 35 O 28 S6) Mass spectrum, the theoretical mass-to-charge ratio of conopeptide is 2373.89; in the figure, 475.9 is the five-charged ion peak of the compound, 594.6 is the four-charged ion peak of the compound, and 792.3 is the three-charged ion peak of the compound.

[0277] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0278] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A large-scale preparation method of conopeptide, comprising: The cono peptide resin is prepared by a solid phase synthesis method, the cono peptide resin is cracked to obtain a crude cono peptide line peptide, and the crude cono peptide line peptide is cyclized and purified to obtain a cono peptide; in the solid phase synthesis method, an amino acid reagent is coupled to the amino resin by a coupling agent; The coupling agent comprises a composition of DIC and compound A; the amount of compound A is 10 to 70 parts by mole; Compound A is a pyridine heterocyclic compound, and the pyridine heterocyclic compound has a pyridine group and an N-heterocyclic group; In the preparation of the pyridine heterocyclic compound, 3-aminopyridine-2-carboxylic acid and benzyloxyamine hydrochloride are first reacted to generate intermediate 1, and then intermediate 1 is reacted with acetaldehyde to generate intermediate 2; finally, benzyloxyamine is removed by catalytic hydrogenation to obtain the pyridine heterocyclic compound; the 3-aminopyridine-2-carboxylic acid and benzyloxyamine hydrochloride are reacted in DCM containing TEA and EDCI, the amount of TEA is 0.5-2 molar parts, the amount of EDCI is 0.05-1 molar parts, and the amount of DCM is 2-8 molar parts; the intermediate 1 is reacted with acetaldehyde in acetic acid, the amount of acetaldehyde is 0.5-2 molar parts, and the amount of acetic acid is 0.5-2 molar parts.

2. The large-scale preparation method of conopeptide according to claim 1, characterized in that: The degree of substitution on the amino resin is 0.4~0.8mmol / g.

3. The large-scale preparation method of conopeptide according to claim 1, characterized in that: The amino resin is AM Resin or MBHA Resin.

4. The large-scale preparation method of conopeptide according to claim 1, characterized in that: The lysis treatment uses a lysis solution, which is composed of TFA, EDT and Tis, and the volume ratio of TFA, EDT and Tis in the lysis solution is 80-90:5-10:5-10.

5. The large-scale preparation method of conopeptide according to claim 1, characterized in that: In the preparation of the crude conopeptide peptide, isopropyl ether is used for precipitation.

6. The large-scale preparation method of conopeptide according to claim 1, characterized in that: The cyclization treatment of the crude cono peptide line peptide uses a buffer solution containing cysteine ​​hydrochloride, the buffer solution contains cysteine ​​hydrochloride, DMSO and water, the molar concentration of cysteine ​​hydrochloride is 0.1-5 mmol / L, and the volume percentage concentration of DMSO is 5-25%.

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