Fatty aldehyde modified conopeptide and preparation method thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to fatty aldehyde modified conopeptide and a preparation method thereof. The fatty aldehyde modified conopeptide is prepared by modifying amino acid side chains at different sites of conopeptide with fatty aldehyde to obtain single-site, double-site and three-site modified conopeptide. According to the invention, conopeptide, fatty aldehyde and sodium cyanoborohydride are used as raw materials, and the fatty aldehyde modified conopeptide is obtained through a reductive amination reaction of aldehyde. The preparation steps involved in the method are easy to operate, complex instruments and equipment are not needed, the highest total yield of the preparation method can reach 60%, the purity of the preparation method can reach 90% or above, lipid solubility of the medicine can be improved, combination of the medicine and a receptor is enhanced, and reference is provided for research of polypeptide modification.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a class of fatty aldehyde-modified conopeptides and a preparation method thereof. Technical Background

[0002] Cono peptide is a small molecule active peptide with unique pharmacological activity secreted by the marine organism cone snail. It is an inhibitor of voltage-gated sodium ion channels (VGSCs) and nicotinic acetylcholine receptors (nAChRs). It can block the transmission of nerve signals between nerves and muscles, causing muscle relaxation and thus reducing muscle tension and spasms. It is currently added to beauty and skin care products as an anti-wrinkle ingredient under the trade name XEP TM -018.

[0003] However, cono peptides have the disadvantages of short half-life and large side effects of peptide drugs. In addition, peptide substances themselves have low structural stability and are easily degraded and inactivated by biological enzymes. Therefore, it is necessary to structurally modify peptide drugs to improve their physical and chemical properties, thereby improving their stability and making them have higher biological activity. In order to efficiently utilize peptide drugs, people have tried to modify and transform peptide drugs, and to impart the properties of the modifying groups to the drugs, which will help to develop more therapeutic drugs with practical significance and alleviate the pain of patients. Functional modification of peptides is an effective means to improve the efficacy of peptide drugs, including strategies such as glycosylation modification, fusion of long-acting fragments, and polyethylene glycol modification.

[0004] The present invention aims to solve the modification of conopeptide and its preparation method, prolong the action time and reduce the toxic side effects, and is completed by modifying the amino acid side chains at different sites by fatty aldehydes. Fatty acids are important components of human fat, lipids and cell membrane phospholipids, which can help improve the fat solubility of drugs and enhance the binding of drugs to receptors. Summary of the invention

[0005] The invention aims to solve the insufficiency of the modification preparation technology of conopeptide and broaden its application, and provides a kind of conopeptide in which the amino acid side chains at different sites are modified by using fatty aldehyde and a preparation method thereof, and a method for obtaining conopeptide modified by single site, double site and triple site fatty aldehyde by using conopeptide, fatty aldehyde and sodium cyanoborohydride as raw materials through the reductive amination reaction of aldehyde.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides a class of fatty aldehyde-modified conopeptides, including K13-Cm-CnIIIC, K8-Cm-CnIIIC, K8+K13-Cm-CnIIIC and N5+K8+K13-Cm-CnIIIC, whose amino acid sequences are represented by SEQ ID NO.1 to NO.4, as shown in Table 1 below:

[0008] Table 1 Amino acid sequences of fatty aldehyde-modified conopeptides

[0009] name Amino acid sequence K13-Cm-CnIIIC (SEQ ID NO. 1) <![CDATA[ZGCCNGPKGCSSK(Cm)WCRDHARCC * ]]> K8-Cm-CnIIIC (SEQ ID NO. 2) <![CDATA[ZGCCNGPK(Cm)GCSSKWCRDHARCC * ]]> K8+K13-Cm-CnIIIC (SEQ ID NO. 3) <![CDATA[ZGCCNGPK(Cm)GCSSK(Cm)WCRDHARCC * ]]> N5+K8+K13-Cm-CnIIIC (SEQ ID NO.4) <![CDATA[ZGCCN(Cm)GPK(Cm)GCSSK(Cm)WCRDHARCC * ]]>

[0010] The first - before the modified amino acid indicates the type and number of the modified amino acid; the Cm between the two - indicates alkyl chains of different carbon chain lengths; the CnIIIC after the second - indicates conopeptide;

[0011] Cm represents an alkyl chain of different carbon chain lengths, m represents the number of carbon atoms, * represents C-terminal amidation, and three pairs of disulfide bonds are formed between the six cysteines C3 and C15, C4 and C21, and C10 and C22 in the sequence; the alkyl chain is selected from a straight chain alkyl and a branched chain alkyl.

[0012] In a second aspect, the present invention provides a method for preparing a type of fatty aldehyde-modified conopeptide according to the first aspect, comprising the following steps:

[0013] Step 1: weigh cono peptide and fatty aldehyde in a container according to a molar mass ratio of 1:1 to 1:10, dissolve them respectively in a methanol solution containing acetic acid, stir and mix them evenly, and obtain a mixed solution 1; after stirring evenly, add 3 to 5 times the molar equivalent of cono peptide sodium cyanoborohydride, and continue to stir until evenly, to obtain a mixed solution 2; quench the sodium cyanoborohydride in the mixed solution 2 and stir evenly, and filter it using a filter membrane;

[0014] Step 2: Purify the filtered solution using a preparative liquid phase and collect the purified eluate;

[0015] Step 3: After removing the organic solvent from the collected eluate, freeze-drying is performed to obtain a fatty aldehyde-modified conopeptide-modified peptide.

[0016] Preferably, in step 1, the mass fraction of acetic acid in the methanol containing acetic acid is 0.5% to 3%.

[0017] Preferably, in step 1, the mixed solution 1 is magnetically stirred at 15° C. to 35° C. for 3 to 5 hours; and sodium cyanoborohydride powder in an amount of 4 times the molar equivalent of conopeptide is added to the mixed solution 1 and the stirring is continued for 2 to 3 hours.

[0018] Preferably, in step 1, a saturated aqueous ammonium chloride solution or an acid solution is added to quench the sodium cyanoborohydride.

[0019] Furthermore, in step 1, after adding 50 mM saturated aqueous ammonium chloride solution to the mixed solution 2 to quench the sodium cyanoborohydride, the mixture is magnetically stirred for 15 to 30 minutes.

[0020] Furthermore, in step 1, the pore size of the filter membrane is 0.22 μm.

[0021] Preferably, in step 2, in the mobile phase used for the preparative liquid phase purification, phase A is water + 0.1% TFA, and phase B is acetonitrile + 0.1% TFA.

[0022] Furthermore, in step 2, the injection volume is 1-2 mL, gradient elution is performed at a B phase ratio of 20%-80%, and the eluate with a peak time of 15-45 min is collected.

[0023] Preferably, in step 3, the organic solvent is removed by rotary evaporation, and the freeze-drying condition is -40°C to -80°C.

[0024] Beneficial effects of the present invention:

[0025] (1) The preparation method is simple to operate and does not require complicated equipment.

[0026] (2) The maximum total yield of the preparation method can reach 60%, and the purity can reach more than 90%.

[0027] (3) By connecting fatty chains, it helps to improve the lipid solubility of drugs, enhance the binding of drugs to receptors, and provide a reference for the study of peptide modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Liquid chromatograms of octanal-modified conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, and K8+K13-C8-CnIIIC

[0029] Figure 2 Mass spectra of octanal-modified conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, and K8+K13-C8-CnIIIC. In the figure:

[0030] (a) is the mass spectrum of K13-C8-CnIIIC peptide

[0031] (b) is the mass spectrum of K8-C8-CnIIIC peptide

[0032] (c) is the mass spectrum of K8+K13-C8-CnIIIC

[0033] Figure 3 The modification site detection results of conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, and K8+K13-C8-CnIIIC modified with n-octanal are shown in the figure:

[0034] (a) is the secondary mass spectrum database search matching image of K13-C8-CnIIIC peptide

[0035] (b) The secondary mass spectrum database search matching diagram of K8-C8-CnIIIC peptide

[0036] (c) The secondary mass spectrum database search matching diagram of K8+K13-C8-CnIIIC peptide

[0037] Figure 4 Liquid chromatograms of octanal-modified conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, K8+K13-C8-CnIIIC, and K8+K13+N5-C16-CnIIIC

[0038] Figure 5 Mass spectrum of octanal-modified conopeptide K8+K13+N5-C16-CnIIIC

[0039] Figure 6 Secondary mass spectrometry database search matching image of conopeptide K8+K13+N5-C16-CnIIIC modified with n-octanal

[0040] Figure 7 Liquid chromatograms of palmitaldehyde-modified conopeptides K13-C16-CnIIIC, K8-C16-CnIIIC, and K8+K13-C16-CnIII

[0041] Figure 8 Mass spectra of palmitaldehyde-modified conopeptides K13-C16-CnIIIC, K8-C16-CnIIIC, and K8+K13-C16-CnIII.

[0042] (a) is the mass spectrum of K13-C16-CnIIIC peptide

[0043] (b) is the mass spectrum of K8-C16-CnIIIC peptide

[0044] (c) is the mass spectrum of K8+K16-C8-CnIIIC

[0045] Fig. 9 The modification site detection results of palmitaldehyde-modified conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, and K8+K13-C8-CnIIIC are shown in the figure:

[0046] (a) is the secondary mass spectrum database search matching diagram of K13-C16-CnIIIC peptide

[0047] (b) The secondary mass spectrum database search matching diagram of K8-C16-CnIIIC peptide

[0048] (c) The secondary mass spectrum database search matching diagram of K8+K16-C8-CnIIIC peptide DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings and Examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0050] Example 1: Preparation of n-octanal-modified conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, and K8+K13-C8-CnIIIC

[0051] The present invention utilizes the aldehyde group of n-octanal to react with the amino group on the amino acid side chain in the conopeptide sequence, and then further reduces it with sodium cyanoborohydride NaBH3CN. The specific implementation steps are as follows:

[0052] Step 1: Weigh 1.5 mg of conopeptide and 0.162 mg of n-octanal sample in a transparent screw-mouth bottle at a molar mass ratio of 1:2, dissolve them in 3 ml of methanol solution containing 1% acetic acid, mix and vortex, stir magnetically at room temperature for 3 hours, add 0.16 mg of sodium cyanoborohydride and stir for 2 hours. Use a disposable pipette to add 3-5 drops of saturated ammonium chloride aqueous solution to quench the sodium cyanoborohydride, and filter using a 0.22 μm filter membrane.

[0053] Step 2: Use high performance liquid chromatography for analysis and mass spectrometry identification. The liquid phase analysis conditions are as follows: a) Mobile phase: Phase A is pure water + 0.1% TFA, and phase B is acetonitrile + 0.1% TFA. After filtering with a 0.22μm water membrane / organic membrane, ultrasonically remove bubbles for 30 minutes; b) Selection of chromatographic column: The analytical chromatographic column model is ChromCore Phenyl, 4.6×250mm, 5μm; c) Detection wavelength: 214nm; d) Flow rate: 1mL / min; e) Column temperature: 25℃; f) Injection volume: 20μL. With a gradient elution of 10% to 80% of phase B, the liquid chromatography analysis results are as follows Figure 1 As shown, the eluents with peak times of 26min, 27min, and 35.5min were collected. The collected eluents were subjected to mass spectrometry analysis. The mass spectrometry detection conditions were ESI ion source, positive ion, Scan full scan detection mode, and Fragmentor voltage of 135V. The mass spectrometry results are shown in Figure 2 As shown. Figure 2 (a) Available Figure 1The mass spectrometry analysis of the eluate at 26 min detected divalent parent ions of 1244.6, trivalent parent ions of 830.1, tetravalent parent ions of 622.9, and pentavalent parent ions of 498.4, which were calculated to be consistent with the relative molecular mass of the K13-C8-CnIIIC peptide of 2487.91Da. Figure 2 (b) Available Figure 1 The mass spectrometry analysis of the eluate at 27 min detected divalent parent ions of 1244.4, trivalent parent ions of 830.1, tetravalent parent ions of 622.8, and pentavalent parent ions of 498.5, which were calculated to be consistent with the relative molecular mass of K8-C8-CnIIIC peptide, 2487.91Da. Figure 2 (c) Available Figure 1 The mass spectrometry analysis of the eluate at 35.5 min detected a divalent parent ion of 1300.8, a trivalent parent ion of 867.5, a tetravalent parent ion of 650.9, and a pentavalent parent ion of 521.0, which were calculated to be consistent with the relative molecular mass of the K8+K13-C8-CnIIIC peptide of 2600.12 Da;

[0054] Step 3: Purify by preparative high performance liquid chromatography using a ChromCore Phenyl column with an injection volume of 2 mL and gradient elution at a ratio of 20% to 60% of phase B. Collect the eluate with a peak time of 15 to 30 min and freeze-dry at -80 °C to obtain a powder sample. The purity of the powder sample is greater than 90% by liquid phase analysis.

[0055] Step 4: Identify the modification sites of the obtained peptide powder by secondary mass spectrometry Figure 3 (a)(b)(c) correspond to Figure 1 The samples at 26min, 27min, and 35.5min were analyzed to obtain modified peptides K13-C8-CnIIIC, K8-C8-CnIIIC, and K8+K13-C8-CnIIIC, respectively, and their amino acid sequences were ZGCCNGPKGCSSK(C8)WCRDHARCC * 、ZGCCNGPK(C8)GCSSKWCRDHARCC * ,ZGCCNGPK(C8)GCSSK(C8)WCRDHARCC * , three pairs of disulfide bonds are formed between the six cysteines C3 and C15, C4 and C21, and C10 and C22 in the sequence.

[0056] Example 2: Preparation of n-octanal-modified conopeptides K13-C8-CnIIIC, K8-C8-CnIIIC, K8+K13-C8-CnIIIC, and K8+K13+N5-C16-CnIIIC

[0057] Step 1: Weigh 1.5 mg of conopeptide and 0.648 mg of n-octanal sample in a transparent screw-mouth bottle at a molar mass ratio of 1:8, dissolve them in a methanol solution containing 1% acetic acid, vortex the two, stir magnetically at room temperature for 3 hours, add 0.16 mg of sodium cyanoborohydride and stir for 2 hours. Use a disposable pipette to drop 3-5 drops of saturated ammonium chloride aqueous solution to quench the sodium cyanoborohydride, and filter using a 0.22 μm filter membrane.

[0058] Step 2: Use high performance liquid chromatography for analysis and mass spectrometry identification. The liquid phase analysis conditions are as follows: a) Mobile phase: Phase A is pure water + 0.1% TFA, and phase B is acetonitrile + 0.1% TFA. Ultrasonic bubble removal for 30 minutes after 0.22μm water membrane / organic membrane filtration; b) Selection of chromatographic column: The analytical chromatographic column model is ChromCore Phenyl, 4.6×250mm, 5μm; c) Detection wavelength: 214nm; d) Flow rate: 1mL / min; e) Column temperature: 25℃; f) Injection volume: 20μL. With a gradient elution of 10% to 80% of phase B, the liquid chromatography analysis results are as follows Figure 4 As shown, the eluents at the peak times of 26min, 27min, 35.5min, and 44.5min were collected respectively. The collected eluents were subjected to mass spectrometry identification, and the mass spectrometry detection conditions were ESI ion source, positive ion, Scan full scan detection mode, and Fragmentor voltage of 135V. It was identified that the eluents at 26min and 27min were consistent with the theoretical molecular weight of K13-C8-CnIIIC and K8-C8-CnIIIC, which was 2487.91Da. The eluent at 35.5min was consistent with the theoretical molecular weight of K8+K13-C8-CnIIIC, which was 2600.12Da. The mass spectrometry results of the eluent at 44.5min are as follows Figure 5 As shown, after mass spectrometry analysis, a trivalent parent ion of 904.9, a tetravalent parent ion of 678.9, and a pentavalent parent ion of 543.4 were detected, which were calculated to be consistent with the relative molecular mass of the N5+K8+K13-C16-CnIIIC peptide of 2713.53Da.

[0059] Step 3: Purify by preparative high performance liquid chromatography using a ChromCore Phenyl column with an injection volume of 2 mL, perform gradient elution at a ratio of 20% to 60% of phase B, collect the eluate with a peak time of 15 to 35 min, and freeze-dry at -80 °C to obtain a powder sample. The purity of the powder sample is greater than 90% by liquid phase analysis.

[0060] Step 4: Identify the modification sites of the obtained three-site modified peptide powder by secondary mass spectrometry Figure 6 Corresponding liquid phase Figure 4The sample at 44.5 min was analyzed to be the modified peptide N5+K8+K13-C16-CnIIIC, with the amino acid sequence of ZGCCN(C8)GPK(C8)GCSSK(C8)WCRDHARCC * , three pairs of disulfide bonds are formed between the six cysteines C3 and C15, C4 and C21, and C10 and C22 in the sequence.

[0061] Example 3: Preparation of palmitaldehyde-modified conopeptides K13-C16-CnIIIC, K8-C16-CnIIIC, and K8+K13-C16-CnIIIC

[0062] Step 1: Weigh 50 mg of conopeptide and 10.12 mg of palmitaldehyde sample in a transparent screw-mouth bottle at a molar mass ratio of 1:2, dissolve them in a methanol solution containing 1% acetic acid, vortex the two, stir magnetically at room temperature for 3 hours, add 5.29 mg of sodium cyanoborohydride and stir for 2 hours. Use a disposable pipette to drop 5 drops of saturated ammonium chloride aqueous solution to quench the sodium cyanoborohydride, and filter using a 0.22 μm filter membrane.

[0063] Step 2: Use high performance liquid chromatography for analysis and mass spectrometry identification. The specific operation steps are the same as step 2 in Example 1. The liquid chromatography analysis results are as follows: Figure 7 The eluents with peak times of 42min, 43min, and 57min were collected for mass spectrometry identification, and the mass spectrometry results are shown in Figure 8 As shown. Figure 8 (a) Available Figure 7 The mass spectrometry analysis of the eluate at 42 min detected divalent parent ions of 1300.4, trivalent parent ions of 867.5, tetravalent parent ions of 650.8, and pentavalent parent ions of 520.8, which were calculated to be consistent with the relative molecular mass of the K13-C16-CnIIIC peptide of 2600.13 Da. Figure 8 (b) Available Figure 7 The mass spectrometry analysis of the eluate at 43 min detected a trivalent parent ion of 867.0, a tetravalent parent ion of 651.0, and a pentavalent parent ion of 520.9, which were calculated to be consistent with the relative molecular mass of the K8-C16-CnIIIC peptide of 2600.13 Da. Figure 8 (c) Available Figure 7 The mass spectrometry analysis of the eluate at 57 min detected a divalent parent ion of 1412.9, a trivalent parent ion of 942.2, a tetravalent parent ion of 707.0, and a pentavalent parent ion of 565.8, which were calculated to be consistent with the relative molecular mass of the K8+K13-C16-CnIIIC peptide of 2824.56 Da;

[0064] Step 3: Purify by preparative high performance liquid chromatography using a ChromCore Phenyl column with an injection volume of 2 mL, perform gradient elution at a ratio of 35% to 80% of phase B, collect the eluate with a peak time of 15 to 35 min, and freeze-dry at -80 ° C to obtain a powder sample. The purity of the powder sample is greater than 90% by liquid phase analysis.

[0065] Step 4: Identify the modification sites of the obtained peptide powder by secondary mass spectrometry Fig. 9 (a)(b)(c) correspond to Figure 7 The samples at 42min, 43min, and 57min were analyzed to obtain modified peptides K13-C16-CnIIIC, K8-C16-CnIIIC, and K8+K13-C16-CnIIIC, respectively, and their amino acid sequences were ZGCCNGPKGCSSK(C16)WCRDHARCC * ,ZGCCNGPK(C16)GCSSKWCRDHARCC * ,ZGCCNGPK(C16)GCSSK(C16)WCRDHARCC * , three pairs of disulfide bonds are formed between the six cysteines C3 and C15, C4 and C21, and C10 and C22 in the sequence.

[0066] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A type of fatty aldehyde-modified conopeptide, characterized in that: Including K13-Cm-CnIIIC, K8-Cm-CnIIIC, K8+K13-Cm-CnIIIC and N5+K8+K13-Cm-CnIIIC, whose amino acid sequences are represented by SEQ ID NO.1~NO.4: SEQ ID NO.1:ZGCCNGPKGCSSK(Cm)WCRDHARCC * ; SEQ ID NO.2:ZGCCNGPK(Cm)GCSSKWCRDHARCC * ; SEQ ID NO.3:ZGCCNGPK(Cm)GCSSK(Cm)WCRDHARCC * ; SEQ ID NO.4:ZGCCN(Cm)GPK(Cm)GCSSK(Cm)WCRDHARCC * ; The first - before the amino acid indicates the type of amino acid to be modified and its serial number in the amino acid sequence; the Cm between the two - indicates alkyl chains of different carbon chain lengths; the CnIIIC after the second - indicates conopeptide; Cm represents alkyl chains of different carbon chain lengths, m represents the number of carbon atoms, and * represents C-terminal amidation; Three pairs of disulfide bonds are formed between the six cysteines C3 and C15, C4 and C21, and C10 and C22 in the amino acid sequence.

2. A method for preparing a type of fatty aldehyde-modified conopeptide as claimed in claim 1, characterized in that: The preparation method comprises the following steps: Step 1: weigh cono peptide and fatty aldehyde respectively according to a molar mass ratio of 1:1 to 1:10, dissolve them respectively in a methanol solution containing acetic acid, stir and mix them evenly, and obtain a mixed solution 1; after stirring evenly, add 3 to 5 times the molar equivalent of cono peptide sodium cyanoborohydride, and continue to stir until uniform, to obtain a mixed solution 2; quench the sodium cyanoborohydride in the mixed solution 2 and stir evenly, and filter it using a filter membrane; Step 2: Purify the filtered solution using a preparative liquid phase and collect the purified eluate; Step 3: After removing the organic solvent from the collected eluate, freeze-drying is performed to obtain the fatty aldehyde-modified conopeptide.

3. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 2, characterized in that: In the step 1, the mass fraction of acetic acid in the methanol containing acetic acid is 0.5% to 3%.

4. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 2, characterized in that: In the step 1, the mixed solution 1 is magnetically stirred at 15° C. to 35° C. for 3 to 5 hours; sodium cyanoborohydride in an amount of 4 times the molar equivalent of conopeptide is added to the mixed solution 2 and the stirring is continued for 2 to 3 hours.

5. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 2, characterized in that: In the step 1, a saturated aqueous ammonium chloride solution or an acid solution is added to quench the sodium cyanoborohydride.

6. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 5, characterized in that: In the step 1, 40-60 mM saturated aqueous ammonium chloride solution is added to the mixed solution 2 to quench the sodium cyanoborohydride, and then the mixture is magnetically stirred for 15-30 minutes.

7. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 2, characterized in that: In the step 1, the pore size of the filter membrane is 0.22 μm.

8. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 2, characterized in that: In the step 2, the mobile phase used for the preparative liquid phase purification is that phase A is water + 0.1% TFA, and phase B is acetonitrile + 0.1% TFA.

9. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 8, characterized in that: In the step 2, the injection volume during preparation is 1-2 mL, and gradient elution is performed at a ratio of 20%-80% of the B phase, and the eluate with a peak time of 15-45 min is collected.

10. The method for preparing a type of fatty aldehyde-modified conopeptide according to claim 2, characterized in that: In the step 3, the freeze-drying condition is -40°C to -80°C.

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