CN112851747A

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Publication number: CN112851747A
Application number: CN201911101870.7A
Authority: CN
Inventors: 江峰; 熊战魁; 尹传龙; 陶安进; 余品香
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2021-05-28
Anticipated expiration: 2039-11-12
Also published as: CN112851747B

Abstract

The application belongs to the technical field of polypeptide synthesis and discloses a method for synthesizing quaternary ammonium base modified polypeptide. The synthesis method of the invention prepares polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method; coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin; and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide. The synthesis method has the characteristics of high synthesis yield, mild synthesis conditions, simple process, easily obtained raw materials, stable process and the like, is suitable for large-scale industrial production of the quaternary ammonium base modified polypeptide, and the prepared quaternary ammonium base modified polypeptide has high purity and low impurity content and has wide application prospect in the industrial or medical field.

Description

Method for synthesizing quaternary ammonium base modified polypeptide

Technical Field

The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing a quaternary ammonium base modified polypeptide.

Background

Quaternary ammonium bases are a class of quaternary ammonium (-N) -containing bases⁺(CH₃)₃) The organic alkali with the structure, wherein carboxyl terminal (-COOH) is also present in molecules such as betaine, L-carnitine and the like, so that the molecules have the characteristic of two ions with different charges.

Substances with zwitterionic characteristics mostly have the characteristics of surfactants, and generally have the functions of washing, emulsifying, sterilizing and bacteriostasis in the fields of cosmetics and medicines. The positive charge end in the zwitterion can change the permeability of cell walls to achieve the effects of inhibiting and killing bacteria by adsorbing the zwitterion on the surface of a cell membrane of the negatively charged bacteria.

Under some conditions proteins or polypeptides readily form polymerization, and their low solubility in some organic solvents can create difficulties for their use in industrial or medical fields. In 2008, Xiao et al found that chemical modification of protein or polypeptide by small-molecule betaine (betaine) can increase protein solubility and prevent polypeptide polymerization for the first time. The solubility of the protein is obviously improved by carrying out betaine chemical modification on the N end of the protein. The research result provides a brand-new small molecule modification method for improving the enzyme solubility, and provides a new method for solving the application problem that the low solubility of protein or polypeptide is not beneficial to the industrial or medical field.

Bioconjugate chem.2008,19(6), pp 1113-1118 reported a method for modifying N-terminal cysteine polypeptide by thioesterification of betaine, but the raw materials used in the method are not commercialized and are not easily available. US5958886 reports a series of compounds for modifying L-carnitine with polypeptide, but the synthesis method of L-carnitine modified with polypeptide is not disclosed. EP0514359B1 provides a method for modifying the N end of amino acid by acetyl L-carnitine in a liquid phase, but the synthesis method has longer reaction time and more complicated separation and purification after reaction.

Disclosure of Invention

In view of the above, the present invention provides a method for synthesizing quaternary ammonium hydroxide modified polypeptide derivatives on a solid phase resin, which is easy to operate.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a method for synthesizing a quaternary ammonium base modified polypeptide comprises the following steps:

1) preparing polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method;

2) coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin;

3) and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide.

Further, the solid-phase synthesis in the step 1) is an Fmoc/tBu solid-phase synthesis method, and the carrier resin is any one of Wang resin, Rink Amide-MBHA resin, Rink Amide resin or Rink Amide-AM resin.

Further, the quaternary ammonium derivative in the step 2) has a structure shown in a formula I,

wherein n is 0, 1, R₁H, OH or a saturated or unsaturated fatty acid containing 2 to 18 carbon atoms.

Further, in the step 2), the quaternary ammonium derivatives are L-carnitine, betaine, palmitoyl carnitine, lauroyl carnitine and acetyl L-carnitine.

Further, the molar ratio of the raw materials of the quaternary ammonium hydroxide and the quaternary ammonium hydroxide derivatives in the step 2) to the polypeptide is (1-8): 1.

further, the solvent for the coupling reaction in the step 2) is a mixed solution of any one or more of DMF, NMP, dichloromethane and DMSO in any proportion.

Further, a coupling agent for the coupling reaction in the step 2) is DIPEA + A + B or DIC + A, wherein A is HOBT or HOAT, and B is any one of PyBOP, PyAOP, HATU, HBTU or TBTU.

Further, the reaction temperature of the coupling reaction in the step 2) is 20-30 ℃, and the reaction time is 1-4 h.

Further, the lysis solution obtained in the step 3) is a mixture of more than two of TFA, H2O, PhOMe and TIS.

Further, the purification in the step 3) is reverse phase high performance liquid chromatography purification.

According to the technical scheme, the synthesis method of the quaternary ammonium base modified polypeptide prepares polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method; coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin; and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide. The synthesis method has the characteristics of high synthesis yield, mild synthesis conditions, simple process, easily available raw materials, environmental friendliness, stable process and the like, is suitable for large-scale industrial production of the quaternary ammonium base modified polypeptide, and the prepared quaternary ammonium base modified polypeptide has high purity and low impurity content and has wide application prospect in the industrial or medical field.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a synthesis scheme for the synthesis method according to the invention;

FIG. 2 shows example 1 beta-GIACL-CONH₂A crude peptide chromatogram;

FIG. 3 shows example 1 beta-GIACL-CONH₂A chromatogram of the semen peptide;

FIG. 4 shows example 1 beta-GIACL-CONH₂Mass spectrogram;

FIG. 5 shows example 2L-carnitine-IIGAC-CONH₂A crude peptide chromatogram;

FIG. 6 shows example 2L-carnitine-IIGAC-CONH₂A chromatogram of the semen peptide;

FIG. 7 shows example 2L-carnitine-IIGAC-CONH₂Mass spectrogram;

FIG. 8 shows a chromatogram of Palmitoyl-L-carnitine-LCQATL-COOH of example 3;

FIG. 9 shows the mass spectrum of Palmitoyl-L-carnitine-LCQATL-COOH of example 3.

Detailed Description

The invention discloses a method for synthesizing quaternary ammonium base modified polypeptide. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The synthetic route of the synthetic method of the invention is shown in figure 1.

In some embodiments, the polypeptide-solid phase resin with naked amino groups is prepared by Fmoc/tBu solid phase synthesis using Rink Amide-AM resin as a carrier resin.

In some embodiments, n ═ 0, R in the quaternary ammonium derivatives₁The structure of the quaternary ammonium derivative is shown as a formula II, namely the quaternary ammonium derivative is betaine;

in some embodiments, n ═ 1, R in the quaternary ammonium derivatives₁OH, which is l-carnitine, and the structure of which is shown in formula III, i.e. the quaternary ammonium derivative is l-carnitine;

in some embodiments, n ═ 1, R in the quaternary ammonium derivatives₁＝OCOCH₃Acetyl L-carnitine with a structure shown in a formula IV, namely the quaternary ammonium derivative is acetyl L-carnitine;

in some embodiments, n ═ 1, R in the quaternary ammonium derivatives₁＝OCO(CH₂)₁₄CH₃The structure of the quaternary ammonium derivative is shown as a formula V, namely the quaternary ammonium derivative is lauroyl carnitine;

in some embodiments, n ═ 1 in the quaternary ammonium derivatives,R₁＝OCO(CH₂)₁₄CH₃the structure of the quaternary ammonium derivative is shown as a formula VI, namely the quaternary ammonium derivative is palmitoyl carnitine;

further, the molar ratio of the raw materials of the quaternary ammonium hydroxide and the quaternary ammonium hydroxide derivatives in the step 2) to the polypeptide is (1-8): 1. in some embodiments, is 3: 1.

further, the solvent for the coupling reaction in the step 2) is a mixed solution of any one or more of DMF, NMP, dichloromethane and DMSO in any proportion. In some embodiments, the solvent of the coupling reaction is DMSO: NMP is a 1:1 mixed solution.

Further, the coupling agent in the coupling reaction in step 2) of the synthesis method of the present invention is DIPEA + a + B or DIC + a, wherein a is HOBT or HOAT, and B is any one of PyBOP, PyAOP, HATU, HBTU or TBTU.

In some embodiments, when betaine, acetyl-l-carnitine, palmitoyl-carnitine, or lauroyl-carnitine is coupled, the coupling agent is DIPEA + HOBT + HBTU. The molar ratio of quaternary ammonium base derivatives such as betaine, acetyl L-carnitine, palmitoyl carnitine or lauroyl carnitine, namely DIPEA, HOBT and HBTU is 1: 2.5: 1.5: 1.5.

in some embodiments, where l-carnitine is coupled, the coupling agent is DIC + HOBT, and the molar ratio of l-carnitine to DIC, HOB is 1: 1.5: 1.5.

furthermore, the reaction temperature of the coupling reaction in the step 2) of the synthesis method is 20-30 ℃, and the reaction time is 1-4 h. In some embodiments, the coupling reaction is most effective for 3 h.

Further, the lysis solution cleaved in step 3) of the synthesis method of the present invention is TFA or H₂O, PhOMe and TIS.

In some embodiments, when a polypeptide is modified with a higher molecular weight quaternary ammonium base, such as palmitoyl carnitine, lauroyl carnitine, etc., the cleaved lysate is TFA H₂And O:95: 5(v/v) lysate.

In some embodiments, when modified with a lower molecular weight quaternary ammonium base such as L-carnitine, acetyl L-carnitine, betaine, etc., the cleaved lysate is TFA H₂(vi) lysates from TIS: PhOMe ═ 90:5:4:1 (v/v).

The crude peptide obtained by the cracking in the step 3) of the preparation method is further purified to obtain the refined peptide, and the purification is preferably reversed-phase high performance liquid chromatography purification.

In some embodiments, the purification is specifically: gradient elution is carried out by taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B.

The synthesis method of the quaternary ammonium base modified polypeptide prepares polypeptide-solid phase resin with naked amino by a solid phase synthesis method; coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin; and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide. The synthesis method has the characteristics of high synthesis yield, mild synthesis conditions, simple process, easily available raw materials, environmental friendliness, stable process and the like, is suitable for large-scale industrial production of the quaternary ammonium hydroxide modified polypeptide, and the prepared quaternary ammonium hydroxide modified polypeptide has high purity and low impurity content and has wide application prospects in the industrial or medical field.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Abbreviations and English meanings in the specification and examples are as follows:

abbreviations and English	Means of
		betaine	Betaine
L-carnitine	L-carnitine
		Palmitoyl-L-carnitine	palmitoyl-L-carnitine
SPPS	Solid phase polypeptide synthesis
		HOAt	1-hydroxy-7-azobenzotriazol
Fmoc	9-fluorenylmethoxycarbonyl group
		DIPCDI	Diisopropylcarbodiimide
HOBt	1-hydroxybenzotriazoles
		HATU	2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
HBTU	benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
		DIPEA	N, N-diisopropylethylamine
PyBOP	Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphates
		PyAOP	(3H-1,2, 3-triazolo [4,5-b ]]Pyridin-3-yloxy) tris-1-pyrrolidinophosphonium hexafluorophosphate.
TBTU	O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate
		DMF	N, N-dimethylformamide
DCM	Methylene dichloride
		THF	Tetrahydrofuran (THF)
TFE	Trifluoroethanol
		TFA	Trifluoroacetic acid
TA	Phenylmethyl sulfide
		PhOMe	Phenylmethyl ether
TIS	Tri-isopropyl silane
		DBLK	20% piperidine/DMF (V/V) solution
tBu	Tert-butyl radical
		NMP	N-methyl pyrrolidone

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1: synthesis of beta-GIACL-CONH₂(FW:574.32) for example, the examples are as follows:

step 1): preparation of polypeptide-solid phase resin with naked amino group

Taking 12.2g of Rink Amide-AM resin with the substitution degree of 0.41mmol/g, adding the Rink Amide-AM resin into a reactor, adding DMF to wash twice, then swelling the Rink Amide-AM resin with DMF for 30 minutes, removing Fmoc protective groups twice with 20% piperidine/DMF solution (DBLK), each time for 10 minutes, and washing six times with DMF after deprotection. 5.3g (15mmol) of Fmoc-Leu-OH, 3.05g (22.5mmol) of HOBT and 8.55g of HBTU (22.5mmol) were dissolved in 35ml of a mixed solution of NMP 1:1, and after activation with 4.85g (35.5mmol) of DIPEA in an ice-water bath for 3min, the mixture was loaded on a reaction column and reacted at room temperature for 2 hours. After the reaction is finished, washing the resin with 35mLDMF for 3 times, adding 20mLDBLK for deprotection for 5min +10min, washing the resin with 35mLDMF for 6 times, and sequentially coupling Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH and Fmoc-Gly-OH according to peptide sequence, wherein each amino acid is fed with 15mmol, HOBT is fed with 22.5mmol, HBTU is fed with 22.5mmol and DIPEA is fed with 35.5 mmol. After the coupling is finished, waiting for the next quaternary ammonium base modification reaction.

Step 2): introduction of betaine to the nitrogen terminus of a polypeptide

3.5g (30mmol) betaine, 6.1g (45mmol) HOBT and 17.1g HBTU (45mmol) were weighed out in 35ml DMSO: NMP ═ 1:1, adding 9.7g (75mmol) of DIPEA under the condition of ice water bath for activating for 3min, adding the mixture into a reactor for reacting for three hours, washing the mixture with DMF for three times after the reaction is finished, and washing the mixture with DCM for three times. The resin was shrunk with 35mL of methanol and suction dried to give 16.3g of peptide resin.

Step 3): betaine-polypeptide preparation

16.3g of the resin obtained in step 2) was put into a 250mL three-necked flask, 160mL of the prepared TFA H2O TIS PhOMe 90:5:4:1(v/v) was added, and the mixture was reacted at room temperature for 2 hours, and the resin was filtered under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 1.6L of glacial methyl tert-butyl ether for precipitation, centrifuged, washed 3 times with 1.0L of glacial methyl tert-butyl ether, and dried under reduced pressure to give 2.92g of betaine-polypeptide derivative, with a crude peptide purity of 85.24% (FIG. 2).

Step 4): preparation of refined peptides

The crude peptide obtained in step 3 was purified by high performance liquid chromatography through a 15cm × 25cm preparative column. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution as a mobile phase A, taking acetonitrile as a mobile phase B, and eluting a gradient phase A: 70% -30%, elution preparation flow rate: 70-80 ml/min; detection wavelength: 220 nm; the desired peak fraction was collected, concentrated and lyophilized to obtain 2.41g of pure product with purity of 99.57%, yield of 82.5% and molecular weight FW:574.32 (FIGS. 3 and 4).

Example 2: synthesis of L-carnitine-IIGAC-CONH₂(FW:617.8)

Step 1): preparation of polypeptide-solid phase resin with naked amino group

Taking 24.4g of Rink Amide-AM resin with the substitution degree of 0.41mmol/g, adding the Rink Amide-AM resin into a reactor, adding DMF to wash twice, then swelling the Rink Amide-AM resin with DMF for 30 minutes, removing Fmoc protective groups twice with 20% piperidine/DMF solution (DBLK), each time for 10 minutes, and washing six times with DMF after deprotection. 17.57g (30mmol) of Fmoc-Cys (Trt) -OH and 6.10g (45mmol) of HOBT were dissolved in 60ml of DMF, and after 5.7g (45mmol) of DIC was added in an ice-water bath to activate the mixture for 3min, the mixture was put into a reaction column and reacted at room temperature for 2 hours. And after the reaction is finished, washing the resin by using 70mLDMF for 3 times, adding 70mLDBLK for deprotection for 5min +10min, washing the resin by using 70mLDMF for 6 times, and sequentially coupling, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH and Fmoc-Ile-OH according to the peptide sequence, wherein each amino acid is fed by 30mmol, HOBT is fed by 45mmol, and DIC is fed by 45 mmol. After the coupling is finished, waiting for the next quaternary ammonium base modification reaction.

Step 2): introduction of L-carnitine into the nitrogen end of polypeptide

Weighing 4.84g (30mmol) of L-carnitine, 6.10g (45mmol) of HOBT, 60ml of DMSO, NMP 1:1, adding 5.7g (45mmol) of DIC under ice water bath for activation for 3min, adding the mixture into a reaction column, and reacting at room temperature for 3 h. After the reaction was complete, it was washed three times with DMF and three times with DCM. The resin was shrunk with 70mL of methanol and suction dried to give 31.4g of peptide resin.

Step 3): preparation of L-carnitine-polypeptide

31.4g of the resin obtained in step 3 was put into a 500mL three-necked flask, 350mL of previously prepared TFA, H2O, TIS, PhOMe, 90:5:4:1(v/v) was added, and the mixture was reacted at room temperature for 2 hours, followed by filtration of the resin under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 3.5L of glacial methyl tert-butyl ether for precipitation, centrifuged, washed 3 times with 1.0L of glacial methyl tert-butyl ether and dried under reduced pressure to give 6.5g of L-carnitine-polypeptide derivative in a crude peptide purity of 75.75% (FIG. 5).

Step 4): preparation of refined peptides

The crude peptide obtained in step 4 was purified by high performance liquid chromatography through a 15cm × 25cm preparative column. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution as a mobile phase A, taking acetonitrile as a mobile phase B, and eluting a gradient phase A: 65% -25%, elution preparation flow rate: 70-80 ml/min; detection wavelength: 220 nm; the desired peak fraction was collected, concentrated and lyophilized to obtain 5.8g of a pure product having a purity of 98.35%, a yield of 93.85% and a molecular weight M of 618.34 (fig. 6 and 7).

Example 3: synthesis of Palmitoyl-L-carbonitine-LCQATL-COOH (FW:1031.37)

Step 1): preparation of polypeptide-solid phase resin with naked amino group

Taking 1.22g of Fmoc-Leu-wang resin with the substitution degree of 0.40mmol/g, adding the Fmoc-Leu-wang resin into a reactor, adding DMF to wash twice, then swelling the mixture with DMF for 30 minutes, removing Fmoc protective groups twice with 20% piperidine/DMF solution (DBLK) for 10 minutes each time, and washing the mixture with DMF for six times after deprotection. 0.596g (1.5mmol) of Fmoc-Thr (tBu) -OH and 0.304g (2.25mmol) of HOBT were dissolved in 5ml of DMF, and 0.28g (2.25mmol) of DIC was added thereto in an ice-water bath for 3min of activation, and then the mixture was put into a reaction column and reacted at room temperature for 2 hours. After the reaction is finished, washing the resin by using 10mLDMF for 3 times, adding 10mLDBLK for deprotection for 5min +10min, washing the resin by using 10mLDMF for 6 times, and sequentially coupling Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Cys (Trt) -OH and Fmoc-Leu-OH according to the peptide sequence, wherein each amino acid is fed by 1.5mmol, HOBT is fed by 2.25mmol and DIC is fed by 2.25 mmol. After the coupling is finished, waiting for the next quaternary ammonium base modification reaction.

Step 2): introduction of palmitoyl-L-carnitine into the nitrogen terminal of a polypeptide

0.60g (1.5.mmol) palmitoyl-L-carnitine hydrochloride 0.304g (2.25mmol) HOBT, 5ml DMSO NMP ═ 1:1, adding 0.28g (2.25mmol) of DIC under ice water bath for activation for 3min, adding the mixture into a reaction column, and reacting at room temperature for 3 h. After the reaction was complete, it was washed three times with DMF and three times with DCM. The resin was shrunk with 10mL of methanol and suction dried to give 1.93g of peptide resin.

Step 3): preparation of palmitoyl-L-carnitine-polypeptide

1.93g of the resin obtained in step 3 was put into a 50mL three-necked flask, and prepared TFA, H2O (95: 5(v/v)20mL, was added thereto, and the mixture was reacted at room temperature for 2 hours, and the resin was filtered under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 200mL of glacial methyl tert-butyl ether for precipitation, centrifuged, washed 3 times with 50mL of glacial methyl tert-butyl ether, and dried under reduced pressure to give 0.526g of palmitoyl-L-carnitine-polypeptide derivative, crude peptide purity 96.56% (FIGS. 8 and 9).

Claims

1. A method for synthesizing a quaternary ammonium base modified polypeptide comprises the following steps:

2. The method of claim 1, wherein the solid phase synthesis in step 1) is Fmoc/tBu solid phase synthesis method, and the carrier resin is any one of Wang resin, Rink Amide-MBHA resin, Rink Amide resin, or Rink Amide-AM resin.

3. The method of claim 1, wherein the quaternary ammonium derivative of step 2) has a structure of formula I,

4. The method of claim 3, wherein the quaternary ammonium derivative of step 2) is L-carnitine, betaine, palmitoyl carnitine, lauroyl carnitine, acetyl L-carnitine.

5. The method of claim 1, wherein the molar ratio of the starting quaternary ammonium hydroxide and quaternary ammonium hydroxide derivative in step 2) to the polypeptide is (1-8): 1.

6. the synthesis method according to claim 1, wherein the solvent for the coupling reaction in step 2) is a mixed solution of any one or more of DMF, NMP, dichloromethane and DMSO in any proportion.

7. The synthesis method of claim 1, wherein the coupling reagent in the coupling reaction in step 2) is DIPEA + A + B or DIC + A, wherein A is HOBT or HOAT, and B is any one of PyBOP, PyAOP, HATU, HBTU or TBTU.

8. The synthesis method of claim 1, wherein the coupling reaction in step 2) is carried out at a reaction temperature of 20-30 ℃ for 1-4 h.

9. The method of claim 1, wherein the cleaved lysate of step 3) is a mixture of two or more of TFA, H2O, PhOMe, and TIS.

10. The synthetic method of claim 1 wherein the purification of step 3) is by reverse phase high performance liquid chromatography.