CN112409445A

CN112409445A - Solid-phase synthesis method of snake venom-like tripeptide

Info

Publication number: CN112409445A
Application number: CN202011327827.5A
Authority: CN
Inventors: 黄毅; 邢海英; 王志国; 虞新友
Original assignee: Zhejiang Baitai Biology Co ltd
Current assignee: Zhejiang Baitai Biology Co ltd
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-02-26

Abstract

The invention discloses a solid phase synthesis method of snake venom tripeptide, which comprises the steps of taking MBHA/AM Resin as initial Resin, connecting a Linker, then sequentially connecting amino acids Boc-Dab (Fmoc) -OH, Boc-Pro-OH and Boc-beta-Ala-OH under the action of a condensation reagent by a solid phase synthesis method to obtain a Resin peptide H-beta-Ala-Pro-Dab (Fmoc) -Linker-AM Resin, adding benzylamine after the Resin is obtained, stirring for reaction, cracking polypeptide from the peptide Resin to obtain a product H-beta-Ala-Pro-Dab-NHBzl, and obtaining the product meeting the requirements through related purification processes. The invention uses a solid phase synthesis method to directly obtain the product H-beta-Ala-Pro-Dab (Boc) -NHBzl, the racemization risk of Dab is not involved in the product, the synthesis of the process route is short, the resin is stable, the process is simple, and the large-scale production is easy.

Description

Solid-phase synthesis method of snake venom-like tripeptide

Technical Field

The present invention relates to a method for producing a polypeptide. More particularly, the invention relates to a preparation method of H-beta-Ala-Pro-Dab-NH-Bzl.

Background

The H-beta-Ala-Pro-Dab-NH-Bzl is a small molecular polypeptide simulating the activity of Waglerin I, clinical tests show that the polypeptide can reduce wrinkle generation by inhibiting muscle contraction, has excellent smooth and rapid wrinkle removing performance, acts on a postsynaptic membrane, is a reversible antagonist of muscle nicotinic acetylcholine receptor (mmAChR), and finds a suitable synthetic method, which has important significance for industrialization of the small molecular polypeptide.

The common chemical synthesis methods for polypeptide synthesis comprise liquid phase synthesis and solid phase synthesis, and for small molecular polypeptides, polypeptides with amino acids smaller than 10 amino acids are generally synthesized by a liquid phase synthesis method, but the liquid phase synthesis method has more steps and is relatively complex, the purity of crude products obtained by completely adopting the liquid phase synthesis method is not high, the total yield is not high, the solid phase synthesis method is adopted, the removal difficulty is relatively high, the cost-benefit ratio is extremely low, and the limitation of each method cannot be avoided by completely adopting the solid phase synthesis or completely adopting the liquid phase synthesis method.

The method mentioned in the patent publication No. CN107936108A, entitled liquid phase synthesis method of snake venom tripeptide, uses two hydrolysis reactions, especially in Boc-beta-Ala-Pro-Dab (Boc) -OH and benzylamine connection reaction, racemization of Dab is easy to generate, and great risk is brought to product quality.

The method mentioned in the invention patent with the patent name of CN107857797A as a liquid phase fragment synthesis method of snake venom tripeptide is that Fmoc-Pro-OH is firstly prepared into a reaction process that Fmoc-Pro-OSu reacts with H-dab (Boc) -OH to form Fmoc-Pro-dab (Boc) -OH, the reaction is relatively trapped due to the larger steric hindrance of Pro amino acid, and the yield is relatively low; furthermore, there is also a risk of racemization of Dab in this route.

The invention patent with the publication number of CN103570804B discloses a method for synthesizing polypeptide with skin activity, and the solid-liquid combination method mentioned in the invention patent is used for obtaining the snake venom tripeptide.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a solid-phase synthesis method of snake venom-like tripeptide, which can avoid racemization of Dab, has short process route, very short production period and stable resin and is suitable for large-scale production.

In order to achieve the purpose, the invention provides the following technical scheme:

a solid phase synthesis method of snake venom tripeptide is characterized by comprising the following steps:

(1) MBHA/AM resin swelling

Adding MBHA/AM resin into a reactor, adding piperidine/DMF solution, continuously swelling until swelling is finished, performing suction filtration, and washing by using piperidine/DMF solution.

(2) 4-hydroxymethylphenylacetic acid coupling

Adding 4-hydroxymethylphenylacetic acid and a condensation reagent into a piperidine/DMF solution, dissolving, cooling, slowly adding DIC (Diformic acid) and stirring to obtain an activated solution, adding the activated solution into the reactor in the step (1), blowing nitrogen to start reaction, adding NMM after the reaction starts, continuously reacting till the reaction is complete, performing suction filtration, washing by using the piperidine/DMF solution, and washing by using DCM (DCM).

(3) Boc-dab (Fmoc) -OH hanging resin

a. Adding Boc-dab (Fmoc) -OH into the reactor in the step (1), adding DCM, blowing nitrogen, adding pyridine, controlling the temperature of the reaction liquid to be less than 20 ℃, dropwise adding DCB, keeping the temperature at a low temperature for a period of time after dropwise adding, then heating to 35-30 ℃ until the reaction is finished, performing suction filtration, and washing by using piperidine/DMF solution;

b. adding DIEA into DMF, stirring and mixing uniformly, adding Ac2O, stirring and mixing uniformly to obtain an end-capping solution, adding the end-capping solution into the reactor in the step (1), performing end-capping suction filtration, and washing with DMF;

c. washing with methanol, DCM and methanol in sequence, and shrinking the resin;

d. the degree of substitution of the resin was measured.

(4) Swelling of the resin

a. Adding the removed resin into a reactor, adding DMF, continuously swelling until swelling is finished, performing suction filtration, and washing by using DCM;

b. TFA/DCM was added and the reaction was allowed to go to completion under nitrogen, filtered off with suction and washed with DMF.

(5) Boc-Pro-OH coupling

a. Adding Boc-Pro-OH and a condensation reagent into an activation cup, adding DMF (dimethyl formamide), dissolving, cooling, slowly adding DIC (Diformic acid) while stirring, keeping the temperature at low temperature after the addition is finished to prepare an activation liquid, adding the activation liquid into the reactor in the step (3), blowing nitrogen to start reaction, adding NMM (N-methyl-;

(6) Boc-beta-Ala-OH coupling

a. Adding Boc-beta-Ala-OH and a condensation reagent into an activation cup, adding DMF (dimethyl formamide), dissolving, cooling, slowly adding DIC (Diformic acid) while stirring, keeping low temperature after the addition is finished to prepare an activation liquid, adding the activation liquid into the reactor in the step (3), blowing nitrogen to start reaction, adding NMM (N-methyl pyrrolidone), continuously reacting until the reaction is finished, performing suction filtration, washing with DMF (dimethyl formamide), and washing with DCM (dimethyl formamide);

(7) Benzylamine linkage

a. Washing the resin obtained after the reaction of the step (5) by using DMF/THF;

b. weighing benzylamine, adding the benzylamine into a beaker, adding DMF/THF for dissolving, adding the benzylamine into a reactor, carrying out nitrogen blowing reaction, taking a small resin sample, and cutting to show that the reaction is finished;

c. filtration and washing of the resin with DMF/THF solution;

d. mixing the filtrates, and drying to obtain crude product;

e. and pulping and washing, adding tert-butyl methyl ether into the crude product, pulping, fully dispersing, filtering to obtain a filter cake, pulping the filter cake by using tert-butyl methyl ether for multiple times, and filtering.

(8) Drying

And (3) putting the filter cake into a vacuum drying oven for drying, and drying to constant weight to obtain a crude product of H-beta-Ala-Pro-Dab-NHBzl.

(9) Purification of

And purifying the H-beta-Ala-Pro-Dab-NHBzl crude product by reverse phase preparative chromatography to obtain the H-beta-Ala-Pro-Dab-NHBzl product.

Preferably, the mole ratio of Boc-Pro-OH, HOBT, DIC and NMM in step (5) is 1:1:1: 1.2.

The solid phase synthesis method of a snake venom-like tripeptide according to claim 1, wherein the solid phase synthesis method comprises the following steps: the molar ratio of Boc-beta-Ala-OH, HOBT, DIC and NMM in step (6) was 1:1:1: 1.2.

Preferably, the condensation reagent in steps (2), (5) and (6) is a conventional condensation reagent such as DCB/Py, DIC/HOBt, DIC/HOAt, HATU or HBTU.

Preferably, the deprotecting reagent in steps (2), (5) and (6) is TFA, and the concentration of the deprotecting reagent is in the range of 5% to 100%

Preferably, the deprotection solvent in steps (2), (5) and (6) is any one of DCM, THF, Acetone, MTBE and the like.

In summary, the following steps:

in the technical route, the product H-beta-Ala-Pro-Dab-NHBzl is directly obtained by one step by using a solid phase synthesis method, the racemization risk of Dab is not involved in the product, and in addition, the technological route is short in synthesis, stable in resin, simple in process and easy for large-scale production. .

Drawings

FIG. 1 is a schematic representation of the solid phase synthesis scheme of a snake venom-like tripeptide according to the present invention;

FIG. 2 is a chart showing the detection of hplc of the purified product in the embodiment of the present invention.

Detailed Description

The solid phase synthesis method of a snake venom-like tripeptide of the invention is further illustrated by the following examples:

swelling of AM resin

a) 1.0mmol/g AM Resin (125 mmol, 125 g) was weighed into a reactor, and 20% piperidine/DMF solution (2.0L) was added and swollen for 0.5 h. After swelling, the mixture was filtered off with suction and washed 5 times with 2.0L of DMF each time

Bis, 4-hydroxymethylphenylacetic acid coupling

a) Weighing 4-hydroxymethylphenylacetic acid (375 mmol, 62 g) and HOBT (375 mmol, 51 g), adding the mixture into an activation cup, adding DMF (250 ml), dissolving the mixture clearly, cooling to 4 ℃, slowly adding DIC (375 mmol, 59 ml), stirring while adding, controlling the temperature to be kept at 2-8 ℃, after the addition is finished, keeping the temperature to be 2-8 ℃ for activation for 15min, adding the activation solution into a reactor, blowing nitrogen to start reaction, adding NMM (125 mmol, 14 ml) after 0.5h, continuing the reaction, reacting completely after 3h, performing suction filtration, washing 3 times with DMF, 2.0L each time, washing 2 times with DCM, and 2.0L each time

Tris, Boc-dab (Fmoc) -OH hanging resin

a) Weighing Boc-dab (Fmoc) -OH (250 mmol, 110 g), adding the Boc-dab (Fmoc) -OH (250 mmol, 110 g) into a reactor, adding DCM (250 ml), bubbling nitrogen, adding pyridine (1000 mmol, 81 ml), controlling the temperature of the reaction solution to be less than 20 ℃, dropwise adding DCB (500 mmol, 71 ml), controlling the dropwise adding speed, keeping the temperature of the reaction solution to be less than 20 ℃, continuing to react at low temperature for 10min after dropwise adding is finished, heating, controlling the temperature of the reaction solution to be 25-30 ℃, reacting for 3h, performing suction filtration, washing for 3 times by using DMF (dimethyl formamide), and washing

b) Preparing an end-capping solution (2L): measuring DMF (1680 ml), adding DIEA (120 ml), stirring, adding Ac2O (200 ml), stirring, adding the end capping solution into the reactor, capping for 0.5h, vacuum filtering, washing with DMF for 3 times (2.0L each time)

c) Preparing an end-capping solution (2L): measuring DMF (1680 ml), adding DIEA (120 ml), stirring, adding Ac2O (200 ml), stirring, adding the end capping solution into the reactor, capping for 0.5h, vacuum filtering, washing with DMF for 3 times (2.0L each time)

d) Methanol washing 2 times, DCM washing 2 times, methanol washing 2 times, each time 2.0L, resin shrinkage

Fourthly, measuring the degree of substitution of the resin

a) 192g of resin were obtained, and the degree of substitution by sampling was 0.57mmol/g

Swelling the resin

a) The suspended resin (100 mmol, 176 g) was weighed into a reactor, DMF (2.0L) was added and the mixture was swollen for 0.5 h. After swelling, the mixture was filtered off with suction and washed 2 times with 2.0L of DCM

b) 50% TFA/DCM (2.0L) was added, the mixture was sparged with nitrogen for 0.5h, filtered with suction and washed 5 times with DMF, 2.0L each time

Hexa, Boc-Pro-OH coupling

a) Weighing Boc-Pro-OH (300 mmol, 65 g) and HOBT (300 mmol, 41 g) and adding the mixture into an activation cup, adding DMF (250 ml), dissolving the mixture clearly, cooling to 4 ℃, slowly adding DIC (300 mmol, 48 ml) while stirring, controlling the temperature to be kept at 2-8 ℃, after the addition is finished, keeping the temperature at 2-8 ℃ for activation for 15min, adding the activation solution into a reactor, blowing nitrogen to start reaction, after 5min, adding NMM (120 mmol, 13 ml), after the reaction is finished for 1.5h, performing suction filtration, washing 2 times by using DMF, using 2.0L of DCM for each time, and using 2.0L of DCM for each time

Hepta, Boc-beitaAla-OH coupling

a) Weighing Boc-beitaAla-OH (300 mmol, 57 g) and HOBT (300 mmol, 41 g) and adding the mixture into an activation cup, adding DMF (250 ml), dissolving the mixture clearly, cooling to 4 ℃, slowly adding DIC (300 mmol, 48 ml), stirring while adding, controlling the temperature to be 2-8 ℃, after the addition is finished, keeping the temperature to be 2-8 ℃ for activation for 15min, adding the activation solution into a reactor, blowing nitrogen to start reaction, after 5min, adding NMM (120 mmol, 13 ml) to react for 1.5h, then completing the reaction, performing suction filtration, washing 2 times by DMF, using 2.0L of DCM for each time, and using 2.0L of DCM for each time

Eight, benzylamine ligation

a) The resin was washed 2 times with DMF/THF (50%) 2.0L each time

b) Weighing benzylamine (400 g), adding the benzylamine into a beaker, adding 50% DMF/THF (1.6L) for dissolving, adding the mixture into a reactor, reacting for 3h under nitrogen, taking a small sample of resin, and cutting to show that the reaction is finished

c) Filtration and washing of the resin with DMF/THF (50%) 2 times in 300ml portions

d) The filtrates were combined and then dried to obtain 34g of crude product, yield of crude product: 90.6 percent

e) Pulping and washing:

i. adding 800ml of tert-butyl methyl ether into the crude product, pulping, fully dispersing, and filtering

ii, the filter cake was slurried with tert-butyl methyl ether (500 ml) and filtered again

The filter cake was slurried with tert-butyl methyl ether (500 ml) and filtered again

Nine, drying

a) The filter cake is put into a vacuum drying oven for drying (30 ℃ and-0.08 MPa) to constant weight, 28.5g of crude product is obtained, and the yield of the crude product is 76%.

Ten, purification

a) The 28.5g crude product was purified by reverse phase preparative chromatography to give 23.3g product in 81.8% yield.

The purity is detected by HPLC, and reaches more than 98%, and in the product of our batch, the maximum single impurity of the product is 0.6574%, and the purity is 99.0710%.

The above description is provided for further details of the present invention with reference to specific modified embodiments, and it should not be considered that the present invention is limited to these specific embodiments, and it should be understood that those skilled in the art may make several simple deductions or substitutions without departing from the spirit of the present invention, and all such alternatives are deemed to fall within the scope of the present invention.

Claims

1. A solid phase synthesis method of snake venom tripeptide is characterized by comprising the following steps:

MBHA/AM resin swelling

Adding MBHA/AM resin into a reactor, adding piperidine/DMF solution, continuously swelling until swelling is finished, performing suction filtration, and washing with piperidine/DMF solution;

4-hydroxymethylphenylacetic acid coupling

Adding 4-hydroxymethylphenylacetic acid and a condensation reagent into a piperidine/DMF solution, dissolving the solution clearly, cooling, slowly adding DIC (Diformic acid) and stirring to obtain an activated solution, adding the activated solution into the reactor in the step (1), blowing nitrogen to start reaction, adding NMM after the reaction starts, continuously reacting until the reaction is complete, performing suction filtration, washing by using the piperidine/DMF solution, and washing by using DCM (DCM);

boc-dab (Fmoc) -OH hanging resin

Adding Boc-dab (Fmoc) -OH into the reactor in the step (1), adding DCM, blowing nitrogen, adding pyridine, controlling the temperature of the reaction liquid to be less than 20 ℃, dropwise adding DCB, keeping the temperature at a low temperature for a period of time after dropwise adding, then heating to 35-30 ℃ until the reaction is finished, performing suction filtration, and washing by using piperidine/DMF solution;

adding DIEA into DMF, stirring and mixing uniformly, adding Ac2O, stirring and mixing uniformly to obtain an end-capping solution, adding the end-capping solution into the reactor in the step (1), performing end-capping suction filtration, and washing with DMF;

washing with methanol, DCM and methanol in sequence, and shrinking the resin;

measuring the degree of substitution of the resin;

swelling of the resin

Adding the removed resin into a reactor, adding DMF, continuously swelling until swelling is finished, performing suction filtration, and washing by using DCM;

adding TFA/DCM, blowing nitrogen to react until the reaction is complete, filtering by suction, and washing by using DMF;

Boc-Pro-OH coupling

Adding Boc-Pro-OH and a condensation reagent into an activation cup, adding DMF (dimethyl formamide), dissolving, cooling, slowly adding DIC (Diformic acid) while stirring, keeping the temperature at low temperature after the addition is finished to prepare an activation liquid, adding the activation liquid into the reactor in the step (3), blowing nitrogen to start reaction, adding NMM (N-methyl-;

Boc-beta-Ala-OH coupling

Adding Boc-beta-Ala-OH and a condensation reagent into an activation cup, adding DMF (dimethyl formamide), dissolving, cooling, slowly adding DIC (Diformic acid) while stirring, keeping low temperature after the addition is finished to prepare an activation liquid, adding the activation liquid into the reactor in the step (3), blowing nitrogen to start reaction, adding NMM (N-methyl pyrrolidone), continuously reacting until the reaction is finished, performing suction filtration, washing with DMF (dimethyl formamide), and washing with DCM (dimethyl formamide);

benzylamine linkage

Washing the resin obtained after the reaction of the step (5) by using DMF/THF;

weighing benzylamine, adding the benzylamine into a beaker, adding DMF/THF for dissolving, adding the benzylamine into a reactor, carrying out nitrogen blowing reaction, taking a small resin sample, and cutting to show that the reaction is finished;

filtration and washing of the resin with DMF/THF solution;

mixing the filtrates, and drying to obtain crude product;

pulping and washing, adding tert-butyl methyl ether into the crude product, pulping, fully dispersing, filtering to obtain a filter cake, pulping the filter cake by using tert-butyl methyl ether for multiple times, and filtering;

drying

Putting the filter cake into a vacuum drying oven for drying until the weight is constant, and obtaining a crude product of H-beta-Ala-Pro-Dab-NHBzl;

purification of

2. The solid phase synthesis method of a snake venom-like tripeptide according to claim 1, wherein the solid phase synthesis method comprises the following steps: the molar ratio of Boc-Pro-OH, HOBT, DIC and NMM in step (5) was 1:1:1: 1.2.

3. The solid phase synthesis method of a snake venom-like tripeptide according to claim 1, wherein the solid phase synthesis method comprises the following steps: the molar ratio of Boc-beta-Ala-OH, HOBT, DIC and NMM in step (6) was 1:1:1: 1.2.

4. The solid phase synthesis method of a snake venom-like tripeptide according to claim 1, wherein the solid phase synthesis method comprises the following steps: in the steps (2), (5) and (6), the condensation reagent is conventional condensation reagent such as DCB/Py, DIC/HOBt, DIC/HOAt, HATU, HBTU and the like.

5. The solid phase synthesis method of a snake venom-like tripeptide according to claim 1, wherein the solid phase synthesis method comprises the following steps: in steps (2), (5) and (6), the deprotection reagent is TFA, and the concentration of the deprotection reagent ranges from 5% to 100%.

6. The solid phase synthesis method of a snake venom-like tripeptide according to claim 1, wherein the solid phase synthesis method comprises the following steps: the deprotection solvent in the steps (2), (5) and (6) is any one of DCM, THF, Acetone, MTBE and the like.