Acyl carrier protein ACP65-74 and preparation method thereof

Abstract

The invention provides an acyl carrier protein ACP65-74 and a preparation method of the acyl carrier protein ACP65-74. The preparation method comprises the following steps: carrying out deprotection and first washing on resin, and sequentially carrying out in-situ activation reaction on the resin by taking one amino acid as a unit according to the sequence of an amino acid sequence of acyl carrier protein ACP65-74 as shown in SEQ ID NO: 1; the in-situ activation reaction comprises the following steps: S1, carrying out coupling reaction on a single amino acid solution, an activating agent, a condensing agent and resin, and when the change of the conductivity of the reaction solution is less than 1%, filtering and carrying out secondary washing; and S2, sequentially carrying out deprotection and third washing on the resin subjected to the step S1. On-line monitoring is carried out on the process of solid-phase synthesis of the ACP65-74 from the polypeptide, so that the reaction efficiency can be improved, the purity of the product is improved, unnecessary waste in the reaction process is reduced, and the synthesis cost of the acyl carrier protein ACP65-74 is saved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein synthesis, and in particular to an acyl carrier protein ACP65-74 and a preparation method thereof. Background Art

[0002] In recent years, with the development and maturity of peptide synthesis technology, peptide drugs have become one of the hot spots in drug research and development. Due to its wide indications, high safety and significant efficacy, its clinical treatment status has been continuously improved. Many varieties have been included in the international and domestic treatment guidelines and expert consensus for related diseases. They have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, cardiovascular and cerebrovascular diseases, hepatitis, diabetes, and AIDS, and have broad development prospects. With the rapid development of technologies related to peptide synthesis (such as the emergence of solid-phase peptide synthesis technology), equipment and processes, the cost of peptide drug research and development and production costs have dropped significantly, and the development of peptide drugs continues to heat up.

[0003] Acyl carrier protein (ACP) is a key protein in fatty acid synthesis. It is located in the center of the fatty acid synthase system and acts as a carrier of acyl groups to transfer acyl groups from one enzyme reaction to another. ACP is not only involved in fatty acid synthesis, but also in mevalonate synthesis and unsaturated fatty acid reactions. The content and composition of unsaturated fatty acids in plant storage fatty acids and their proportion in total fatty acids are closely related to the types and differential expressions of ACP isomers. Therefore, acyl carrier protein is an important auxiliary factor in the biosynthesis of fatty acids in higher plants.

[0004] Acyl Carrier Protein (ACP65-74) is an active fragment of acyl carrier protein, ACP. ACP65-74 is recognized worldwide as a difficult peptide to synthesize. This protein fragment has been widely accepted as a model peptide to illustrate the efficiency of reagents, polymer supports, or synthetic conditions. Although the synthesis of this decapeptide appears simple, early attempts to synthesize this peptide using standard procedures have yielded disappointing results, with low yields and purity. Peptide yields gradually decrease after the addition of asparagine, isoleucine, and tyrosine, which may be due to steric hindrance of the amino acids. Peptide conformation is another important factor that leads to low peptide yields.

[0005] At present, the key to the synthesis of difficult peptides is to destroy the β-folded structure caused by the difficult sequence. By using mixed solvents, high-efficiency activators and very expensive new resins to change the reaction system, or by extending the reaction time, increasing the amount of amino acids, increasing the reaction temperature, adjusting the pH value of the reaction system and using high-ion salt solutions to change the reaction conditions, as well as new synthetic strategies such as fragment condensation and introduction of substituents, solutions are provided for the synthesis of "difficult sequence" peptides. However, some measures are highly operational, some processes are more complicated, and some are so expensive that they are not suitable for large-scale industrial production. Some strategies are still only in the laboratory research stage and cannot be achieved in large-scale production.

[0006] Therefore, how to develop a preparation method with high reaction efficiency and high product purity is of great significance for the preparation of acyl carrier protein ACP65-74. Summary of the invention

[0007] The main purpose of the present invention is to provide an acyl carrier protein ACP65-74 and a preparation method thereof, so as to solve the problem of low reaction efficiency of the acyl carrier protein ACP65-74 prepared in the prior art.

[0008] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for preparing acyl carrier protein ACP65-74 is provided, the preparation method comprising: deprotecting and washing the resin in sequence, performing in situ activation reaction on the resin in sequence with one amino acid as a unit in the order of the amino acid sequence of the acyl carrier protein ACP65-74 shown in SEQ ID NO:1, each time the in situ activation reaction is completed, one more amino acid is connected to the resin, and after all the amino acids are connected, the acyl carrier protein ACP65-74 is obtained; the in situ activation reaction comprises: S1, coupling reaction of a single amino acid solution, an activator, a condensing agent and the resin, and filtering and performing a second washing when the conductivity of the reaction solution changes by less than 1%; S2, deprotecting and washing the resin after completing step S1 in sequence.

[0009] Furthermore, the resin includes: Rink amide resin; deprotection is performed using a deprotecting agent, and the deprotecting agent includes: a piperidine solution with a mass fraction of 20-30%.

[0010] Furthermore, the activator includes: 1-hydroxybenzotriazole, N-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate or O-benzotriazole-tetramethyluronium hexafluorophosphate; the condensation agent includes: diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide or N,N-diisopropylethylenediamine.

[0011] Furthermore, the completion of deprotection of the resin is indicated by: after the first washing is completed, the resin is deprotected for a second time, and the eluate obtained after the second deprotection is subjected to ultraviolet detection. If no peak appears, the deprotection is completed.

[0012] Furthermore, the preparation of acyl carrier protein ACP65-74 is carried out using a polypeptide solid phase synthesis device, and the polypeptide solid phase synthesis device includes a deprotection cycle and an amino acid coupling cycle; wherein the deprotection cycle includes: a synthesis column, a third switching valve, a first pump body, a first switching valve, and a second switching valve are sequentially arranged from upstream to downstream, the outlet of the synthesis column is connected to the first inlet a1 of the third switching valve, the first outlet a2 of the third switching valve is connected to the inlet of the first pump body, the outlet of the first pump body is connected to the first inlet b1 of the first switching valve, the first outlet b2 of the first switching valve is connected to the first inlet c1 of the second switching valve, and the first outlet c2 of the second switching valve is connected to the inlet of the synthesis column; the amino acid coupling cycle includes: a fourth switching valve, a second pump body, a first switching valve, an activation reactor, a second switching valve, a synthesis column, a third switching valve, and a first pump body are sequentially arranged from upstream to downstream, the first outlet d2 of the fourth switching valve is connected to the inlet of the second pump body, the first outlet a2 of the third switching valve is connected to the inlet of the first pump body, and the outlet of the second pump body and the outlet of the first pump body are respectively connected to the first inlet of the first switching valve. The first switching valve is connected to the inlet b1, the second outlet b3 of the first switching valve is connected to the inlet of the activation reactor, the outlet of the activation reactor is connected to the second inlet c3 of the second switching valve, and the outlet of the synthesis column is connected to the first inlet a1 of the third switching valve; the resin is arranged in the synthesis column; the preparation method comprises: S1', the deprotection reagent and the detergent enter the deprotection cycle through the second inlet a3 of the third switching valve in sequence, and respectively complete the deprotection and the first washing of the resin; S2', the single amino acid solution is introduced into the second pump body and the first pump body from the first inlet d1 of the fourth switching valve, and the activator and the condensing agent are introduced into the second pump body and the first pump body from the second inlet a3 of the third switching valve, respectively, enter the amino acid coupling cycle, and complete the coupling reaction; S3', the detergent is introduced into the second pump body and the first pump body from the first inlet d1 of the fourth switching valve and the second inlet a3 of the third switching valve, respectively, enter the amino acid coupling cycle, and complete the second washing; S4', the deprotection reagent and the detergent enter the deprotection cycle through the second inlet a3 of the third switching valve in sequence, and respectively complete the deprotection and the third washing of the resin; the in situ activation reaction comprises the process of steps S2'-S4'.

[0013] Furthermore, the deprotection cycle and the amino acid coupling cycle also respectively include: a conductivity detector, which is located between the first pump body and the first switching valve and is used to detect the conductivity of the coupling reaction solution in S2'.

[0014] Furthermore, the deprotection cycle and the amino acid coupling cycle also respectively include: a UV detector, which is located between the synthesis column and the third switching valve and is used to detect whether the deprotection of the resin in S1' is complete.

[0015] Furthermore, after S1' and before S2', the preparation method further includes: after the first washing of the resin, the deprotection reagent enters the deprotection cycle through the second inlet a3 of the third switching valve, and the deprotection solution is ultraviolet detected by a UV detector.

[0016] Furthermore, the deprotection cycle and the amino acid coupling cycle also respectively include: a fifth switching valve, the fifth switching valve has a first inlet e1 and a first outlet e2, the outlet of the synthesis column is connected to the first inlet e1 of the fifth switching valve, and the first outlet e2 of the fifth switching valve is connected to the first inlet a1 of the third switching valve.

[0017] In order to achieve the above object, according to the second aspect of the present invention, an acyl carrier protein ACP65-74 prepared by the above preparation method is provided.

[0018] By applying the technical solution of the present invention, the process of solid phase synthesis of polypeptide ACP65-74 is monitored online, which can improve the reaction efficiency, further improve the degree of completion of the reaction, improve the purity of the product, reduce unnecessary waste in the reaction process, and save the synthesis cost of acyl carrier protein ACP65-74. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 The present invention shows a polypeptide solid phase synthesis device used in the preparation of acyl carrier protein ACP65-74;

[0021] Figure 2 The graph shows the conductivity monitoring result of the solution during the preparation process of the acyl carrier protein ACP65-74 in Example 1 of the present application;

[0022] Figure 3 The figure shows the UV value monitoring result of the solution during the preparation process of the acyl carrier protein ACP65-74 in Example 1 of the present application;

[0023] in, Figure 1 The following reference numerals are included:

[0024] 1. First injection valve; 2. Fourth switching valve; 3. Conductivity detector; 4. Second injection valve; 5. Third switching valve; 6. First switching valve; 7. Fifth switching valve; 8. Activation reactor; 9. Synthesis column; 10. Second switching valve; 11. UV detector; 13. Current limiter; 14. Waste liquid outlet valve; PB, first pump body; PA, second pump body. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0026] As mentioned in the background technology, the existing preparation process of acyl carrier protein ACP65-74 has the problems of difficult synthesis, high cost, long time or low purity. In a first typical embodiment of the present application, a preparation method of acyl carrier protein ACP65-74 is provided, and the preparation method comprises: deprotecting and washing the resin in sequence, and performing in situ activation reaction on the resin in sequence with 1 amino acid as a unit in the order of the amino acid sequence of acyl carrier protein ACP65-74 shown in SEQ ID NO: 1. After each in situ activation reaction is completed, one more amino acid is connected to the resin. After all amino acids are connected, acyl carrier protein ACP65-74 is obtained; the in situ activation reaction comprises: S1, coupling reaction of a single amino acid solution, an activator, a condensing agent and a resin, and filtering and performing a second washing when the conductivity of the reaction solution changes by less than 1%; S2, deprotecting and washing the resin after completing step S1 in sequence.

[0027] SEQ ID NO: 1: NH2-VQAAIDYING-CONH2.

[0028] The present application monitors the preparation method process of the in situ activation of the acyl carrier protein ACP65-74, and can timely obtain the degree of reaction completeness, so as to make timely adjustments to the parameters in the reaction process, further avoid the generation of side reactions, and obtain the acyl carrier protein ACP65-74 with higher purity.

[0029] Any type of reagent suitable for the synthesis of the acyl carrier protein ACP65-74 of the present application is suitable for the present application. In a preferred embodiment, the resin includes: Rink amide resin; preferably, deprotection is performed using a deprotecting agent, and the deprotecting agent includes: a piperidine solution with a mass fraction of 20-30%; preferably, the activating agent includes: N-hydroxy-7-azabenzotriazole (HOAT), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TBTU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT); preferably, the condensing agent includes: diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide (DCC) or N,N-diisopropylethylenediamine (DIPEA); any reagent that can be used for peptide synthesis cleaning is suitable for the present application. In a preferred embodiment, a detergent is used for the first wash, the second wash or the third wash, and the detergent includes: dimethylacetamide DMF.

[0030] In order to further improve the synthesis efficiency of acyl carrier protein ACP65-74, acyl carrier protein ACP65-74 with higher purity can be obtained in a shorter time. In a preferred embodiment, the deprotection time is 5-30min, specifically 5min, 10min, 15min, 20min, 25min; preferably, the coupling reaction time is 45-120min, specifically 45min, 55min, 70min, 80min, 90min, 110min, 115min, 120min. It should be noted that in order to further improve the synthesis efficiency of acyl carrier protein ACP65-74, the reaction degree can be obtained in time by ultraviolet detection of the deprotection reaction solution and conductivity detection of the coupling reaction solution, and the synthesis of acyl carrier protein ACP65-74 can be further efficiently completed by timely adjustment of the reaction time. More preferably, the deprotection time is 5min; preferably, the coupling reaction time is 45min.

[0031] In order to further efficiently obtain the acyl carrier protein ACP65-74 with higher purity, in a preferred embodiment, the amino acids include: valine, glutamine, alanine, isoleucine, aspartic acid, tyrosine, asparagine or glycine; preferably, the concentration of the single amino acid solution is 0.1-1.0 mol / L, specifically 0.1 mol / L, 0.25 mol / L, 0.5 mol / L, 0.65 mol / L, 0.85 mol / L, 0.9 mol / L, 1.0 mol / L. Preferably, the molar ratio of the amount of the single amino acid solution to the synthesis scale of the acyl carrier protein ACP65-74 is 1.5-10:1, specifically 1.5:1, 3:1, 10:1; preferably, the molar ratio of the amount of the activator to the synthesis scale of the acyl carrier protein ACP65-74 is 1.5-10:1, specifically 1.5:1, 3:1, 10:1; preferably, the molar ratio of the amount of the condensing agent to the synthesis scale of the acyl carrier protein ACP65-74 is 1.5-10:1, specifically 1.5:1, 3:1, 10:1; wherein, the synthesis scale of the acyl carrier protein ACP65-74 is the product of the weight of the resin (g) and the amino acid loading (mol / g) of the resin.

[0032] It should be noted that in order to further improve the synthesis efficiency of the acyl carrier protein ACP65-74, the degree of reaction can be obtained in time by performing ultraviolet detection on the deprotection reaction solution and conductivity detection on the coupling reaction solution, and the synthesis of the acyl carrier protein ACP65-74 can be further efficiently completed by timely adjusting the amount of each reactant. More preferably, the molar ratio of the amount of the single amino acid solution to the synthesis scale of the acyl carrier protein ACP65-74 is 1.5:1; preferably, the molar ratio of the amount of the activator to the synthesis scale of the acyl carrier protein ACP65-74 is 1.5:1; preferably, the molar ratio of the amount of the condensing agent to the synthesis scale of the acyl carrier protein ACP65-74 is 1.5:1.

[0033] In order to further determine whether the deprotection and washing of the resin are complete, in a preferred embodiment, the sign of the completion of the deprotection of the resin is: after the first washing is completed, the resin is deprotected for a second time, and the eluent obtained after the second deprotection is subjected to UV detection. If no peak appears, the deprotection is completed. During the deprotection process, 4-aminomethyl (Fmoc group) forms Fmoc-piperidine adduct with the deprotection reagent, which has UV absorption at 289nm and 301nm. The second addition of the deprotection reagent does not produce a peak, indicating that no Fmoc group has been removed, that is, the deprotection is completed. Preferably, the washing liquid obtained by the second washing of S1 and the third washing of S2 in the in-situ activation reaction can also be subjected to UV detection to observe whether there is a peak of the Fmoc group to determine the degree of completeness of the elution.

[0034] In order to further efficiently control the deprotection reaction, in a preferred embodiment, the acyl carrier protein ACP65-74 is prepared by using a polypeptide solid phase synthesis device, and the polypeptide solid phase synthesis device includes a deprotection cycle and an amino acid coupling cycle; wherein the deprotection cycle includes: a synthesis column 9, a third switching valve 5, a first pump body PB, a first switching valve 6, and a second switching valve 10 arranged in sequence from upstream to downstream, the outlet of the synthesis column 9 is connected to the first inlet a1 of the third switching valve 5, the first outlet a2 of the third switching valve 5 is connected to the inlet of the first pump body PB, the outlet of the first pump body PB is connected to the first inlet b1 of the first switching valve 6, the first outlet b2 of the first switching valve 6 is connected to the first inlet c1 of the second switching valve 10, and the first outlet c2 of the second switching valve 10 is connected to the inlet of the synthesis column 9.

[0035] The deprotection cycle in the above device can more conveniently control the deprotection reaction and make the resin contact with the reaction solution more thoroughly and completely, further improving the reaction efficiency.

[0036] In order to further efficiently control the amino acid coupling reaction, in a preferred embodiment, the amino acid coupling cycle includes: a fourth switching valve 2, a second pump body PA, a first switching valve 6, an activation reactor 8, a second switching valve 10, a synthesis column 9, a third switching valve 5, and a first pump body PB, which are arranged in sequence from upstream to downstream, wherein the first outlet d2 of the fourth switching valve 2 is connected to the inlet of the second pump body PA, the first outlet a2 of the third switching valve 5 is connected to the inlet of the first pump body PB, the outlet of the second pump body PA and the outlet of the first pump body PB are respectively connected to the first inlet b1 of the first switching valve 6, the second outlet b3 of the first switching valve 6 is connected to the inlet of the activation reactor 8, the outlet of the activation reactor 8 is connected to the second inlet c3 of the second switching valve 10, and the outlet of the synthesis column 9 is connected to the first inlet a1 of the third switching valve 5.

[0037] The amino acid coupling reaction cycle in the above device can make the resin and the amino acid fully contact, reduce the damage of the carrier and the connected peptide chain caused by the strong shear force, and avoid the problem of low synthesis efficiency caused by the resin sticking to the wall of the synthesis reactor.

[0038] The preparation of acyl carrier protein ACP65-74 using the above device can improve the degree of automation of the reaction, make it easier to control the purity and reaction efficiency of the acyl carrier protein ACP65-74 product, and further improve the repeatability of the reaction, which is more suitable for industrial production. In a preferred embodiment, the resin is arranged in a synthesis column 9; the preparation method comprises: S1', a deprotection reagent and a detergent enter a deprotection cycle through a second inlet a3 of a third switching valve 5 in sequence, and deprotection and a first washing of the resin are respectively completed; S2', a single amino acid solution is introduced from a first inlet d1 of a fourth switching valve 2 , the activator and the condensing agent are respectively introduced into the second pump body PA and the first pump body PB from the second inlet a3 of the third switching valve 5, entering the amino acid coupling cycle to complete the coupling reaction; S3', the detergent is respectively introduced into the second pump body PA and the first pump body PB from the first inlet d1 of the fourth switching valve 2 and the second inlet a3 of the third switching valve 5, entering the amino acid coupling cycle to complete the second washing; S4', the deprotection reagent and the detergent enter the deprotection cycle in turn through the second inlet a3 of the third switching valve 5 to complete the deprotection of the resin and the third washing respectively; the in situ activation reaction includes the process of steps S2'-S4'.

[0039] In order to further quickly obtain the degree of completion of the amino acid coupling reaction, in a preferred embodiment, the deprotection cycle and the amino acid coupling cycle also include: a conductivity detector 3, which is located between the first pump body PB and the first switching valve 6 and is used to detect the conductivity of the coupling reaction solution in S2'.

[0040] In order to further quickly determine whether the washing after the deprotection reaction is complete, in a preferred embodiment, the deprotection cycle and the amino acid coupling cycle also include: a UV detector 11, which is located between the synthesis column 9 and the third switching valve 5, and is used to detect whether the deprotection of the resin in S1' and S4' and the washing of the coupling reaction solution in S3' are complete.

[0041] In order to further improve the accuracy of judging whether the washing after the deprotection reaction is complete, in a preferred embodiment, after S1' and before S2', the preparation method further includes: after the first washing of the resin, the deprotection reagent enters the deprotection cycle through the second inlet a3 of the third switching valve 5, and the deprotection liquid is subjected to ultraviolet detection using the UV detector 11.

[0042] In order to further accurately control the flow rate of the reaction system, in a preferred embodiment, the deprotection cycle and the amino acid coupling cycle also include: a fifth switching valve 7, the fifth switching valve 7 has a first inlet e1 and a first outlet e2, the outlet of the synthesis column 9 is connected to the first inlet e1 of the fifth switching valve 7, and the first outlet e2 of the fifth switching valve 7 is connected to the first inlet a1 of the third switching valve 5.

[0043] In a second typical embodiment of the present application, an acyl carrier protein ACP65-74 prepared using the above-mentioned preparation method is provided.

[0044] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0045] Example 1

[0046] This embodiment uses Figure 1 The polypeptide solid phase synthesis device shown is used to prepare the acyl carrier protein ACP65-74, the sequence of the acyl carrier protein ACP65-74 is NH2-VQAAIDYING-CONH2 (SEQ ID NO: 1), the resin used is Rink amide resin, which is arranged in the synthesis column 9 of the polypeptide solid phase synthesis device, the synthesis scale is: 2g resin, and the amino acid loading capacity of the resin is 0.69mmol / g.

[0047] The reagents used in this example include: 20% piperidine solution (deprotecting agent), dimethylacetamide DMF (detergent), diisopropylcarbodiimide (DIC) (condensing agent), 1-hydroxybenzotriazole (HOBt) (activating agent) and 8 different types of single amino acid solutions (0.25 mol / L) shown in SEQ ID NO: 1.

[0048] In this example, two synthesis methods (pre-activation and in-situ activation) were used to prepare and synthesize the acyl carrier protein ACP65-74.

[0049] The specific steps of in-situ activation are as follows:

[0050] For example Figure 1 The peptide solid phase synthesis device shown in the figure was debugged to confirm that the equipment can operate normally.

[0051] S1', 20% piperidine solution is introduced into the polypeptide solid phase synthesis device through the inlet of the second injection valve 4, and enters the deprotection cycle through the second inlet a3 of the third switching valve 5. The 20% piperidine solution enters the first pump body PB from the first outlet a2 of the third switching valve 5, and passes through the conductivity detector 3, the first switching valve 6, the second switching valve 10, the synthesis column 9, the fifth switching valve 7, the UV detector 11 and the third switching valve 5 in sequence for 5-30 minutes to complete the deprotection of the resin.

[0052] Then, dimethylacetamide DMF is introduced into the deprotection cycle through the second injection valve 4, and dimethylacetamide enters the first pump body PB from the first outlet a2 of the third switching valve 5, and then passes through the conductivity detector 3, the first switching valve 6, the second switching valve 10, the synthesis column 9, the fifth switching valve 7, the UV detector 11 and the third switching valve 5 in sequence to complete the first washing of the resin.

[0053] To detect whether the above deprotection washing is complete, the 20% piperidine solution is passed through the deprotection cycle again, and the 20% piperidine solution passing through is detected by the UV detector 11. If there is no UV peak, that is, there is no Fmoc protecting group, that is, the deprotection is complete, otherwise it is incomplete. The UV value monitoring curve in the preparation process of this application is shown in FIG. Figure 3 shown.

[0054] In the order shown in SEQ ID NO: 1, one single amino acid solution is passed through the amino acid coupling cycle to perform one in situ activation reaction, and then subsequent single amino acid solutions are sequentially passed through to complete subsequent in situ activation reactions.

[0055] S2', the single amino acid solution is introduced into the polypeptide solid phase synthesis device through the inlet of the first injection valve 1, and is introduced into the second pump body PA through the first outlet d2 of the fourth switching valve 2, diisopropylcarbodiimide and 1-hydroxybenzotriazole are introduced into the polypeptide solid phase synthesis device through the inlet of the second injection valve 4, and are introduced into the first pump body PB through the second inlet a3 of the third switching valve 5, the single amino acid solution, diisopropylcarbodiimide and 1-hydroxybenzotriazole enter the amino acid coupling cycle, and flow through the conductivity detector 3, the first switching valve 6, the activation reactor 8, the second switching valve 10, the synthesis column 9, the fifth switching valve 7, the UV detector 11, the third switching valve 5 and the first pump body PB in sequence for 45-120 minutes to complete the coupling reaction.

[0056] The molar amounts of the single amino acid solution, diisopropylcarbodiimide and 1-hydroxybenzotriazole introduced into the peptide solid phase synthesis device are 2.07 mmol, 2.07 mmol and 2.07 mmol respectively. The conductivity of the reaction solution during the above S2' coupling reaction is detected by the conductivity detector 3. When the conductivity change is less than 1% within 5 minutes, the coupling reaction is terminated. The conductivity monitoring curve during the preparation process of this application is shown in FIG. Figure 2 shown.

[0057] S3', dimethylacetamide is introduced into the polypeptide solid phase synthesis device through the inlet of the first injection valve 1 and the first outlet d2 of the fourth switching valve 2, and enters the amino acid coupling cycle through the second pump body PA and the first pump body PB to complete the second washing.

[0058] S4', 20% piperidine solution is introduced into the polypeptide solid phase synthesis device through the inlet of the second injection valve 4, and enters the deprotection cycle through the second inlet a3 of the third switching valve 5. The 20% piperidine solution enters the first pump body PB from the first outlet a2 of the third switching valve 5, and passes through the conductivity detector 3, the first switching valve 6, the second switching valve 10, the synthesis column 9, the fifth switching valve 7, the UV detector 11 and the third switching valve 5 in sequence for 5 minutes to complete the deprotection of the resin.

[0059] Then, dimethylacetamide is introduced into the deprotection cycle through the second injection valve 4, and dimethylacetamide enters the first pump body PB from the first outlet a2 of the third switching valve 5, and then passes through the conductivity detector 3, the first switching valve 6, the second switching valve 10, the synthesis column 9, the fifth switching valve 7, the UV detector 11 and the third switching valve 5 in sequence to complete the third washing of the resin.

[0060] The process of steps S2'-S4' is one in situ activation reaction, and the next amino acid in the amino acid sequence of the acyl carrier protein ACP65-74 is used as the single amino acid solution for the next in situ activation reaction for subsequent in situ activation reactions until the acyl carrier protein ACP65-74 reaches a predetermined length, for a total of 10 in situ activation reactions.

[0061] The waste liquid obtained during the first washing, second washing and third washing processes is discharged from the polypeptide solid phase synthesis device from the waste liquid outlet valve 14 through the restrictor 13 connected to the second outlet a4 of the third switching valve 5 .

[0062] The product of the above 10 in situ activation reactions was cut, and the complete polypeptide chain was peeled off from the carrier. After post-treatment, the acyl carrier protein ACP65-74 prepared by in situ activation was obtained. The sample was sent for purity detection, and the purity of the in situ activated synthetic product was 96.13%.

[0063] The pre-activation reaction is the same as the in-situ activation reaction except that the S20' amino acid pre-activation step is added between S1' and S2'. The specific S20' amino acid pre-activation step is as follows:

[0064] S20', the single amino acid solution is introduced into the polypeptide solid phase synthesis device through the inlet of the first injection valve 1, and is introduced into the second pump body PA through the first outlet d2 of the fourth switching valve 2. 1-Hydroxybenzotriazole and N,N-diisopropylcarbodiimide are introduced into the polypeptide solid phase synthesis device through the inlet of the second injection valve 4, and are introduced into the first pump body PB through the second inlet a3 of the third switching valve 5. The single amino acid solution and 1-hydroxybenzotriazole flow through the conductivity detector 3, the first switching valve 6, the activation reactor 8, the second switching valve 10, the fourth switching valve 2 and the second pump body PA in sequence, wherein the second outlet c4 of the second switching valve 10 is connected to the second inlet d3 of the fourth switching valve 2, for 5 minutes to complete the amino acid pre-activation reaction.

[0065] The molar masses of the single amino acid solution, 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide introduced into the peptide solid phase synthesis device were 2.07 mmol, 2.07 mmol and 2.07 mmol, respectively.

[0066] After the amino acid pre-activation reaction is completed, dimethylacetamide is introduced into the polypeptide solid phase synthesis device through the inlet of the first injection valve 1 and the first outlet d2 of the fourth switching valve 2, respectively, and flows through the conductivity detector 3, the first switching valve 6, the activation reactor 8, the second switching valve 10, the fourth switching valve 2 and the second pump body PA in sequence through the second pump body PA and the first pump body PB to complete the washing after the amino acid pre-activation reaction.

[0067] The above-mentioned S2'-S4' is subsequently carried out, i.e., S20', S2', S3' and S4' are one pre-activation reaction, which can complete the synthesis of one amino acid in the acyl carrier protein ACP65-74, and a total of 10 pre-activation reactions are carried out. The product of the 10 pre-activation reactions of S20'-S4' is cut, and the complete polypeptide chain is peeled off from the carrier. After post-treatment, the acyl carrier protein ACP65-74 prepared by pre-activation is obtained, and the sample is sent for purity detection. The purity of the pre-activated synthetic product is 94.67%. It can be seen that the purity of the acyl carrier protein ACP65-74 obtained by the in-situ activation preparation method of the present application is higher.

[0068] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: by optimizing the reaction parameters of each reaction stage, the acyl carrier protein ACP65-74 is synthesized using a polypeptide solid phase synthesis device, the reagent equivalents required for synthesis are reduced, the generation of by-products is reduced, the reaction efficiency and product purity are improved, and the reaction cost is saved.

[0069] In addition, the peptide solid phase synthesis device of the present application adopts a circulation pipeline instead of traditional mechanical stirring, which reduces the damage caused by strong shear force to the carrier and the connected peptide chain. At the same time, the resin in the synthesis column is completely immersed in the reaction solution, avoiding the problem of low synthesis efficiency caused by the resin sticking to the side wall of the synthesis reactor, further improving the mixing efficiency and shortening the time required for synthesis. And by real-time monitoring of processes such as deprotection, washing and amino acid coupling, the reaction parameters can be automatically adjusted in time, making the synthesis process more controllable, reducing the difficulty of operation, and improving the repeatability and reliability of the experiment. And the method of the present application is not only limited to laboratory research, but also suitable for large-scale peptide production, providing a more efficient and economical peptide synthesis solution for the fields of pharmaceuticals and biotechnology.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing acyl carrier protein ACP65-74, characterized in that: The preparation method comprises: The resin is sequentially deprotected and first washed, and an in situ activation reaction is sequentially performed on the resin in the order of the amino acid sequence of the acyl carrier protein ACP65-74 shown in SEQ ID NO: 1, with one amino acid as a unit, and each time the in situ activation reaction is completed, one more amino acid is connected to the resin, and after all the amino acids are connected, the acyl carrier protein ACP65-74 is obtained; The in situ activation reaction comprises: S1, coupling reaction of a single amino acid solution, an activating agent, a condensing agent and the resin, and filtering and performing a second washing when the conductivity of the reaction solution changes by less than 1%; S2, sequentially performing the deprotection and the third washing on the resin after completing the S1 step.

2. The preparation method according to claim 1, characterized in that: The resin includes: Rink amide resin; The deprotection is performed using a deprotecting agent, wherein the deprotecting agent comprises: a piperidine solution with a mass fraction of 20-30%.

3. The preparation method according to claim 1, characterized in that: The activator includes: 1-hydroxybenzotriazole, N-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate or O-benzotriazole-tetramethyluronium hexafluorophosphate; The condensing agent includes: diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide or N,N-diisopropylethylenediamine.

4. The preparation method according to claim 1, characterized in that: The completion of the deprotection of the resin is indicated by: After the first washing is completed, the resin is subjected to a second deprotection, and the eluate obtained after the second deprotection is subjected to ultraviolet detection. If no peak appears, the deprotection is completed.

5. The preparation method according to claim 1, characterized in that: The acyl carrier protein ACP65-74 is prepared by using a polypeptide solid phase synthesis device. The polypeptide solid phase synthesis device includes a deprotection cycle and an amino acid coupling cycle; Wherein, the deprotection cycle comprises: A synthesis column (9), a third switching valve (5), a first pump body (PB), a first switching valve (6), and a second switching valve (10) are arranged in sequence from upstream to downstream, wherein the outlet of the synthesis column (9) is connected to the first inlet a1 of the third switching valve (5), the first outlet a2 of the third switching valve (5) is connected to the inlet of the first pump body (PB), the outlet of the first pump body (PB) is connected to the first inlet b1 of the first switching valve (6), the first outlet b2 of the first switching valve (6) is connected to the first inlet c1 of the second switching valve (10), and the first outlet c2 of the second switching valve (10) is connected to the inlet of the synthesis column (9); The amino acid coupling cycle comprises: A fourth switching valve (2), a second pump body (PA), a first switching valve (6), an activation reactor (8), a second switching valve (10), a synthesis column (9), a third switching valve (5), and a first pump body (PB) are arranged in sequence from upstream to downstream, wherein a first outlet d2 of the fourth switching valve (2) is connected to an inlet of the second pump body (PA), a first outlet a2 of the third switching valve (5) is connected to an inlet of the first pump body (PB), an outlet of the second pump body (PA) and an outlet of the first pump body (PB) are respectively connected to a first inlet b1 of the first switching valve (6), a second outlet b3 of the first switching valve (6) is connected to an inlet of the activation reactor (8), an outlet of the activation reactor (8) is connected to a second inlet c3 of the second switching valve (10), and an outlet of the synthesis column (9) is connected to a first inlet a1 of the third switching valve (5); The resin is arranged in the synthesis column (9); The preparation method comprises: S1', the deprotection reagent and the washing agent enter the deprotection cycle in sequence through the second inlet a3 of the third switching valve (5), respectively completing the deprotection and the first washing of the resin; S2', the single amino acid solution is introduced into the second pump body (PA) and the first pump body (PB) from the first inlet d1 of the fourth switching valve (2), and the activator and the condensing agent are introduced into the second pump body (PA) and the first pump body (PB) from the second inlet a3 of the third switching valve (5), respectively, to enter the amino acid coupling cycle, and complete the coupling reaction; S3', the detergent is respectively passed through the first inlet d1 of the fourth switching valve (2) and the second inlet a3 of the third switching valve (5), into the second pump body (PA) and the first pump body (PB), enters the amino acid coupling cycle, and completes the second washing; S4', the deprotection reagent and the washing agent enter the deprotection cycle through the second inlet a3 of the third switching valve (5) in sequence, and respectively complete the deprotection and the third washing of the resin; The in-situ activation reaction includes steps S2'-S4'.

6. The preparation method according to claim 5, characterized in that: The deprotection cycle and the amino acid coupling cycle also respectively include: a conductivity detector (3), the conductivity detector (3) is located between the first pump body (PB) and the first switching valve (6), and is used to detect the conductivity of the coupling reaction solution in the S2'.

7. The preparation method according to claim 5, characterized in that: The deprotection cycle and the amino acid coupling cycle also respectively include: a UV detector (11), wherein the UV detector (11) is located between the synthesis column (9) and the third switching valve (5) and is used to detect whether the deprotection of the resin in the S1' is complete.

8. The preparation method according to claim 7, characterized in that: After S1' and before S2', the preparation method further includes: After the first washing of the resin, the deprotection reagent enters the deprotection cycle through the second inlet a3 of the third switching valve (5), and the deprotection solution is subjected to ultraviolet detection using the UV detector (11).

9. The preparation method according to claim 5, characterized in that: The deprotection cycle and the amino acid coupling cycle further include: a fifth switching valve (7), wherein the fifth switching valve (7) has a first inlet e1 and a first outlet e2, the outlet of the synthesis column (9) is connected to the first inlet e1 of the fifth switching valve (7), and the first outlet e2 of the fifth switching valve (7) is connected to the first inlet a1 of the third switching valve (5).

10. Acyl carrier protein ACP65-74 prepared by the preparation method according to any one of claims 1 to 9.

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