A method for synthesizing an antifreeze glycopeptide polypeptide

Abstract

The application relates to a synthesis method of an anti-freezing glycopeptide polypeptide and belongs to the technical field of polypeptide drug synthesis. The method comprises the following steps: 2-CL-Resin is used as a carrier resin, under the condition of adding an activating agent, the carrier resin and alanine are coupled to obtain Fmoc-Ala-2-CL-Resin; according to the amino acid sequence of the anti-freezing glycopeptide, other amino acids are sequentially coupled through a solid-phase synthesis method; after a protecting group is removed and the carrier resin is cleaved, the anti-freezing glycopeptide crude peptide is obtained; and after purification, salt conversion and freeze-drying, the anti-freezing glycopeptide polypeptide is obtained. The method has the advantages of short synthesis period, low cost, easy post-treatment, few by-products, high product yield, facilitation of large-scale production of the anti-freezing glycopeptide, and considerable economic applicative value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis technology, specifically, it relates to a method for synthesizing an antifreeze glycopeptide polypeptide. Background Technology

[0002] Antifreeze glycopeptides are a class of biological agents that can survive at temperatures below the freezing point of body fluids. They are typically composed of repeating tripeptide units (-Ala-Ala-Thr*-) with threonine side chains. With the growth of global trade and the widening of production and sales distances, the demand for cold-chain processed foods is increasing. Frozen foods are also playing a growing role in the food industry.

[0003] Antifreeze peptides, as a new type of food additive, can effectively reduce the formation and recrystallization of ice crystals in food during the cold chain process, thereby improving the quality of low-temperature cold chain food.

[0004] A better understanding of the mechanism of action of antifreeze glycopeptides will help apply these materials to fields such as the food industry and biomedicine.

[0005] Antifreeze glycopeptides are a type of antifreeze peptide, and their amino acid sequence abbreviation is as follows:

[0006] Ala-Ala-Thr(OGalNAcAc3)-Ala-Ala-Thr(OGalNAcAc3)-Ala-Ala-Thr(OGalNAcAc3)-Ala-Ala.

[0007] The single-letter symbol is represented as: AAT(OGalNAcAc3)AAT(OGalNAcAc3)AAT(OGalNAcAc3)AA.

[0008] In existing technologies, the synthesis of antifreeze glycopeptides often employs a conventional method of sequentially coupling amino acids followed by sugar modification. However, this synthesis method results in incomplete sugar modification of the antifreeze glycopeptide chain, leading to poor peptide chain purity and low product yield at the end of the synthesis. Summary of the Invention

[0009] The purpose of this invention is to provide a method for synthesizing antifreeze glycopeptides. This method is simple to operate, has a short synthesis cycle, low cost, reduces waste liquid generation, is easy to process, produces few by-products, and has a high product yield.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] A method for synthesizing an antifreeze glycopeptide specifically includes the following steps:

[0012] Step S1: Preparation of antifreeze glycopeptide precursor resin: Starting with the carrier resin, the carrier resin is added to a solid-phase reaction column, washed and swollen with DMF, and then Fmoc-Ala-OH is dissolved in DMF and mixed with activator and condensing agent, and added to the above-mentioned reaction column containing the carrier resin to carry out a condensation reaction, so that the solid-phase carrier resin and the N-terminal Fmoc-protected Fmoc-Ala-OH are coupled to obtain Fmoc-Ala-OH-carrier resin;

[0013] Step S2: Based on the amino acid sequence of the antifreeze glycopeptide, following the coupling order from C-terminus to N-terminus, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled to Fmoc-Ala-carrier resin using a solid-phase synthesis method to obtain Fmoc antifreeze glycopeptide-carrier resin.

[0014] Step S3: After removing Fmoc protection from the antifreeze glycopeptide-carrier resin, the Fmoc-antifreeze glycopeptide-carrier resin is cleaved and lysed using a mixture and trifluoroacetic acid to obtain crude antifreeze glycopeptide. The crude antifreeze glycopeptide is then placed in a NaOH aqueous solution (50 mg / L) and stirred at a warm temperature. Mass spectrometry analysis shows that the protection removal can be completed within 20 minutes.

[0015] Step S4: After purifying, converting, and freeze-drying the crude antifreeze glycopeptide, the final antifreeze glycopeptide polypeptide product is obtained.

[0016] Furthermore, the carrier resin is 2-CL resin with a degree of substitution of 0.40 mmol / g.

[0017] Furthermore, Fmoc is a 9-fluorenemethyloxycarbonyl group, and Fmoc is attached to an amino group or to a nitrogen atom on the carbon atom attached to a carboxyl group.

[0018] Furthermore, the solid-phase synthesis method in step S2 includes the following steps:

[0019] Step A1: The Fmoc protecting group of the Fmoc-Ala-carrier resin is removed by DBLK to obtain NH-Ala-carrier resin. The DBLK is composed of piperidine and DMF mixed in a volume ratio of 1:3.

[0020] Step A2: Under the condition of adding an activator and a condensing agent system, NH-Ala-carrier resin and Fmoc-protected and side-chain-protected threonine are coupled to obtain Fmoc-Ala-Ala-carrier resin.

[0021] Step A3: Repeat steps A1 and A2 based on the amino acid sequence of the antifreeze glycopeptide.

[0022] Step A4: Based on the amino acid sequence of the antifreeze glycopeptide, remove the AC3 protecting group from Thr(OGalNAcAc3) using hydrazine hydrate.

[0023] Furthermore, the activator is DIC, the condensing agent is HOBt, and the condensation reaction time is 2 hours.

[0024] Furthermore, the amino acid T* in the antifreeze glycopeptide is Fmoc-Thr(OGalNAcAc3)-OH.

[0025] Furthermore, the coupling sequence from C-terminus to N-terminus is Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH.

[0026] Further, the purification conditions in step S4 are as follows: reversed-phase high-performance liquid chromatography is used, with reversed-phase octadecylsilane as the stationary phase, the column temperature is 35℃, mobile phase A is 0.1% acetic acid / water solution by volume, mobile phase B is acetonitrile, mobile phase A and mobile phase B are eluted at a gradient of 80:20 by volume, the flow rate is 60-70 mL / min, and the detection wavelength is 214 nm.

[0027] The beneficial effects of this invention are as follows: The antifreeze glycopeptides obtained by the above-described synthesis method inhibit ice crystal recrystallization and protect the cell integrity and structural characteristics of food under low-temperature conditions. By adsorbing onto the surface of ice crystals, the antifreeze peptides inhibit ice crystal growth and recrystallization, thereby reducing mechanical damage to food cells and maintaining the high quality of the food.

[0028] This invention is simple to operate, has a short synthesis cycle, low cost, reduces waste liquid generation, is easy to process, produces few by-products, and has a high product yield. It is conducive to the large-scale production of antifreeze glycopeptides and polypeptides, and has considerable economic and practical value and broad application prospects. Attached Figure Description

[0029] Figure 1 This is a flowchart of the synthesis method of the present invention;

[0030] Figure 2 This is a structural diagram of the crude antifreeze glycopeptide of the present invention;

[0031] Figure 3 This is a purity diagram of the antifreeze glycopeptide acetate of the present invention;

[0032] Figure 4 This is a mass spectrometry chromatogram of the antifreeze glycopeptide acetate of the present invention;

[0033] Figure 5 This is a structural diagram of the crude antifreeze glycopeptide from another embodiment of the present invention. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] The English abbreviations used in this invention and their meanings are explained in Table 1 below:

[0036]

[0037] In this article, "substitution rate" refers to the amount of substance loaded per unit volume of resin, expressed in mmol / g.

[0038] Example 1:

[0039] like Figure 1 As shown, the synthesis of Fmoc-Ala-2-CL-Resin:

[0040] Weigh 10g of 2-CL-Resin with a substitution degree of 0.40mmol / g and add it to a solid-phase reaction column. Wash twice with DMF and swell 2-CL-Resin with DCM for 25min. Then weigh 1.24g of Fmoc-Ala-OH and dissolve it in DCM. Mix thoroughly with 3.70mL of DIEA and add it to the above reaction column containing 2-CL-Resin. Perform condensation reaction for 2h, then wash with DMF. After washing, add 8mL of methanol to block for 1h. Then wash three times with DMF and three times with DCM. The methanol is condensed and dried to obtain Fmoc-Ala-2-CL-Resin.

[0041] Example 2:

[0042] Elongation reaction of Fmoc-Ala-2-CL-Resin peptide chain:

[0043] Weigh 5g of Fmoc-Ala-2-CL-Resin and add it to the solid-phase reaction column. Wash twice with DMF, swell Fmoc-Ala-2-CL-Resin with DMF for 20 min, remove Fmoc protection with 20% DBLK, then wash four times with DMF and twice with DCM. Detect the resin color using the ninhydrin method. If the resin is colored, it indicates that Fmoc has been removed.

[0044] Dissolve 1.55g Fmoc-Ala-OH, 0.68g HOBt, and 0.63g DIC in a 1:1 volume ratio of DCM and DMF mixed solution, then add the solution to a solid-phase reaction column and react at room temperature for 2 hours. The reaction endpoint is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin shows color, the reaction is incomplete and requires an additional 1 hour of coupling reaction. This criterion applies to the determination of the reaction endpoint by the ninhydrin method in the following content.

[0045] Repeat the steps of removing Fmoc protection and adding corresponding amino acid couplings, following the main chain peptide sequence of the antifreeze glycopeptide, sequentially performing the coupling of Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH to obtain Fmoc-antifreeze glycopeptide-2-cl-Resin. By optimizing the coupling sequence, side reactions that may occur between adjacent amino acid residues can be avoided. At the same time, this coupling sequence can make the reaction steps smoother, reduce the accumulation of intermediate products, and thus improve the overall synthesis efficiency.

[0046] Example 3:

[0047] Drying of Fmoc-antifreeze glycopeptide-2-cl-Resin with fully protected side chains:

[0048] The Fmoc-antifreeze glycopeptide-2-cl-Resin obtained in Example 2 was used to remove Fmoc protection with 20% DBLK, followed by washing with DMF four times and DCM twice. The resin color was detected by the ninhydrin method; the presence of color in the resin indicated that Fmoc had been removed. Then, the AC3 protecting group in Thr(OGalNAcAc3) was removed using hydrazine hydrate, followed by washing with methanol twice, and the resin was vacuum dried overnight.

[0049] The antifreeze glycopeptide-2-cl-Resin was weighed to obtain 8.5g (resin weight gain rate 70%).

[0050] Example 4:

[0051] Furthermore, to address the issue of conventional sequential amino acid coupling followed by final sugar modification, which results in incomplete sugar modification of the antifreeze glycopeptide chain and poor peptide purity at the end of synthesis, leading to low product yield, a method was proposed: first modifying the sugar and amino acids, then coupling them in one step, and using solid-phase removal of acetylcholine (Ac). This method requires hydrazine hydrate, which generates sugar hydrolysis impurities, making purification difficult. Hydrazine hydrate is a highly explosive and hazardous chemical with a high risk factor, significant toxicity to aquatic organisms, and a long-term pollution impact on the aquatic environment.

[0052] like Figure 1 As shown, the present invention also provides a method for synthesizing antifreeze glycopeptides, the steps of which are as follows:

[0053] Step S1: Add the carrier resin to the solid-phase reaction column for washing and swelling. Then, dissolve Fmoc-Ala-OH in DMF and mix it with the activator and condensing agent. Add the mixture to the solid-phase reaction column to carry out the condensation reaction, so that the carrier resin and Fmoc-Gly-OH are coupled to obtain Fmoc-Gly carrier resin.

[0054] Step S2: Following the coupling sequence from C-terminus to N-terminus, amino acids are coupled with Fmoc-Ala-carrier resin using a solid-phase synthesis method to obtain Fmoc-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Carrier Resin.

[0055] Step S3: After removing Fmoc protection from the Fmoc-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-carrier resin, the Fmoc-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-carrier resin is cleaved and lysed using a mixture and trifluoroacetic acid to obtain crude Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro antifreeze glycosides.

[0056] Step S4: After purifying, converting to salt, and lyophilizing the crude antifreeze glycopeptide, the antifreeze glycopeptide is obtained.

[0057] The solid-phase synthesis method includes the following steps:

[0058] Step A1: Use DBLK to remove the Fmoc protecting group from the Fmoc-Ala-carrier resin to obtain NH-Ala-carrier resin. The DBLK is composed of piperidine and DMF mixed in a volume ratio of 1:3.

[0059] Step A2: Under the condition of adding activator and condensing agent system, NH-Pro-carrier resin and Fmoc protected alanine are coupled to obtain Fmoc-Gly-Pro-carrier resin.

[0060] Step A3: Repeat steps A1 and A2 based on the amino acid sequence of the antifreeze glycopeptide.

[0061] Step A4: Based on the amino acid sequence of the antifreeze glycopeptide, we used TFA to remove the BZL3 ​​protecting group from Thr(OGalNAc-BZL3).

[0062] The amino acid T* is Fmoc-Thr(OGalNAc-BZL3)-OH.

[0063] The coupling sequence from C to N is Fmoc-PRO-OH, Fmoc-GLY-OH, Fmoc-Thr(OGalNAc-BZL3)-OH, Fmoc-Pro-OH, Fmoc-GLY-OH, Fmoc-Thr(OGalNAc-BZL3)-OH, Fmoc-Pro-OH, Fmoc-Ala-O.

[0064] This invention employs a one-step TFA deprotection method, which removes BZL simultaneously during the TFA cleavage of the peptide and resin.

[0065] Elongation reaction of Fmoc-GLY-2-CL-Resin peptide chain:

[0066] Weigh 5g of Fmoc-GLY-2-CL-Resin and add it to the solid-phase reaction column. Wash twice with DMF, swell Fmoc-GLY-2-CL-Resin with DMF for 20 min, remove Fmoc protection with 20% DBLK, then wash four times with DMF and twice with DCM. Detect the resin color using the ninhydrin method. If the resin is colored, it indicates that Fmoc has been removed.

[0067] Dissolve 1.55g Fmoc-PRO-OH, 0.68g HOBt, and 0.63g DIC in a 1:1 volume ratio of DCM and DMF mixed solution, then add the solution to a solid-phase reaction column and react at room temperature for 2 hours. The reaction endpoint is determined using the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin shows color, the reaction is incomplete and requires an additional 1 hour of coupling reaction. This criterion applies to the determination of the reaction endpoint using the ninhydrin method in subsequent sections.

[0068] Repeat the above process of removing Fmoc protection and adding the corresponding Fmoc-GLY-OH, Fmoc-Pro-OH, Fmoc-Thr(OGalNAc-BZL3)-OH, Fmoc-GLY-OH, Fmoc-Pro-OH, Fmoc-Thr(OGalNAc-BZL3)-OH, Fmoc-GLY-OH, Fmoc-Pro-OH, Fmoc-Thr(OGalNAc-BZL3)-OH, Fmoc-GLY-OH, and Fmoc-Pro-OH to obtain Fmoc-antifreeze glycopeptide-2-cl-Resin.

[0069] Example 5:

[0070] Preparation of crude antifreeze glycopeptides:

[0071] 8.5 g of the fully protected antifreeze glycopeptide-2-cl-Resin obtained in Example 3 was added to a 1 L three-necked flask, along with 100 mL of a pre-prepared TFA:H2O:PhOMe:PhSMe = 90:5:4:1 (V:V). The mixture was reacted at room temperature for 2 h, filtered through a resin under reduced pressure, and the filtrate was collected. The residue was washed with a small amount of TFA, and the filtrates were combined. The combined filtrate was slowly added to 10 L of ice-cold diethyl ether to precipitate the precipitate. After centrifugation, the precipitate was washed 5 times (5 L / time) with ice-cold diethyl ether and dried under reduced pressure to obtain 2.99 g of crude antifreeze glycopeptide. The crude antifreeze glycopeptide was then placed in a NaOH aqueous solution (50 mg / L) and stirred at a low temperature. Mass spectrometry showed that the protection removal was completed within 20 min. High-performance liquid chromatography analysis showed that the purity of the crude antifreeze glycopeptide was 84.6%. The structure is as follows. Figure 2 As shown.

[0072] Example 6:

[0073] Preparation of antifreeze glycopeptide acetate:

[0074] Weigh 2.99g of crude antifreeze glycopeptide and dissolve it in 500mL of 50% acetonitrile / water solution. Then, directly load the mixture onto the sample.

[0075] Purification conditions: Reverse-phase octadecylsilane was used as the stationary phase; mobile phase A was 0.1% (v / v) acetic acid / water solution, mobile phase B was acetonitrile, and the A:B ratio was 80:20 (v:v) with an isogradient elution; flow rate: 65 mL / min; detection wavelength: 214 nm.

[0076] The target peak fraction was collected, concentrated and freeze-dried in liquid nitrogen to obtain 2.2g of pure product with a purity of 96%. Figure 3 ); Mw=1499.54, mass spectrometry detection showed 1499.40 ( Figure 4 The peak values ​​are shown in Table 2, which meet the requirements, with a yield of 73.57%.

[0077] Table 2 Mass Spectrometry Detection Peak Values

[0078]

[0079] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0080] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method of synthesis of an antifreeze glycopeptide polypeptide, characterized by: The method includes the following steps: Step 1: Synthesis of Fmoc-Ala-2-CL-Resin: 10 g of 2-CL-Resin with a degree of substitution of 0.40 mmol / g was weighed and added to a solid-phase reaction column. The column was washed twice with DMF and swollen with DCM for 25 min. Then, 1.24 g of Fmoc-Ala-OH was weighed and dissolved in DCM. After thorough mixing with 3.70 mL of DIEA, the solution was added to the reaction column containing 2-CL-Resin. The condensation reaction was carried out for 2 h. The column was then washed with DMF and 8 mL of methanol was added to block the reaction for 1 h. The column was then washed three times with DMF and three times with DCM. The methanol was then dried under vacuum to obtain Fmoc-Ala-2-CL-Resin. Step 2: Elongation reaction of Fmoc-Ala-2-CL-Resin peptide chain: Weigh 5g of Fmoc-Ala-2-CL-Resin and add it to the solid-phase reaction column. Wash twice with DMF, swell Fmoc-Ala-2-CL-Resin with DMF for 20min, remove Fmoc protection with 20% DBLK, then wash 4 times with DMF and 2 times with DCM. Detect the resin color using the ninhydrin method. If the resin is colored, it indicates that Fmoc has been removed. Dissolve 1.55g Fmoc-Ala-OH, 0.68g HOBt, and 0.63g DIC in a 1:1 volume ratio of DCM and DMF mixed solution, then add the solution to a solid-phase reaction column and react at room temperature for 2 hours. The reaction endpoint is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin shows color, the reaction is incomplete and requires an additional 1 hour of coupling reaction. This criterion applies to the determination of the reaction endpoint by the ninhydrin method in the following content. Repeat the steps of removing Fmoc protection and adding corresponding amino acid conjugation, and according to the main chain peptide sequence of the antifreeze glycopeptide, sequentially complete the conjugation of Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH to obtain Fmoc-antifreeze glycopeptide-2-cl-Resin; Step 3: Drying of Fmoc-antifreeze glycopeptide-2-cl-Resin with full side chain protection: Take the Fmoc-antifreeze glycopeptide-2-cl-Resin obtained in Step 2, remove Fmoc protection with 20% DBLK, then wash 4 times with DMF and 2 times with DCM. Detect the resin color using the ninhydrin method; color in the resin indicates that Fmoc has been removed. Then, remove the AC3 protecting group in Thr(OGalNAcAc3) using hydrazine hydrate, wash twice with methanol, and vacuum dry the resin overnight. Weigh to obtain antifreeze glycopeptide-2-cl-Resin. Step 4: Preparation of crude antifreeze glycopeptide: 8.5g of the fully protected antifreeze glycopeptide-2-cl-Resin obtained in Step 3 was added to a 1L three-necked flask. 100mL of a pre-prepared volume ratio of TFA:H2O:PhOMe:PhSMe = 90:5:4:1 was added. The mixture was reacted at room temperature for 2h. The mixture was filtered through a resin under reduced pressure, and the filtrate was collected. The residue was washed with a small amount of TFA, and the filtrates were combined. The combined filtrate was slowly added to 10L of ice-cold ether to precipitate the residue. The residue was centrifuged, washed 5 times with ice-cold ether, and dried under reduced pressure to obtain crude antifreeze glycopeptide. The crude antifreeze glycopeptide was then placed in a 50mg / L NaOH aqueous solution and stirred at a low temperature. Mass spectrometry showed that the protection removal was completed within 20min.

2. The method for synthesizing an antifreeze glycopeptide according to claim 1, characterized in that: Fmoc is 9-fluorenemethyloxycarbonyl, and Fmoc is attached to an amino group or to a nitrogen atom on the carbon atom attached to a carboxyl group.

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