Synthesis method of antifreeze glycopeptide polypeptide

Abstract

The invention relates to a synthesis method of antifreeze glycopeptide polypeptide, and belongs to the technical field of polypeptide drug synthesis, the method comprises the following steps: taking 2-CL-Resin as carrier resin, under the condition of adding an activator, coupling the carrier resin with alanine to obtain Fmoc-Ala-2-CL-Resin; sequentially coupling other amino acids according to the amino acid sequence of the antifreeze glycopeptide through a solid-phase synthesis method; removing the protecting group and cracking the carrier resin to obtain crude antifreeze glycopeptide; and performing purification, salt conversion and freeze-drying to obtain the antifreeze glycopeptide polypeptide. The method has the advantages of short synthesis period, low cost, easiness in post-treatment, few byproducts and high product yield, is beneficial to large-scale production of the antifreeze glycopeptide, and has considerable economic applicable value and wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide drug synthesis, and in particular relates to a method for synthesizing an antifreeze glycopeptide polypeptide. Background Art

[0002] Antifreeze glycopeptides are a class of biological agents that can survive at temperatures below the freezing point of body fluids. Antifreeze glycopeptides are usually composed of repeating tripeptide units (-Ala-Ala-Thr*-) glycosylated with threonine side chains. With the growth of global trade and the expansion of the distance between production and sales, the demand for cold chain processed foods has also increased. The share of frozen foods in the food industry is also growing.

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

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

[0005] Antifreeze glycopeptide polypeptide is a type of antifreeze polypeptide, and its amino acid sequence is abbreviated as:

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

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

[0008] In the prior art, the synthesis of antifreeze glycopeptides mostly adopts the conventional method of sequentially coupling amino acids and finally performing sugar modification. However, this synthesis method will result in incomplete sugar modification of the antifreeze glycopeptide peptide chain, resulting in poor peptide chain purity and low product yield at the end of the synthesis. Summary of the invention

[0009] The object of the present invention is to provide a method for synthesizing an antifreeze glycopeptide polypeptide, which has the advantages of simple operation, short synthesis cycle, low cost, reduced generation of waste liquid, easy post-treatment, few by-products and high product yield.

[0010] The purpose of the present invention can be achieved through the following technical solutions:

[0011] A method for synthesizing an antifreeze glycopeptide polypeptide comprises the following steps:

[0012] Step S1, preparing an antifreeze glycopeptide polypeptide precursor resin: starting with a carrier resin, adding the carrier resin to a solid phase reaction column, washing and swelling with DMF, then dissolving Fmoc-Ala-OH with DMF and mixing it with an activator and a condensing agent, and then adding it to the reaction column containing the carrier resin, and performing a condensation reaction to couple the solid phase carrier resin with the N-terminal Fmoc-protected Fmoc-Ala-OH to obtain Fmoc-Ala-OH-carrier resin;

[0013] Step S2, according to the amino acid sequence of the antifreeze glycopeptide polypeptide, in accordance with the coupling order from C-terminus to N-terminus, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled with Fmoc-Ala-carrier resin by solid phase synthesis method to obtain Fmoc antifreeze glycopeptide-carrier resin;

[0014] Step S3, after removing the Fmoc protection from the antifreeze glycopeptide-carrier resin, use the mixed solution and trifluoroacetic acid to cut and cleave the Fmoc-antifreeze glycopeptide-carrier resin to obtain a crude antifreeze glycopeptide peptide, and place the crude antifreeze glycopeptide peptide in a NaOH aqueous solution (50 mg / L) and stir to react at room temperature, and the protection removal can be completed within 20 minutes by mass spectrometry detection;

[0015] Step S4, purifying, converting salts and freeze-drying the crude antifreeze glycopeptide peptide to obtain a finished antifreeze glycopeptide polypeptide.

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

[0017] Furthermore, Fmoc is 9-fluorenylmethoxycarbonyl, and Fmoc is connected to the amino group or to the nitrogen atom on the carbon atom to which the carboxyl group is connected.

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

[0019] Step A1: using DBLK to remove the Fmoc protecting group of the Fmoc-Ala-carrier resin to obtain NH-Ala-carrier resin, wherein the DBLK is a mixture of piperidine and DMF in a volume ratio of 1:3;

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

[0021] Step A3: Repeat steps A1 and A2 according to the amino acid sequence of the antifreeze glycopeptide polypeptide.

[0022] Step A4: According to the amino acid sequence of the antifreeze glycopeptide polypeptide, the AC3 protecting group in Thr (OGalNAcAc3) is removed by using hydrazine hydrate.

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

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

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

[0026] Furthermore, the purification conditions in step S4 are: using reverse-phase high performance liquid chromatography, with reverse-phase octadecylsilane as the stationary phase, the column temperature is 35°C, the mobile phase A is an acetic acid / water solution with a volume fraction of 0.1%, the mobile phase B is acetonitrile, the mobile phase A and the mobile phase B are eluted in an isocratic gradient according to a volume ratio of 80:20, the flow rate is 60-70 mL / min, and the detection wavelength is 214 nm.

[0027] Beneficial effects of the present invention: The antifreeze glycopeptide polypeptide prepared by the above-mentioned synthesis method has the characteristics of inhibiting ice crystal recrystallization and protecting the cell integrity and structure of food in a low temperature environment. The antifreeze peptide is adsorbed on the surface of ice crystals to inhibit the growth and recrystallization of ice crystals, thereby reducing mechanical damage to food cells and maintaining the high quality of food.

[0028] The invention has simple operation, short synthesis cycle, low cost, reduced generation of waste liquid, easy post-treatment, few by-products, high product yield, is conducive to large-scale production of antifreeze glycopeptide polypeptides, and has considerable economic applicability value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The synthetic method flow chart of the present invention is as follows;

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

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

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

[0033] Figure 5 This is a crude peptide structure diagram of an antifreeze glycopeptide according to another embodiment of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The English abbreviations used in the present invention and their meanings are described in Table 1 below:

[0036]

[0037] Herein, "substitution degree" refers to the amount of a substance loaded per unit amount of resin, and the unit is "mmol / g".

[0038] Embodiment 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, add it to the solid phase reaction column, wash it with DMF twice, swell 2-CL-Resin with DCM for 25min, weigh 1.24g of Fmoc-Ala-OH and dissolve it with DCM, mix it thoroughly with 3.70mL of DIEA, add it to the reaction column containing 2-CL-Resin, condense react for 2h, then wash it with DMF, add 8mL of methanol after washing to seal it for 1h, then wash it with DMF 3 times, wash it with DCM 3 times, shrink it with methanol and drain it to obtain Fmoc-Ala-2-CL-Resin.

[0041] Embodiment 2:

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

[0043] Weigh 5 g of Fmoc-Ala-2-CL-Resin, add it to the solid phase reaction column, wash it with DMF twice, swell Fmoc-Ala-2-CL-Resin with DMF for 20 min, remove Fmoc protection with 20% DBLK, then wash it with DMF 4 times, wash it with DCM 2 times, detect the color of the resin with the ninhydrin method, and the resin has color, indicating that Fmoc has been removed;

[0044] 1.55g Fmoc-Ala-OH, 0.68g HOBt and 0.63g DIC were dissolved in a mixed solution of DCM and DMF with a volume ratio of 1:1, and then added to the solid phase reaction column and reacted at room temperature for 2h; the reaction endpoint was determined by the ninhydrin method. If the resin was colorless and transparent, it indicated that the reaction was complete; if the resin showed color, it indicated that the reaction was incomplete and the coupling reaction was required for another 1h. This judgment standard is applicable to the determination of the reaction endpoint by the ninhydrin method in the subsequent content;

[0045] Repeat the above steps of removing Fmoc protection and adding corresponding amino acids for coupling, and complete 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-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH in sequence according to the main chain peptide sequence of the antifreeze glycopeptide polypeptide to obtain Fmoc-antifreeze glycopeptide-2-cl-Resin. By optimizing the coupling order, possible side reactions between adjacent amino acid residues can be avoided. At the same time, the coupling order can make the reaction steps smoother and reduce the accumulation of intermediates, thereby improving the overall synthesis efficiency.

[0046] Embodiment three:

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

[0048] Take the Fmoc-antifreeze glycopeptide-2-cl-Resin obtained in Example 2, remove the Fmoc protection with 20% DBLK, then wash with DMF 4 times, wash with DCM 2 times, and detect the color of the resin by the ninhydrin method. The resin has color, indicating that Fmoc has been removed. Then use hydrazine hydrate to remove the AC3 protecting group in Thr (OGalNAcAc3), then wash with methanol 2 times, and vacuum dry the resin overnight.

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

[0050] Embodiment 4:

[0051] Furthermore, in order to solve the conventional method of sequentially coupling amino acids, sugar modification is finally performed, but this synthesis method will cause incomplete sugar modification of the antifreeze glycopeptide peptide chain, resulting in poor peptide chain purity and low product yield at the end of the synthesis. First modify the sugar amino acid, then couple once, and take solid phase to remove Ac. This method requires the use of hydrazine hydrate to remove sugar hydrolysis impurities, which is difficult to purify. Hydrazine hydrate is an explosive and dangerous chemical with a high risk factor. It is very toxic to aquatic organisms and causes long-term pollution to the aquatic environment. Technical problems.

[0052] like Figure 5 As shown, the present invention also provides a method for synthesizing an antifreeze glycopeptide, the steps of which are as follows: Step S1, adding the carrier resin to a solid phase reaction column to wash and swell, then dissolving Fmoc-Ala-OH with DMF and mixing it with an activator and a condensing agent, and then adding it to the solid phase reaction column to perform a condensation reaction, so that the carrier resin and Fmoc-Gly-OH are coupled to obtain Fmoc-Gly carrier resin; Step S2, coupling the amino acid with the Fmoc-Ala-carrier resin by solid phase synthesis in the coupling order from the C-terminus to the N-terminus to obtain Fmoc-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-carrier resin; Step S3, after removing Fmoc protection from the Fmoc-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-carrier resin, use the mixed solution and trifluoroacetic acid to cut and cleave the Fmoc-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-carrier resin to obtain a crude Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro-Thr*-Gly-Pro antifreeze sugar peptide; Step S4, purifying, converting salts and freeze-drying the crude antifreeze glycopeptide to obtain the antifreeze glycopeptide.

[0053] The solid phase synthesis method comprises the following steps: Step A1: using DBLK to remove the Fmoc protecting group of the Fmoc-Ala-carrier resin to obtain the NH-Ala-carrier resin, wherein the DBLK is a mixture of piperidine and DMF in a volume ratio of 1:3; Step A2: Under the condition of adding an activator and a condensing agent system, NH-Pro-support resin and Fmoc-protected alanine are coupled to obtain Fmoc-Gly-Pro-support resin; Step A3: Repeat steps A1 and A2 according to the amino acid sequence of the antifreeze glycopeptide polypeptide.

[0054] Step A4: Based on the amino acid sequence of the antifreeze glycopeptide polypeptide, we used TFA to remove the BZL3 ​​protecting group in Thr(OGalNAc-BZL3).

[0055] The amino acid T* was Fmoc-Thr(OGalNAc-BZL3)-OH.

[0056] The coupling order from C-terminus to N-terminus 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.

[0057] The present invention adopts a one-step TFA deprotection method, and BZL is removed simultaneously when Tfa is cut to separate the polypeptide and the resin.

[0058] Elongation reaction of Fmoc-GLY-2-CL-Resin peptide chain: Weigh 5 g of Fmoc-GLY-2-CL-Resin, add it to the solid phase reaction column, wash it with DMF twice, swell Fmoc-GLY-2-CL-Resin with DMF for 20 min, remove Fmoc protection with 20% DBLK, then wash it with DMF 4 times, wash it with DCM 2 times, detect the color of the resin with the ninhydrin method, and the resin has color, indicating that Fmoc has been removed; Dissolve 1.55g Fmoc-PRO-OH, 0.68g HOBt and 0.63g DIC in a mixed solution of DCM and DMF with a volume ratio of 1:1, then add it to the solid phase reaction column and react at room temperature for 2h. Use the ninhydrin method to determine the reaction end point. If the resin is colorless and transparent, it means that the reaction is complete; if the resin is colored, it means that the reaction is incomplete and needs to be coupled for another 1h. This judgment standard is applicable to the subsequent content to determine the reaction end point by the ninhydrin method; Repeat the above removal of Fmoc protection and the addition of 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, Fmoc-Pro-OH, Fmoc-Thr(OGalNAc-BZL3)-OH, Fmoc-GLY-OH, Fmoc-Pro-OH coupling to obtain Fmoc-antifreeze glycopeptide-2-cl-Resin.

[0059] Embodiment five:

[0060] Preparation of crude antifreeze glycopeptide peptide:

[0061] 8.5g of the side chain fully protected antifreeze glycopeptide-2-cl-Resin obtained in Example 3 was added to a 1L three-necked flask, and 100mL of pre-prepared TFA:H2O:PhOMe:PhSMe=90:5:4:1(V:V) was added. The mixture was reacted at room temperature for 2h. The resin was filtered under reduced pressure, the filtrate was collected, and the residue was washed with a small amount of TFA. The filtrate was combined, and the combined filtrate was slowly added to 10L of ice ether for precipitation, centrifuged, and washed with ice ether 5 times (5L / time). The crude antifreeze glycopeptide peptide was dried under reduced pressure to obtain 2.99g. The crude antifreeze glycopeptide peptide was placed in a NaOH aqueous solution (50mg / L) and stirred for warm reaction. The protection removal was completed within 20min by mass spectrometry detection. The purity of the crude antifreeze glycopeptide peptide was 84.6% by high performance liquid chromatography detection and analysis. The structure is as follows Figure 2 shown.

[0062] Embodiment six:

[0063] Preparation of antifreeze glycopeptide peptide acetate:

[0064] Weigh 2.99 g of crude antifreeze glycopeptide peptide and dissolve it in 500 mL of 50% acetonitrile / water solution, and directly load the mixture onto the sample;

[0065] Purification conditions: reversed-phase octadecylsilane as stationary phase; mobile phase A is 0.1% (v / v) acetic acid / water solution, mobile phase B is acetonitrile, A:B=80:20 (v:v) isocratic elution; flow rate: 65 mL / min; detection wavelength: 214 nm;

[0066] The target peak fractions were collected and concentrated by liquid nitrogen and freeze-dried to obtain 2.2 g of pure product with a purity of 96% ( Figure 2 ); Mw = 1499.54 mass spectrometry detection is 1499.40 ( Figure 3 ) The peak value table is shown in Table 2, which meets the requirements and the yield is 73.57%.

[0067] Table 2 Mass spectrometry detection peak table

[0068]

[0069] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0070] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for synthesizing an antifreeze glycopeptide polypeptide, characterized in that: The following steps are involved: Step S1, adding the carrier resin to a solid phase reaction column to wash and swell, then dissolving Fmoc-Ala-OH with DMF and mixing it with an activator and a condensing agent, and then adding it to the solid phase reaction column to perform a condensation reaction, so that the carrier resin and Fmoc-Ala-OH are coupled to obtain Fmoc-Ala carrier resin; Step S2, coupling the amino acid with the Fmoc-Ala-carrier resin by solid phase synthesis in a coupling order from the C-terminus to the N-terminus to obtain Fmoc-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-carrier resin; Step S3: After removing Fmoc protection from Fmoc-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-carrier resin, use the mixed solution and trifluoroacetic acid to cut and cleave Fmoc-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-carrier resin to obtain Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala-Thr*-Ala-Ala antifreeze glycopeptide crude peptide, and place the antifreeze glycopeptide crude peptide in a NaOH aqueous solution (50 mg / L) with stirring for warm reaction, and the protection removal can be completed within 20 minutes by mass spectrometry detection; Step S4, purifying, converting salts and freeze-drying the crude antifreeze glycopeptide to obtain the antifreeze glycopeptide.

2. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 1, characterized in that: The carrier resin is 2-cl resin, and the degree of substitution is 0.42 mmol / g.

3. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 1, characterized in that: Fmoc is 9-fluorenylmethoxycarbonyl, and Fmoc is connected to the amino group or to the nitrogen atom on the carbon atom to which the carboxyl group is connected.

4. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 1, characterized in that: The solid phase synthesis method comprises the following steps: Step A1: using DBLK to remove the Fmoc protecting group of the Fmoc-Ala-carrier resin to obtain the NH-Ala-carrier resin, wherein the DBLK is a mixture of piperidine and DMF in a volume ratio of 1:3; Step A2: Under the condition of adding an activator and a condensing agent system, NH-Ala-support resin and Fmoc-protected alanine are coupled to obtain Fmoc-Ala-Ala-support resin; Step A3: repeating steps A1 and A2 according to the amino acid sequence of the antifreeze glycopeptide polypeptide; Step A4: According to the amino acid sequence of the antifreeze glycopeptide polypeptide, the AC3 protecting group in Thr (OGalNAcAc3) is removed by hydrazine hydrate.

5. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 4, characterized in that: The activator is DIC, the condensation agent is HOBt, and the condensation reaction time is 2h.

6. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 4, characterized in that: The amino acid T* was Fmoc-Thr(OGalNAcAc3)-OH.

7. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 6, characterized in that: The coupling order 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-Thr(OGalNAcAc3)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH.

8. The method for synthesizing an antifreeze glycopeptide polypeptide according to claim 1, characterized in that: The purification conditions are as follows: reversed-phase octadecylsilane is used as the stationary phase, the column temperature is 35°C, the mobile phase A is a 0.1% volume fraction acetic acid / water solution, the mobile phase B is acetonitrile, the mobile phase A and the mobile phase B are eluted in an isocratic manner at a volume ratio of 80:20, the flow rate is 60-70 mL / min, and the detection wavelength is 214 nm.

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