Method for preparing hyaluronic acid peptide

Abstract

The invention relates to the technical field of polypeptide product preparation, in particular to a method for preparing hyaluronic acid peptide. According to the method disclosed by the invention, a hyaluronic acid peptide product is prepared by adopting a solid-liquid phase combined method. The preparation method comprises the following steps: synthesizing an intermediate 1 by using a liquid phase, sequentially coupling the intermediate 1 to resin from a C terminal to an N terminal according to a product sequence, cutting peptide from the resin to obtain a crude product, and performing reversed-phase preparation on the crude product to obtain the high-purity hyaluronic acid peptide. The preparation process is high in product quality and yield, low in cost, easy to realize industrialization and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide product preparation, and in particular to a method for preparing hyaluronic acid peptide. Background Art

[0002] Hyaluronic acid peptide, English name Syn-Hycan, CAS number 934368-60-2, the specific peptide sequence is as follows: CH3-(CH3)13-NH-CO-Dab-Val-Dab-COOH.2TFA.

[0003] The amino acid polysaccharides that hyaluronic acid peptides can promote can also serve as raw materials for the synthesis of core proteins. Core proteins can bind to collagen fibrils. When there is a large amount of protein in this connective tissue that binds collagen fibrils into bundles, collagen fibers are formed. Hyaluronic acid peptides can stimulate the synthesis of a larger mass of collagen, and its effect of promoting the synthesis of hyaluronic acid will also play a synergistic role in the moisture retention of connective tissue, making the skin more hydrated and elastic. At the same time, hyaluronic acid peptides promote the production of endogenous hyaluronic acid. The core effects can be summarized as: promoting the synthesis of hyaluronic acid; stimulating the production of collagen fibers; initiator of endogenous hyaluronic acid synthesis.

[0004] Hyaluronic acid peptide is in the form of trifluoroacetate, with a tetradecene structure at the N-terminus and a tripeptide structure at the C-terminus. The tripeptide structure and the tetradecene structure are connected by a carbonyl group to form a urea structure. Considering the particularity of the urea structure, there are currently few reports or patents on the preparation of this product.

[0005] Patent CN107987123A adopts a liquid phase synthesis method to synthesize hyaluronic acid peptides, which has the characteristics of long steps and a long industrialization period; Patent CN108047305A adopts a solid phase synthesis method to synthesize hyaluronic acid peptides, which also has the characteristics of many steps and a long industrialization period. At the same time, CTC Resin is used as a solid phase carrier for solid phase coupling. Due to the unstable characteristics of the solid phase resin during coupling, the yield fluctuates greatly, which is not conducive to industrial production.

[0006] How to prepare high-purity, high-quality, and low-cost hyaluronic acid peptides in a simple way is the focus of process development. Summary of the invention

[0007] In order to overcome the process problems in the preparation of hyaluronic acid peptides, the purpose of the present invention is to provide a process for preparing hyaluronic acid peptides. The hyaluronic acid peptides prepared by the present invention have high quality, stable yield, short preparation cycle, and are easy to realize industrial scale-up production.

[0008] The specific steps are as follows:

[0009] Step 1: After dissolving tetradecylamine in tetrahydrofuran, add CDI or DSC and then dropwise add alkali, react for 4 hours, and then continue to dropwise add H-Dab(Boc)-OH under temperature control to obtain intermediate 1 after the reaction is completed;

[0010] Step 2: The solid phase carrier is sequentially coupled with Fmoc-Dab(Boc)-OH, Fmoc-Val-OH and intermediate 1 in the presence of a condensing agent by solid phase synthesis method to obtain a peptide resin;

[0011] Step 3: After the peptide resin is cleaved by a cleavage reagent, it is washed with isopropyl ether and precipitated to obtain a crude hyaluronic acid peptide;

[0012] Step 4: The crude hyaluronic acid peptide is purified by reverse phase preparation, concentrated under reduced pressure, and freeze-dried to obtain high-purity hyaluronic acid peptide.

[0013] Furthermore, in step 1, the base is selected from triethylamine or diisopropylethylamine.

[0014] Furthermore, in step 1, the solid phase carrier is selected from wang resin, and the substitution degree of the solid phase resin is 0.4 mmol / g to 1.5 mmol / g. As a preferred embodiment, the substitution degree of the solid phase carrier wang resin is 1.08 mmol / g.

[0015] Furthermore, in step 2, the condensing agent is selected from HOBt / DIC, HBTU / DIEA, and as a preferred solution, HBTU / DIEA is selected as the condensing agent.

[0016] Furthermore, in actual production applications, the amino acid feed ratio is 2.0eq, and the ratio of amino acid to condensing agent is preferably 1:1.1.

[0017] Furthermore, in step 3, the lysis reagent is a TFA / TIS / H2O mixture. As a preferred embodiment, the volume ratio of TFA / TIS / H2O is 95 / 2.5 / 2.5.

[0018] Furthermore, the reverse phase preparative chromatography purification method described in step 4 is: the crude peptide is dissolved in a 35% acetonitrile / water mixed solution, filtered with a 0.45um organic filter membrane, and purified by high performance liquid preparative chromatography C18, the mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is acetonitrile, the flow rate is 400mL / min, the detection wavelength is 230nm, and the gradient is: 34%-54%; the required fractions are tracked and collected by a liquid chromatograph, and the qualified sample peaks are merged.

[0019] The present invention has the following beneficial effects:

[0020] According to the structural characteristics of hyaluronic acid peptide, the present invention adopts a solid-liquid combination preparation method to easily obtain hyaluronic acid peptide. The process of the present invention, through a specific process design, can obtain intermediate 1 in one step; the cheap wang resin is used to obtain peptide resin in 3 steps, and the hyaluronic acid peptide crude product is obtained after cutting, and the product is obtained by reversed-phase high-performance liquid chromatography purification, concentration, and freeze-drying. The process has a short preparation cycle, a high total yield, and a high product purity, and is easy to realize industrialized scale-up production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is the HPLC chart of the hyaluronic acid peptide product of Example 7;

[0022] Figure 2 Shown is the MS graph of the hyaluronic acid peptide product of Example 7;

[0023] Figure 3 Shown is the HPLC chart of the hyaluronic acid peptide product of Example 12;

[0024] Figure 4 Shown is the MS graph of the hyaluronic acid peptide product of Example 12. DETAILED DESCRIPTION

[0025] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

[0026] The Chinese names corresponding to the English abbreviations involved in the claims and the specification of the present invention are as follows:

[0027] Abbreviations and English meaning Fmoc 9-Fluorenylmethoxycarbonyl HOB 1-Hydroxybenzotriazole DIC Diisopropylcarbodiimide HBTU Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate DIEA Diisopropylethylamine DMF N,N-Dimethylformamide DMAP 4-Dimethylaminopyridine Dab 2,4-Diaminobutyric acid Boc Tert-butyl CDI N,N-Carbonyldiimidazole DSC N,N'-Disuccinimidyl carbonate TIS Triisopropylsilane TFA Trifluoroacetic acid DCM Dichloromethane DCC N,N′-Dicyclohexylcarbodiimide

[0028] Example 1: Synthesis of Intermediate 1CH3(CH3)13NH-CO-Dab(Boc)-OH

[0029] 3L tetrahydrofuran, 300.0g tetradecylamine, and 227.9g CDI were added to a 10L reactor in sequence, and 181.7g DIEA was added dropwise at 0-5°C. After the addition was completed, the reaction continued for 4h at room temperature. The reaction was complete when detected by HPLC. 337.6g H-Dab(Boc)-OH was added to the above reaction solution, and after stirring and dissolving, 566.1g DIEA was added dropwise at 0-5°C. After the addition was completed, the reaction was reacted for 4h at room temperature. After HPLC detection, the reaction was complete and concentrated under reduced pressure. 2L of dichloromethane and 2L of purified water were added to the concentrated viscous liquid, and after dissolving and clarifying, the organic phase was taken out, and 2L of 2% dilute hydrochloric acid was added to wash the organic phase. After standing and stratifying, the organic phase was collected, washed once with 2L of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. After filtering, the white solid of intermediate 1 was concentrated under reduced pressure to obtain 611.2g, with a yield of 94.99%.

[0030] Example 2: Synthesis of Intermediate 1CH3(CH3)13NH-CO-Dab(Boc)-OH

[0031] 3L tetrahydrofuran, 300.0g tetradecylamine, and 360.18g DSC were added to a 10L reactor in sequence, and 142.26g triethylamine was added dropwise at 0-5°C. After the addition was completed, the reaction continued for 4h at room temperature. The reaction was complete when detected by HPLC. 337.6g H-Dab(Boc)-OH was added to the above reaction solution, and after stirring and dissolving, 443.22g DIEA was continued to be added dropwise at 0-5°C. After the addition was completed, the reaction was reacted for 4h at room temperature. After HPLC detection, the reaction was complete and concentrated under reduced pressure. 2L of dichloromethane and 2L of purified water were added to the concentrated viscous liquid, and after dissolving and clarifying, the organic phase was taken out, and 2L of 2% dilute hydrochloric acid was added to wash the organic phase. After standing and stratifying, the organic phase was collected, washed once with 2L of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. After filtering, the white solid of intermediate 1 was 602.7g, and the yield was 93.67%.

[0032] Example 3: Synthesis of Fmoc-Dab(Boc)-wang resin

[0033] 555.6 g of Wang Resin (substitution degree: 1.08 mmol / g) was weighed and washed once with 3.6 L DMF, and then dried. Then, the mixture was swelled with 3.6 L DMF for 0.5 h and dried.

[0034] Weigh 528.6g Fmoc-Dab(Boc)-OH, 14.7g DMAP and 178.3g HOBt, dissolve them in 1.8L DMF, add 196.9g DIC to activate for about 3-5min, add to the above solid phase resin, stir and react at room temperature for 6h, and remove the reaction solution after coupling for 6h. Wash the resin with DMF 3 times, each time with 3.6L DMF. After draining the resin, add 3.6L DMF, 183.6g DIEA and 232.6g acetic anhydride, continue to react for 2h under stirring at room temperature, remove the reaction solution, wash 3 times with 3.6LDMF, and the substitution degree is 0.57mmol / g. The subsequent resin feed scale is 460mmol.

[0035] Example 4: Synthesis of Fmoc-Val-Dab(Boc)-wang resin

[0036] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 3.6 L / time, 5 min + 15 min. After deprotection, wash with DMF 6 times, 3.6 L / time / min, drain, and detect with ninhydrin, K+.

[0037] Weigh 312.2g Fmoc-Val-OH and 383.79g HBTU and dissolve them in 3.6L DMF. Add 130.8g DIEA to the solution under ice bath and activate for about 5 minutes. Pour into the resin and react with stirring at room temperature for 2 hours. Take samples and detect K- with ninhydrin. Wash the resin 3 times with DMF at 3.6L / time / min and drain.

[0038] Example 5: Synthesis of peptide resin

[0039] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 3.6 L / time, 5 min + 15 min. After deprotection, wash with DMF 6 times, 3.6 L / time / min, drain, and detect with ninhydrin, K+.

[0040] Weigh 421.0 g of the intermediate 1CH3(CH3)13NH-CO-Dab-OH and 383.79 g of HBTU, dissolve them in 3.6 L DMF, add 130.8 g of DIEA to the solution for activation for about 5 min under an ice bath, pour into the resin, stir and react at room temperature for 2 h, take a sample, and detect K- with ninhydrin; wash the resin 3 times with DMF at 3.6 L / time / min, and drain.

[0041] The mixture was washed with dichloromethane for three times at 3.6 L / time / min; the mixture was drained and vacuum dried at room temperature for 24 hours until constant weight was reached. 949.2 g of the dry peptide resin was weighed.

[0042] Example 6: Preparation of crude product

[0043] Prepare 7.6 L of lysis solution (TFA / TIS / H2O, V / V / V, 95 / 2.5 / 2.5), cool to 0°C, add 949.2 g of the above-mentioned peptide resin, stir at 0-5°C for 30 min, return to room temperature and stir to react for 1.5 h. After the reaction is complete, concentrate under reduced pressure, precipitate with isopropyl ether, filter, and dry to obtain 359.6 g of a crude white solid.

[0044] Example 7: Purification of hyaluronic acid peptide

[0045] 359.6 g of the crude product was dissolved in 300 L of 35% acetonitrile water, filtered through a 0.45 um organic filter membrane, and purified by high performance liquid preparative chromatography C18, with mobile phase A being 0.1% trifluoroacetic acid aqueous solution, phase B being ethanol, flow rate 400 mL / min, detection wavelength 230 nm, gradient: 34%-54%; the required fractions were collected by liquid chromatograph, qualified sample peaks were combined, concentrated and freeze-dried, and 321.3 g of white flocculent product was obtained, with a total yield of 89.0%; purity: 99.4%; MS detection: 557.32 (M+H).

[0046] Example 8: Synthesis of Fmoc-Dab(Boc)-wang resin

[0047] 555.6 g of Wang Resin (substitution degree: 1.08 mmol / g) was weighed and washed once with 3.6 L DMF, and then dried. Then, the mixture was swelled with 3.6 L DMF for 0.5 h and dried.

[0048] Weigh 528.6g Fmoc-Dab(Boc)-OH, 14.7g DMAP and 178.3g HOBt, dissolve them in 1.8L DMF, add 196.9g DIC to activate for about 3-5min, add to the above solid phase resin, stir and react at room temperature for 6h, and remove the reaction solution after coupling for 6h. Wash the resin with DMF 3 times, each time with 3.6L DMF. After draining the resin, add 3.6L DMF, 183.6g DIEA and 232.6g acetic anhydride, continue to react for 2h under stirring at room temperature, remove the reaction solution, wash 3 times with 3.6LDMF, and the substitution degree is 0.55mmol / g. The subsequent resin feed scale is 450mmol.

[0049] Example 9: Synthesis of Fmoc-Val-Dab(Boc)-wang resin

[0050] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 3.6 L / time, 5 min + 15 min. After deprotection, wash with DMF 6 times, 3.6 L / time / min, drain, and detect with ninhydrin, K+.

[0051] Weigh 305.4g Fmoc-Val-OH and 133.8g HOBt, dissolve in 3.6L DMF, add 124.9g DIC to the solution under ice bath and activate for about 5min, pour into the resin, stir and react at room temperature for 2h, take samples, detect K- with ninhydrin; wash the resin 3 times with DMF, 3.6L / time / min, and drain.

[0052] Example 10: Synthesis of peptide resin

[0053] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 3.6 L / time, 5 min + 15 min. After deprotection, wash with DMF 6 times, 3.6 L / time / min, drain, and detect with ninhydrin, K+.

[0054] Weigh 411.7 g of the intermediate 1CH3(CH3)13NH-CO-Dab-OH and 133.8 g of HOBt, dissolve them in 3.6 L DMF, add 124.9 g of DIC to the solution under ice bath and activate for about 5 min, pour into the resin, react with stirring at room temperature for 2 h, take samples, detect with ninhydrin, K-; wash the resin 3 times with DMF, 3.6 L / time / min, and drain.

[0055] The mixture was washed with dichloromethane for three times at 3.6 L / time / min; the mixture was drained and vacuum dried at room temperature for 24 hours until constant weight was reached. 928.7 g of the dry peptide resin was weighed.

[0056] Example 11: Preparation of crude product

[0057] Prepare 7.5 L of lysis solution (TFA / TIS / H2O, V / V / V, 95 / 2.5 / 2.5), cool to 0°C, add 929.8 g of the above-mentioned peptide resin, stir at 0-5°C for 30 min, return to room temperature and stir to react for 1.5 h. After the reaction is complete, concentrate under reduced pressure, precipitate with isopropyl ether, filter, and dry to obtain 325.7 g of a crude white solid.

[0058] Example 12: Purification of hyaluronic acid peptide

[0059] 325.7 g of the crude product was dissolved in 290 L of 35% acetonitrile water, filtered through a 0.45 um organic filter membrane, and purified by high performance liquid preparative chromatography C18, with mobile phase A being 0.1% trifluoroacetic acid aqueous solution, phase B being ethanol, flow rate 400 mL / min, detection wavelength 230 nm, gradient: 34%-54%; the required fractions were collected by liquid chromatograph, qualified sample peaks were combined, concentrated and freeze-dried to obtain 302.7 g of white flocculent product, total yield: 85.7%; purity: 98.4%; MS detection: 557.37 (M+H), 279.36 (1 / 2M+H).

Claims

1. A method for preparing hyaluronic acid peptide, characterized in that: The following steps are involved: Step 1: After tetradecylamine is dissolved in tetrahydrofuran, CDI or DSC is added, and then a base is added dropwise. After reacting for 4 hours, H-Dab(Boc)-OH is continuously added dropwise under temperature control conditions. After the reaction is completed, intermediate 1 is obtained; Step 2: The solid phase carrier is sequentially coupled with Fmoc-Dab(Boc)-OH, Fmoc-Val-OH and intermediate 1 in the presence of a condensing agent by solid phase synthesis method to obtain a peptide resin; Step 3: After the peptide resin is cleaved by a cleavage reagent, it is washed with isopropyl ether and precipitated to obtain a crude hyaluronic acid peptide; Step 4: The crude hyaluronic acid peptide is purified by reverse phase preparation, concentrated under reduced pressure, and freeze-dried to obtain high-purity hyaluronic acid peptide.

2. The method according to claim 1, characterized in that: In step 1, the base to be added dropwise may be triethylamine or diisopropylethylamine.

3. The method according to claim 1, characterized in that: The solid phase carrier in step 2 is selected from wang resin.

4. The method according to claim 1, characterized in that: The condensing agent in step 2 is selected from HBTU / DIEA.

5. The method according to claim 1, characterized in that: The lysis reagent in step 3 is a TFA / TIS / H2O mixture, wherein the volume ratio of TFA / TIS / H2O is 95 / 2.5 / 2.

5.

6. The method according to claim 1, characterized in that: The purification method in step 4 is: the crude product is dissolved in a 35% acetonitrile / water mixed solution, filtered through a 0.45um organic filter membrane, and purified by high performance liquid preparative chromatography C18, with mobile phase A being 0.1% trifluoroacetic acid aqueous solution, mobile phase B being acetonitrile, and gradient: 34%-54%.

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