CN101704878A - Polypeptide derivatives for generating stable micro-molecular hydrogel - Google Patents

Abstract

该式中，R₁是芳环衍生物，R₂是20种天然氨基酸中一种的侧链基团，R₃是H或者卤素中的任意一种，R₄是OMe基或者NHMe基，n＝0～3。基于该多肽衍生物的小分子水凝胶在形成后能在大量水溶液中稳定存在，外形能得到很好地保持，具有生物相容度高，在组织工程和化妆品等领域得到广泛的实际应用。The present invention is used to generate a polypeptide derivative of a stable small molecule hydrogel, and relates to a compound library containing a polypeptide, and its molecular composition and structure are shown in the following compound structural formula:

In this formula, R ₁ is an aromatic ring derivative, R ₂ is a side chain group of one of 20 kinds of natural amino acids, R ₃ is any one of H or halogen, R ₄ is OMe group or NHMe group, n =0~3. The small-molecule hydrogel based on the polypeptide derivative can exist stably in a large amount of aqueous solution after formation, can keep its shape well, has high biocompatibility, and has been widely used in the fields of tissue engineering and cosmetics.

Description

Peptide Derivatives for Forming Stable Small Molecule Hydrogels

technical field

The technical scheme of the present invention relates to a compound library containing polypeptides, in particular to polypeptide derivatives used to generate stable small molecule hydrogels.

Background technique

Peptide-based small molecule hydrogels have high biocompatibility and have high potential application value in tissue engineering materials, cosmetics and other fields. However, so far, the known small molecule hydrogels based on polypeptides cannot exist stably in a large amount of aqueous solution and are easily dissolved into a uniform solution, which prevents such hydrogels from being widely used in practical applications. This has been reported in existing literature (Genove E, Shen C, Zhang SG, Semino CE, BIOMATERIALS, 2005, 16, 3341-3351; Ghanaati S, Webber MJ, Unger RE, Orth C, Hulvat JF, Kiehna SE, BarbeckM, Rasic A , Stupp SI, Kirkpatrick CJ, BIOMATERIALS, 2009, 31, 6202-6212) have been reported. So far, there have been no literature reports on polypeptides used to generate stable small-molecule hydrogels, or polypeptide-based small-molecule hydrogels that can stably exist in a large amount of aqueous solution.

Contents of the invention

The technical problem to be solved by the present invention is to provide polypeptide derivatives used to generate stable small molecule hydrogels, the small molecule hydrogels based on the polypeptide derivatives can exist stably in a large amount of aqueous solution after formation, and the shape can be greatly improved. It is well maintained and has high biocompatibility, and has been widely used in the fields of tissue engineering and cosmetics.

The technical solution adopted by the present invention to solve the technical problem is: a polypeptide derivative used to generate a stable small molecule hydrogel, its molecular composition and structure are shown in the following compound structural formula:

In this formula, R ₁ is an aromatic ring derivative, R ₂ is a side chain group of one of 20 kinds of natural amino acids, R ₃ is any one of H or halogen, R ₄ is OMe group or NHMe group, n =1~3.

The above-mentioned polypeptide derivatives used to generate stable small molecule hydrogels, said R ₁ is

In the above-mentioned polypeptide derivatives for generating stable small molecule hydrogels, the R ₂ is a side chain group of glycine or lysine.

The beneficial effects of the present invention are: the use of the polypeptide derivatives of the present invention for generating stable small-molecule hydrogels can obtain small-molecule hydrogels that exist stably in aqueous solution, are not easily dissociated, and have high biocompatibility. The reason is that: since the polypeptide of the present invention has phosphorylated amino acids, such molecules can be well dissolved in aqueous solution. With the addition of phosphatase, the phosphate group will be cleaved to obtain molecules that can be assembled into gels, eventually leading to the formation of small molecule hydrogels. And because the amino and carboxyl groups of the polypeptide have been removed by derivatization, the small molecule hydrogel prepared by this method can well maintain its original shape, and it is not easy to dissociate into a uniform aqueous solution in the presence of a large amount of aqueous solution. . Therefore, the polypeptide derivatives used to generate stable small molecule hydrogels of the present invention can obtain extremely high application prospects in the field of tissue engineering materials as carriers of biological individuals such as cells, and have been widely used in the fields of tissue engineering and cosmetics. .

Detailed ways

Example 1

The synthetic method step of the polypeptide for generating stable small molecule hydrogel of the present invention is as follows:

Wherein, DIPEA _is N, N-diisopropylethylamine, Acetone is acetone, I is iodine, triethyl phosphite is triethyl phosphite, DCM is dichloromethane, pyridine is pyridine, TFA is trifluoroacetic acid, Spps is the English abbreviation of "polypeptide solid-phase synthesis", TMSBr is trimethylbromosilane, and MeOH is methanol.

The first step, the synthesis of Fmoc-L-Tyr-O ^t Bu

10mmol L-tert-butyl tyrosine and 10 molecules of N,N-diisopropylethylamine are dissolved in 75ml of acetone, and the acetone solution of 75ml of Fmoc-OSu containing 9.8mmol is added under stirring conditions, Stir at room temperature for 12 hours, then separate with silica gel column to obtain 4.4g of product Fmoc-L-Tyr-O ^t Bu (i.e. compound 3 in the above synthesis method step), its yield is 95.8%, ¹ H NMR (300MHz , DMSO-d ₆ ) δ8.03-8.05 (d, 2H), 7.81-7.85 (t, 2H), 7.56-7.59 (t, 2H), 7.46-7.48 (m, 2H), 7.19-7.21 (d, 2H), 6.81-6.83(d, 2H), 4.30-4.40(m, 3H), 4.2-4.24(m, 1H), 3.30-3.36(m, 2H), 1.50(s, 9H), MS: calc. M ⁺ = 459.2, obsvd.(M+1) ⁺ = 459.78.

The second step, the synthesis of Fmoc-L-Tyr(PO(OEt) ₂ )-O ^t Bu

Dissolve 8 mmol of triethyl phosphite in 80 ml of dichloromethane, add 7.5 mmol of iodine, place in an ice bath, react for 10 minutes, and return the temperature to room temperature, then add dropwise to 5.0 mmol of Compound 3 and 20.0mmol of pyridine in 50ml of dichloromethane, placed in an ice bath, reacted for 2h, then extracted with 100ml of ether and 300ml of ethyl acetate, and the organic phase was washed three times with 5% potassium bisulfate of 100ml each time and then washed once with saturated sodium chloride, then the organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and separated by silica gel column to obtain Fmoc-L-Tyr(PO(OEt) ₂ )-O ^t Bu (ie Compound 4) in the steps of the above synthetic method has a yield of 85.5%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.03-8.05 (d, 2H), 7.82-7.84 (d, 2H), 7.57-7.59 (t, 2H), 7.42-7.48 (m, 4H), 7.25 -7.28(d, 2H), 4.10-4.40(m, 8H), 3.11-3.13(m, 1H), 3.03-3.07(m, 1H), 1.49(s, 9H), 1.37-1.41(t, 6H) . ³¹ P NMR (δ-6.366ppm). MS: calc. M ⁺ = 595.2, obsvd. (M + 1) ⁺ = 596.01.

The third step, the synthesis of Fmoc-L-Tyr(PO(OEt) ₂ -OH

Add 1 mmol of the compound 4 synthesized in the second step to a mixture of 5 ml of dichloromethane and 10 ml of trifluoroacetic acid, place it in an ice bath, stir for 4 hours, then evaporate to dryness, and then co-rotate with toluene twice, Fmoc-L-Tyr(PO(OEt) ₂ -OH (that is, compound 5 in the steps of the above synthetic method) was obtained with a yield of 98.3%, ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.23-8.25 ( d, 2H), 8.01-8.05(t, 2H), 7.76-7.79(t, 2H), 7.59-7.62(m, 4H), 7.47-7.52(d, 2H), 4.30-4.60(m, 8H), 3.45-3.48 (m, 1H), 3.21-3.27 (m, 1H), 1.58-1.61 (t, 6H). ³¹ P NMR (δ-6.348ppm). MS: calc.M ⁺ =539.2, obsvd.(M +H) ⁺ = 540.14.

The fourth step, the synthesis of compound 6 in the above-mentioned synthetic method step shown in the following structural formula

The compound 5 synthesized in the third step was synthesized into the compound 6 in the above step by the common solid-phase synthesis method of polypeptide at room temperature. For the solid phase synthesis method, see Fmoc solid phase peptide synthesis-A practical approach edited by Weng c.chan and peterd.white OXFORD UNIVERSITY PRESS, wherein the raw materials for peptide synthesis are 20 kinds of natural amino acids and aromatic ring derivatives protected by Fmoc, using The condensing agent is 1 molar equivalent of HBTU, and 2 molar equivalents of DIEA are added as a catalyst.

The fifth step is the synthesis of polypeptide derivatives (i.e. compound 1 in the steps of the above-mentioned synthetic method) used to generate stable small molecule hydrogels as shown in the following structural formula

Dissolve 0.4mmol of compound 6 prepared in the fourth step in 10ml of dry dichloromethane, add 10mmol of trimethylbromosilane, stir at room temperature for 24 hours, then spin evaporate to dryness to obtain a solid, then add 10ml of anhydrous methanol, this method Obtained R ₄ is an OMe group, or adding a methanol solution containing 20% methylammonia in mass percent, the R ₄ obtained by this method is an NHMe group, stirred and reacted at room temperature for 2 hours, and then spun to dryness, The product is separated by high-performance liquid chromatography to obtain the polypeptide derivative shown in the above structural formula for generating a stable small molecule hydrogel (ie, compound 1 in the steps of the above synthesis method).

Example 2

Synthesis of the polypeptide compound Nap-GFFY(p)-OMe shown in the following structural formula (I).

The first step to the third step are all the same as in Example 1.

基团，R₂为H，R₃为H)的合成In the fourth step, the compound Nap-GFFY(PO(OFt) ₂ )-OMe (that is, in the general formula of compound 6 in Example 1, R ₁ is

group, R ₂ is H, R ₃ is H) synthesis

Adopt the method for Fmoc solid phase synthesis, step is:

Step 1, weigh 0.5mmol 2-chlorotrityl chloride resin in a solid-phase synthesizer, add 2.5mL of anhydrous dichloromethane, and feed nitrogen for 5min to make the 2-chlorotrityl chloride resin fully swelling;

In the 2nd step, dichloromethane is removed from the solid-phase synthesizer that 2-chlorotrityl chloride resin is housed with nitrogen;

In the third step, 1 mmol of the compound 5 synthesized in the third step was dissolved in 2 mL of anhydrous dichloromethane, and 1 mL (ie 0.5 mmol) was taken, and 0.5 mmol of N,N-diisopropylethylamine was added, Then transfer to the above-mentioned solid-phase synthesizer, add 0.5mmol of N,N-diisopropylethylamine, feed nitrogen, and react at room temperature for 1h;

In the 4th step, the liquid in the solid-phase synthesizer is removed, and then washed with 5mL of anhydrous dichloromethane, each time for 1min, and washed 5 times altogether, and the volume ratio of adding is anhydrous dichloromethane: N, N - 3 mL of a solution of diisopropylethylamine: anhydrous methanol = 17: 1: 2, blowing in nitrogen gas, and reacting at room temperature for 10 min;

Step 5: Press out the liquid in the solid-phase synthesizer, wash with 5 mL of anhydrous dichloromethane, 1 min each time, wash 5 times in total, and then wash with 5 mL N, N-dimethylformamide, 1 min each time , washed 5 times in total, added 5 mL of N, N-dimethylformamide solution containing 20% by volume of piperidine, and reacted for 30 min with nitrogen gas, and then used 5 mL of N, N-dimethylformamide containing 20% by volume of piperidine, Wash once with N-dimethylformamide solution, then wash with 5mL N,N-dimethylformamide, 1 min each time, wash 5 times in total, and proceed to the next reaction;

In the 6th step, 2.5mmol of Fmoc-phenylalanine, 2.5mmol of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2.5mmol of N,N-di 5 mmol of isopropylethylamine and 3 ml of anhydrous N, N-dimethylformamide were prepared into a solution, and the prepared solution was added to the above-mentioned solid-phase synthesizer, and nitrogen gas was passed through to react for 2 h;

In the 7th step, replace the raw material Fmoc-phenylalanine for polypeptide synthesis with Fmoc-phenylalanine, Fmoc-glycine and naphthalene acetic acid for polypeptide synthesis in turn, repeat steps 5 and 6, and finally Nap-GFFY(PO(OEt)2)-COO attached to 2-chlorotrityl chloride resin was obtained;

The 8th step, according to the volume ratio of trifluoroacetic acid and anhydrous dichloromethane is 1: 99, is prepared into the trifluoroacetic acid solution that the volume percentage concentration is 1%, takes this trifluoroacetic acid solution every 3mL and joins in above-mentioned solid phase In the synthesizer, add a total of ten times, each reaction time is 1min, cut the product from the 2-chlorotrityl chloride resin, concentrate, add toluene twice to remove the residual trifluoroacetic acid, and drain it with an oil pump , to obtain the compound Nap-GFFY(PO(OEt)2)-COOH;

The fifth step is used to generate the synthesis of polypeptide Nap-GFFY(p)-OMe shown in the structural formula (I) of stable small molecule hydrogel

Dissolve 0.4mmol of the compound Nap-GFFY(PO(OEt) ₂ )-COOH obtained in the fourth step in 10ml of dry dichloromethane, add 10mmol of bromotrimethylsilane, stir at room temperature for 24 hours, and then evaporate to dryness to obtain solid, then add 10ml of anhydrous methanol, stir and react at room temperature for 2 hours, then evaporate to dryness, and separate the product with high performance liquid chromatography, thereby synthesizing the polypeptide Nap-GFFY(p)- as shown in the above structural formula (I)- OMe, its yield is 95.0%, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.48 (d, 1H, NH), 8.19-8.28 (m, 2H, NH), 8.06 (d, 1H, NH) , 7.75-7.88(m, 3H), 7.754(s, 1H), 7.41-7.48(m, 3H), 7.07-7.25(m, 14H), 4.46-4.58(m, 3H), 3.70-3.76(m, 1H), 3.6-3.68(s, 4H), 3.58(s, 3H), 2.92-3.03(m, 4H), 2.76-2.81(m, 1H), 2.63-2.69(m, 1H). ³¹ P NMR( δ-6.008ppm).MS: calc.M ⁺ =794.27, obsvd.(M+Na) ⁺ =817.30 and (M+2Na) ⁺ =839.25, (MH) ^- =793.25.

The Nap-GFFY(p)-OMe molecule of the polypeptide shown in the above structural formula (I) prepared in this example can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase, thereby obtaining a hydrogel. The hydrogel has good stability, and the minimum gel-forming concentration is extremely low-reaching one ten-thousandth. It can be used as a carrier for three-dimensional cell culture. Since the polypeptide derivatives with low solubility are used as gel-forming molecules, the amount of compounds used to obtain colloids is greatly reduced, so it can improve the biocompatibility of the prepared small-molecule hydrogel. Cells can grow well in this type of hydrogel.

Example 3

Synthesis of the polypeptide Nap-FFY(p)-OMe shown in the following structural formula (II).

The operation methods and parameters of other synthesis steps are the same as those in Example 2, except that the raw materials for polypeptide synthesis in steps 6-7 of the fourth step are replaced by Fmoc-phenylalanine and naphthaleneacetic acid.

Thus, the polypeptide Nap-FFY(p)-OMe shown in the above structural formula (II) was synthesized with a yield of 94%, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.63 (d, J=7.324 , 1H), 8.39(d, J=8.518, 1H), 8.28(d, J=8.216, 1H), 7.90-8.00(m, 3H), 7.74(s, 1H), 7.62(m, 2H), 7.35 -7.40(m, 3H), 7.25-7.40(m, 10H), 7.23-7.25(m, 2H), 4.6-4.8(m, 3H), 3.745(s, 3H), 3.60-3.72(m, 3H) , 3.000-3.120 (m, 3H), 2.3-2.80 (m, 2H). MS: calc. M ⁺ = 737.25, obsvd. (M + Na) ⁺ = 760.35.

The Nap-FFY(p)-OMe molecule of the polypeptide shown in the above structural formula (II) prepared in this example can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 4

Synthesis of the polypeptide Nap-GGFFY(p)-OMe shown in the following structural formula (III).

Except that the raw materials of the peptide synthesis in steps 6 to 7 of the fourth step are replaced by Fmoc-phenylalanine, Fmoc-glycine, Fmoc-glycine and naphthaleneacetic acid, the operation methods and parameters of other synthesis steps are the same as those in the examples 2.

Thus, the polypeptide Nap-GGFFY(p)-OMe shown in the above structural formula (III) was synthesized with a yield of 88%, ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.587 (d, J=7.287 , 1H), 8.495-8.520(m, 1H), 8.227-8.280(m, 2H), 8.147(d, J=8.348, 1H), 7.970-8.030(m, 3H), 7.922(s, 1H), 7.590- 7.650(m, 3H), 7.400-7.420(m, 4H), 7.320-7.360(m, 8H), 7.234(d, J=8.3022H), 4.614-4.741(m, 4H) 3.881-3.890(m, 4H ), 3.819(m, 3H), 3.075-3.186(m, 5H), 2.936-2.975(m, 1H), 2.813-2.853(m, 1H).MS: calc.M ⁺ =851.29, obsvd.(M+ Na) ⁺ = 874.35.

The polypeptide Nap-GGFFY(p)-OMe molecule prepared in this example as shown in the above structural formula (III) can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 5

Synthesis of the polypeptide Nap-GFY(p)-OMe shown in the following structural formula (IV).

The operating methods and parameters of other synthesis steps are the same as those in Example 2, except that the raw materials for polypeptide synthesis in steps 6-7 of the fourth step are replaced by Fmoc-glycine and naphthaleneacetic acid.

Thus, the polypeptide Nap-GFY(p)-OMe shown in the above structural formula (IV) was synthesized with a yield of 96%, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.46 (d, 1H), 8.16-8.25(m, 1H), 8.02(d, 1H), 7.65-7.90(m, 2H), 7.754(s, 1H), 7.41-7.48(m, 3H), 7.13-7.32(m, 10H), 4.42-4.56(m, 2H), 3.63(s, 3H), 3.58(s, 4H), 2.89-3.01(m, 2H), 2.67-2.78(m, 1H), 2.62-2.67(m, 1H). MS: calc. M ⁺ = 647.20, obsvd. (M + Na) ⁺ = 670.35.

The Nap-GFY(p)-OMe molecule of the polypeptide shown in the above structural formula (IV) prepared in this example can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 6

Synthesis of the polypeptide Nap-GFFFY(p)-OMe shown in the following structural formula (V).

Operation methods and parameters of other synthesis steps, except that the raw materials of peptide synthesis in steps 6 to 7 of the fourth step are replaced by Fmoc-phenylalanine, Fmoc-phenylalanine, Fmoc-glycine and naphthaleneacetic acid With embodiment 2.

Thus, the polypeptide Nap-GFY(p)-OMe shown in the above structural formula (V) was synthesized with a yield of 92%, ¹ HNMR (400 MHz, DMSO-d ₆ ) δ8.641 (d, J=6.998, 1H), 8.373(s, 1H), 8.234-8.373(m, 2H), 8.143-8.157(m, 1H), 7.965-8.025(m, 3H), 7.902(s, 1H), 7.564-7.638(m, 4H), 7.210-7.561(m, 18H), 4.623-4.766(m, 4H), 3.735-3.887(m, 3H), 3.731(s, 3H), 2.979-3.163(m, 5H), 2.889-2.917( m, 2H), 2.803-2.876 (m, 1H), 2.650-2.763 (m, 1H). MS: calc. M ⁺ = 941.34, obsvd. (M + Na) ⁺ = 964.44.

The polypeptide Nap-GFY(p)-OMe molecule as shown in the above structural formula (V) prepared in this example can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase, thereby obtaining a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 7

Synthesis of the polypeptide Nap-GFFY(p)-NHMe represented by the following structural formula (VI).

Except that the methanol solution containing 20% by mass of methylammonia was used to replace the anhydrous methanol in the fifth step, the operating methods and parameters of other synthesis steps were the same as those in Example 2.

Thus, the polypeptide Nap-GFFY(p)-NHMe shown in the above structural formula (VI) was synthesized with a yield of 85%, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.45 (d, 1H), 8.16-8.25(m, 2H), 8.03(d, 1H), 7.72-7.85(m, 4H), 7.751(s, 1H), 7.38-7.45(m, 3H), 7.04-7.22(m, 14H), 4.43-4.55(m, 3H), 3.67-3.73(m, 1H), 3.60(s, 2H), 3.55(s, 4H), 2.88-3.29(m, 4H), 2.73-2.78(m, 1H), 2.60-2.66 (m, 1H). ³¹ P NMR (δ-6.008 ppm). MS: calc. M ⁺ = 793.29, obsvd. (M + 2Na) ⁺ = 838.22.

The polypeptide Nap-GFFY(p)-NHMe molecule prepared in this example as shown in the above structural formula (VI) can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 8

Synthesis of the polypeptide Nap-GF(f)F(f)Y(p)-OMe shown in the following structural formula (VII).

Except that the Fmoc-amino acid in the third step is Fmoc-4-fluoro-phenylalanine, the raw materials for peptide synthesis in steps 6 to 7 of the fourth step are replaced by Fmoc-4-fluoro-phenylalanine, Fmoc -Except for glycine and naphthaleneacetic acid, the operating methods and parameters of other synthetic steps are the same as in Example 2.

Thus, the polypeptide Nap-GF(f)F(f)Y(p)-OMe shown in the above structural formula (VII) was synthesized with a yield of 92%, ¹ H NMR (400MHz, DMSO-d ₆ )δ8 .48(d, 1H), 8.21-8.26(m, 2H), 8.10(d, 1H), 7.70-7.81(m, 3H), 7.76(s, 1H), 7.40-7.48(m, 3H), 7.10 -7.25(m, 12H), 4.47-4.58(m, 3H), 3.71-3.76(m, 1H), 3.65(s, 2H), 3.60(s, 4H), 2.95-3.03(m, 4H), 2.80 -2.85 (m, 1H), 2.65-2.70 (m, 1H). ³¹ P NMR (δ-6.012 ppm). MS: calc. M ⁺ = 830.25, obsvd. (M + 2Na) ⁺ = 853.35.

The polypeptide Nap-GF(f)F(f)Y(p)-OMe molecule prepared in this embodiment as shown in the above structural formula (VII) can be well dissolved in aqueous solution, and the phosphate radical is absorbed by the phosphatase under the action of phosphatase excised to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 9

Synthesis of the polypeptide biPhenyl-GFFY(p)-OMe shown in the following structural formula (VIII).

The operating methods and parameters of other synthesis steps are the same as those in Example 2, except that the raw materials for polypeptide synthesis in steps 6-7 of the fourth step are replaced by Fmoc-phenylalanine, Fmoc-glycine and felbinac.

Thus, the polypeptide biPhenyl-GFFY(p)-OMe shown in the above structural formula (VIII) was synthesized with a yield of 93%, ¹ H NMR (600 MHz, DMSO-d ₆ ) δ8.590 (d, J=7.011 , 1H), 8.303-8.367(m, 2H), 8.171(d, J=8.301, 1H), 7.771-7.785(d, 2H), 7.707-7.722(d, 2H), 7.602(m, 2H), 7.486 -7.501(m, 3H), 7.301-7.402(m, 11H), 7.227-7.242(m, 2H), 4.620-4.720(m, 3H), 3.857-3.895(m, 1H), 3.729(m, 4H) , 3.649(m, 2H), 3.143-3.178(m, 2H), 3.086-3.113(m, 2H), 2.930-2.980(m, 1H), 2.80-2.85(m, 1H). MS: calc.M ⁺ 820.29, obsvd. (M+Na) ⁺ = 843.39.

The polypeptide biPhenyl-GFFY(p)-OMe molecule prepared in this example as shown in the above structural formula (VIII) can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 10

Synthesis of the polypeptide biOMePhenyl-GFFY(p)-OMe shown in the following structural formula (IX).

In addition to the replacement of raw materials for polypeptide synthesis in steps 6 to 7 of the fourth step to Fmoc-phenylalanine, Fmoc-glycine and 3,4-dimethoxyphenylacetic acid, the operating methods and methods of other synthesis steps Parameter is with embodiment 2.

Thus, the polypeptide biOMePhenyl-GFFY(p)-OMe shown in the above structural formula (IX) was synthesized with a yield of 82%, ¹ H NMR (600 MHz, DMSO-d ₆ ) δ8.298 (d, 1H), 8.11-8.16(m, 3H), 7.308-7.451(m, 10H), 7.226-7.242(m, 3H), 6.912-7.029(m, 2H), 6.775-6.847(m, 2H), 4.705-4.713(m , 1H), 4.61-4.65(m, 2H), 3.852-3.884(m, 8H), 3.730-3.775(m, 5H), 3.084-3.175(m, 4H), 2.283-2.289(d, 2H).MS : calc.M ⁺ =804.28, obsvd.(M+Na) ⁺ =827.39.

The polypeptide biOMePhenyl-GFFY(p)-OMe molecule prepared in this example as shown in the above structural formula (IX) can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 11

Synthesis of the polypeptide NitroPhenyl-GFFY(p)-OMe shown in the following structural formula (X).

In addition to the replacement of raw materials for polypeptide synthesis in steps 6 to 7 of the fourth step to Fmoc-phenylalanine, Fmoc-glycine and 4-nitrophenylacetic acid, the operation methods and parameters of other synthesis steps are the same as those in the examples 2.

Thus, the polypeptide NitroPhenyl-GFFY(p)-OMe shown in the above structural formula (X) was synthesized with a yield of 80%, ¹ H NMR (600MHz, DMSO-d ₆ ) δ8.402(d, 2H)8.293 (d, 1H), 8.124-8.162(m, 3H), 7.942(d, 2H), 7.551-7.675(m, 10H), 7.372-7.386(m, 2H), 6.908-7.027(m, 2H), 4.452 -4.621(m, 3H), 3.682-3.758(m, 1H), 3.626(s, 2H), 3.579(s, 4H), 2.918-3.032(m, 4H), 2.758-2.805(m, 1H), 2.629 -2.685 (m, 1H). MS: calc. M ⁺ = 789.24, obsvd. (M + Na) ⁺ = 811.34.

The polypeptide NitroPhenyl-GFFY(p)-OMe molecule prepared in this example as shown in the above structural formula (X) can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 12

Synthesis of the polypeptide biClPhenyl-GFFY(p)-OMe shown in the following structural formula (XI).

Except that the raw materials of peptide synthesis in steps 6 to 7 of the fourth step are replaced by Fmoc-phenylalanine, Fmoc-glycine and 3,4-dichlorophenylacetic acid, the operation methods and parameters of other synthesis steps are the same as Example 2.

Thus, the polypeptide biClPhenyl-GFFY(p)-OMe shown in the above structural formula (XI) was synthesized with a yield of 81%, ¹ H NMR (600 MHz, DMSO-d ₆ ) δ8.304 (d, 1H), 8129-8.172(m, 3H), 7.312-7.458(m, 10H), 7.230-7.245(m, 3H), 6.909-7.034(m, 2H), 6.772-6.851(m, 2H), 4.701-4.716(m , 1H) 4.609-4.634(m, 2H), 3.848-3.875(m, 7H), 3.728(s, 3H), 3.124-3.180(m, 4H), 2.281-2.287(d, 2H). MS: calc. M ⁺ = 812.18, obsvd. (M+2Na) ⁺ = 857.20, (MH) ^- = 811.30.

The polypeptide biClPhenyl-GFFY(p)-OMe molecule prepared in this example as shown in the above structural formula (XI) can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel. The hydrogel has good stability and can be prepared into various shapes.

Example 13

Synthesis of the polypeptide Nap-KFFY(p)-NHMe represented by the following structural formula (XII).

In addition to the replacement of raw materials for polypeptide synthesis in steps 6 to 7 of the fourth step, Fmoc-phenylalanine, Fmoc-lysine and naphthalene acetic acid; The operating methods and parameters of the other synthesis steps are the same as in Example 2 except that the methanol solution of ammonia replaces anhydrous methanol.

Thus, the polypeptide Nap-KFFY(p)-NHMe shown in the above structural formula (XII) was synthesized with a yield of 78%, ¹ H NMR (600 MHz, DMSO-d ₆ ) δ8.478 (d, 1H), 8.182-8.276(m, 2H), 8.053(d,, 1H), 7.746-7.875(m, 3H), 7.754(s, 1H), 7.387-7.462(m, 3H), 7.065-7.243(m, 15H) , 4.923(m, 2H), 4.458-4.575(m, 3H), 3.689-3.754(m, 1H), 3.625(s, 2H), 3.564(s, 4H), 2.910-3.018(m, 4H), 2.724 -2.791(m, 1H), 2.673-2.715(m, 2H), 1.953-1.976(m, 2H), 1.435-1.468(m, 2H), 1.258-1.360(m, 2H).MS: calc.M- =863.50, obsvd.(M+Na)+=887.45.

The Nap-KFFY(p)-NHMe molecule of the polypeptide shown in the above structural formula (XII) prepared in this example can be well dissolved in aqueous solution, and the phosphate group is excised under the action of phosphatase to obtain a hydrogel.

The English full name of the condensing agent HBTU used in the above examples is O-Benzotriazole-N, N, N', N'-tetramethyl-uronium-hexafluoro-phosphate, which is known to those skilled in polypeptide synthesis. The Chinese name of the catalyst DIEA is diisopropylethylamine. These two chemicals, as well as all Fmoc-protected amino acids, were purchased from Jill Biochemical.

Claims (3)

1. For generating the polypeptide derivative of stable small molecule hydrogel, it is characterized in that its molecular composition and structure are as shown in the following compound structural formula:

In this formula, R ₁ is an aromatic ring derivative, R ₂ is a side chain group of one of 20 natural amino acids, R ₃ is any one of H or halogen, and R ₄ is Ome group or NHMe group. n=1~3. 2. according to claim 1, be used to generate the polypeptide derivative of stable small molecule hydrogel, it is characterized in that: described R ₁ is

3. The polypeptide derivative for generating stable small molecule hydrogel according to claim 1, characterized in that: said R ₂ is a side chain group of glycine or lysine.