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

Polypeptide derivatives for generating stable micro-molecular hydrogel Download PDF

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CN101704878A
CN101704878A CN200910229186A CN200910229186A CN101704878A CN 101704878 A CN101704878 A CN 101704878A CN 200910229186 A CN200910229186 A CN 200910229186A CN 200910229186 A CN200910229186 A CN 200910229186A CN 101704878 A CN101704878 A CN 101704878A
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杨志谋
王玲
王怀民
王景玉
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Nankai University
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Abstract

本发明用于生成稳定小分子水凝胶的多肽衍生物,涉及含多肽的化合物库,其分子组成和结构如下化合物结构式所示:

Figure 200910229186.7_AB_0
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:
Figure 200910229186.7_AB_0
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

用于生成稳定小分子水凝胶的多肽衍生物 Peptide Derivatives for Forming Stable Small Molecule Hydrogels

技术领域technical field

本发明的技术方案涉及含多肽的化合物库,具体地说是用于生成稳定小分子水凝胶的多肽衍生物。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.

背景技术Background technique

基于多肽的小分子水凝胶生物相容度高,在组织工程材料、化妆品等领域具有极高的潜在应用价值。但是,目前为止,公知的基于多肽的小分子水凝胶在大量水溶液中无法稳定存在,易于溶解成均一的溶液,从而使得这类水凝胶无法得到广泛的实际应用。这在已有文献(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)中已经有过报道。至今尚未见到关于用于生成稳定小分子水凝胶的多肽,或者说在大量水溶液中能够稳定存在的基于多肽的小分子水凝胶的文献报道。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.

发明内容Contents of the invention

本发明所要解决的技术问题是:提供用于生成稳定小分子水凝胶的多肽衍生物,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 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.

本发明解决该技术问题所采用的技术方案是:用于生成稳定小分子水凝胶的多肽衍生物,其分子组成和结构如下化合物结构式所示: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:

Figure G2009102291867D0000011
Figure G2009102291867D0000011

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=1~3。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.

上述用于生成稳定小分子水凝胶的多肽衍生物,所述R1The above-mentioned polypeptide derivatives used to generate stable small molecule hydrogels, said R 1 is

Figure G2009102291867D0000021
Figure G2009102291867D0000021

上述用于生成稳定小分子水凝胶的多肽衍生物,所述R2是甘氨酸或赖氨酸的侧链基团。In the above-mentioned polypeptide derivatives for generating stable small molecule hydrogels, the R 2 is a side chain group of glycine or lysine.

本发明的有益效果是:应用本发明用于生成稳定小分子水凝胶的多肽衍生物能够获得在水溶液中稳定存在、不易解离且生物相容度高的小分子水凝胶。其原因在于:由于本发明的多肽具有磷酸化的氨基酸,该类分子能在水溶液中良好的溶解。在加入磷酸酶的情况下,磷酸根将被切除,从而获得能白组装成胶的分子,最终导致小分子水凝胶的生成。又由于多肽的氨基和羧基都通过衍生化已经去除,通过这种方法制备获得的小分子水凝胶能很好的保持其初始的外形,不易在大量水溶液存在的情况下解离成均一的水溶液。因此,本发明用于生成稳定小分子水凝胶的多肽衍生物能作为细胞等生物个体的载体在组织工程材料领域获得极高的应用前景,并且在组织工程和化妆品等领域得到广泛的实际应用。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. .

具体实施方式Detailed ways

实施例1Example 1

本发明的用于生成稳定小分子水凝胶的多肽的合成方法步骤如下所示:The synthetic method step of the polypeptide for generating stable small molecule hydrogel of the present invention is as follows:

Figure G2009102291867D0000022
Figure G2009102291867D0000022

其中,DIPEA为N,N-二异丙基乙基胺,Acetone为丙酮,I2为碘,triethyl phosphite为亚磷酸三乙酯,DCM为二氯甲烷,pyridine为吡啶,TFA为三氟乙酸,spps为“多肽固相合成”的英文简称,TMSBr为三甲基溴硅烷,MeOH为甲醇。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.

第一步,Fmoc-L-Tyr-OtBu的合成The first step, the synthesis of Fmoc-L-Tyr-O t Bu

将10mmolL-酪氨酸叔丁酯和10分子的N,N-二异丙基乙基胺溶解在75ml的丙酮中,在搅拌的条件下加入含有9.8mmol的Fmoc-OSu的75ml的丙酮溶液,室温下搅拌12小时,然后用硅胶色谱柱分离,得到4.4g产物Fmoc-L-Tyr-OtBu(即上述合成方法步骤中的化合物3),其产率为95.8%,1H NMR(300MHz,DMSO-d6)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。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.

第二步,Fmoc-L-Tyr(PO(OEt)2)-OtBu的合成The second step, the synthesis of Fmoc-L-Tyr(PO(OEt) 2 )-O t Bu

将8mmol亚磷酸三乙酯溶解在80ml二氯甲烷中,加入7.5mmol碘,置于冰浴中,反应10min后使之温度恢复到室温,然后滴加到含有5.0mmol的由第一步合成得到的化合物3和20.0mmol吡啶的50ml的二氯甲烷中,置于冰浴中,反应2h,然后用100ml乙醚和300ml乙酸乙酯萃取,有机相用每次100ml的5%的硫酸氢钾洗三遍,再用饱和氯化钠洗一遍,然后该有机相用无水硫酸镁干燥,过滤,浓缩,硅胶色谱柱分离,得到Fmoc-L-Tyr(PO(OEt)2)-OtBu(即上述合成方法步骤中的化合物4),其产率为85.5%。1H NMR(300MHz,DMSO-d6)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).31P NMR(δ-6.366ppm).MS:calc.M+=595.2,obsvd.(M+1)+=596.01。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.

第三步,Fmoc-L-Tyr(PO(OEt)2-OH的合成The third step, the synthesis of Fmoc-L-Tyr(PO(OEt) 2 -OH

在5ml二氯甲烷和10ml三氟乙酸的混合液中加入1mmol第二步合成得到的化合物4,再置于冰浴中,搅拌4小时,然后旋转蒸干,再用甲苯共旋转蒸发两次,得到Fmoc-L-Tyr(PO(OEt)2-OH(即上述合成方法步骤中的化合物5),其产率为98.3%,1H NMR(300MHz,DMSO-d6)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).31P NMR(δ-6.348ppm).MS:calc.M+=539.2,obsvd.(M+H)+=540.14。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.

第四步,如下结构式所示的上述合成方法步骤中的化合物6的合成The fourth step, the synthesis of compound 6 in the above-mentioned synthetic method step shown in the following structural formula

Figure G2009102291867D0000031
Figure G2009102291867D0000031

将第三步合成得到的化合物5通过在常温下常用的多肽固相合成的方法合成上述步骤中的化合物6。该固相合成的方法见Fmoc solid phase peptide synthesis-A practicalapproach edited by Weng c.chan and peterd.white OXFORD UNIVERSITY PRESS,其中,多肽合成的原料为Fmoc保护的20种天然氨基酸和芳环衍生物,使用的缩合剂为1摩尔当量的HBTU,加入2摩尔当量的DIEA做催化剂.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.

第五步,如下结构式所示的用于生成稳定小分子水凝胶的多肽衍生物(即上述合成方法步骤中的化合物1)的合成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

Figure G2009102291867D0000041
Figure G2009102291867D0000041

将0.4mmol第四步制得的化合物6溶于10ml干燥二氯甲烷中,加入10mmol三甲基溴硅烷,室温下搅拌24小时,然后旋转蒸干得到固体,再加入10ml无水甲醇,此法得到的R4是OMe基团,或再加入含质量百分数为20%的甲基氨的甲醇溶液,此法得到的R4是NHMe基团,在室温下搅拌反应2小时,再旋转蒸干,用高效液相色谱分离产物,得到如上面结构式所示的用于生成稳定小分子水凝胶的多肽衍生物(即上述合成方法步骤中的化合物1)。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).

实施例2Example 2

如下结构式(I)所示多肽化合物Nap-GFFY(p)-OMe的合成。Synthesis of the polypeptide compound Nap-GFFY(p)-OMe shown in the following structural formula (I).

Figure G2009102291867D0000042
Figure G2009102291867D0000042

第一步~第三步,均同实施例1。The first step to the third step are all the same as in Example 1.

第四步,化合物Nap-GFFY(PO(OFt)2)-OMe(即在实施例1的化合物6通式中,R1

Figure G2009102291867D0000043
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
Figure G2009102291867D0000043
group, R 2 is H, R 3 is H) synthesis

采用Fmoc固相合成的方法,步骤是:Adopt the method for Fmoc solid phase synthesis, step is:

第1步,称取0.5mmol 2-氯三苯甲基氯树脂于固相合成器中,加入2.5mL的无水二氯甲烷,通入氮气5min,使2-氯三苯甲基氯树脂充分溶胀;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;

第2步,用氮气把二氯甲烷从装有2-氯三苯甲基氯树脂的固相合成器中压除干净;In the 2nd step, dichloromethane is removed from the solid-phase synthesizer that 2-chlorotrityl chloride resin is housed with nitrogen;

第3步,将1mmol第三步合成得到的化合物5溶解在2mL的无水二氯甲烷里,取其中1mL(即0.5mmol),加入0.5mmol的N,N-二异丙基乙基胺,然后转移到上述固相合成器中,再补加0.5mmol的N,N-二异丙基乙基胺,通入氮气,在室温下反应1h;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;

第4步,把该固相合成器中液体除净,然后用5mL无水二氯甲烷洗涤,每次1min,共洗5次,加入配好的体积比为无水二氯甲烷∶N,N-二异丙基乙基胺∶无水甲醇=17∶1∶2的溶液3mL,通入氮气,在室温下反应10min;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;

第5步,把该固相合成器中液体压净,用5mL无水二氯甲烷洗涤,每次1min,共洗5次,再用5mL N,N-二甲基甲酰胺洗涤,每次1min,共洗5次,加入5mL含体积百分比为20%的哌啶的N,N-二甲基甲酰胺溶液,通入氮气反应30min,再用5mL含体积百分比为20%的哌啶的N,N-二甲基甲酰胺溶液洗涤一次,然后用5mL N,N-二甲基甲酰胺洗涤,每次1min,共洗5次,进行下一步反应;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;

第6步,取多肽合成的原料Fmoc-苯丙氨酸2.5mmol、苯并三氮唑-N,N,N’,N’-四甲基脲六氟磷酸酯2.5mmol、N,N-二异丙基乙基胺5mmol和3ml无水N,N-二甲基甲酰胺配制成溶液,把配好的溶液加入到上述固相合成器中,通入氮气反应2h;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;

第7步,依次用多肽合成的原料Fmoc-苯丙氨酸、Fmoc-甘氨酸和多肽合成的原料萘乙酸替换多肽合成的原料Fmoc-苯丙氨酸,重复第5步和第6步操作,最终获得连接在2-氯三苯甲基氯树脂上的Nap-GFFY(PO(OEt)2)-COO;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;

第8步,按三氟乙酸与无水二氯甲烷的体积比为1∶99,配制成体积百分比浓度为1%的三氟乙酸溶液,取该三氟乙酸溶液每次3mL加入到上述固相合成器中,共加十次,每次反应时间为1min,把产物从2-氯三苯甲基氯树脂上切下,浓缩,加入两次甲苯以除去残留的三氟乙酸,用油泵抽干,得到化合物Nap-GFFY(PO(OEt)2)-COOH;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;

第五步,用于生成稳定小分子水凝胶的结构式(I)所示多肽Nap-GFFY(p)-OMe的合成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

将0.4mmol第四步制得的化合物Nap-GFFY(PO(OEt)2)-COOH溶于10ml干燥二氯甲烷中,加入10mmol三甲基溴硅烷,室温下搅拌24小时,然后旋转蒸干得到固体,再加入10ml无水甲醇,在室温下搅拌反应2小时,再旋转蒸干,用高效液相色谱分离产物,由此合成得到如上面结构式(I)所示多肽Nap-GFFY(p)-OMe,其产率为95.0%,1H NMR(400MHz,DMSO-d6)δ8.48(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).31P NMR(δ-6.008ppm).MS:calc.M+=794.27,obsvd.(M+Na)+=817.30and(M+2Na)+=839.25,(M-H)-=793.25。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.

本实施例制得的如上面结构式(I)所示多肽Nap-GFFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,最低成胶浓度极低-达到万分之一。它能作为三维细胞培养的载体,由于使用了溶解度较低的多肽衍生物作为成胶分子,要获得胶体时化合物的用量大大降低,因此能提高所制得的小分子水凝胶的生物相容度,细胞能在该类水凝胶中得到很好的生长。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.

实施例3Example 3

如下结构式(II)所示多肽Nap-FFY(p)-OMe的合成。Synthesis of the polypeptide Nap-FFY(p)-OMe shown in the following structural formula (II).

Figure G2009102291867D0000061
Figure G2009102291867D0000061

除在第四步的第6~7步中的多肽合成的原料替换为Fmoc-苯丙氨酸和萘乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(II)所示多肽Nap-FFY(p)-OMe,其产率为94%,1H NMR(400MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(II)所示多肽Nap-FFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例4Example 4

如下结构式(III)所示多肽Nap-GGFFY(p)-OMe的合成。Synthesis of the polypeptide Nap-GGFFY(p)-OMe shown in the following structural formula (III).

Figure G2009102291867D0000062
Figure G2009102291867D0000062

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-甘氨酸、Fmoc-甘氨酸和萘乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(III)所示多肽Nap-GGFFY(p)-OMe,其产率为88%,1H NMR(400MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(III)所示多肽Nap-GGFFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例5Example 5

如下结构式(IV)所示多肽Nap-GFY(p)-OMe的合成。Synthesis of the polypeptide Nap-GFY(p)-OMe shown in the following structural formula (IV).

Figure G2009102291867D0000071
Figure G2009102291867D0000071

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-甘氨酸和萘乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(IV)所示多肽Nap-GFY(p)-OMe,其产率为96%,1H NMR(400MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(IV)所示多肽Nap-GFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例6Example 6

如下结构式(V)所示多肽Nap-GFFFY(p)-OMe的合成。Synthesis of the polypeptide Nap-GFFFY(p)-OMe shown in the following structural formula (V).

Figure G2009102291867D0000072
Figure G2009102291867D0000072

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-苯丙氨酸、Fmoc-甘氨酸和萘乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(V)所示多肽Nap-GFY(p)-OMe,其产率为92%,1HNMR(400MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(V)所示多肽Nap-GFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶.该水凝胶稳定性好,可制备成各种形状.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.

实施例7Example 7

如下结构式(VI)所示多肽Nap-GFFY(p)-NHMe的合成。Synthesis of the polypeptide Nap-GFFY(p)-NHMe represented by the following structural formula (VI).

Figure G2009102291867D0000081
Figure G2009102291867D0000081

除第五步中用含质量百分数为20%的甲基氨的甲醇溶液替换无水甲醇之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(VI)所示多肽Nap-GFFY(p)-NHMe,其产率为85%,1H NMR(400MHz,DMSO-d6)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).31P NMR(δ-6.008ppm).MS:calc.M+=793.29,obsvd.(M+2Na)+=838.22。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.

本实施例制得的如上面结构式(VI)所示多肽Nap-GFFY(p)-NHMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例8Example 8

如下结构式(VII)所示多肽Nap-GF(f)F(f)Y(p)-OMe的合成。Synthesis of the polypeptide Nap-GF(f)F(f)Y(p)-OMe shown in the following structural formula (VII).

Figure G2009102291867D0000091
Figure G2009102291867D0000091

除在第三步的Fmoc-氨基酸为Fmoc-4-氟-苯丙氨酸,第四步的第6~7步中多肽合成的原料替换依次为Fmoc-4-氟-苯丙氨酸、Fmoc-甘氨酸和萘乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(VII)所示多肽Nap-GF(f)F(f)Y(p)-OMe,其产率为92%,1H NMR(400MHz,DMSO-d6)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).31P NMR(δ-6.012ppm).MS:calc.M+=830.25,obsvd.(M+2Na)+=853.35。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.

本实施例制得的如上面结构式(VII)所示多肽Nap-GF(f)F(f)Y(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例9Example 9

如下结构式(VIII)所示多肽biPhenyl-GFFY(p)-OMe的合成。Synthesis of the polypeptide biPhenyl-GFFY(p)-OMe shown in the following structural formula (VIII).

Figure G2009102291867D0000092
Figure G2009102291867D0000092

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-甘氨酸和联苯乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(VIII)所示多肽biPhenyl-GFFY(p)-OMe,其产率为93%,1H NMR(600MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(VIII)所示多肽biPhenyl-GFFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例10Example 10

如下结构式(IX)所示多肽biOMePhenyl-GFFY(p)-OMe的合成。Synthesis of the polypeptide biOMePhenyl-GFFY(p)-OMe shown in the following structural formula (IX).

Figure G2009102291867D0000101
Figure G2009102291867D0000101

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-甘氨酸和3,4-二甲氧基苯乙酸之外,其他合成步骤的操作方法和参数同实施例2.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.

由此合成得得到如上面结构式(IX)所示多肽biOMePhenyl-GFFY(p)-OMe,其产率为82%,1H NMR(600MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(IX)所示多肽biOMePhenyl-GFFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例11Example 11

如下结构式(X)所示多肽NitroPhenyl-GFFY(p)-OMe的合成。Synthesis of the polypeptide NitroPhenyl-GFFY(p)-OMe shown in the following structural formula (X).

Figure G2009102291867D0000102
Figure G2009102291867D0000102

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-甘氨酸和4-硝基苯乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(X)所示多肽NitroPhenyl-GFFY(p)-OMe,其产率为80%,1H NMR(600MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(X)所示多肽NitroPhenyl-GFFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例12Example 12

如下结构式(XI)所示多肽biClPhenyl-GFFY(p)-OMe的合成。Synthesis of the polypeptide biClPhenyl-GFFY(p)-OMe shown in the following structural formula (XI).

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-甘氨酸和3,4-二氯苯乙酸之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(XI)所示多肽biClPhenyl-GFFY(p)-OMe,其产率为81%,1H NMR(600MHz,DMSO-d6)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,(M-H)-=811.30。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.

本实施例制得的如上面结构式(XI)所示多肽biClPhenyl-GFFY(p)-OMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。该水凝胶稳定性好,可制备成各种形状。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.

实施例13Example 13

如下结构式(XII)所示多肽Nap-KFFY(p)-NHMe的合成。Synthesis of the polypeptide Nap-KFFY(p)-NHMe represented by the following structural formula (XII).

Figure G2009102291867D0000112
Figure G2009102291867D0000112

除在第四步的第6~7步中多肽合成的原料替换依次为Fmoc-苯丙氨酸、Fmoc-赖氨酸和奈乙酸;并且第五步中用含质量百分数为20%的甲基氨的甲醇溶液替换无水甲醇之外,其他合成步骤的操作方法和参数同实施例2。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.

由此合成得得到如上面结构式(XII)所示多肽Nap-KFFY(p)-NHMe,其产率为78%,1H NMR(600MHz,DMSO-d6)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。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.

本实施例制得的如上面结构式(XII)所示多肽Nap-KFFY(p)-NHMe分子能很好的溶解在水溶液中,在磷酸酶的作用下磷酸根被切除,从而获得水凝胶。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.

上述实施例中所用缩合剂HBTU的英文全名为O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate,是做多肽合成的技术人员都知道的。催化剂DIEA的中文名称为二异丙基乙级胺。这两个化学品以及所有的Fmoc保护的氨基酸都从吉尔生化购买获得。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.用于生成稳定小分子水凝胶的多肽衍生物,其特征在于其分子组成和结构如下化合物结构式所示: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:
Figure F2009102291867C0000011
Figure F2009102291867C0000011
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=1~3。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.根据权利要求1所述的用于生成稳定小分子水凝胶的多肽衍生物,其特征在于:所述R12. 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
Figure F2009102291867C0000012
Figure F2009102291867C0000012
3.根据权利要求1所述的用于生成稳定小分子水凝胶的多肽衍生物,其特征在于:所述R2是甘氨酸或赖氨酸的侧链基团。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.
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