CN108017690B - 具有抗菌活性的柱[5]芳烃类人工跨膜通道及其制备方法和应用 - Google Patents
具有抗菌活性的柱[5]芳烃类人工跨膜通道及其制备方法和应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
本发明公开了一种具有抗菌活性的柱[5]芳烃类人工跨膜通道及其制备方法和应用,属于具有抗菌活性化合物的合成技术领域。本发明的技术方案要点为:具有抗菌活性的柱[5]芳烃类人工跨膜通道,其结构通式为:
Description
技术领域
本发明属于具有抗菌活性化合物的合成技术领域,具体涉及一种具有抗菌活性的柱[5]芳烃类人工跨膜通道及其制备方法和应用。
背景技术
由细菌、病毒、真菌等引起的病原菌感染性疾病对人类及动物的健康造成严重的威胁。目前为止,传统的抗生素仍是治疗这类感染性疾病的首选方法。然而,随着抗生素的使用,人们逐渐发现抗生素易残留、产生过敏反应和污染环境等问题。特别是近些年来,多耐药性病原菌的迅速增加成为人类社会所面临的一个严重威胁,开发新型抗菌剂成为迫在眉睫的任务。
抗菌肽(Antimicrobial peptides,AMPs)是在生物界中广泛存在的一类由生物机体产生的活性肽,它是动物免疫防御***为对抗外源性病原体而生成的一类多肽活性物质,在调节机体免疫功能方面具有重要作用。抗菌肽一般由12-80个氨基酸组成的多肽类物质,对细菌、真菌等具有广谱的抗菌活性,并且具有稳定性强、无免疫原性等特点。与传统抗生素通常作用于生物合成的某一特定步骤不同,目前主流研究认为抗菌肽的杀菌机理主要是通过作用于细菌的细胞膜,破坏其完整性并产生穿孔现象,造成细胞内容物流出胞外而死亡,这一独特的作用机理使细菌很难产生耐药性。虽然抗菌肽具有抗菌活性强、抗菌谱广等优点,但是抗菌肽同样会影响哺乳动物细胞(如:血红细胞等),从而引起溶血毒性。所以进入临床应用的抗菌肽(如:gramicidin A等)主要作为外用药物,用与皮肤表面感染和角膜炎等疾病的治疗。因此,如何发展可全身应用抗菌肽是广大药物化学工作者亟待解决的问题。
人工跨膜通道是一类通过人工合成得到的有机分子,通过合理的设计,这类分子可以在磷脂双分子层上形成贯穿脂双层的纳米孔道,并且可以实现对阴阳离子和极性物质的跨膜输送。由于人工跨膜通道和抗菌肽的作用机制类似,都可以在脂双层上形成跨膜通道。但是,人工跨膜通道要通过在脂双层形成稳定纳米级的孔道才能实现跨膜输送功能,所以其结构往往非常复杂。如何高效的构筑人工跨膜通道是目前的研究难点。人工跨膜通道具有的高稳定性、高输送效率以及结构多样性等特点。所以近年来,关于人工跨膜通道在抗菌、抗癌等生物活性方面的研究引起了人们的关注。目前为止,虽然个别人工跨膜通道表现出类似抗菌肽的抗菌活性,但都面临着同样的问题:(1)抗菌活性不高;(2)溶血毒性难以消除。
综上所述,目前关于人工跨膜通道的制备方法步骤冗长,可以高效制备人工跨膜的方法鲜有报道。并且,同时具有高抗菌活性和低溶血毒性的人工跨膜通道还未见报道。
发明内容
本发明解决的技术问题是提供了一种具有抗菌活性的柱[5]芳烃类人工跨膜通道及其制备方法,该方法制得的柱[5]芳烃类人工跨膜通道能够用于制备治疗或预防因细菌(如枯草芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌)所致病害的药物。
本发明为解决上述技术问题采用如下技术方案,具有抗菌活性的柱[5]芳烃类人工跨膜通道,其特征在于其结构通式为:
其中R为多肽侧链,该多肽侧链的多肽序列为:
Val-Gly-Ala-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH。
本发明所述的具有抗菌活性的柱[5]芳烃类人工跨膜通道的制备方法,其特征在于具体步骤为:将双炔基修饰的柱[5]芳烃类化合物、叠氮修饰的多肽类化合物、催化剂五水硫酸铜和抗坏血酸钠依次加入到反应容器中,再加入DMSO溶解,于室温反应12h,TLC监控原料反应完全后过滤,干燥,柱层析纯化得到目标产物具有抗菌活性的柱[5]芳烃类人工跨膜通道,制备过程中的反应方程式为:
进一步优选,所述双炔基修饰的柱[5]芳烃类化合物、叠氮修饰的多肽类化合物、催化剂五水硫酸铜与抗坏血酸钠的投料摩尔比为8:25:0.8:4。
本发明所述的具有抗菌活性的柱[5]芳烃类人工跨膜通道在制备治疗或预防因细菌所致的病害药物中的应用,其中细菌为枯草芽孢杆菌、金黄色葡萄球菌或表皮葡萄球菌。
本发明通过“Click Chemistry”对柱[5]芳烃进行化学修饰,将具有β螺旋构象的多肽侧链引入到柱[5]芳烃骨架中,高效构筑了一类具有管状结构的单分子人工跨膜通道,该类化合物可以高效的嵌入到细菌细胞膜中,改变细胞膜的通透性,从而抑制细菌生长;该类化合物具有优异的抗菌性能,能够较好地应用于因细菌(如枯草芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌等)所致的动植物病害的防治,并且该类化合物的溶血毒性很低,可作为全身应用的离子通道类抗生素的候选药物。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
(1)柱[4]芳烃[1]醌的制备
将柱[5]芳烃(3g,4mmol)溶于二氯甲烷(50mL)中,加入硝酸铈铵(2.19g,4mmol),室温搅拌30min,加水萃取,然后用无水Na2SO4干燥,过滤,滤液旋干。柱层析分离(乙酸乙酯:石油醚)后得到固体1.44g,收率为50%。
1H NMR(DMSO-d6,600MHz)δ:8.22(s,2H),6.84(s,2H),6.83(s,2H),6.79(s,2H),6.78(s,2H),6.54(s,2H),3.71(s,6H),3.68(s,6H),3.65(s,12H),3.63(s,6H),3.55(s,4H).HRMS:Calcd for C43H44NaO10[M+Na]+:743.2832.Found:743.2867。
(2)二羟基柱[5]芳烃的制备
将柱[4]芳烃[1]醌(0.5g,0.693mmol)、硼氢化钠(0.13g,3.448mmol)溶于四氢呋喃中,室温搅拌。待反应完全,用乙酸乙酯萃取,有机相用饱和氯化钠溶液洗,加入无水Na2SO4干燥,过滤,滤液旋干。柱层析分离得到0.27g浅黄色固体,收率为54%。
1H NMR(DMSO-d6,600MHz)δ:8.23(s,2H),6.85(s,2H),6.84(s,2H),6.80(s,4H),3.72(s,6H),3.69(s,6H),3.66(s,12H),3.64(s,2H),3.56(s,2H).ESI-HRMS forC43H46NaO10[M+Na]+calcd:745.2989,found:745.2997。
(3)二炔基柱[5]芳烃的制备
称取化合物2-19(0.1g,0.139mmol)和碳酸钾(0.3g,0.694mmol)于烧瓶中,加入乙腈(2.5mL)、溴丙炔(0.12mL,1.108mmol),反应2h。待反应完全,抽滤,滤液旋干。柱层析分离(乙酸乙酯:石油醚)得到0.06g红色固体化合物2-20,收率为52%。
1H NMR(DMSO-d6,600MHz)δ:6.87(s,2H),6.82(s,2H),6.79(s,4H),6.78(s,2H),4.66(s,2H),3.70-3.66(m,34H),3.31(s,2H).ESI-HRMS for C49H50O10[M+Na]+calcd:821.3302,found:821.3261。
实施例2
双八肽侧链修饰的柱[5]芳烃的制备
将叠氮修饰的八肽(48mg,0.05mmol,3equiv)溶于DMSO(3mL)中,加入二炔基柱[5]芳烃(13mg,0.016mmol),加入抗坏血酸钠(1.5mg,0.008mmol,0.5equiv)和CuSO4·5H2O(0.4mg,1.6μmol,0.1equiv)。搅拌12h后,旋干。所得粗产物经HPLC分离提纯,最终得到30mg白色固体,收率为67%。
1H NMR(DMSO-d6,600MHz)δ:12.55(s,2H),10.76(s,4H),8.87-7.96(m,20H),7.61(t,J=6.0Hz,4H),7.30(t,J=6.0Hz,4H),7.13-6.92(m,14H),6.78(d,J=6.0Hz,4H),6.75(d,J=6.0Hz,4H),5.29-4.97(m,9H),4.55-3.79(m,22H),3.51(d,J=6.0Hz,8H),3.20-2.87(m,11H),2.02-1.97(m,3H),1.47-1.44(m,5H),1.24-1.06(m,27H),0.88(m,12H),0.69-0.59(m,25H).ESI-HRMS for C145H180N26O30[M+2H]2+calcd:1384.1769,found:1384.1838。
实施例3
双十肽侧链修饰的柱[5]芳烃的制备
将叠氮修饰的十肽(64mg,0.05mmol,3equiv)溶于DMSO(3mL)中,加入二炔基柱[5]芳烃(13mg,0.016mmol),加入抗坏血酸钠(1.5mg,0.008mmol,0.5equiv)和CuSO4·5H2O(0.4mg,1.6μmol,0.1equiv)。搅拌12h后,旋干。所得粗产物经HPLC分离提纯,最终得到32mg白色固体,收率为59%。
1H NMR(DMSO-d6,600MHz)δ:10.76(s,2H),10.72(s,2H),10.71(s,2H),8.48(d,2H),8.38(t,2H),8.34-8.31(m,4H),8.18(s,2H),8.15(d,4H),8.04(d,2H),7.99(d,2H),7.92(d,2H),7.83(d,2H),7.59(q,6H),7.30-7.27(m,6H),7.12(s,2H),7.08(s,4H),7.02-6.99(m,8H),6.96-6.91(m,6H),6.78(d,4H),6.75(d,4H),5.32(t,2H),5.28-5.19(m,4H),5.02-4.96(m,4H),4.60-4.52(m,6H),4.40-4.35(m,2H),4.25-4.15(m,8H),3.80(d,4H),3.66-3.65(m,24H),3.51(d,6H),3.21-3.18(m,4H),3.09-3.07(m,4H),2.93-2.86(m,6H),2.02-1.96(m,6H),1.82-1.77(m,2H),1.48-1.43(m,2H),1.30-1.28(m,4H),1.16(d,6H),1.08-1.04(m,6H),1.01-0.97(m,2H),0.93-0.90(m,2H),0.87(d,12H),0.63(d,6H),0.58-0.56(m,16H),0.53(d,10H).HRMS:calcd for C177H220N32O34[M+2H]2+1669.3252,found1669.3451。
实施例4
双十二肽侧链修饰的柱[5]芳烃的制备
将叠氮修饰的十二肽(74mg,0.05mmol,3equiv)溶于DMSO(3mL)中,加入二炔基柱[5]芳烃(13mg,0.016mmol),加入抗坏血酸钠(1.5mg,0.008mmol,0.5equiv)和CuSO4·5H2O(0.4mg,1.6μmol,0.1equiv)。搅拌12h后,旋干。所得粗产物经HPLC分离提纯,最终得到41mg白色固体,收率为68%。
1H NMR(DMSO-d6,600MHz)δ:10.79(s,2H),10.76(s,2H),10.71(s,2H),8.51(d,2H),8.42(t,2H),8.36-8.33(m,4H),8.20(br,6H),8.07-8.06(m,2H),8.00-7.89(m,8H),7.81(d,2H),7.59(d,4H),7.55(d,2H),7.30-7.26(m,6H),7.12(s,2H),7.08(s,4H),7.04-6.99(m,8H),6.96-6.90(m,6H),6.78(d,4H),6.75(d,4H),5.33-5.21(m,6H),5.02-4.97(m,4H),4.59-4.52(m,6H),4.41-4.39(m,2H),4.32-4.30(m,4H),4.23(br,4H),4.18-4.15(m,4H),3.80(d,4H),3.67-3.66(m,24H),3.52(d,6H),3.19(d,2H),3.10-3.08(m,4H),2.92-2.86(m,6H),2.03-1.98(m,8H),1.80-1.75(m,2H),1.29-1.27(m,2H),1.17(d,6H),1.10-1.04(m,10H),0.98-0.94(m,2H),0.88(br,12H),0.85-0.83(m,4H),0.80-0.78(m,24H),0.62(d,6H),0.57-0.53(m,22H),0.50(d,6H).HRMS:calcd for C197H256N36O38[M+2H]2+1867.9637,found 1867.9733。
实施例5
双十四肽侧链修饰的柱[5]芳烃的制备
将叠氮修饰的十四肽(83mg,0.05mmol,3equiv)溶于DMSO(3mL)中,加入二炔基柱[5]芳烃(13mg,0.016mmol),加入抗坏血酸钠(1.5mg,0.008mmol,0.5equiv)和CuSO4·5H2O(0.4mg,1.6μmol,0.1equiv)。搅拌12h后,旋干。所得粗产物经HPLC分离提纯,最终得到38mg白色固体,收率为57%。
1H NMR(DMSO-d6,600MHz)δ:12.56(br,2H),10.76(br,4H),10.68(s,2H),8.51(d,2H),8.37(br,4H),8.32(br,2H),8.19(s,2H),8.18-8.15(m,4H),8.04(br,2H),7.95-7.91(m,8H),7.81(br,2H),7.73(br,2H),7.58(d,4H),7.54(d,2H),7.30-7.26(m,6H),7.11(s,2H),7.08(s,4H),7.04-6.99(m,8H),6.96-6.89(m,6H),6.78(d,4H),6.75(d,4H),5.33-5.21(m,4H),5.02-4.97(m,4H),4.55-4.53(m,6H),4.31-4.17(m,18H),3.79-3.75(m,6H),3.70-3.64(m,30H),3.52(d,6H),3.21-3.16(m,2H),3.09(br,4H),2.90-2.88(m,6H),2.03-1.94(m,8H),1.81(br,2H),1.57-1.52(m,2H),1.46-1.44(m,4H),1.22(s,4H),1.20(d,6H),1.16(d,6H),1.09(br,8H),0.96(br,2H),0.89(d,12H),0.84(d,8H),0.81-0.77(m,28H),0.63(d,6H),0.57-0.52(m,28H).HRMS:calcd for C215H288N40O42[M+2H]2+2052.0849,found2052.1042。
实施例6
双十六肽侧链修饰的柱[5]芳烃的制备
将叠氮修饰的十六肽(97mg,0.05mmol,3equiv)溶于DMSO(3mL)中,加入二炔基柱[5]芳烃(13mg,0.016mmol),加入抗坏血酸钠(1.5mg,0.008mmol,0.5equiv)和CuSO4·5H2O(0.4mg,1.6μmol,0.1equiv)。搅拌12h后,旋干。所得粗产物经HPLC分离提纯,最终得到46mg白色固体,收率为61%。
1H NMR(DMSO-d6,600MHz)δ:12.55(br,2H),10.76(br,6H),10.69(s,2H),8.51(d,2H),8.37(br,6H),8.19-8.14(m,10H),7.99-7.93(m,12H),7.77-7.74(m,4H),7.58-7.52(m,8H),7.29-7.27(m,8H),7.11(s,2H),7.08(s,6H),7.03-7.00(m,10H),6.95-6.89(m,8H),6.78(br,4H),6.75(d,4H),5.33-5.21(m,4H),5.03-4.97(m,4H),4.2(br,8H),4.30-4.15(m,20H),3.79-3.75(m,6H),3.70-3.64(m,30H),3.52(d,6H),3.25-3.21(m,2H),3.17-3.12(m,6H),2.90(br,8H),2.03-1.97(m,6H),1.81-1.77(m,2H),1.56-1.52(m,2H),1.46(br,4H),1.22-1.12(m,28H),0.89-0.77(m,52H),0.63-0.51(m,46H).HRMS:calcd forC249H329KN46O46[M+H+K]2+2370.2262,found 2370.2216。
实施例7
对细菌的抑菌活性测试
(1)液体培养基的配制
用电子天平分别称取0.2515g酵母提取液、0.5020g胰化蛋白胨和0.5030g NaCl于玻璃瓶中,加入二次蒸馏水50mL至完全溶解,用NaOH调节pH=7.51。用装有500μL枪头的移液抢分别移取10mL培养液到试管中。
(2)菌种的活化及菌液的制备
在试验前两日,将四种供试菌分别接种于供试斜面上,置28℃恒温培养48h,每支斜面用0.85wt%的灭菌生理盐水2mL将菌苔洗下,摇匀备用。
(3)铺板
取一96孔板,用移液枪取适量液体培养基加入其中,轻轻摇晃使混合均匀,加入200μL菌液,分别加入2.5μL的纯DMSO和不同浓度的样品液。
(4)测量方法
将准备好的96孔板于恒温振荡器中(37℃)震荡12h后,用酶标仪进行吸光度测定。每次设置三个重复,同样条件做三次。
细菌存活率(%)=(ODsample+bac-ODbroth only)/(ODDMSO+bac-ODbroth only)×100%。
实施例8
对血红细胞的溶血毒性测试
(1)血红细胞悬浮液的制备
取新鲜的SD大鼠血液,通过在3500转离心5min分离大鼠血红细胞。分离出的血红细胞用PBS缓冲液洗至上清液澄清,然后将其重新分散到PBS缓冲液中(1%,v/v)备用。
(2)铺板
取一96孔板,在每个孔中加入200μL血红细胞悬浮液,随后加入一式三份2.5μL样品分子的DMSO溶液或纯DMSO。轻微晃动该96孔板,并在37℃条件下培养30min。
(3)测量方法
将上述96孔板在3500转离心10min,在每个孔中取等量(50μL)的上清液加入到新的96孔板中,随后在用PBS缓冲液稀释到100μL。随后使用酶标仪在562nm下对该96孔板进行测量。
溶血率(%)=(Asample-ADMSO)/(Atriton X-100-ADMSO)×100%。
表1典型化合物的编号、化学结构、产率
表2为典型化合物对具有代表性细菌的抑菌活性测试结果,表2中化合物编号1-5与表1中的相对应。
表2典型化合物对具有代表性细菌的抑菌活性
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
SEQUENCE LISTING
<110> 河南师范大学
<120> 具有抗菌活性的柱[5]芳烃类人工跨膜通道及其制备方法和应用
<130> 2017
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 1
Val Gly Ala Leu Trp Leu Trp Gly
<210> 2
<211> 10
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 1
Val Gly Ala Val Trp Leu Trp Leu Trp Gly
<210> 3
<211> 12
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 1
Val Gly Ala Val Val Val Trp Leu Trp Leu Trp Gly
<210> 4
<211> 14
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 1
Val Gly Ala Leu Ala Val Val Val Trp Leu Trp Leu Trp Gly
<210> 5
<211> 16
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 1
Val Gly Ala Leu Ala Val Val Val Trp Leu Trp Leu Trp Leu Trp Gly
Claims (4)
1.具有抗菌活性的柱[5]芳烃类人工跨膜通道,其特征在于其结构通式为:
其中R为多肽侧链,该多肽侧链的多肽序列为:
Val-Gly-Ala-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH;
Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-D-Leu-Trp-Gly-COOH。
3.根据权利要求2所述的具有抗菌活性的柱[5]芳烃类人工跨膜通道的制备方法,其特征在于:所述双炔基修饰的柱[5]芳烃类化合物、叠氮修饰的多肽类化合物、催化剂五水硫酸铜与抗坏血酸钠的投料摩尔比为8:25:0.8:4。
4.权利要求1所述的具有抗菌活性的柱[5]芳烃类人工跨膜通道在制备治疗或预防因细菌所致的病害药物中的应用,所述细菌为枯草芽孢杆菌、金黄色葡萄球菌或表皮葡萄球菌。
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