CN114539083B - 脂质纳米粒及其在核酸递送中的应用 - Google Patents

脂质纳米粒及其在核酸递送中的应用 Download PDF

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CN114539083B
CN114539083B CN202210094484.5A CN202210094484A CN114539083B CN 114539083 B CN114539083 B CN 114539083B CN 202210094484 A CN202210094484 A CN 202210094484A CN 114539083 B CN114539083 B CN 114539083B
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lipid
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nucleic acid
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CN114539083A (zh
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张灿
鞠曹云
黄滢霜
陶雨
薛玲静
龙秋林
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China Pharmaceutical University
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Abstract

本发明公开了脂质纳米粒及其在核酸递送中的应用。本发明的功能脂质以叔胺或PEG片段作为亲水部分，以氨基酸作为骨架，以酯键作为连接键，以烷烃链作为疏水部分，具有生物相容性好、安全性高、合成方法简便快捷等优势。基于本发明设计的功能脂质制备的脂质纳米粒能稳定荷载核酸药物，且在原代细胞和肿瘤细胞上转染效果显著。本发明将提供一类能够有效转染细胞的新型脂质材料，并为细胞转染提供安全有效的非病毒载体平台。

Description

脂质纳米粒及其在核酸递送中的应用
技术领域
本发明涉及化学与制剂领域，具体涉及脂质纳米粒及其在核酸递送中的应用。
背景技术
核酸药物是指各种具有不同功能的核糖核酸(RNA)或脱氧核糖核酸(DNA)，能够直接作用于致病靶基因或者靶mRNA，在基因水平上发挥治疗疾病的作用。由于核酸药物具有负电性强、亲水性强、易被降解等特性，无法直接穿透生物膜屏障，不能被细胞有效摄取并到达靶细胞器发挥作用。因此，如何安全高效地将核酸药物递送至细胞内并成功发挥作用，成为核酸药物进一步发展的核心问题。
目前研究最广泛的核酸递送载体包括病毒载体和非病毒载体。与病毒载体相比，非病毒载体具有安全性高，荷载量大，并且合成制备简单等优点。其中，脂质纳米粒(Lipidnanoparticle,LNP)是目前发展最为迅速的非病毒递送载体。2018年，FDA批准的基因疗法Onpattro，使LNP成为FDA批准的首个用于递送核酸药物siRNA的非病毒载体。2020年新型冠状病毒肺炎在世界范围内的爆发加速推进了核酸药物的研发，其中BNT162b2 mRNA疫苗(Pfizer-BioNTech)与mRNA-1273疫苗(Moderna)均采用LNP作为mRNA递送载体，进一步验证了LNP的巨大潜力。LNP的处方主要包括四种主要成分：可离子化脂质、聚乙二醇化脂质、中性磷脂、胆固醇。可离子化脂质是促进基因药物溶酶体逃逸的关键成分，其结构中的叔胺头基在酸性环境下可被质子化带正电，通过静电作用复合核酸；在生理pH下几乎不带电，能够减少对细胞的损伤；在溶酶体酸性环境中恢复带正电，帮助核酸药物进行溶酶体逃逸。聚乙二醇化脂质也会影响LNP的递送效率，在LNP处方中加入聚乙二醇化脂质能够防止颗粒聚集，控制LNP的粒径，增加体系的稳定性。聚乙二醇化脂质的亲水端容易修饰各种靶向基团，合成路线简单且产率高，为制备靶向性LNP提供了一种相对简单的方法。
RNA药物被内吞入胞后经历内涵体逃逸至胞质从而发挥作用，而质粒DNA(PlasmidDNA，pDNA)需从内涵体/溶酶体逃逸并转运至细胞核才能发挥作用，内涵体/溶酶体逃逸和pDNA入核率是决定LNP转染效果的关键。LNP通过在酸性环境中带正电的可离子化脂质与内涵体/溶酶体膜上负电性磷脂相互作用而破坏膜稳定性，使荷载的pDNA逃逸进入胞质。然而pDNA在黏稠的细胞质中几乎无法移动，成为限制LNP转染能力的主要原因之一。细胞可以利用动力蛋白将内涵体沿微管从细胞膜附近运输至靠近细胞核的溶酶体中，若能利用细胞自身的转运功能，设计具有适宜pKa的可离子化脂质，使LNP在内涵体(pH 5.0～6.0)中保持结构的完整性，仅在溶酶体(pH 4.5～5.0)中快速逃逸并释放pDNA，可以提高核周pDNA浓度，这可能是增加pDNA入核表达的有效策略。此外，一些活化的免疫细胞或肿瘤细胞为了满足快速生长增殖的需要，会显著提高细胞膜上葡萄糖转运体1的表达水平以增大葡萄糖摄取量。在LNP处方中引入葡萄糖转运体1靶向脂质具有增加细胞摄取的可能性。
发明内容
本发明的目的是针对现有技术的上述不足，提供一种功能脂质。
本发明的另一目的是提供该功能脂质的应用。
本发明的目的可通过以下技术方案实现：
通式(I)所示的功能脂质：
其中，
n＝1或2；
m代表0-5的整数；
其中X＝CH2或NH；
R2选自中的任意一种，其中u代表1-17的整数，优选5-10的整数；
p代表1-4的整数；q代表1-3的整数；r代表1-2的整数；s＝10、23、45、78或113，t代表0-5的整数；R5代表甲基、乙基、羟甲基、羟乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、苯基或苄基；R6代表甲基或葡萄糖胺甲酰基。
作为本发明的一种优选，当所述的通式(I)所示的功能脂质为可离子化脂质时，
n＝2；m代表1-3的整数；
R2选自中的任意一种；
p代表1-4的整数；q代表1-3的整数；r代表1-2的整数；R5代表甲基、乙基、羟乙基。
在可离子化脂质的设计中，选取具有适宜pKa的叔胺作为亲水头基，酯键作为连接键，直链烷烃作为疏水尾链。其中，叔胺的pKa多为8～9，既能在酸性缓冲液中被质子化而带有较强的正电性，又能在生理pH 7.4下去质子化而表现为微弱的正电性；当内吞入胞后，叔胺头基在内涵体/溶酶体(pH 4.5～6.0)中恢复质子化而带有正电荷，可与膜上阴离子磷脂静电结合直接破坏膜的稳定性，或利用Lα-HⅡ的相转变发生膜融合作用，实现溶酶体逃逸。此外，研究表明头基较小且尾链较大的脂质易于自组装形成负曲率的HⅡ结构并促进膜融合，因此选取具有适宜pKa和较小尺寸的叔胺基团作为亲水头基。为了提高LNP的生物相容性，选用氨基酸和酯键分别作为骨架和连接键。选用直链烷烃作为脂质材料的疏水尾链，利用其与细胞膜的高度亲和力，有利于促进阳离子脂质体跨过细胞膜。增加烷烃的不饱和度不仅可以降低LNP的相转变温度，还能增大脂质疏水区的有效截面面积并提高纳米粒的负曲率，从而增强LNP的膜融合能力。
作为本发明的一种优选，当所述的通式(I)所示的功能脂质为聚乙二醇化脂质时，n＝2；m代表0-3的整数；X＝CH2；R2选自/>中的任意一种，其中u代表10-17的整数，优选14；
s＝10、23、45、78或113，优选45；t代表0-5的整数，优选1；
R6代表甲基。
作为本发明的一种优选，当所述的通式(I)所示的功能脂质为葡萄糖转运体1靶向脂质时，n＝2；m代表1-4的整数；X＝NH；R2选自/>中的任意一种，其中u代表10-17的整数，优选14；
s＝10、23、45、78或113，优选45；t代表0-5的整数，优选4；R6代表葡萄糖胺甲酰基。
在聚乙二醇化脂质和葡萄糖转运体1靶向脂质的设计中，选择PEG片段或含有PEG的氨基葡萄糖分别作为亲水头基，赋予脂质材料良好的两亲性或葡萄糖转运体1的靶向性。
本发明提供一种上述脂质衍生物的合成方法。该合成方法高效快捷，通用性好，收率高，合成成本低，合成过程环境友好，适宜工业化放大生产。
作为本发明的一种优选，当所述的通式(I)所示的功能脂质为可离子化脂质或聚乙二醇化脂质时，其合成步骤如下：
a.将二元羧基氨基酸(I-1)和对甲苯磺酸溶解于无水甲苯(或苯，或环己烷)，升温至110～150℃，回流反应1～3h。停止加热后，将混合溶液冷却至室温，分批加入脂肪醇，升温至100～150℃，回流反应6～20h。反应结束后，旋蒸除去甲苯(或苯，或环己烷)。将粗产物溶于二氯甲烷(或氯仿，或乙酸乙酯)，用适量水洗涤两次，饱和碳酸氢钠水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，经石油醚/乙酸乙酯柱层析纯化，得到脂肪醇-二元羧基氨基酸(I-2)。
脂肪醇-二元羧基氨基酸(I-2)合成反应式：
b.将脂肪醇-二元羧基氨基酸(I-2)溶解于二氯甲烷(或四氢呋喃，或氯仿)，在搅拌状态下向反应液中先后加入三乙胺(或4-二甲氨基吡啶，或N,N-二异丙基乙胺)和二元酸酐，室温反应6～20h。反应结束后，旋蒸除去溶剂，将粗产物经二氯甲烷/甲醇柱层析纯化，得到羧基化-脂肪醇-二元羧基氨基酸(I-3)。
羧基化-脂肪醇-二元羧基氨基酸(I-3)合成反应式：
c.将羧基化-脂肪醇-二元羧基氨基酸(I-3)或羧基化聚乙二醇溶解于氯仿(或四氢呋喃，或二氯甲烷)，在0℃下依次加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCl)和1-羟基苯并三唑(HOBt)并搅拌5～10min，随后将混合溶液移至室温反应1～3h，得到反应液A；将R3NH2或R4OH溶解于氯仿(或四氢呋喃，或二氯甲烷)，在室温下加入三乙胺，搅拌反应1～3h，得到反应液B。将反应液B缓慢滴加进反应液A，室温搅拌6～20h。反应结束后，反应液用适量水洗涤两次，10％柠檬酸水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，粗产物经二氯甲烷/甲醇柱层析或用去离子水透析(MWCO2000Da)纯化，得到叔胺-羧基化-脂肪醇-二元羧基氨基酸或羧基化聚乙二醇-脂肪醇-二元羧基氨基酸(I)。
叔胺-羧基化-脂肪醇-二元羧基氨基酸或羧基化聚乙二醇-脂肪醇-二元羧基氨基酸(I)合成反应式：
作为本发明的一种优选，当所述的通式(I)所示的功能脂质为葡萄糖靶头脂质时，其合成步骤如下：
a.将N-叔丁氧羰基氨基单元酸溶解于二氯甲烷(或四氢呋喃，或氯仿)，在0℃下依次加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)并搅拌5～10min，随后将混合溶液移至室温反应1～3h，得到反应液A；将脂肪醇-二元羧基氨基酸(I-2)溶解于二氯甲烷(或四氢呋喃，或氯仿)，在室温下加入三乙胺，搅拌反应1～3h，得到反应液B。将反应液B缓慢滴加进反应液A，室温搅拌6～20h。反应结束后，反应液用适量水洗涤两次，10％柠檬酸水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，粗产物经石油醚/乙酸乙酯柱层析纯化，得到脂肪醇-N-叔丁氧羰基氨基单元酸(I-4)。
脂肪醇-N-叔丁氧羰基氨基单元酸(I-4)合成反应式：
b.在0℃下向脂肪醇-N-叔丁氧羰基氨基单元酸中缓慢滴加氯化氢-二氧六环溶液，搅拌反应6～20h。反应结束后，旋蒸除去溶液，得到脂肪醇-氨基盐酸盐(I-5)。
脂肪醇-氨基盐酸盐(I-5)合成反应式：
c.将脂肪醇-氨基盐酸盐(I-5)溶解于二氯甲烷(或四氢呋喃，或氯仿)，滴加三乙胺，室温搅拌反应0.5～1h。向反应液中加入NHS-PEGs-NHS，室温反应24～48h。反应结束后，旋蒸除去溶剂，得到活性酯-聚乙二醇-脂肪醇(I-6)。
活性酯-聚乙二醇-脂肪醇(I-6)合成反应式：
d.将D-氨基葡萄糖盐酸盐溶解于N,N-二甲基甲酰胺(DMF)，滴加N,N-二异丙基乙胺(DIPEA)，室温活化1～3h。将活性酯-聚乙二醇-脂肪醇(I-6)溶解于N,N-二甲基甲酰胺(DMF)，加入活化后的D-氨基葡萄糖溶液中，室温搅拌6～20h。反应结束后，反应液用去离子水透析后冻干，得到氨基葡萄糖-聚乙二醇-脂肪醇(I)。
氨基葡萄糖-聚乙二醇-脂肪醇(I)合成反应式：
本发明所述的功能脂质在制备荷载核酸药物的脂质纳米粒中的应用。
一种荷载核酸药物的脂质纳米粒，包含第一脂质、第二脂质、胆固醇以及第三脂质；所述的第一脂质选自权利要求2中所述的可离子化脂质，所述的第二脂质选自中性磷脂，第三脂质选自权利要求3中所述的聚乙二醇化脂质或权利要求4中所述的葡萄糖转运体1靶向脂质中的任一种或两种，其中第一脂质：第二脂质：胆固醇：第三脂质的摩尔比为25～60:10～30:30～60:0.5～10，优选摩尔比为30～50:10～20:40～60:0.5～2.5。本发明脂质纳米粒具有30～220nm的平均粒径，+5～+40mV的表面电位。
作为本发明的一种优选，其中中性磷脂为1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)、大豆磷脂(SPC)、1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺(DSPE)、1-硬脂酰-2-油酰-sn-甘油-3-磷酸胆碱(SOPC)、1-棕榈酰基-2-油酰基卵磷脂(POPC)、二芥酰基卵磷脂(DEPC)、1,2-二油酰-sn-甘油-3-磷酸胆碱(DOPC)或二棕榈酰基卵磷脂(DPPC)中的一种或多种，优选DOPE。
作为本发明的一种优选，所述的核酸药物选自pDNA、siRNA、shRNA、microRNA和mRNA中的任意一种，优选pDNA。
作为本发明的一种优选，所述的荷载核酸药物的脂质纳米粒中可离子化脂质和核酸药物的氮磷比为1:1～30:1，优选3:1～8:1。
本发明所述的荷载核酸药物的脂质纳米粒在pDNA转染、siRNA基因沉默、mRNA疫苗或CRISPR/Cas9基因编辑中的应用。
荷载核酸药物的脂质纳米粒用于原代免疫细胞或肿瘤细胞，其中免疫细胞包括T细胞、中性粒细胞、巨噬细胞、树突细胞等，肿瘤细胞包括乳腺癌MCF-7细胞、胰腺癌PANC-1细胞、肝癌HepG2细胞、肺癌A549细胞等。
本发明公开的脂质纳米粒制备方法包括：乙醇注入法，微流控法，T型管混合法，过膜挤压法。脂质纳米粒制备方法优选乙醇注入法和微流控法。
采用乙醇注入法制备脂质纳米粒的过程如下：称取适量可离子化脂质、中性磷脂、胆固醇和聚乙二醇化脂质或葡萄糖转运体1靶向脂质，溶解于无水乙醇。同时，将核酸药物溶解于10mM柠檬酸盐缓冲液(pH＝4.0)中。在剧烈搅拌状态下，将乙醇相快速注入水相，水相和乙醇相的体积比为1:1～5:1。注入完成后，在室温下对超纯水透析2～8h即可得到脂质纳米粒溶液，4℃保存备用。
采用微流控法制备脂质纳米粒的过程下：称取适量可离子化脂质、中性磷脂、胆固醇和聚乙二醇化脂质或葡萄糖转运体1靶向脂质，溶解于无水乙醇，作为乙醇相。将核酸药物溶解于10mM柠檬酸盐缓冲液(pH＝4.0)中，作为水相。两相以0.02～6mL/min的流速和1:1～5:1的比例同时通过微流控设备混合，在室温下对超纯水透析2～8h即可得到脂质纳米粒溶液，4℃保存备用。
本发明以改善溶酶体逃逸能力和提高核周pDNA浓度、增强载体稳定性、提高细胞摄取率为目的，设计不同的功能脂质，包括：一系列含有不同叔胺头基、不同连接链长度和不同尾链的可离子化脂质，用于改善溶酶体逃逸能力并提高核周pDNA分布；聚乙二醇化脂质——用于增强LNP稳定性；葡萄糖转运体1靶向脂质——用于提高LNP的入胞效率，进而改善LNP对细胞的转染能力。
有益效果：
本发明公开的脂质纳米粒粒径均一，电位为正(见实施例36)，能稳定荷载siRNA(见实施例37)、mRNA(见实施例38)和pDNA(见实施例39)，且在体外有良好的稳定性(见实施例40)。本发明的脂质纳米粒能有效转染人原代T细胞(见实施例41)和肿瘤细胞(见实施例42)。本发明所有脂质纳米粒在正常生理条件(pH 7.4)和内涵体环境(pH 5.0)中的膜融合能力都较低，而在溶酶体环境(pH 4.5)中的融合能力显著增强，表明本发明的脂质纳米粒在生理环境中具有良好的安全性，并且在晚期内涵体(pH 5.0)中可保持结构的完整性，仅在溶酶体(pH 4.5)中可通过膜融合作用逃逸进入胞质(见实施例43)。
本发明的创新之处为从头基结构、连接链长度、尾链不饱和度三方面入手，以改善溶酶体逃逸能力和提高核周pDNA浓度、增强载体稳定性、提高细胞摄取率为目的，设计合成了一系列新型可离子化脂质、聚乙二醇化脂质以及葡萄糖转运体1靶向脂质，为核酸递送提供转染效率高、安全性好且合成简便的脂质材料。
附图说明
图1是本发明的脂质纳米粒LA1NP～LA9NP和OA1NP～OA9NP荷载pDNA后在不同氮磷比下的粒径和电位；
图2是本发明的脂质纳米粒LA11NP按不同氮磷比荷载siRNA后的琼脂糖凝胶电泳图；
图3是本发明的脂质纳米粒LA11NP按不同氮磷比荷载mRNA后的琼脂糖凝胶电泳图；
图4是本发明的脂质纳米粒LA1NP～LA9NP和OA1NP～OA9NP按不同氮磷比荷载pDNA后的琼脂糖凝胶电泳图；
图5是本发明的脂质纳米粒LA1NP～LA9NP和OA1NP～OA9NP的体外稳定性；
图6是本发明的靶向脂质纳米粒荷载pDNA转染人源T细胞后的绿色荧光蛋白(GFP)表达情况(倒置荧光显微镜，比例尺：100μm)；
图7是本发明的靶向脂质纳米粒荷载pDNA转染人源T细胞后的绿色荧光蛋白(GFP)表达情况(流式细胞仪)；
图8是本发明的脂质纳米粒荷载pDNA转染人乳腺癌MCF-7细胞后的绿色荧光蛋白(GFP)表达情况(倒置荧光显微镜，比例尺：100μm)；
图9是本发明的脂质纳米粒荷载pDNA转染人乳腺癌MCF-7细胞后的绿色荧光蛋白(GFP)表达情况(流式细胞仪)；
图10是本发明的脂质纳米粒LA1NP～LA11NP和OA1NP～OA11NP的膜融合实验结果；
图11是本发明的脂质纳米粒LA1NP～LA11NP和OA1NP～OA11NP的溶血实验结果。
具体实施方式
通过以下实施例对本发明进一步解释，但这些实施例不对本发明构成任何限制。
实施例1
制备谷氨酸双亚麻醇酯(LA2-NH2)，化学结构式如下：
向250mL反应瓶中加入L-谷氨酸(3.00g，20.60mmol)、对甲苯磺酸(4.30g，22.66mmol)和无水甲苯(150mL)，随后将反应液升温至140℃，回流反应3h。停止加热后，将混合溶液冷却至室温，分批加入亚麻醇(11.53g，43.25mmol)，140℃回流反应过夜。反应结束后，旋蒸除去甲苯，得到深棕色油状物。将粗产物溶于二氯甲烷，用适量水洗涤两次，饱和碳酸氢钠水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到深黄色油状物。经柱层析纯化(石油醚︰乙酸乙酯＝6︰1)，得到淡黄色油状物8.23g，收率：62.0％。1H NMR(300MHz,CDCl3):δ(ppm)5.42-5.28(m,8H,CHCH),4.10(t,J＝6.7Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.52-3.46(m,1H,NH2CH),2.76(t,J＝6.6Hz,4H,CHCH2CH),2.38(dd,J＝16.3,8.4Hz,2H,CHCH2CH2,2H,OCOCH2),2.04(m,8H,CHCHCH2),1.61(dd,J＝12.7,6.4Hz,4H,COOCH2CH2),1.35-1.25(m,32H,CH2(linoleoyl)),0.88(t,J＝6.9Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd for C41H74NO4[M+H]+,644.5612；found,644.5608.
实施例2
制备羧基化谷氨酸双亚麻醇酯(LA2-COOH)，化学结构式如下：
将LA2-NH2(1.21g，1.88mmol)溶解于二氯甲烷，在搅拌状态下向其中先后加入三乙胺(1.04mL，7.51mmol)和丁二酸酐(188mg，1.88mmol)，室温反应过夜。反应结束后，旋蒸除去溶剂，得到淡黄色油状粗品，经柱层析纯化(二氯甲烷︰甲醇＝50︰1)，得到淡黄色油状物1.20g，收率：85.7％。1H NMR(300MHz,CDCl3):δ(ppm)7.53(d,J＝8.7Hz,1H,CONHCH),5.43-5.26(m,8H,CHCH),4.51(d,J＝5.4Hz,1H,CONHCH),4.11(t,J＝6.7Hz,2H,COOCH2),4.05(t,J＝6.8Hz,2H,COOCH2),2.96(s,2H,CH2COOH),2.83(s,2H,NHCOCH2),2.75(t,J＝6.6Hz,4H,CHCH2CH),2.39(dd,J＝16.3,8.4Hz,2H,CHCH2CH2,2H,OCOCH2),2.05(m,8H,CHCHCH2),1.62(dd,J＝12.7,6.4Hz,4H,COOCH2CH2),1.36-1.26(m,32H,CH2(linoleoyl)),0.88(t,J＝6.9Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd for C45H78NO7[M+H]+,744.5773；found,744.5767.
实施例3
制备化合物LA1，化学结构式如下：
将LA2-COOH(500mg，0.67mmol)溶解于二氯甲烷，在0℃下依次加入EDCI(206mg，1.08mmol)、HOBt(145mg，1.08mmol)并搅拌5min，随后将混合溶液移至室温反应3h，得到反应液A；将1-(3-氨基丙基)吡咯烷(85μL，0.67mmol)溶解于二氯甲烷，在室温下加入三乙胺(280μL，2.02mmol)，搅拌反应1h，得到反应液B。将反应液B缓慢滴加进反应液A，室温搅拌过夜。反应结束后，反应液用适量水洗涤两次，10％柠檬酸水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝15︰1)，得到淡黄色油状物254mg，收率：44.3％。1H NMR(500MHz,CDCl3):δ(ppm)7.56-7.42(m,1H,CONHCH),6.95(d,J＝6.4Hz,1H,CONHCH2),5.42-5.28(m,8H,CHCH),4.51(d,J＝5.4Hz,1H,CONHCH),4.10(t,J＝6.7Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.78(s,2H,CONHCH2),3.40(s,2H,NCH2),3.16(s,2H,NHCOCH2),2.83(s,2H,NHCOCH2),2.76(t,J＝6.6Hz,4H,CHCH2CH),2.57(s,4H,NCH2CH2),2.38(dd,J＝16.3,8.4Hz,2H,CHCH2CH2,2H,OCOCH2),2.19(d,J＝19.1Hz,2H,NCH2CH2),2.04(dd,J＝13.9,6.9Hz,8H,CHCHCH2,4H,NCH2CH2),1.61(dd,J＝12.7,6.4Hz,4H,COOCH2CH2),1.35-1.25(m,32H,CH2(linoleoyl)),0.88(t,J＝6.9Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)173.10(1C,NHCOCH2),172.88(1C,NHCOCH2),172.38(1C,COOCH2),171.98(1C,COOCH2),130.20(2C,CHCH),130.02(2C,CHCH),128.03(2C,CHCH),127.89(2C,CHCH),65.73(1C,COOCH2),64.92(1C,COOCH2),53.78(2C,N(CH2CH2)2),52.86(1C,NHCHCH2),51.93(1C,CONHCH2CH2CH2),36.18(1C,CONHCH2CH2CH2),31.49(2C,NHCOCH2),31.40(1C,CH2(linoleoyl)),31.37(1C,CH2(linoleoyl)),31.33(1C,OCOCH2),30.36(1C,CONHCH2CH2),30.19(1C,CH2(linoleoyl)),29.63(2C,CH2(linoleoyl)),29.41(1C,CH2(linoleoyl)),29.31(2C,CH2(linoleoyl)),29.22(2C,CH2(linoleoyl)),29.18(2C,CH2(linoleoyl)),28.58(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.25(1C,NHCHCH2),27.20(2C,CH2CHCH),27.18(2C,CH2CHCH),25.87(1C,COOCH2CH2CH2),25.80(1C,COOCH2CH2CH2),25.62(2C,CHCH2CH),23.27(2C,N(CH2CH2)2),22.53(2C,CH2CH3),14.02(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H92N3O6[M+H]+,854.6981；found,854.6975.
实施例4
制备化合物LA2，化学结构式如下：
标题化合物LA2以化合物LA2-COOH(500mg，0.67mmol)和3-二甲胺基丙胺(85μL，0.67mmol)为原料，按照制备LA1的方法，得到浅黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝15︰1)，得到无色透明油状物264mg，收率：47.4％。1H NMR(500MHz,CDCl3):δ(ppm)7.53(d,J＝8.7Hz,1H,CONHCH),6.86(d,J＝7.6Hz,1H,CONHCH2),5.35(ddd,J＝19.3,11.0,5.3Hz,8H,CHCH),4.52(d,J＝5.3Hz,1H,CONHCH),4.11(t,J＝6.8Hz,2H,COOCH2),4.04(dd,J＝6.9,2.7Hz,2H,COOCH2),3.42(dd,J＝10.1,5.6Hz,2H,CONHCH2),3.14-3.10(m,2H,(CH3)2NCH2),2.81(s,4H,CHCH2CH),2.70-2.48(m,4H,NHCOCH2,2H,OCOCH2),2.44-2.31(m,2H,NHCHCH2),2.13-1.95(m,6H,N(CH3)2,8H,CHCHCH2),1.61(dd,J＝12.7,6.6Hz,4H,COOCH2CH2,2H,NCH2CH2),1.33-1.27(m,32H,CH2(linoleoyl)),0.88(t,J＝6.9Hz,6H,CH2CH3).13CNMR(126MHz,CDCl3):δ(ppm)173.06(1C,NHCOCH2),172.87(1C,NHCOCH2),172.27(1C,COOCH2),171.98(1C,COOCH2),130.19(2C,CHCH),130.01(2C,CHCH),128.03(2C,CHCH),127.89(2C,CHCH),65.71(1C,COOCH2),64.91(1C,COOCH2),55.46(1C,(CH3)2NCH2),51.93(1C,CONHCH),43.02(2C,N(CH3)2),36.01(1C,CONHCH2),31.49(2C,NHCOCH2),31.40(1C,CH2(linoleoyl)),31.32(1C,CH2(linoleoyl)),31.26(1C,OCOCH2),30.34(1C,CH2(linoleoyl)),30.19(1C,CH2(linoleoyl)),29.62(2C,CH2(linoleoyl)),29.40(1C,CH2(linoleoyl)),29.39(1C,CH2(linoleoyl)),29.31(2C,CH2(linoleoyl)),29.22(1C,CH2(linoleoyl)),29.17(1C,CH2(linoleoyl)),28.57(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.23(1C,NHCHCH2),27.19(2C,CH2CHCH),27.17(2C,CH2CHCH),25.87(1C,COOCH2CH2CH2),25.79(1C,COOCH2CH2CH2),25.61(2C,CHCH2CH),24.45(1C,(CH3)2NCH2CH2),22.52(2C,CH2CH3),14.01(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C50H90N3O6[M+H]+,828.6824；found,828.6825.
实施例5
制备化合物LA3，化学结构式如下：
标题化合物LA3以化合物LA2-COOH(500mg，0.67mmol)和1-(3-氨基丙基)哌啶(107μL，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝25︰1)，得到淡黄色油状物467mg，收率：80.0％。1H NMR(500MHz,CDCl3):δ(ppm)7.55(d,J＝11.5Hz,1H,CONHCH),6.98(s,1H,CONHCH2),5.35(dt,J＝12.7,6.6Hz,8H,CHCH),4.52(dd,J＝13.1,7.6Hz,1H,CONHCH),4.10(t,J＝6.8Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.41-3.31(m,2H,CONHCH2),2.95(t,J＝6.9Hz,4H,NCH2CH2,2H,NCH2CH2CH2NH),2.76(t,J＝6.6Hz,4H,CHCH2CH),2.57(t,J＝8.0Hz,4H,NHCOCH2),2.46-2.33(m,2H,OCOCH2),2.21-2.06(m,2H,NHCHCH2),2.04-1.99(m,8H,CHCHCH2),1.97-1.91(m,2H,CONHCH2CH2),1.62-1.57(m,4H,COOCH2CH2),1.29(dd,J＝23.1,14.6Hz,4H,NCH2CH2,2H,NCH2CH2CH2,32H,CH2(linoleoyl)),0.87(d,J＝6.9Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.83(1C,NHCOCH2),172.19(1C,NHCOCH2),171.98(2C,COOCH2),130.20(2C,CHCH),130.04(2C,CHCH),128.03(2C,CHCH),127.90(2C,CHCH),65.67(1C,COOCH2),64.86(1C,COOCH2),53.69(2C,NCH2),53.37(1C,NHCHCH2),51.89(2C,CONHCH2CH2CH2),31.50(2C,NHCOCH2),31.44(2C,CH2(linoleoyl)),31.41(1C,OCOCH2),30.36(1C,CONHCH2CH2),30.14(2C,CH2(linoleoyl)),29.63(2C,CH2(linoleoyl)),29.41(2C,CH2(linoleoyl)),29.32(1C,CH2(linoleoyl)),29.23(2C,CH2(linoleoyl)),29.18(1C,CH2(linoleoyl)),28.59(1C,COOCH2CH2),28.50(1C,COOCH2CH2),27.31(1C,NHCHCH2),27.20(4C,CH2CHCH),25.88(2C,NCH2CH2),25.80(2C,COOCH2CH2CH2),25.62(2C,CHCH2CH),24.01(1C,NCH2CH2CH2),22.54(2C,CH2CH3),14.02(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C53H94N3O6[M+H]+,868.7137；found,868.7135.
实施例6
制备化合物LA4，化学结构式如下：
标题化合物LA4以化合物LA2-COOH(500mg，0.67mmol)和4-甲基-1-哌嗪乙胺(101μL，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到白色凝胶状物456mg，收率：78.1％。1H NMR(500MHz,CDCl3):δ(ppm)7.34(s,1H,CONHCH),6.99(s,1H,CONHCH2),5.34(ddd,J＝15.8,10.5,3.9Hz,8H,CHCH),4.52-4.45(m,1H,CONHCH),4.08(t,J＝6.7Hz,2H,COOCH2),4.03(t,J＝6.8Hz,2H,COOCH2),3.52(s,2H,CONHCH2),3.29(s,8H,CH3NCH2CH2),2.97(s,2H,NCH2CH2NH),2.75(t,J＝6.7Hz,4H,CHCH2CH),2.73(s,2H,NHCOCH2),2.66-2.57(m,2H,NHCOCH2),2.55(s,3H,NCH3),2.39(dt,J＝17.1,8.5Hz,2H,OCOCH2),2.19-2.04(m,2H,NHCHCH2),2.04-1.95(m,8H,CHCHCH2),1.60(dd,J＝12.1,6.5Hz,4H,COOCH2CH2),1.26(dd,J＝14.2,8.3Hz,32H,CH2(linoleoyl)),0.86(d,J＝7.0Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.86(1C,NHCOCH2),172.76(1C,NHCOCH2),172.31(1C,COOCH2),171.98(1C,COOCH2),130.18(2C,CHCH),130.00(1C,CHCH),129.99(1C,CHCH),128.03(2C,CHCH),127.88(2C,CHCH),65.75(1C,COOCH2),64.96(1C,COOCH2),56.52(2C,CH3NCH2),52.03(2C,CH3NCH2CH2),50.03(1C,CONHCH),43.70(1C,NCH2CH2NH),34.96(1C,NCH3),31.54(1C,CONHCH2),31.48(2C,NHCOCH2),31.40(1C,CH2(linoleoyl)),31.26(1C,CH2(linoleoyl)),30.30(1C,OCOCH2),30.18(1C,CH2(linoleoyl)),29.72(1C,CH2(linoleoyl)),29.62(2C,CH2(linoleoyl)),29.40(1C,CH2(linoleoyl)),29.30(2C,CH2(linoleoyl)),29.22(2C,CH2(linoleoyl)),29.18(1C,CH2(linoleoyl)),28.57(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.18(2C,CH2CHCH),27.17(2C,CHCHCH2),27.13(1C,NHCHCH2),25.87(1C,COOCH2CH2CH2),25.80(1C,COOCH2CH2CH2),25.61(2C,CHCH2CH),22.52(2C,CH2CH3),14.01(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H93N4O6[M+H]+,869.7090；found,869.7085.
实施例7
制备化合物LA5，化学结构式如下：
标题化合物LA5以化合物LA2-COOH(500mg，0.67mmol)和3-二乙胺基丙胺(106μL，0.67mmol)为原料，按照制备LA1的方法，得到橙黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝12︰1)，得到无色透明油状物266mg，收率：46.2％。1H NMR(500MHz,CDCl3):δ(ppm)7.52(d,J＝8.6Hz,1H,CONHCH),7.00(s,1H,CONHCH2),5.41-5.27(m,8H,CHCH),4.51(d,J＝5.3Hz,1H,CONHCH),4.08(dd,J＝17.8,11.0Hz,2H,COOCH2),4.03(d,J＝6.9Hz,2H,COOCH2),3.39(s,2H,CONHCH2),3.19(s,2H,NCH2CH2),3.09(s,4H,NCH2CH3),2.76(t,J＝6.6Hz,4H,CHCH2CH),2.65-2.52(m,4H,NHCOCH2),2.38(dt,J＝18.5,9.4Hz,2H,OCOCH2),2.16(dt,J＝13.2,6.5Hz,2H,NHCHCH2),2.05-1.92(m,8H,CHCHCH2,2H,NCH2CH2),1.61(dd,J＝13.3,6.7Hz,4H,COOCH2CH2),1.40-1.29(m,32H,CH2(linoleoyl)),1.25(s,6H,NCH2CH3),0.87(dd,J＝7.0,5.0Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.85(2C,NHCOCH2),172.02(2C,COOCH2),130.20(2C,CHCH),130.03(2C,CHCH),128.03(2C,CHCH),127.90(2C,CHCH),65.69(1C,COOCH2),64.88(1C,COOCH2),51.94(1C,(CH3CH2)2NCH2),49.44(1C,CONHCH),46.20(2C,N(CH2CH3)2),36.17(1C,CONHCH2),31.50(2C,NHCOCH2),31.45(1C,CH2(linoleoyl)),31.41(1C,CH2(linoleoyl)),31.35(1C,OCOCH2),30.38(1C,CONHCH2CH2),30.19(1C,CH2(linoleoyl)),29.63(2C,CH2(linoleoyl)),29.41(1C,CH2(linoleoyl)),29.31(2C,CH2(linoleoyl)),29.22(2C,CH2(linoleoyl)),29.18(2C,CH2(linoleoyl)),28.59(1C,COOCH2CH2),28.50(1C,COOCH2CH2),27.23(1C,NHCHCH2),27.20(2C,CH2CHCH),27.18(2C,CHCHCH2),25.87(1C,COOCH2CH2CH2),25.80(1C,COOCH2CH2CH2),25.62(2C,CHCH2CH),23.99(1C,CH2CH3),22.53(1C,CH2CH3),14.02(2C,CH2CH3),8.28(2C,N(CH2CH3)2).HRMS,ESI+,m/z:Calcd for C52H94N3O6[M+H]+,856.7137；found,856.7144.
实施例8
制备化合物LA6，化学结构式如下：
标题化合物LA6以化合物LA2-COOH(500mg，0.67mmol)和(1-甲基-4-哌啶)甲胺(96μL，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝15︰1)，得到黄白色凝胶状物341mg，收率：59.4％。1H NMR(500MHz,CDCl3):δ(ppm)7.34(s,1H,CONHCH),7.22(s,1H,CONHCH2),5.41-5.28(m,8H,CHCH),4.48(d,J＝5.6Hz,1H,CONHCH),4.12-4.07(m,2H,COOCH2),4.03(t,J＝6.8Hz,2H,COOCH2),3.53(d,J＝10.6Hz,2H,CONHCH2CH),3.28-3.04(m,3H,NCH3),2.79-2.72(m,4H,CH3NCH2,4H,CHCH2CH),2.65-2.54(m,4H,NHCOCH2),2.40(dd,J＝15.0,7.1Hz,2H,OCOCH2),2.15(td,J＝13.9,7.4Hz,1H,CONHCH2CH),2.03(q,J＝6.8Hz,8H,CH2CHCHCH2),2.00(s,2H,NHCHCH2),1.89(s,4H,CH3NCH2CH2),1.61(dt,J＝13.5,6.8Hz,4H,COOCH2CH2H),1.32-1.24(m,32H,CH2(linoleoyl)),0.88-0.84(m,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)173.06(1C,NHCOCH2),172.91(1C,NHCOCH2),172.60(1C,COOCH2),172.00(1C,COOCH2),130.21(2C,CHCH),130.03(2C,CHCH),128.05(2C,CHCH),127.90(2C,CHCH),65.72(1C,COOCH2),64.94(1C,COOCH2),54.75(1C,CH3NCH2),53.39(1C,CH3NCH2),52.07(1C,CONHCH),43.72(1C,NCH3),33.48(1C,CONHCH2CH),31.69(1C,CONHCH2CH),31.51(2C,NHCOCH2),31.42(1C,OCOCH2),30.45(1C,CH2(linoleoyl)),30.20(1C,CH2(linoleoyl)),29.64(2C,CH2(linoleoyl)),29.43(1C,CH2(linoleoyl)),29.41(1C,CH2(linoleoyl)),29.32(2C,CH2(linoleoyl)),29.24(2C,CH2(linoleoyl)),29.20(2C,CH2(linoleoyl)),28.59(1C,COOCH2CH2),28.51(1C,COOCH2CH2),27.21(2C,CH2CHCH),27.19(2C,CHCHCH2),27.09(1C,NHCHCH2),26.92(1C,CH3NCH2CH2),26.84(1C,CH3NCH2CH2),25.89(1C,COOCH2CH2CH2),25.82(1C,COOCH2CH2CH2),25.63(2C,CHCH2CH),22.54(2C,CH2CH3),14.03(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H92N3O6[M+H]+,854.6981；found,854.6978.
实施例9
制备化合物LA7，化学结构式如下：
标题化合物LA7以化合物LA2-COOH(500mg，0.67mmol)和N-甲基-2-(2-氨乙基)-吡咯烷(87μL，0.67mmol)为原料，按照制备LA1的方法，得到深黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到深黄色凝胶状物400mg，收率：69.7％。1H NMR(500MHz,CDCl3):δ(ppm)7.45(s,1H,CONHCH),7.11(dd,J＝8.6,2.5Hz,1H,CONHCH2),5.42-5.28(m,8H,CHCH),4.53-4.47(m,1H,CONHCH),4.09(d,J＝6.0Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.79(s,1H,CH3NCH2),3.42(s,1H,CH3NCH2),3.29(s,2H,CONHCH2),3.15-3.07(m,1H,CH3NCH2CH2),2.94(d,J＝15.3Hz,1H,CH3NCH2CH2),2.87(s,3H,NCH3),2.76(t,J＝6.6Hz,4H,CHCH2CH),2.56(d,J＝18.9Hz,4H,NHCOCH2),2.38(dd,J＝15.8,8.3Hz,4H,OCOCH2CH2),2.24(s,1H,CH3NCH),2.18-2.09(m,2H,CONHCH2CH2),2.06-1.98(m,8H,CHCHCH2,2H,CH3NCHCH2),1.60(dd,J＝11.8,6.3Hz,4H,OCOCH2CH2),1.27(dd,J＝14.0,8.1Hz,32H,CH2(linoleoyl)),0.89-0.85(m,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.95(1C,NHCOCH2),172.76(1C,NHCOCH2),172.25(1C,COOCH2),171.95(1C,COOCH2),130.21(2C,CHCH),130.03(2C,CHCH),128.05(2C,CHCH),127.90(2C,CHCH),73.22(1C,CH3NCH),67.53(1C,CH3NCH2),65.81(1C,COOCH2),65.01(1C,COOCH2),56.15(1C,NHCHCH2),51.97(1C,NCH3),45.99(1C,CONHCH2),39.80(1C,CONHCH2CH2),31.51(2C,NHCOCH2),31.42(1C,CH2(linoleoyl)),31.34(1C,CH2(linoleoyl)),30.33(1C,OCOCH2),30.20(1C,CH3NCHCH2),29.75(1C,CH2(linoleoyl)),29.64(2C,CH2(linoleoyl)),29.43(1C,CH2(linoleoyl)),29.42(1C,CH2(linoleoyl)),29.32(1C,CH2(linoleoyl)),29.24(2C,CH2(linoleoyl)),29.20(2C,CH2(linoleoyl)),28.59(1C,COOCH2CH2),28.50(1C,COOCH2CH2),27.21(2C,CH2CHCH),27.19(2C,CHCHCH2),25.88(1C,COOCH2CH2CH2),25.82(1C,COOCH2CH2CH2),25.63(2C,CHCH2CH),22.66(1C,NHCHCH2),22.54(2C,CH2CH3),21.64(1C,CH3NCH2CH2),14.03(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H92N3O6[M+H]+,854.6981；found,854.6980.
实施例10
制备化合物LA8，化学结构式如下：
标题化合物LA8以化合物LA2-COOH(500mg，0.67mmol)和N-(3-氨基丙基)二乙醇胺(102μL，0.67mmol)为原料，按照制备LA1的方法，得到深黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝12︰1)，得到淡黄色凝胶状物210mg，收率：35.2％。1H NMR(500MHz,CDCl3):δ(ppm)7.53(d,J＝8.6Hz,1H,CONHCH),7.17(d,J＝32.3Hz,1H,CONHCH2),5.41-5.28(m,8H,CHCH),4.48(d,J＝5.7Hz,1H,CONHCH),4.10(d,J＝6.0Hz,4H,COOCH2),4.04(t,J＝6.8Hz,4H,CH2OH),3.44(d,J＝41.7Hz,6H,NCH2CH2,2H,CONHCH2),2.77(t,J＝6.6Hz,4H,CHCH2CH),2.61(s,4H,NHCOCH2),2.40(dd,J＝15.0,7.7Hz,2H,OCOCH2),2.22-2.06(m,2H,NHCHCH2,2H,NCH2CH2),2.06-1.95(m,8H,CHCHCH2),1.61(dd,J＝12.4,6.3Hz,4H,OCOCH2CH2),1.28(dd,J＝14.0,8.3Hz,32H,CH2(linoleoyl)),0.90-0.83(m,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.97(2C,NHCOCH2),172.00(2C,COOCH2),130.20(2C,CHCH),130.02(2C,CHCH),128.04(2C,CHCH),127.90(2C,CHCH),65.81(2C,COOCH2),65.01(2C,CH2CH2OH,1C,(HOCH2CH2)2NCH2),56.16(2C,CH2OH),53.37(1C,NHCHCH2),52.04(1C,CONHCH2),31.50(2C,NHCOCH2),31.41(1C,CH2(linoleoyl)),31.21(1C,CH2(linoleoyl)),30.35(1C,OCOCH2),30.19(1C,CONHCH2CH2),29.65(2C,CH2(linoleoyl)),29.42(2C,CH2(linoleoyl)),29.32(2C,CH2(linoleoyl)),29.25(2C,CH2(linoleoyl)),29.22(2C,CH2(linoleoyl)),28.58(1C,COOCH2CH2),28.50(1C,COOCH2CH2),27.21(2C,CH2CHCH),27.18(2C,CH2CHCH),27.10(1C,NHCHCH2),25.89(1C,COOCH2CH2CH2),25.83(1C,COOCH2CH2CH2),25.62(2C,CHCH2CH),22.53(2C,CH2CH3),14.02(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H94N3O8[M+H]+,888.7035；found,888.7051.
实施例11
制备化合物LA9，化学结构式如下：
标题化合物LA9以化合物LA2-COOH(500mg，0.67mmol)和1-甲基-3-氨基吡咯烷盐酸盐(98μL，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到白色凝胶状物390mg，收率：70.2％。1H NMR(500MHz,CDCl3):δ(ppm)8.04(s,1H,CONHCH),7.30(d,J＝7.5Hz,1H,CONHCH2),5.41-5.29(m,8H,CHCH),4.84(s,1H,CONHCH),4.57(d,J＝7.2Hz,1H,CH3NCH2CH),4.06(dt,J＝19.8,6.8Hz,4H,COOCH2),3.97(s,1H,CH3NCH2),3.82(d,J＝9.9Hz,1H,CH3NCH2),3.03(s,2H,CH3NCH2CH2),2.91(d,J＝11.3Hz,4H,CHCH2CH,2H,CH3NCH2CH2),2.77(d,J＝6.7Hz,3H,NCH3),2.58(dd,J＝25.4,8.0Hz,4H,NHCOCH2),2.45-2.32(m,2H,OCOCH2),2.20(dd,J＝20.7,12.5Hz,2H,NHCHCH2),2.06-1.99(m,8H,CHCHCH2),1.61(dd,J＝11.5,6.5Hz,4H,OCOCH2CH2),1.33-1.25(m,32H,CH2(linoleoyl)),0.88(t,J＝6.8Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.82(1C,NHCOCH2),172.32(1C,NHCOCH2),171.97(2C,COOCH2),130.20(2C,CHCH),130.04(2C,CHCH),128.03(2C,CHCH),127.91(2C,CHCH),65.67(1C,CH3NCH2),64.88(2C,COOCH2),54.78(1C,CH3NCH2),51.74(1C,NHCHCH2),47.77(1C,CONHCH,1C,NCH3),40.34(1C,CONHCHCH2),31.50(2C,NHCOCH2),31.41(1C,OCOCH2),29.64(2C,CH2(linoleoyl)),29.42(2C,CH2(linoleoyl)),29.32(2C,CH2(linoleoyl)),29.24(4C,CH2(linoleoyl)),29.20(2C,CH2(linoleoyl)),28.58(2C,COOCH2CH2),28.48(1C,NHCHCH2),27.21(2C,CHCHCH2),27.18(2C,CHCHCH2),25.88(1C,COOCH2CH2CH2),25.82(1C,COOCH2CH2CH2),25.62(2C,CHCH2CH),22.53(2C,CH2CH3),14.02(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C50H88N3O6[M+H]+,826.6668；found,826.6656.
实施例12
制备化合物LA10，化学结构式如下：
标题化合物LA10以化合物LA2-COOH(500mg，0.67mmol)和1-(3-氨丙基)-4-甲基哌嗪(114μL，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到淡黄色凝胶状物267mg，收率：45.0％。1H NMR(300MHz,CDCl3):δ(ppm)7.56-7.43(m,1H,CONHCH),7.27-7.11(m,1H,CONHCH2),5.35(q,J＝9.4,8.6Hz,8H,CHCH),4.50(q,J＝6.9Hz,1H,CONHCH),4.17-3.97(m,4H,COOCH2),3.31(m,8H,CH3NCH2CH2),2.98(s,2H,CONHCH2),2.77(t,J＝6.2Hz,4H,COCH2CH2CO),2.71(s,2H,CH2NCH2),2.66-2.47(m,4H,CHCH2CH),2.40(q,J＝6.9,6.3Hz,2H,OCOCH2),2.08(s,2H,NHCHCH2),2.08(s,3H,NCH3),2.06-1.84(m,8H,CHCHCH2),1.62(q,J＝7.2Hz,4H,OCOCH2CH2),1.46(d,J＝22.7Hz,2H,NCH2CH2),1.28(m,32H,CH2(linoleoyl)),0.89-0.85(m,6H,CH2CH3).13CNMR(75MHz,CDCl3):δ(ppm)172.80(2C,NHCOCH2),172.42(1C,COOCH2),171.93(1C,COOCH2),130.15(2C,CHCH),129.97(2C,CHCH),128.01(2C,CHCH),127.87(2C,CHCH),65.67(1C,COOCH2),64.89(1C,COOCH2),54.81(1C,NHCHCH2),51.92(2C,CH3NCH2),51.79(2C,CH3NCH2CH2),50.13(1C,CH2NCH2),43.91(1C,NCH3),36.88(1C,CONHCH2),31.85(1C,NCH2CH2),31.46(2C,NHCOCH2),30.34(1C,OCOCH2),29.61(2C,CH2(linoleoyl)),29.39(2C,CH2(linoleoyl)),29.28(4C,CH2(linoleoyl)),29.21(4C,CH2(linoleoyl)),28.57(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.16(4C,CHCHCH2),25.86(1C,COOCH2CH2CH2),25.81(1C,COOCH2CH2CH2),25.59(2C,CHCH2CH),24.56(1C,NHCHCH2),22.50(2C,CH2CH3),13.98(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C53H95N4O6[M+H]+,883.7246；found,883.7229.
实施例13
制备化合物LA11，化学结构式如下：
标题化合物LA11以化合物LA2-COOH(500mg，0.67mmol)和11b(115mg，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝12︰1)，得到黄色凝胶状物210mg，收率：34.8％。1H NMR(300MHz,CDCl3):δ(ppm)7.55(s,1H,CONHN),6.83(s,1H,CONHCH),5.36(d,J＝5.6Hz,8H,CHCH),4.48(d,J＝8.6Hz,1H,CONHCH),4.09(dd,J＝15.8,7.5Hz,4H,COOCH2),3.95-3.47(m,8H,CH3NCH2CH2),3.34(t,J＝16.4Hz,2H,CONHCH2),3.19(t,J＝7.6Hz,2H,CH2NCH2),2.87(s,4H,CHCH2CH),2.77(t,J＝6.4Hz,4H,COCH2CH2CO),2.62(d,J＝18.0Hz,4H,OCOCH2CH2),2.42(t,J＝7.6Hz,3H,NCH3),2.11-2.00(m,8H,CHCHCH2),1.88(s,2H,CH2NCH2CH2),1.64(s,2H,CH2NCH2CH2CH2,4H,OCOCH2CH2),1.39-1.29(m,32H,CH2(linoleoyl)),0.96-0.87(m,6H,CH2CH3).13C NMR(75MHz,CDCl3):δ(ppm)172.89(1C,NHCOCH2),172.72(1C,COOCH2),172.64(1C,COOCH2),172.00(1C,NHCOCH2),130.10(2C,CHCH),129.93(2C,CHCH),127.96(2C,CHCH),127.84(2C,CHCH),65.60(1C,COOCH2),64.83(1C,COOCH2),53.45(2C,CH3NCH2),51.92(2C,CH3NCH2CH2),50.68(1C,NHCHCH2),49.10(1C,NCH3),43.40(1C,CH2NCH2),38.11(1C,CONHCH2),31.43(2C,NHCOCH2,2C,CH2(linoleoyl)),30.37(1C,OCOCH2),29.59(2C,CH2(linoleoyl)),29.38(2C,CH2(linoleoyl)),29.26(3C,CH2(linoleoyl)),29.20(3C,CH2(linoleoyl)),28.54(1C,COOCH2CH2),28.47(1C,COOCH2CH2),27.13(2C,NCH2CH2CH2),27.12(4C,CHCHCH2),27.00(1C,NHCHCH2),25.82(2C,COOCH2CH2CH2),25.56(2C,CHCH2CH),22.48(2C,CH2CH3),13.99(2C,CH2CH3).
实施例14
制备化合物LA12，化学结构式如下：
标题化合物LA12以化合物LA2-COOH(500mg，0.67mmol)和1-氨基-4-甲基哌嗪(81μL，0.67mmol)为原料，按照制备LA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝15︰1)，得到黄白色凝胶状物183mg，收率：32.4％。1H NMR(300MHz,CDCl3):δ(ppm)8.72(s,1H,CONHN),7.04(s,1H,CONHCH),5.35(d,J＝5.6Hz,8H,CHCH),4.53(q,J＝6.5Hz,1H,CONHCH),4.07(m,4H,COOCH2),3.35(m,8H,CH3NCH2CH2),2.84(s,4H,CHCH2CH),2.76(d,J＝5.9Hz,4H,COCH2CH2CO),2.60(s,2H,OCOCH2),2.39(s,3H,NCH3),2.12(m,2H,NHCHCH2),2.04(t,J＝6.6Hz,8H,CHCHCH2),1.61(t,J＝7.2Hz,4H,OCOCH2CH2),1.30(s,32H,CH2(linoleoyl)),0.88(d,J＝6.7Hz,6H,CH2CH3).13C NMR(75MHz,CDCl3):δ(ppm)172.76(1C,CONHN),172.24(1C,CONHCH),171.96(1C,COOCH2),171.95(1C,COOCH2),130.15(2C,CHCH),129.98(2C,CHCH),128.02(2C,CHCH),127.88(2C,CHCH),65.63(1C,COOCH2),64.85(1C,COOCH2),53.38(1C,NCH3),53.07(1C,CH3NCH2),52.99(1C,CH3NCH2),51.88(1C,NHCHCH2),51.38(1C,CH3NCH2CH2),51.29(1C,CH3NCH2CH2),43.26(1C,NHCOCH2),43.15(1C,NHCOCH2),31.84(1C,OCOCH2),31.46(2C,CH2(linoleoyl)),30.41(1C,NHCHCH2),29.61(2C,CH2(linoleoyl)),29.39(2C,CH2(linoleoyl)),29.28(2C,CH2(linoleoyl)),29.20(4C,CH2(linoleoyl)),28.58(1C,COOCH2CH2),28.50(1C,COOCH2CH2),27.16(4C,CHCHCH2),25.86(1C,COOCH2CH2CH2),25.81(1C,COOCH2CH2CH2),25.60(2C,CHCH2CH),22.49(2C,CH2CH3),13.97(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C50H89N4O6[M+H]+,841.6777；found,841.6769.
实施例15
制备谷氨酸双油醇酯(OA2-NH2)，化学结构式如下：
向500mL反应瓶中加入L-谷氨酸(8.00g，54.37mmol)、对甲苯磺酸(11.38g，59.83mmol)和无水甲苯(300mL)，随后将反应液升温至140℃，回流反应3h。停止加热后，将混合溶液冷却至室温，分批加入油醇(30.66g，114.18mmol)，140℃回流反应过夜。反应结束后，旋蒸除去甲苯，得到深棕色油状物。将粗产物溶于二氯甲烷，用适量水洗涤两次，饱和碳酸氢钠水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到深黄色油状物。经柱层析纯化(石油醚︰乙酸乙酯＝3︰1)，得到淡黄色油状物21.5g，收率：61.0％。1H NMR(300MHz,CDCl3):δ(ppm)5.41-5.29(m,4H,CHCH),4.12(t,J＝6.8Hz,2H,COOCH2),4.05(d,J＝6.9Hz,2H,COOCH2),3.41(m,1H,NH2CH),2.44-2.30(m,2H,OCOCH2),2.07(m,2H,NHCHCH2),2.02-1.95(m,8H,CH2CHCHCH2),1.61(dd,J＝13.5,6.8Hz,4H,COOCH2CH2),1.31-1.24(m,44H,CH2(oleoyl)),0.87(d,J＝7.1Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd forC41H78NO4[M+H]+,648.5925；found,648.5929.
实施例16
制备羧基化谷氨酸双油醇酯(OA2-COOH)，化学结构式如下：
将OA2-NH2(2.65g，4.10mmol)溶解于二氯甲烷，在搅拌状态下向其中先后加入三乙胺(2.28mL，16.39mmol)和丁二酸酐(410mg，4.10mmol)，室温反应过夜。反应结束后，旋蒸除去溶剂，得到淡黄色油状粗品，经柱层析纯化(二氯甲烷︰甲醇＝50︰1)，得到淡黄色油状物2.56g，收率：83.4％。1H NMR(300MHz,CDCl3):δ(ppm)7.40(s,1H,CONHCH),5.40-5.29(m,4H,CHCH),4.54(m,1H,CONHCH),4.12(t,J＝6.8Hz,2H,COOCH2),4.03(d,J＝6.9Hz,2H,COOCH2),2.97(s,2H,CH2COOH),2.45-2.31(m,2H,OCOCH2),2.30(s,2H,NHCOCH2),2.17(m,2H,NHCHCH2),2.03-1.96(m,8H,CH2CHCHCH2),1.62(dd,J＝13.5,6.8Hz,4H,COOCH2CH2),1.30-1.25(m,44H,CH2(oleoyl)),0.87(d,J＝7.1Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd forC45H82NO7[M+H]+,748.6086；found,748.6080.
实施例17
制备化合物OA1，化学结构式如下：
将OA2-COOH(500mg，0.67mmol)溶解于二氯甲烷，在0℃下依次加入EDCI(205mg，1.07mmol)、HOBt(145mg，1.07mmol)并搅拌5min，随后将混合溶液移至室温反应3h，得到反应液A；将1-(3-氨基丙基)吡咯烷(85μL，0.67mmol)溶解于二氯甲烷，在室温下加入三乙胺(280μL，2.02mmol)，搅拌反应1h，得到反应液B。将反应液B缓慢滴加进反应液A，室温搅拌过夜。反应结束后，反应液用适量水洗涤两次，10％柠檬酸水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到橙黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到无色油状物238mg，收率：41.5％。1H NMR(500MHz,CDCl3):δ(ppm)7.39(s,1H,CONHCH),6.85(d,J＝7.6Hz,1H,CONHCH2),5.41-5.30(m,4H,CHCH),4.53(dt,J＝13.0,6.5Hz,1H,CONHCH),4.11(t,J＝6.8Hz,2H,COOCH2),4.04(d,J＝6.9Hz,2H,COOCH2),3.41(d,J＝5.4Hz,2H,CONHCH2),3.15(t,J＝6.9Hz,2H,NCH2CH2),2.57(d,J＝4.8Hz,4H,N(CH2CH2)2),2.44-2.30(m,2H,NHCOCH2),2.17(dd,J＝13.3,6.4Hz,2H,NHCOCH2),2.07(dd,J＝12.1,5.9Hz,4H,OCOCH2CH2),2.02-1.95(m,8H,CH2CHCHCH2),1.77(s,4H,N(CH2CH2)2,2H,NCH2CH2),1.61(dd,J＝13.5,6.8Hz,4H,COOCH2CH2),1.31-1.24(m,44H,CH2(oleoyl)),0.87(d,J＝7.1Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.94(1C,NHCOCH2),172.84(1C,NHCOCH2),172.22(1C,COOCH2),171.98(1C,COOCH2),129.96(2C,CHCH),129.72(2C,CHCH),65.68(1C,COOCH2),64.88(1C,COOCH2),53.65(2C,N(CH2CH2)2),52.82(1C,CONHCH),51.90(1C,(CH2CH2)2NCH2),36.16(1C,CONHCH2CH2),31.86(2C,NHCOCH2),31.47(1C,CONHCH2CH2),31.36(1C,CH2(oleoyl)),31.32(1C,CH2(oleoyl)),30.35(1C,OCOCH2),29.73(2C,CH2(oleoyl)),29.66(2C,CH2(oleoyl)),29.48(2C,CH2(oleoyl)),29.41(2C,CH2(oleoyl)),29.28(2C,CH2(oleoyl)),29.27(2C,CH2(oleoyl)),29.21(2C,CH2(oleoyl)),29.18(2C,CH2(oleoyl)),28.58(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.27(1C,NHCHCH2),27.19(2C,CHCHCH2),27.17(2C,CHCHCH2),25.87(1C,COOCH2CH2CH2),25.80(1C,COOCH2CH2CH2),23.25(2C,N(CH2CH2)2),22.63(2C,CH2CH3),14.05(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H96N3O6[M+H]+,858.7294；found,858.7291.
实施例18
制备化合物OA2，化学结构式如下：
标题化合物OA2以化合物OA2-COOH(500mg，0.67mmol)和3-二甲胺基丙胺(84μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝12︰1)，得到黄白色凝胶状物327mg，收率：58.7％。1H NMR(500MHz,CDCl3):δ(ppm)7.38(s,1H,CONHCH),6.82(d,J＝7.4Hz,1H,CONHCH2),5.42-5.29(m,4H,CHCH),4.52(dd,J＝13.0,7.7Hz,1H,CONHCH),4.11(t,J＝6.8Hz,2H,COOCH2),4.05(t,J＝6.8Hz,2H,COOCH2),3.41(d,J＝5.8Hz,2H,CONHCH2),3.12(t,J＝7.0Hz,2H,(CH3)2NCH2),2.81(s,6H,NCH3),2.58(dd,J＝11.5,6.8Hz,4H,NHCOCH2),2.44-2.32(m,2H,OCOCH2),2.08(d,J＝6.8Hz,2H,NHCHCH2),2.01(dd,J＝12.6,6.4Hz,8H,CH2CHCHCH2),1.92(d,J＝32.2Hz,2H,(CH3)2NCH2CH2),1.61(dd,J＝12.8,6.8Hz,4H,COOCH2CH2),1.34-1.25(m,44H,CH2(oleoyl)),0.88(t,J＝6.9Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.96(1C,NHCOCH2),172.84(1C,NHCOCH2),172.23(1C,COOCH2),171.99(1C,COOCH2),129.97(2C,CHCH),129.71(2C,CHCH),65.71(1C,COOCH2),64.90(1C,COOCH2),55.56(1C,(CH3)2NCH2),51.93(1C,CONHCH),43.10(2C,N(CH3)2),36.14(1C,CONHCH2),31.87(2C,NHCOCH2),31.35(1C,CH2(oleoyl)),31.27(1C,CH2(oleoyl)),30.34(1C,OCOCH2CH2),29.73(2C,CH2(oleoyl)),29.66(1C,CH2(oleoyl)),29.63(1C,CH2(oleoyl)),29.48(2C,CH2(oleoyl)),29.41(1C,CH2(oleoyl)),29.39(1C,CH2(oleoyl)),29.28(2C,CH2(oleoyl)),29.27(2C,CH2(oleoyl)),29.22(2C,CH2(oleoyl)),29.18(2C,CH2(oleoyl)),28.58(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.19(2C,CH2CHCH),27.17(2C,CHCHCH2),25.87(1C,NHCHCH2),25.80(1C,(CH3)2NCH2CH2),24.51(2C,COOCH2CH2CH2),22.63(2C,CH2CH3),14.05(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C50H94N3O6[M+H]+,832.7137；found,832.7149.
实施例19
制备化合物OA3，化学结构式如下：
标题化合物OA3以化合物OA2-COOH(500mg，0.67mmol)和1-(3-氨基丙基)哌啶(106μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到淡黄色油状物419mg，收率：71.8％。1H NMR(500MHz,CDCl3):δ(ppm)7.62(t,J＝5.6Hz,1H,CONHCH),7.03(d,J＝7.6Hz,1H,CONHCH2),5.41-5.30(m,4H,CHCH),4.51(td,J＝7.8,5.4Hz,1H,CONHCH),4.09(t,J＝6.8Hz,2H,COOCH2),4.03(t,J＝6.8Hz,2H,COOCH2),3.36(dd,J＝11.3,5.6Hz,2H,CONHCH2),2.98(t,J＝7.2Hz,4H,NCH2CH2),2.57(dd,J＝9.0,6.4Hz,4H,NHCOCH2),2.45-2.32(m,2H,NCH2CH2CH2NH),2.03-1.94(m,4H,OCOCH2CH2,8H,CHCHCH2),1.67-1.54(m,2H,NCH2CH2CH2NH,4H,COOCH2CH2),1.40-1.12(m,44H,CH2(oleoyl),6H,NCH2CH2CH2),0.86(t,J＝6.9Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.95(1C,NHCOCH2),172.84(1C,NHCOCH2),172.28(1C,COOCH2),172.01(1C,COOCH2),129.96(2C,CHCH),129.72(2C,CHCH),65.67(1C,COOCH2),64.87(1C,COOCH2),54.84(1C,NHCHCH2),53.44(2C,NCH2CH2),51.90(1C,NCH2CH2CH2NHCO),36.43(1C,NCH2CH2CH2NHCO),32.56(1C,NCH2CH2CH2NHCO),31.86(2C,CH2CH2CH3),31.43(1C,NHCOCH2),31.39(1C,NHCOCH2),30.37(1C,OCOCH2),29.73(2C,CH2(oleoyl)),29.66(1C,CH2(oleoyl)),29.63(1C,CH2(oleoyl)),29.48(2C,CH2(oleoyl)),29.40(1C,CH2(oleoyl)),29.38(1C,CH2(oleoyl)),29.28(2C,CH2(oleoyl)),29.26(2C,CH2(oleoyl)),29.21(2C,CH2(oleoyl)),29.18(2C,CH2(oleoyl)),28.58(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.25(2C,NCH2CH2CH2),27.19(2C,CH2CHCH),27.17(2C,CHCHCH2),25.87(1C,COOCH2CH2CH2),25.79(1C,COOCH2CH2CH2),23.86(1C,NHCHCH2),22.99(1C,CH2CH3),22.63(1C,CH2CH3),22.36(1C,NCH2CH2CH2),14.05(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C53H98N3O6[M+H]+,872.7450；found,872.7447.
实施例20
制备化合物OA4，化学结构式如下：
标题化合物OA4以化合物OA2-COOH(500mg，0.67mmol)和4-甲基-1-哌嗪乙胺(100μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝18︰1)，得到淡黄色凝胶状物460mg，收率：78.7％。1H NMR(500MHz,CDCl3):δ(ppm)6.73-6.67(m,1H,CONHCH),6.34(s,1H,CONHCH2),5.43-5.26(m,4H,CHCH),4.56(dd,J＝13.0,7.8Hz,1H,CONHCH),4.11(d,J＝6.9Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.37-3.31(m,2H,CONHCH2),2.62-2.55(m,4H,NHCOCH2),2.50(dd,J＝10.2,4.7Hz,4H,CH3NCH2CH2,2H,NHCHCH2,2H,OCOCH2),2.37(dd,J＝15.2,7.5Hz,2H,CH2CH2NCH2),2.32(d,J＝10.6Hz,4H,CH3NCH2),2.03-1.94(m,3H,NCH3,8H,CHCHCH2),1.60(dt,J＝13.5,6.8Hz,4H,COOCH2CH2),1.26(dd,J＝12.7,4.8Hz,44H,CH2(oleoyl)),0.84(d,J＝5.4Hz,6H,CH2CH3).13CNMR(126MHz,CDCl3):δ(ppm)172.79(1C,NHCOCH2),172.08(1C,NHCOCH2),171.82(2C,COOCH2),129.96(2C,CHCH),129.73(2C,CHCH),65.71(1C,COOCH2),64.90(1C,COOCH2),56.42(2C,CH3NCH2CH2),54.78(2C,CH3NCH2),52.54(1C,NHCHCH2),51.80(1C,NCH2CH2NH),45.76(1C,NCH3),35.97(1C,NCH2CH2NH),31.87(2C,CH2CH2CH3),31.54(1C,NHCOCH2),31.50(1C,NHCOCH2),30.28(1C,NHCHCH2CH2),29.74(2C,CH2(oleoyl)),29.67(2C,CH2(oleoyl)),29.49(2C,CH2(oleoyl)),29.41(2C,CH2(oleoyl)),29.38(2C,CH2(oleoyl)),29.29(2C,CH2(oleoyl)),29.21(2C,CH2(oleoyl)),29.17(2C,CH2(oleoyl)),28.59(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.20(2C,CH2CHCH),27.17(2C,CHCHCH2),25.87(1C,NHCHCH2),25.79(2C,COOCH2CH2CH2),22.64(1C,CH2CH3),22.57(1C,CH2CH3),14.05(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H97N4O6[M+H]+,873.7403；found,873.7389.
实施例21
制备化合物OA5，化学结构式如下：
标题化合物OA5以化合物OA2-COOH(500mg，0.67mmol)和3-二乙胺基丙胺(105μL，0.67mmol)为原料，按照制备OA1的方法，得到橙黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到无色透明油状物383mg，收率：66.5％。1H NMR(500MHz,CDCl3):δ(ppm)7.54-7.45(m,1H,CONHCH),6.98(d,J＝7.5Hz,1H,CONHCH2),5.40-5.26(m,4H,CHCH),4.51(dd,J＝13.0,7.8Hz,1H,CONHCH),4.09(t,J＝6.8Hz,2H,COOCH2),4.03(t,J＝6.9Hz,2H,COOCH2),3.38(dd,J＝12.4,6.1Hz,2H,CONHCH2),3.09(q,J＝7.3Hz,4H,NCH2CH3),3.05(t,J＝7.3Hz,2H,(CH3CH2)2NCH2),2.64-2.49(m,4H,NHCOCH2),2.38(dd,J＝16.7,8.7Hz,2H,OCOCH2),2.16(dt,J＝20.8,7.0Hz,2H,NHCHCH2),2.00(dd,J＝12.6,6.5Hz,8H,CHCHCH2),1.61(dt,J＝13.3,6.8Hz,4H,COOCH2CH2),1.29(ddd,J＝13.5,13.1,6.6Hz,44H,CH2(oleoyl),2H,(CH3CH2)2NCH2CH2,6H,NCH2CH3),0.87(t,J＝6.9Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)173.04(1C,NHCOCH2),172.85(1C,NHCOCH2),172.21(1C,COOCH2),172.02(1C,COOCH2),129.97(2C,CHCH),129.74(2C,CHCH),65.69(1C,COOCH2),64.89(1C,COOCH2),51.93(1C,CH2N(CH2CH3)2),49.50(1C,CONHCH),46.12(2C,N(CH2CH3)2),36.26(1C,CONHCH2),31.87(1C,NHCOCH2),31.48(1C,NHCOCH2),31.42(1C,CH2(oleoyl)),31.36(1C,CH2(oleoyl)),30.37(1C,OCOCH2CH2),30.13(1C,CONHCH2CH2),29.74(2C,CH2(oleoyl)),29.67(2C,CH2(oleoyl)),29.49(2C,CH2(oleoyl)),29.39(2C,CH2(oleoyl)),29.29(2C,CH2(oleoyl)),29.27(2C,CH2(oleoyl)),29.22(2C,CH2(oleoyl)),29.18(2C,CH2(oleoyl)),28.59(1C,COOCH2CH2),28.50(1C,COOCH2CH2),27.20(2C,CH2CHCH),27.18(2C,CHCHCH2),25.88(1C,COOCH2CH2CH2),25.80(1C,COOCH2CH2CH2),24.00(1C,NHCHCH2),22.64(2C,CH2CH3),14.06(2C,CH2CH3),8.37(2C,N(CH2CH3)2).HRMS,ESI+,m/z:Calcd for C52H98N3O6[M+H]+,860.7450；found,860.7437.
实施例22
制备化合物OA6，化学结构式如下：
标题化合物OA6以化合物LA2-COOH(500mg，0.67mmol)和(1-甲基-4-哌啶)甲胺(96μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到无色凝胶状物429mg，收率：74.7％。1H NMR(500MHz,CDCl3):δ(ppm)7.08(s,1H,CONHCH),6.79(s,1H,CONHCH2),5.42-5.30(m,4H,CHCH),4.50(dd,J＝13.0,7.7Hz,1H,CONHCH),4.11(d,J＝7.2Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.38(d,J＝11.0Hz,2H,CONHCH2CH),3.29-3.13(m,2H,CH3NCH2),2.68(s,3H,NCH3),2.67-2.62(m,2H,CH3NCH2),2.60(dd,J＝10.1,4.8Hz,2H,NHCOCH2),2.56(s,2H,NHCOCH2),2.40(dd,J＝15.2,8.1Hz,2H,OCOCH2),2.05-1.98(m,8H,CHCHCH2,1H,CONHCH2CH),1.96(s,2H,NHCHCH2),1.88(s,2H,CH3NCH2CH2),1.86-1.76(m,2H,CH3NCH2CH2),1.61(dt,J＝14.1,7.0Hz,4H,COOCH2CH2),1.33-1.23(m,44H,CH2(oleoyl)),0.87(t,J＝6.9Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.88(1C,NHCOCH2),172.80(1C,NHCOCH2),172.38(1C,COOCH2),171.94(1C,COOCH2),,129.97(2C,CHCH),129.72(2C,CHCH),65.73(1C,COOCH2),64.93(1C,COOCH2),60.34(2C,CH3NCH2),52.05(1C,NHCHCH2),43.82(1C,NCH3),32.57(1C,CONHCH2CH),31.87(2C,NHCOCH2),31.69(1C,CH2CH2CH3),31.64(1C,CH2CH2CH3),31.40(1C,CONHCH2CH),30.41(1C,OCOCH2),29.73(2C,CH2(oleoyl)),29.66(1C,CH2(oleoyl)),29.63(1C,CH2(oleoyl)),29.49(2C,CH2(oleoyl)),29.41(1C,CH2(oleoyl)),29.39(1C,CH2(oleoyl)),29.29(2C,CH2(oleoyl)),29.27(2C,CH2(oleoyl)),29.22(2C,CH2(oleoyl)),29.18(2C,CH2(oleoyl)),28.58(1C,COOCH2CH2),28.49(1C,COOCH2CH2),27.20(2C,CH2CHCH),27.18(2C,CHCHCH2),27.11(2C,CH3NCH2CH2),26.87(1C,NHCHCH2),25.88(1C,COOCH2CH2CH2),25.80(1C,COOCH2CH2CH2),22.64(2C,CH2CH3),14.05(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H96N3O6[M+H]+,858.7294；found,858.7280.
实施例23
制备化合物OA7，化学结构式如下：
标题化合物OA7以化合物OA2-COOH(500mg，0.67mmol)和N-甲基-2-(2-氨乙基)-吡咯烷(87μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝25︰1)，得到无色透明凝胶状物290mg，收率：50.5％。1H NMR(300MHz,CDCl3):δ(ppm)7.39(s,1H,CONHCH),7.00(s,1H,CONHCH2),5.40-5.28(m,4H,CHCH),4.49(dd,J＝13.0,7.6Hz,1H,CONHCH),4.11-3.99(m,4H,COOCH2),3.65-3.37(m,2H,CONHCH2),3.28(d,J＝4.4Hz,1H,CH3NCH2),3.15(s,1H,CH3NCH2),2.92-2.80(m,1H,CH3NCH),2.74(s,3H,NCH3),2.53(d,J＝5.6Hz,4H,NHCOCH2),2.36(dd,J＝7.6,4.3Hz,2H,OCOCH2),2.18-2.07(m,2H,NHCHCH2),1.99(d,J＝5.5Hz,8H,CH2CHCHCH2,2H,CH3NCHCH2),1.66-1.52(m,4H,COOCH2CH2),1.29(t,J＝15.0Hz,44H,CH2(oleoyl),4H,CH3NCH2CH2CH2),0.86(t,J＝6.6Hz,6H,CH2CH3).13C NMR(75MHz,CDCl3):δ(ppm)172.78(1C,NHCOCH2),172.57(1C,NHCOCH2),172.22(1C,COOCH2),171.86(1C,COOCH2),129.91(2C,CHCH),129.66(2C,CHCH),66.43(1C,CH3NCH),65.65(1C,COOCH2),64.87(1C,COOCH2),56.04(1C,CH3NCH2),53.33(1C,NHCHCH2),51.84(1C,NCH3),39.10(1C,CONHCH2),36.26(1C,CONHCH2CH2),32.51(1C,NHCOCH2),31.81(1C,NHCOCH2),31.32(2C,CH2(oleoyl)),30.28(1C,OCOCH2CH2,1C,NCHCH2),29.67(4C,CH2(oleoyl)),29.43(2C,CH2(oleoyl)),29.35(4C,CH2(oleoyl)),29.22(2C,CH2(oleoyl)),29.17(4C,CH2(oleoyl)),28.53(1C,COOCH2CH2),28.44(1C,COOCH2CH2),27.13(4C,CH2CHCHCH2),25.82(1C,NHCHCH2),25.75(2C,COOCH2CH2CH2),22.58(2C,CH2CH3),21.54(1C,CH3NCH2CH2),14.00(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H96N3O6[M+H]+,858.7294；found,858.7288.
实施例24
制备化合物OA8，化学结构式如下：
标题化合物OA8以化合物OA2-COOH(500mg，0.67mmol)和N-(3-氨基丙基)二乙醇胺(101μL，0.67mmol)为原料，按照制备OA1的方法，得到黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝13︰1)，得到淡黄色凝胶状物264mg，收率：44.2％。1H NMR(500MHz,CDCl3):δ(ppm)7.78(s,1H,CONHCH),7.59-7.44(m,1H,CONHCH2),5.46-5.29(m,4H,CHCH),4.46(s,1H,CONHCH),4.09(dd,J＝25.8,18.7Hz,2H,CH2OH,4H,COOCH2),3.44(d,J＝31.7Hz,2H,CONHCH2,10H,CH2NCH2CH2OH,),2.59(s,4H,NHCOCH2),2.40(s,2H,OCOCH2),2.12(s,2H,NHCHCH2),2.06-1.92(m,8H,CH2CHCHCH2),1.61(s,4H,COOCH2CH2),1.40-1.15(m,2H,NCH2CH2CH2,44H,CH2(oleoyl)),0.88(t,J＝6.8Hz,6H,CH2CH3).13C NMR(126MHz,CDCl3):δ(ppm)172.97(2C,NHCOCH2),172.18(2C,COOCH2),129.94(2C,CHCH),129.72(2C,CHCH),65.81(1C,COOCH2),65.02(1C,COOCH2),56.12(2C,CH2CH2OH),56.06(2C,CH2OH),52.38(1C,HOCH2CH2NCH2),52.02(1C,CONHCH),36.39(1C,CONHCH2),32.60(2C,NHCOCH2),31.88(1C,CH2CH2CH3),31.48(1C,CH2CH2CH3),31.31(1C,OCOCH2),30.45(1C,CONHCH2CH2),30.14(1C,CH2(oleoyl)),29.77(2C,CH2(oleoyl)),29.75(2C,CH2(oleoyl)),29.71(2C,CH2(oleoyl)),29.66(2C,CH2(oleoyl)),29.51(2C,CH2(oleoyl)),29.30(4C,CH2(oleoyl)),29.18(1C,CH2(oleoyl)),28.61(1C,COOCH2CH2),28.54(1C,COOCH2CH2),27.21(4C,CH2CHCHCH2),27.07(1C,NHCHCH2),25.93(1C,COOCH2CH2CH2),25.90(1C,COOCH2CH2CH2),22.65(2C,CH2CH3),14.06(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C52H98N3O8[M+H]+,892.7348；found,892.7354.
实施例25
制备化合物OA9，化学结构式如下：
标题化合物OA9以化合物OA2-COOH(500mg，0.67mmol)和1-甲基-3-氨基吡咯烷盐酸盐(98μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色凝胶状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到白色凝胶状物412mg，收率：74.2％。1H NMR(500MHz,CDCl3):δ(ppm)7.22-7.08(m,1H,CONHCH),7.03-6.91(m,1H,CONHCH2),5.37(s,2H,CHCH),5.33(s,2H,CHCH),4.55(d,J＝5.6Hz,1H,CONHCH),4.09(dd,J＝8.1,5.2Hz,2H,COOCH2),4.04(t,J＝6.8Hz,2H,COOCH2),3.29(s,1H,NCH2CHNH),3.05(dd,J＝34.0,10.4Hz,1H,CH3NCH2),2.82-2.73(m,1H,CH3NCH2CH),2.55(dd,J＝20.9,12.0Hz,4H,NHCOCH2,4H,OCOCH2CH2),2.43-2.28(m,3H,NCH3),2.16(td,J＝13.9,7.3Hz,1H,CH3NCH2),2.06-1.93(m,8H,CHCHCH2),1.88(s,1H,CH3NCH2),1.68-1.53(m,4H,COOCH2CH2),1.35-1.20(m,2H,CH3NCH2CH2,44H,CH2(oleoyl)),0.86(t,J＝6.6Hz,6H,CH2CH3).13CNMR(126MHz,CDCl3):δ(ppm)172.77(1C,NHCOCH2),171.99(1C,NHCOCH2),171.69(1C,COOCH2),171.57(1C,COOCH2),129.94(2C,CHCH),129.71(2C,CHCH),65.64(1C,CH3NCH2CH),64.85(1C,COOCH2),62.03(1C,COOCH2),54.75(1C,CH3NCH2CH2),51.75(1C,CONHCH),48.86(1C,CONHCHCH2),48.70(1C,NCH3),31.98(1C,CH3NCH2CH2),31.85(1C,NHCOCH2),31.51(1C,NHCOCH2),31.32(1C,CH2CH2CH3),31.29(1C,CH2CH2CH3),30.35(1C,NHCHCH2CH2),29.72(1C,CH2(oleoyl)),29.71(1C,CH2(oleoyl)),29.65(2C,CH2(oleoyl)),29.61(2C,CH2(oleoyl)),29.47(2C,CH2(oleoyl)),29.39(1C,CH2(oleoyl)),29.37(1C,CH2(oleoyl)),29.26(2C,CH2(oleoyl)),29.20(2C,CH2(oleoyl)),29.16(2C,CH2(oleoyl)),28.57(1C,COOCH2CH2),28.47(1C,COOCH2CH2),27.38(1C,CH2CHCH),27.33(1C,CH2CHCH),27.18(1C,CHCHCH2),27.16(1C,CHCHCH2),25.86(1C,NHCHCH2),25.79(2C,COOCH2CH2CH2),22.62(2C,CH2CH3),14.04(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C50H92N3O6[M+H]+,830.6981；found,830.6996.
实施例26
制备化合物OA10，化学结构式如下：
标题化合物OA10以化合物OA2-COOH(500mg，0.67mmol)和1-(3-氨丙基)-4-甲基哌嗪(114μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到淡黄色凝胶状物245mg，收率：41.3％。1H NMR(300MHz,CDCl3):δ(ppm)7.37(s,1H,CONHCH),7.07(s,1H,CONHCH2),5.34(q,J＝7.5,4.8Hz,4H,CHCH),4.49(q,J＝7.0Hz,1H,CONHCH),4.16-4.01(m,4H,COOCH2),3.26(s,8H,CH3NCH2CH2),2.96(s,2H,CONHCH2),2.69(s,4H,COCH2CH2CO),2.60-2.43(m,4H,OCOCH2CH2),2.39(d,J＝8.1Hz,2H,CH2NCH2),2.16(m,3H,NCH3),2.00(t,J＝6.4Hz,8H,CHCHCH2),1.66-1.55(m,4H,COOCH2CH2),1.43(s,2H,NCH2CH2),1.28(m,44H,CH2(oleoyl)),0.88(t,J＝7.3Hz,6H,CH2CH3).13C NMR(75MHz,CDCl3):δ(ppm)172.82(2C,NHCOCH2),172.41(1C,COOCH2),171.93(1C,COOCH2),129.95(2C,CHCH),129.71(2C,CHCH),65.73(1C,COOCH2),64.94(1C,COOCH2),51.97(2C,CH3NCH2),51.75(2C,CH3NCH2CH2),50.23(1C,CH2NCH2),50.13(1C,CONHCH),44.43(1C,NCH3),43.97(1C,CONHCH2),31.86(1C,NCH2CH2,2C,NHCOCH2,1C,OCOCH2),31.54(1C,CH2CH2CH3),31.35(1C,CH2CH2CH3),29.73(4C,CH2(oleoyl)),29.64(2C,CH2(oleoyl)),29.48(2C,CH2(oleoyl)),29.42(2C,CH2(oleoyl)),29.27(6C,CH2(oleoyl)),28.59(1C,COOCH2CH2),28.51(1C,COOCH2CH2),27.19(4C,CH2CHCHCH2),26.89(1C,NHCHCH2),25.89(1C,COOCH2CH2CH2),25.83(1C,COOCH2CH2CH2),22.63(2C,CH2CH3),14.03(2C,CH2CH3).HRMS,ESI+,m/z:Calcd forC53H99N4O6[M+H]+,887.7559；found,887.7556.
实施例27
制备化合物OA11，化学结构式如下：
标题化合物OA11以化合物OA2-COOH(500mg，0.67mmol)和11b(114mg，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝12︰1)，得到白色凝胶状物380mg，收率：63.1％。1H NMR(300MHz,CDCl3):δ(ppm)7.40(s,1H,CONHCH),6.93(s,1H,CONHCH2),5.35(p,J＝7.5,6.9Hz,4H,CHCH),4.61-4.41(m,1H,CONHCH),4.09(dt,J＝15.7,7.4Hz,4H,COOCH2),3.69(s,8H,CH3NCH2CH2),3.36(s,2H,CONHCH2),3.21(s,2H,CH2NCH2),2.89(s,3H,NCH3),2.63(s,4H,COCH2CH2CO),2.43(d,J＝8.2Hz,2H,OCOCH2),2.25-2.08(m,2H,NHCHCH2),2.07-1.95(m,8H,CHCHCH2),1.89(s,2H,CH2NCH2CH2),1.72-1.51(m,2H,CH2NCH2CH2CH2,4H,OCOCH2CH2),1.37-1.23(m,44H,CH2(oleoyl)),0.89(t,J＝7.2Hz,6H,CH2CH3).13C NMR(75MHz,CDCl3):δ(ppm)172.73(2C,CONHN),172.53(1C,COOCH2),172.04(1C,COOCH2),129.88(2C,CHCH),129.64(2C,CHCH),65.60(1C,COOCH2),64.85(1C,COOCH2),51.94(2C,CH3NCH2),50.62(2C,CH3NCH2CH2),49.11(1C,NHCHCH2),49.05(1C,NCH3),43.34(1C,CH2NCH2),38.01(1C,CONHCH2),32.51(1C,NHCOCH2),31.81(2C,CH2(oleoyl)),31.43(1C,NHCOCH2),30.37(1C,OCOCH2),29.68(4C,CH2(oleoyl)),29.43(4C,CH2(oleoyl)),29.38(2C,CH2(oleoyl)),29.38(2C,CH2(oleoyl)),29.21(4C,CH2(oleoyl)),28.54(1C,COOCH2CH2),28.46(1C,COOCH2CH2),27.13(4C,CHCHCH2),27.01(2C,CONHCH2CH2CH2),26.27(1C,NHCHCH2),25.84(1C,COOCH2CH2CH2),25.82(1C,COOCH2CH2CH2),22.58(2C,CH2CH3),14.02(2C,CH2CH3).
实施例28
制备化合物OA12，化学结构式如下：
标题化合物OA12以化合物OA2-COOH(500mg，0.67mmol)和1-氨基-4-甲基哌嗪(81μL，0.67mmol)为原料，按照制备OA1的方法，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝15︰1)，得到黄白色凝胶状物300mg，收率：52.8％。1H NMR(300MHz,CDCl3):δ(ppm)8.72(s,1H,CONHN),7.02(s,1H,CONHCH),5.34(d,J＝5.7Hz,4H,CHCH),4.53(q,J＝6.7Hz,1H,CONHCH),4.07(dt,J＝13.9,6.9Hz,4H,COOCH2),3.48(s,8H,CH3NCH2CH2),3.24(s,2H,NHCOCH2),2.82(s,2H,NHCOCH2,2H,OCOCH2),2.65-2.49(m,3H,NCH3),2.39(q,J＝6.7,5.9Hz,2H,NHCHCH2),2.00(p,J＝9.1,7.6Hz,8H,CHCHCH2),1.62(q,J＝7.0Hz,4H,COOCH2CH2),1.35-1.21(m,44H,CH2(oleoyl)),0.93-0.84(m,6H,CH2CH3).13C NMR(75MHz,CDCl3):δ(ppm)172.63(1C,CONHN),172.27(1C,COOCH2),172.12(1C,COOCH2),171.90(1C,CONHCH),129.86(2C,CHCH),129.62(2C,CHCH),65.47(1C,COOCH2),64.74(1C,COOCH2),53.38(1C,NCH3),52.89(2C,CH3NCH2),51.81(1C,NHCHCH2),51.41(2C,CH3NCH2CH2),43.45(1C,NHCOCH2),43.17(1C,NHCOCH2),32.47(1C,OCOCH2),31.79(2C,CH2(oleoyl)),30.40(1C,NHCHCH2),29.65(4C,CH2(oleoyl)),29.56(2C,CH2(oleoyl)),29.40(2C,CH2(oleoyl)),29.34(2C,CH2(oleoyl)),29.19(4C,CH2(oleoyl)),29.14(2C,CH2(oleoyl)),28.55(1C,COOCH2CH2),28.47(1C,COOCH2CH2),27.12(4C,CH2CHCHCH2),25.82(1C,COOCH2CH2CH2),25.77(1C,COOCH2CH2CH2),22.54(2C,CH2CH3),13.95(2C,CH2CH3).HRMS,ESI+,m/z:Calcd for C50H93N4O6[M+H]+,845.7090；found,845.7077.
实施例29
制备谷氨酸双十四醇酯(TA2-NH2)，化学结构式如下：
向500mL反应瓶中加入L-谷氨酸(9.80g，66.68mmol)、对甲苯磺酸(12.60g，73.35mmol)和无水甲苯(300mL)，随后将反应液升温至140℃，回流反应3h。停止加热后，将混合溶液冷却至室温，分批加入十四醇(30.00g，139.93mmol)，140℃回流反应过夜。反应结束后，旋蒸除去甲苯，得到黄色油状物。将粗产物溶于二氯甲烷，用适量水洗涤两次，饱和碳酸氢钠水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到乳白色油状物。经柱层析纯化(石油醚︰乙酸乙酯＝6︰1)，得到白色固体21.60g，收率：60.0％。1HNMR(300MHz,CDCl3):δ(ppm)4.12(t,J＝6.8Hz,2H,COOCH2),4.08(t,J＝6.8Hz,2H,COOCH2),3.53-3.44(m,1H,NH2CH),2.47(t,J＝7.6Hz,2H,CH2CO),2.16-2.02(m,1H,NH2CHCH2),1.91-1.82(m,1H,NH2CHCH2),1.65-1.59(m,4H,COOCH2CH2),1.35-1.24(m,44H,CH2(myristoyl)),0.87(t,J＝6.9Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd for C33H66NO4[M+H]+,540.4986；found,540.4982.
实施例30
制备羧基化聚乙二醇2000(PEG2000-Suc)，化学结构式如下：
将mPEG2000-OH(400mg，0.20mmol)溶解于二氯甲烷，在室温下向反应瓶中依次加入丁二酸酐(200mg，2.00mmol)、DMAP(489mg，4.00mmol)，升温至45℃，回流反应48h。反应结束后，旋蒸除去溶剂，经柱层析纯化(二氯甲烷︰甲醇＝20︰1)，得到淡黄色油状物372mg，收率：88.6％。1H NMR(300MHz,CDCl3):δ(ppm)3.65(d,J＝14.9Hz,182H,OCH2CH2),3.26(s,3H,OCH3),2.64(t,J＝3.4Hz,2H,CH2COOH),2.05(t,J＝3.4Hz,2H,CH2CH2COOH).
实施例31
制备聚乙二醇2000丁二酸双十四醇酯(PEG2000-Suc-TA2)，化学结构式如下：
将PEG2000-Suc(200mg，0.10mmol)溶解于二氯甲烷，在0℃下依次加入EDCI(29mg，0.15mmol)、NHS(18mg，0.15mmol)并搅拌5min，随后将混合溶液移至室温反应3h，得到反应液A；将TA2-NH2(154mg，0.29mmol)溶解于二氯甲烷中，在室温下加入三乙胺(119μL，0.86mmol)，搅拌反应1h，得到反应液B。将反应液B缓慢滴加进反应液A，室温搅拌过夜。反应结束后，反应液用适量水洗涤两次，10％柠檬酸水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到淡黄色油状粗产物，经柱层析纯化(二氯甲烷︰甲醇＝25︰1)，得到淡黄色凝胶状物209mg，收率：83.9％。1H NMR(300MHz,CDCl3):δ(ppm)6.40(d,J＝7.7Hz,1H,CONHCH),4.57(dd,J＝12.9,7.8Hz,1H,CONHCH),4.23(dd,J＝9.3,3.4Hz,2H,COOCH2CH2O),4.10(s,2H,COOCH2),4.04(s,2H,COOCH2),3.65(d,J＝14.9Hz,182H,OCH2CH2),3.56-3.46(m,2H,COOCH2CH2O),3.36(s,3H,OCH3),2.72-2.60(m,2H,OCOCH2),2.51(t,J＝6.6Hz,2H,NHCOCH2),2.36(dd,J＝15.7,8.4Hz,2H,OCOCH2),2.17(d,J＝7.6Hz,2H,NHCHCH2),1.60(d,J＝6.4Hz,4H,COOCH2CH2),1.24(s,44H,CH2(myristoyl)),0.86(s,6H,CH2CH3).
实施例32
制备N-叔丁氧羰基氨基己酸双十四醇(TA2-Boc)，化学结构式如下：
将N-叔丁氧羰基-6-氨基己酸(643mg，2.78mmol)溶解于二氯甲烷，在0℃下依次加入EDCI(852mg，4.45mmol)、HOBt(601mg，4.45mmol)并搅拌5min，随后将混合溶液移至室温反应3h，得到反应液A；将TA2-NH2(1.50g，2.78mmol)溶解于二氯甲烷，在室温下加入三乙胺(1158μL，8.34mmol)，搅拌反应1h，得到反应液B。将反应液B缓慢滴加进反应液A，室温搅拌过夜。反应结束后，反应液用适量水洗涤两次，10％柠檬酸水溶液洗涤两次，饱和食盐水洗涤一次，无水硫酸钠干燥后，抽滤浓缩，得到淡黄色油状粗产物，经柱层析纯化(石油醚︰乙酸乙酯＝2︰1)，得到无色透明油状物1.33g，收率：63.6％。1H NMR(300MHz,CDCl3):δ(ppm)6.22(d,J＝7.7Hz,1H,OCONH),4.58(m,1H,OCOCHNH),4.14(t,J＝6.8Hz,2H,COOCH2),4.09(t,J＝6.8Hz,2H,COOCH2),3.09(d,J＝6.2Hz,2H,OCONHCH2),2.47-2.24(m,2H,OCOCH2),2.19(d,J＝7.6Hz,2H,NHCHCH2),1.90-1.69(m,2H,NHCOCH2),1.69-1.53(m,4H,OCH2CH2,2H,NHCH2CH2),1.43(s,9H,C(CH3)3),1.38-1.05(m,4H,COCH2CH2CH2,44H,CH2(myristoyl)),0.87(t,J＝6.5Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd for C44H84N2O7Na[M+Na]+,775.6171；found,775.6170.
实施例33
制备氨基己酸双十四醇盐酸盐(TA2-NH2·HCl)，化学结构式如下：
在0℃下向TA2-Boc(1.33g，1.77mmol)中缓慢滴加氯化氢-二氧六环溶液(53mL)，搅拌反应6h。反应结束后，旋蒸除去溶液，得到白色固体1.12g，收率：91.8％。1H NMR(300MHz,CDCl3):δ(ppm)6.37(d,J＝7.5Hz,1H,CONH),4.59(m,1H,OCOCHNH),4.12(t,J＝6.8Hz,2H,COOCH2),4.07(t,J＝6.8Hz,2H,COOCH2),2.49-2.30(m,2H,NH2CH2),2.20(m,2H,OCOCH2,2H,CHCH2,2H,NHCOCH2),1.64(m,4H,OCH2CH2,2H,NH2CH2CH2),1.53-1.43(m,2H,NH2),1.27(m,4H,COCH2CH2CH2,44H,CH2(myristoyl)),0.87(t,J＝6.4Hz,6H,CH2CH3).HRMS,ESI+,m/z:Calcd for C39H77N2O5[M+H]+,653.5827；found,653.5821.
实施例34
制备活性酯聚乙二醇2000双十四醇(NHS-TA2)，化学结构式如下：
将TA2-NH2·HCl(115mg，0.17mmol)溶解于二氯甲烷，滴加三乙胺(97μL，0.70mmol)，室温搅拌反应0.5h。向反应液中加入NHS-PEG2000-NHS，室温反应24h。反应结束后，旋蒸除去溶剂，得到棕色粗产物424mg，无进一步处理，直接进行下一步反应。
实施例35
制备氨基葡萄糖聚乙二醇2000双十四醇(GlcN-PEG2000-TA2)，化学结构式如下：
将D-氨基葡萄糖盐酸盐(40mg，0.184mmol)溶解于N,N-二甲基甲酰胺(DMF)中，滴加DIPEA(91μL，0.55mmol)，室温活化1h。将上一步中得到到的NHS-TA2溶解于DMF，加入活化后的D-氨基葡萄糖溶液中，室温搅拌过夜。反应结束后，反应液用去离子水透析(MWCO2000Da)3天后冻干，得到淡黄色棉絮状固体320mg。1H NMR(500MHz,DMSO-d6):δ(ppm)8.13(s,1H,C1H(GlcN)),4.22(s,1H,CONHCH),4.22-4.18(m,1H,C4OH(GlcN)),4.13(s,1H,C3OH(GlcN)),4.08(s,1H,C3H(GlcN)),4.05(s,1H,C6OH(GlcN)),4.03(s,1H,C1OH(GlcN)),4.00(d,J＝6.6Hz,2H,COOCH2),3.98(s,2H,COOCH2),3.70(s,1H,C2H,1H,C4H,1H,C5H,2H,C6H2,2H,NHCOOCH2),3.68-3.62(m,2H,NHCOOCH2CH2,2H,OCONHCH2,2H,NHCOCH2),3.52(d,J＝2.6Hz,182H,OCH2CH2),2.35(s,2H,OCOCH2),2.10(t,J＝7.4Hz,2H,OCOCH2CH2),1.53(d,J＝6.5Hz,4H,COOCH2CH2),1.50-1.44(m,2H,OCONHCH2CH2),1.23(s,4H,NHCOCH2CH2CH2,44H,CH2(myristoyl)),0.85(t,J＝6.9Hz,6H,CH2CH3).
实施例36
脂质纳米粒的制备与表征
制备方法：按照35:15:48:2的摩尔比精密称取不同氮磷比(N/P＝1，3，5，7，9，11)所需的可离子化脂质、DOPE、胆固醇、聚乙二醇化脂质和葡萄糖转运体1靶向脂质，具体处方如表1所示。
表1本发明LA1NP-LN9NP和OA1NP～OA9NP的处方
将所有脂质材料溶解于无水乙醇作为乙醇相。将pDNA溶于10mM柠檬酸盐缓冲液(pH＝4.0)作为水相。在剧烈搅拌状态下，将乙醇相快速注入水相，水相和乙醇相的体积比为5:1。注入完成后，样品使用超纯水在室温下透析4h，以除去体系中的乙醇。透析后的样品放置于4℃保存备用。使用Omni粒度电位分析仪测定LNP的粒径和电位，其粒径和电位如图1所示。
上述数据表明，本发明的脂质纳米粒粒径在30～220nm之间，符合作为基因载体粒径大小的要求；电位在+5～+40mV之间，表明所制备的脂质纳米粒表面电位较低，具有良好的安全性。
实施例37
脂质纳米粒对siRNA的荷载能力考察
按上述乙醇注入法按照不同的氮磷比(N/P＝3、5、7)制备荷载siRNA的脂质纳米粒siRNA/LA11NP，其处方为LA11/DOPE/Chol/PEG2000-Suc-TA2＝35/15/48/2(mol/mol)。通过琼脂糖凝胶电泳实验考察脂质纳米粒荷载siRNA的能力，如图2所示。结果表明，所有脂质纳米粒均能在一定N/P条件下稳定荷载siRNA，不发生泄漏，可进一步用于细胞转染实验。
实施例38
脂质纳米粒对mRNA的荷载能力考察
按上述乙醇注入法按照不同的氮磷比(N/P＝3、5、7、9)制备荷载mRNA的脂质纳米粒mRNA/LA11NP。通过琼脂糖凝胶电泳实验考察脂质纳米粒荷载mRNA的能力，如图3所示。结果表明，所有脂质纳米粒均能在一定N/P条件下稳定荷载mRNA，不发生泄漏，可进一步用于细胞转染实验。
实施例39
脂质纳米粒对pDNA的荷载能力考察
按上述方法按照不同的氮磷比(N/P＝1、3、5、7、9、11、13)制备荷载pDNA的脂质纳米粒pDNA/LA1NP-LN9NP和pDNA/OA1NP～OA9NP。通过琼脂糖凝胶电泳实验考察脂质纳米粒荷载pDNA的能力，如图4所示。结果表明，所有脂质纳米粒均能在一定N/P条件下稳定荷载pDNA，不发生泄漏，可进一步用于细胞转染实验。
实施例40
脂质纳米粒的稳定性
按上述方法制备荷载pDNA的脂质纳米粒pDNA/LA1NP～LA9NP和pDNA/OA1NP～OA9NP，分别加入T细胞培养基和超纯水并置于37℃分别静置0、2、4、6、8、24h后，通过粒径仪测量其粒径变化(图5)。结果显示脂质纳米粒在细胞培养基和超纯水中孵育24h后粒径基本不变，表明脂质纳米粒具有良好的体外稳定性。
实施例41
靶向脂质纳米粒转染人源T细胞
按上述方法制备荷载pDNA的靶向脂质纳米粒，GlcN-LANP和GlcN-OANP的处方分别为LA/DOPE/Chol/PEG2000-Suc-TA2/GlcN-PEG2000-TA2＝35/15/48/1/1(mol/mol)或OA/DOPE/Chol/PEG2000-Suc-TA2/GlcN-PEG2000-TA2＝35/15/48/1/1(mol/mol),其中LA选自LA4、LA10、LA11，OA选自OA4、OA10和OA11。转染时以1×106cells/mL的密度将人源T细胞接种于24孔板中，每孔加入0.5mL含CD3/CD28刺激剂和人源IL-2的T细胞培养基，最后加入100μL脂质纳米粒(含1μg pDNA)。将孔板放置于含5％二氧化碳的37℃细胞孵箱中培养48h后，通过倒置荧光显微镜观察人源T细胞中绿色荧光蛋白的表达情况，并用流式细胞仪定量考察人源T细胞中的绿色荧光强度。实验结果如图6和7所示，GlcN-LA4NP、GlcN-LA10NP、GlcN-LA11NP和GlcN-OA11NP转染人源T细胞有较明显的GFP表达。其中，本发明的脂质纳米粒GlcN-LA11NP在人源T细胞上的转染效果显著优于阳性对照Lipofectamine 2000。
实施例42
脂质纳米粒转染MCF-7细胞
按上述方法制备荷载pDNA的脂质纳米粒LA11NP，其处方为LA11/DOPE/Chol/PEG2000-Suc-TA2＝35/15/48/2(mol/mol)。取对数生长期的MCF-7细胞，调整细胞密度为2×105个/mL的细胞悬液，接种于24孔细胞板中，每孔接种细胞悬液500μL，置于37℃，5％CO2恒温培养箱内继续培养到细胞密度达80％左右。弃去培养液，用磷酸缓冲盐溶液(PBS)洗涤两次，各孔依次加入100μl脂质纳米粒400μl DMEM高糖培养基，使每孔pDNA的质量为1μg(n＝3)，继续培养6h后弃去旧培养基，用PBS洗涤三次，加入500μl含10％胎牛血清的DMEM高糖培养基继续培养48h后，通过倒置荧光显微镜观察MCF-7中绿色荧光蛋白的表达情况，并用流式细胞仪定量考察MCF-7中的阳性率。实验结果如图8和9所示，本发明的脂质纳米粒转染MCF-7有明显的绿色荧光表达，且阳性率显著高于阳性对照lipofectamine 2000。
实施例43
脂质纳米粒的膜融合能力测定
分别采用荧光共振能量转移(Fluorescence resonance energy transfer，FRET)实验和溶血实验考察了本发明的脂质纳米粒在不同pH条件下的膜融合能力和生理环境中的安全性。将NBD-Rhodamine荧光分子对掺入模拟内涵体/溶酶体膜的阴离子脂质体中。按上述方法制备脂质纳米粒LA1NP～LA11NP和OA1NP～OA11NP，分别使用PBS缓冲液(pH 7.4，1×PBS溶液)和柠檬酸盐缓冲液(pH 5.0和4.5，20mM柠檬酸盐，130mM NaCl)将其稀释，使可离子化脂质浓度均为95μM。向96孔板中依次加入100μL LNP溶液和1.0μL阴离子脂质体溶液，37℃下共孵5min后，用全功能微孔板检测仪测定荧光强度F，激发波长：480nm，发射波长：538nm。以50μL 1％Triton X-100溶液处理的阴离子脂质体溶液作为阳性对照，测得的荧光强度记为Fmax，未经任何处理的阴离子脂质体溶液作为阴性对照，测得的荧光强度记为Fmin，每个样品均设置三个平行实验，按照公式Lipid fusion(％)＝(F-Fmin)/(Fmax-Fmin)×100％计算各脂质纳米粒的膜融合效率。另外，从C57BL/6小鼠新鲜血液中提取红细胞，使红细胞浓度为4％(v/v)。用上述不同pH的缓冲液稀释空白LNP溶液，使其等同于含有0.0034μg/μL质粒的浓度。将100μL红细胞重悬液和100μL空白LNP溶液在37℃培养箱中共孵1h，随后在4℃下以1000g的转速离心5min；吸取100μL上清液，用全功能微孔板检测仪测定540nm处血红蛋白吸光度(Optical density，OD)。用100μL 0.1％Triton X-100溶液处理的红细胞作为阳性对照，测得的吸光度记为ODmax，未经任何处理的红细胞作为阴性对照，测得的吸光度记为ODmin，每个样品均设置三个平行实验，按照公式Hemolysis(％)＝(OD-ODmin)/(ODmax-ODmax)×100％计算各脂质纳米粒的溶血能力。
结果如图10和11所示，FRET实验和溶血实验结果均表明，本发明的所有脂质纳米粒在在正常生理条件(pH 7.4)和pH 5.0的条件下的膜融合能力都较低，在pH 4.5条件下的膜融合能力显著增强，说明其在生理环境中具有良好的安全性，并在晚期内涵体中能够保持结构的相对完整，而在溶酶体中实现有效的逃逸。据此可以推测，本发明可离子化脂质制备的脂质纳米粒可借助内涵体-溶酶体运输途径，由动力蛋白沿微管运输至细胞核附近，从溶酶体中逃逸后将pDNA释放于细胞核周围，具有增加pDNA入核效率的可能性。

Claims (11)

1.通式(I)所示的功能脂质：
其中，
当所述的通式(I)所示的功能脂质为聚乙二醇化脂质时，n＝2；m代表0-3的整数；X＝CH2；R2选自/>中的任意一种，其中u代表10-17的整数；
s＝10、23、45、78或113；t代表0-5的整数；
R6代表甲基；
当所述的通式(I)所示的功能脂质为葡萄糖转运体1靶向脂质时，n＝2；m代表1-4的整数；R1＝X＝NH；R2选自/>中的任意一种，其中u代表10-17的整数；
s＝10、23、45、78或113；t代表0-5的整数；
R6代表葡萄糖胺甲酰基。
2.权利要求1所述的功能脂质在制备荷载核酸药物的脂质纳米粒或制备基因药物中的应用。
3.一种荷载核酸药物的脂质纳米粒，其特征在于包含第一脂质、第二脂质、胆固醇以及第三脂质；所述的第一脂质选自可离子化脂质，所述的第二脂质选自中性磷脂，第三脂质选自权利要求1中所述的聚乙二醇化脂质或葡萄糖转运体1靶向脂质中的任一种或两种，其中第一脂质：第二脂质：胆固醇：第三脂质的摩尔比为25～60:10～30:30～60:0.5～10；
所述的可离子化脂质结构如通式(I)所示，
其中，n＝2；m代表1-3的整数；
R2选自中的任意一种；
p代表1-4的整数；q代表1-3的整数；r代表1-2的整数；R5代表甲基、乙基、羟乙基、正丙基、异丙基、叔丁基、苯基或苄基。
4.根据权利要求3所述的荷载核酸药物的脂质纳米粒，其特征在于第一脂质：第二脂质：胆固醇：第三脂质的摩尔比为30～50:10～20:40～60:0.5～2.5。
5.根据权利要求3所述的荷载核酸药物的脂质纳米粒，其特征在于所述的中性磷脂为1,2-二油酰-sn-甘油-3-磷酸乙醇胺、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱、二肉豆蔻酰磷脂酰胆碱、大豆磷脂、1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺、1-硬脂酰-2-油酰-sn-甘油-3-磷酸胆碱、1-棕榈酰基-2-油酰基卵磷脂、二芥酰基卵磷脂、1,2-二油酰-sn-甘油-3-磷酸胆碱、二棕榈酰基卵磷脂。
6.根据权利要求5所述的荷载核酸药物的脂质纳米粒，其特征在于所述的中性磷脂为DOPE。
7.根据权利要求3所述的荷载核酸药物的脂质纳米粒，其特征在于所述的核酸药物选自pDNA、siRNA、shRNA、microRNA和mRNA中的任意一种。
8.根据权利要求7所述的荷载核酸药物的脂质纳米粒，其特征在于所述的核酸药物为pDNA。
9.根据权利要求3所述的荷载核酸药物的脂质纳米粒，其特征在于所述的荷载核酸药物的脂质纳米粒中可离子化脂质和核酸药物的氮磷比为1:1～30:1。
10.根据权利要求9所述的荷载核酸药物的脂质纳米粒，其特征在于所述的荷载核酸药物的脂质纳米粒中可离子化脂质和核酸药物的氮磷比为3:1～8:1。
11.权利要求3-10中任一项所述的荷载核酸药物的脂质纳米粒在pDNA转染、siRNA基因沉默、mRNA疫苗或CRISPR/Cas9基因编辑中的应用。
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