摘要
以氨基酸为配体，树枝状大分子聚酰胺-胺（PAMAM）为间隔臂，根据点击化学的原理设计和制备对免疫球蛋白（IgG）具有高选择性，且成本低廉、安全、吸附性能与蛋白A吸附材料相当的新型免疫吸附材料。通过系统地考察含不同间隔臂的免疫吸附材料对人血浆中IgG的吸附性能，研究免疫吸附材料的间隔臂长度、结构和刚/柔性等对配体偶联和免疫吸附材料的吸附性能的影响，从而探索一条能简单，高效、可控地制备仿生物特异性的非蛋白类免疫吸附材料的新途径。
用廉价、稳定、安全且具有可修饰性的的氨基酸小分子为仿生配体代替常用的蛋白A制备免疫吸附材料，可避免蛋白类免疫吸附材料价格高昂、易脱落，存在安全隐患等缺陷；采用点击化学——Huisgen1,3-偶极环加成反应来实现配体与活性载体的偶联，可大大增强这两者间的反应选择性，从而增加配体的固载量和稳定性。此外，点击反应条件温和，在较宽范围的溶剂、温度和pH下均可发生，且对很多官能团呈惰性，因而很少甚至没有副产物，能尽量保持配体官能团的完整性和活性，从而保持其对抗体的亲和力。另一方面，选择外层带有大量活性官能团的PAMAM树枝状大分子为间隔臂，则可望利用这些官能团键接比线型间隔臂多得多的配体，从而提高所设计和制备的免疫吸附材料的配体含量，并改善其吸附性能。
基于上述研究背景和思路，本课题主要开展了以下几个方面的研究和探索：
（1）以琼脂糖凝胶（Sep）为载体，用常规法制备了分别以不同氨基酸（AA）为仿生配体的免疫吸附材料Sep-AA；测定了它们的配体含量，并通过对人血浆中IgG的吸附试验，对比分析了Sep-AA和用相同方法制备的蛋白A免疫吸附材料Sep-PA的吸附性能。
（2）基于Huisgen1,3-偶极环加成反应的原理和要求，分别将载体Sep和配体AA通过适宜的化学修饰制备成―可点击‖的叠氮化活性载体Sep-N_3与含炔基的配体；将二者偶联制备了仿生物特异性的非蛋白类免疫吸附材料Sep-triazole-AA；表征了Sep-triazole-AA的配体含量和对人血浆中IgG的吸附性能，并与常规法制备的免疫吸附材料Sep-AA进行对比分析，研究了点击化学对吸附材料的结构和性能的影响。
（3）以炔丙胺为核心，采用发散法合成了0.5～4.0代端基炔化的PAMAM树状大分子，并表征和分析了所得产物的结构，为将PAMAM用作免疫吸附材料的间隔臂奠定了基础。
（4）通过树状大分子PAMAM外层的官能团与配体AA的反应，制得带端炔基树状间隔臂的“可点击”配体，并将这一配体偶联到―可点击‖的活性载体Sep-N_3上，以制备带树状间隔臂的免疫吸附材料Sep-PAMAM-AA；表征了其结构、配体含量和吸附性能，并与间隔臂为线型分子链的相应的吸附材料Sep-triazole-AA进行对比分析，研究了树枝状间隔臂PAMAM的结构与免疫吸附材料的IgG吸附性能之间的关系。从本课题的研究可得到的主要研究结果如下：
（1）在免疫吸附材料Sep-AA的常规法制备中，琼脂糖载体上的活性基团含量随着环氧化、胺化、醛基化和配体的偶联等一系列反应的进行而逐步减少，使得所制备的分别以L-组氨酸（His）、L-苯丙氨酸（Phe）和L-色氨酸（Trp）为配体的免疫吸附材料Sep-His、Sep-Phe和Sep-Trp的配体含量较低。它们对IgG的吸附容量分别为2.88、2.62和2.53mg/g，远低于蛋白A吸附材料Sep-PA的IgG吸附容量22.97mg/g；所制备的Sep-AA对人血浆中的IgG有较高的选择性，几乎没有非特异性吸附。这一结果证明了氨基酸作为仿生配体的可行性。
（2）用点击化学的方法成功地合成了三种以不同氨基酸为配体的免疫吸附材料Sep-triazole-His、Sep-triazole-Phe和Sep-triazole-Trp。它们的配体含量和对人血浆中IgG的吸附容量均明显地优于相应的用常规法制备的免疫吸附材料Sep-AA；其中，Sep-triazole-His的IgG吸附容量最高，达到16.49mg/g，但仍比不上Sep-PA。研究进一步证明，Sep-triazole-His具有与Sep-PA相当的对IgG的高吸附选择性；其间隔臂结构中由点击反应生成的三唑环能够促进IgG的结合而不引起非特异性吸附。
（3）成功地设计与合成了G1.0～G4.0共4代外层带大量氨基的炔化PAMAM；并用点击化学的方法制备了带有不同的树状间隔臂和不同的氨基酸配体的免疫吸附材料Sep-PAMAM-AA。对产物结构的分析表明，在四种间隔臂G1.0～G4.0PAMAM中，以G3.0PAMAM键接的配体数最多，键接效率也最高。氨基酸修饰的PAMAM与活性载体Sep-N_3的点击反应选择性很高，反应效率接近100%；所得免疫吸附材料Sep-PAMAM-AA对人血浆中IgG的吸附容量均较高，大部分能与蛋白A免疫吸附材料相媲美。其中，用G3.0PAMAM作间隔臂的免疫吸附材料Sep-G3-His具有优异的吸附选择性和高达28.43mg/g的IgG吸附容量，是较理想的免疫吸附材料。
总之，本研究探索了一种简单、高效、可控的方法来设计和制备更安全、更有效、更经济的免疫吸附材料。该方法不仅能克服常规制备法固有的缺陷，还能提高免疫吸附材料的偶联效率和吸附性能。研究结果为新型免疫吸附材料的设计、制备以及在IgG分离和血液净化方面的应用奠定了坚实的理论和实验基础，可望产生极大的社会效益和经济效益。
In this work, using amino acids as ligands and polyamidoamine (PAMAM) dendrimersas spacer-arms, a novel immunoadsorbent possessing pseudo-biospecific affinity forimmunoglobulin (IgG), which is cheap, safe and reliable and can have an adsorptionperformance comparable to protein A immunoadsorbent, was designed and prepared via clickchemistry. The IgG adsorption performance of the immunoadsorbents with different spacer-arms from human plasma were systematically investigated and the effects of the length,structures and rigidity/flexibility of the spacer-arms on their adsorption performance werealso studied, thus, exploring a novel route to preparing pseudo-biospecific non-proteinousimmunoadsorbents by a simple, efficient and controllable method.
As biomimetic or pseudo-biospecific ligands, small molecular amino acids with low cost,high chemical and physical stability, good safety and modifiability are promising alternativesto protein A ligand for preparing immunoadsorbents, which can help to overcome the defectsof proteinaceous immunoadsorbents in the cost and potential safety hazards. Click chemistry—the Huisgen1,3-dipolar cycloaddition reaction used to achieve the coupling between theligand and the activated support can greatly enhance the reaction selectivity, therebyincreasing the density and stability of the ligands immobilized onto the support. Besides, clickreaction can be conducted under mild conditions and it is inert to most chemicalfunctionalities and stable to wide ranges of solvent, temperature and pH. Consequently, itgenerates little or no by-products and the integrity and activity of the functional group of theligand immobilized onto the support via the click reaction can be maintained as far as possible,so keeping the affinity of the ligand. On the other hand, choosing the PAMAM dendrimerswith a great number of reactive groups on the peripheral ends as spacer-arms will make itpossible to bond much more ligands by use of these functional groups than the linear spacer-arms with one reactive end groups, thus leading to an increase in the ligand density and animprovement in the adsorption performance of the designed and prepared immunoadsorbent.
Based on the above-mentioned research background and ideas, the main research workincludes the following aspects:(1) A group of immunoadsorbents, Sep-AA, were prepared by a conventional method using
sepharose (Sep) as a support and different amimo acids (AA) as pseudo-biospecific
ligands, respectively. The ligand density of the prepared immunoadsorbents was
determined, and the adsorption performance of them and the protein A immunoadsorbent Sep-PA prepared by the same method was compared and analyzed through an adsorptionexperiment of IgG from human plasma.
(2) According to the principle of Huisgen1,3-dipolar cycloaddition reaction, sepharose as asupport and amino acids as ligands were transformed into the―clickable‖reactive supportor azidated sepharose (Sep-N_3) and the―clickable‖alkyne-containing ligands via suitablechemical modifications, respectively; and then each of the―clickable‖ligands wascoupled with the―clickable‖reactive support to prepare pseudo-biospecific non-proteinous immunoadsorbents Sep-triazole-AA. The ligand density and IgG adsorptionperformance of Sep-triazole-AA were characterized and compared with those of theimmunoadsorbents Sep-AA prepared by the conventional method. The influence of clickchemistry on the structure and the properties of the immunoadsorbents was also discussed.
(3) A series of PAMAM dendrimers with terminal alkyne from generation0.5(G0.5) togeneration4.0(G4.0) was synthesized by the divergent strategy using2-propynylamineas the core; and the structure of the products was characterized and analyzed, therebylaying the foundation for using the PAMAM dendrimers as the spacer-arms ofimmunoadsorbents.
(4) The―clickable‖ligands combined with alkyne-containing dendritic spacer-arms wereprepared by the reaction between the functional groups on the peripheral ends of thePAMAM dendrimers and an amino acid ligand, and coupled with the―clickable‖reactivesupport Sep-N_3to prepare the immunoadsorbents Sep-PAMAM-AA bearing dendriticspacer-arms. Their structure, ligand density and adsorption performance werecharacterized and compared with those of the corresponding immunoadsorbents Sep-triazole-AA whose spacer-arm is a linear molecular chain. The relationship between thestructure of the PAMAM dendritic spacer-arms and the IgG adsorption performance of theimmunoadsorbents Sep-PAMAM-AA was also studied.
The main conclusions drawn from this study are as follows:
(1) In the conventional preparation of immunoadsorbents Sep-AA, the content of activegroups in the sepharose support will decrease gradually as a series of reactions, includingthe epoxidization of Sep and the amination, hydroformylation and coupling reaction ofmodified Sep, are conducted, thus resulting in low ligand density of the products Sep-His,Sep-Phe and Sep-Trp, whose ligands are L-histidine (His), L-phenylalanine (Phe) and L-tryptophan (Trp), respectively. The IgG adsorption capacity of Sep-His, Sep-Phe and Sep-Trp was measured to be2.88,2.62and2.53mg/g, respectively, which are much lower than that of the protein A immunoadsorbent Sep-PA, or22.97mg/g. All of the preparedimmunoadsorbents Sep-AA, Sep-His, Sep-Phe and Sep-Trp, can exhibit high adsorptionselectivity for IgG from human plasma and almost have no non-specific adsorption. Itproves the feasibility of using amino acids as the pseudo-biospecific ligand ofimmunoadsorbents.
(2) The immunoadsorbents Sep-triazole-His, Sep-triazole-Phe and Sep-triazole-Trp weresuccessfully prepared by click chemistry using three kinds of amino acids His, Phe andTrp as the ligand, respectively. They are obviously superior in the ligand density and theadsorption capacity to the corresponding immunoadsorbents Sep-AA prepared by aconventional method. Among the three immunoadsorbents, Sep-triazole-His shows thehighest IgG adsorption capacity, which is as high as16.49mg/g, but still lower than thatof Sep-PA. The further studies prove that Sep-triazole-His can exhibit high adsorptionselectivity for IgG comparable to Sep-PA and that the1,2,3-triazole ring in its spacer-arm, which is produced via the click reaction between Sep-N_3and L-histidine withterminal alkyne, can facilitate the binding of IgG without non-specific adsorption.
(3) Generation1.0~4.0(G1.0~4.0) PAMAM dendrimers with terminal alkyne and multipleamino groups on the peripheral ends were successfully designed and synthesized; and thenthe immunoadsorbents with different dendritic spacer-arms and different amino acids asligands, Sep-PAMAM-AA, were prepared via click chemistry. The structure analyses ofthe products show that among the four spacer-arms, G1.0~4.0PAMAM dendrimers, G3.0PAMAM can covalently combine with the most number of ligands and achieve thehighest bonding efficiency; and that the click reaction between the dendritic PAMAMmodified amino acids, PAMAM-AA, and the activated support Sep-N_3possesses veryhigh selectivity and the reaction efficiency is almost close to100%. The preparedimmunoadsorbents Sep-PAMAM-AA all possess high adsorption capacity of IgG fromhuman plasma, and most of them can exhibit an IgG adsorption capacity comparable tothat of Sep-PA. By comparison, the immunoadsorbent Sep-G3-His, whose spacer-arm isG3.0PAMAM, possesses an excellent adsorption selectivity for IgG and the adsorptioncapacity up to28.43mg/g, so it is an ideal immunoadsorbent.
In a word, a simple, efficient and controllable method has been explored in this study todesign and prepare safe, effective and inexpensive immunoadsorbents. This method can notonly overcome the inherent defects of conventional methods, but also improve theimmobilization efficiency and adsorption performance of the immunoadsorbents. Theobtained research results lay a solid theoretical and experimental foundation for the design and preparation of novel immunoadsorbents and their applications in the fields of IgGseparation and blood purification, which will be expected to generate great social andeconomic benefits.
引文
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