NEW LIGANDS FOR BORONATE AFFINITY-CHROMATOGRAPHY - SYNTHESIS AND PROPERTIES
NEW LIGANDS FOR BORONATE AFFINITY-CHROMATOGRAPHY - SYNTHESIS AND PROPERTIES
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DOI：
10.1016/s0021-9673(01)95752-8
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发表时间：
1991-04-26
期刊：
影响因子：
--
通讯作者：
SARWAR, Y
中科院分区：
文献类型：
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作者：
SINGHAL, RP;RAMAMURTHY, B;SARWAR, Y
为了使硼酸盐配体在纯化生物分子的亲和层析中有用，它必须能够在亲和分子稳定的环境（pH）中形成稳定的复合物。广泛使用的配体3-氨基苯基硼酸盐的一个主要限制是其高电离常数（pK(A) 8.75）。为了使该配合物在更有利的pH条件下，本文探索了不同的方法，以便在苯基环上引入一个吸电子（硝基）基团。介绍了用三氟甲烷磺酸硝基制备丁二脒苯硼酸邻、间和对硝基衍生物的试剂和方法。通过选择性地使用乙酸酐作为反应介质，利用硝基官能团优先取代硼酸的邻位。在选定的反应条件下，该方法主要生成邻硝基衍生物（pK(a) 7.4）。用B-11核磁共振和分光光度法研究了几种苯硼酸盐在溶液中的电离和溶质-配体相互作用。结果表明，中性离子（δ 30）、硼酸阴离子（δ 3）和顺式二醇络合硼酸盐（δ 7.5）存在特定的化学位移。在有顺式二醇衍生物的情况下，络合物的形成比中性物质的阴离子化更有利。此外，该配合物的形成比配体的电离常数低约一个pH单位，并且是稳定的，即即使溶液pH值明显提高，也不会在硼酸阴离子中分解。考察了两种硼酸亲和柱基质的结合能力和表观解离常数。结果清楚地表明，硼酸盐配体中吸电子基团的存在极大地促进了配合物的形成和分解。结果进一步表明，亲和分子的微小结构差异对其结合能力有显著差异。烷基顺式二醇和芳基顺式二醇与不同硼酸基质的结合比较表明，芳基亲和分子不仅能形成络合物，而且能很有效地形成络合物。这项工作的意义在于证明了在溶液中进行研究可以建立配体-溶质相互作用的最佳环境，而无需事先固定配体。溶液研究的结果与亲和色谱柱的结果非常吻合。新的硝基苯基硼酸盐基质提供了增强的结合大多数亲和分子比那些检查与苯基硼酸盐基质。此外，新的基质提供了碱不稳定生物分子的色谱分离。
In order for a boronate ligand to be useful in affinity chromatography for the purification of biomolecules, it must be able to form a stable complex in an environment (pH) in which the affinity molecule is stable. A major limitation of the widely used ligand, 3-aminophenylboronate, is its high ionization constant (pK(a) 8.75). To make this complex under more favorable pH conditions, different methods have been explored here in order to introduce an electron-withdrawing (nitro) group in the phenyl ring. Reagents and procedures for the preparation of ortho-, meta- and para-nitro derivatives of succinamidophenylboronic acid using nitronium trifluoromethanesulfonate are described. Preferential substitution of the nitro functionality into the ortho position of the boronic acid is exploited by selective use of acetic anhydride for the reaction medium. This method yields mostly an ortho-nitro derivative (pK(a) 7.4) under selected reaction conditions. The ionization and solute-ligand interaction of several phenylboronates are studied in solution by using B-11 NMR and spectrophotometric methods. The results indicate the presence of specific chemical shifts for the neutral (delta 30), the boronate anions (delta 3), and the cis-diol-complexed boronate species (delta 7.5). In the presence of a cis-diol derivative, the complex formation is favored over anionization of neutral species. Moreover, the complex is formed approximately one pH unit below the ionization constant of the ligand and is stable, i.e. fails to break down in boronate anion, even when the solution pH is raised appreciably. Two boronate affinity column matrices were examined for their binding capacity and apparent dissociation constant. The results clearly indicate that the formation and also the breakdown of the complex are greatly enhanced because of the presence of the electron-withdrawing group in the boronate ligand. The results further demonstrate that small structural differences in affinity molecules have significant differences on their binding capacities. A comparison of binding between alkyl-cis-diols and aryl-cis-diols to different boronate matrices indicates that the aryl affinity molecules not only form a complex but do so very effectively. The significance of this work lies in the demonstration that the best environment for the ligand-solute interaction can be established by carrying out studies in solution, without prior immobilization of the ligand. The results derived from in-solution studies and those from the affinity columns are in very good agreement. The new nitrophenylboronate matrix offers enhanced binding of most affinity molecules over those examined with the phenylboronate matrix. In addition, the new matrix offers chromatographic separations of alkali-unstable biomolecules.