Conjugation of unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, to monoclonal antibody CC49 by an improved active ester protocol.
Conjugation of unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, to monoclonal antibody CC49 by an improved active ester protocol.
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通过改进的活性酯方案将未保护的三琥珀酸 N-[三[2-[(N-羟基氨基)羰基]乙基]甲基]琥珀酰胺酸与单克隆抗体 CC49 缀合。
DOI：
10.1021/bc970127m
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发表时间：
1997
期刊：
影响因子：
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通讯作者：
Buchsbaum,DJ
中科院分区：
文献类型：
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作者：
Safavy,A;Sanders,A;Qin,H;Buchsbaum,DJ
为了将三羟甲酸酯双功能螯合agentN-[tris[2-[[N-(benzyloxy)amino]carbonyl]ethyl]methyl]succinamic酸(1)偶联到抗体上，我们最初使用了相应的2，3，5，6-四氟苯基活性酯，然后通过催化氢化去除了苄基保护基团。我们感兴趣的是设计一种能够将解封的异羟甲酸酯掺入多肽和蛋白质中的接合方案。根据产品缺乏188Re放射标记能力的判断，报告的程序被预期与三糖素中存在的功能相兼容，但没有成功。然后利用未保护的三糖素的邻硝基苯酚活化酯N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic酸3开发了一种简单的偶联方法，该方法消除了接合后解封的需要。建立了一种间接估计活性酯含量的方法，该方法基于其分解副产物ONP-OH的浓度。将间接估计的浓度与直接从纯化产品中获得的浓度进行比较，表明该检测方法的准确率为90%。这一过程的优点是快速使用未纯化的活性酯，消除其通过溶剂分解或未保护的异羟甲酸官能团自我缩合的可能性。建立了一种比色法来估计每个蛋白质分子的配体数量。本实验和所有的偶联物均用188Re放射性标记的事实表明，该程序将未受保护的羟基甲酸酯配体偶联到CC49单抗上。这些结果表明，这项技术在无保护的羟基甲酸酯衍生物与其他蛋白质和多肽的偶联方面具有潜在的适用性。
For the conjugation of the trihydroxamate bifunctional chelating agentN-[tris[2-[[N-(benzyloxy)amino]carbonyl]ethyl]methyl]succinamic acid (trisuccin,1) to antibodies, we originally used the corresponding 2,3,5,6-tetrafluorophenyl active ester followed by the postconjugation removal of the benzyl protecting groups by catalytic hydrogenation. It was of interest to us to design a conjugation protocol capable of incorporating deblocked hydroxamates into peptides and proteins. Reported procedures that were expected to be compatible with the functionalities present in trisuccin were used with no success, as judged by the lack of ability of the products to radiolabel with188Re. A simple conjugation method was then developed utilizing theo-nitrophenol (ONP) activated ester of the unprotected trisuccin,N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid,3, which eliminates the need for the postconjugation deblocking. An assay for indirect estimation of the active ester content, based on the concentration of its decomposition byproduct, ONP-OH, was developed. Comparison of the indirectly estimated concentrations with those obtained directly from purified products showed >90% accuracy for this assay. This procedure has the advantage of rapidly using the unpurified active ester, eliminating the possibilities of its decomposition through solvolysis or self-condensation by the unprotected hydroxamate functions. A colorimetric assay was developed for estimation of the number of ligands per molecule of protein. This assay and the fact that all conjugates consistently radiolabeled with188Re show that this procedure conjugated the unprotected hydroxamate ligands to the CC49 monoclonal antibody. These results indicate the potential applicability of this technique to conjugation of unprotected hydroxamate derivatives with other proteins and peptides.