Further studies on the protein conjugation of hydroxamic acid bifunctional chelating agents: group-specific conjugation at two different loci.
Further studies on the protein conjugation of hydroxamic acid bifunctional chelating agents: group-specific conjugation at two different loci.
复制标题
异羟肟酸双功能螯合剂蛋白质缀合的进一步研究：两个不同位点的基团特异性缀合。
DOI：
10.1021/bc980045d
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Buchsbaum,DJ
中科院分区：
文献类型：
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作者：
Safavy,A;Khazaeli,MB;Kirk,M;Coward,L;Buchsbaum,DJ
最近报道了一种利用活化酯方法将未受保护的异羟肟酸与抗体和肽缀合的方法。这里描述了一种比活化酯策略具有优势的替代方法。该方案利用三异羟肟酸配体三琥珀酸的酰肼衍生物与抗体的酮衍生物或通过碳水化合物残基氧化产生的醛基之间形成腙。因此，三琥珀酸(1)用肼基甲酸叔丁酯衍生为酰肼2，并通过催化氢化和TFA酸解除去保护基，得到异羟肟酰肼4。 4与单克隆抗体CC49的缀合通过两种方法实现：通过抗体的胺(例如，赖氨酸)残基连接或碳水化合物残基的氧化。通过评估缀合物与未缀合抗体相比分子量的增加，MALDI 监测缀合程度。本报告介绍了第一种利用酮连接体（6-氧代庚酸，OHA）作为酰肼锚的方法，作为酰肼衍生物与蛋白质缀合的新技术。事实证明，在操作简便性和蛋白质回收率方面，OHA 方法是优于醛方法的策略。它还具有在调节配体与蛋白质比率方面产生更多控制的优点，因此被选择用于方案优化。由这两种方法产生的所有缀合物均用125 I进行放射性标记并筛选其免疫反应性。此外，通过优化的OHA方案制备的缀合物用99mTc和125I进行放射性标记，报告了放射性标记产量和免疫反应性。
A procedure utilizing an activated ester approach for conjugation of unprotected hydroxamic acids to antibodies and peptides was recently reported. Here, an alternative method with advantages over the activated ester strategy is described. This protocol utilizes the hydrazone formation between a hydrazide derivative of the trihydroxamate ligand trisuccin and either a ketone derivative of antibody or the aldehyde groups, generated by oxidation of the carbohyrate residues. Thus, the trisuccin carboxylic acid (1) was derivatized withtert-butyl carbazate to the hydrazide2, and the protecting groups were removed by catalytic hydrogenation and acidolysis with TFA to afford the hydroxamate hydrazide4. Conjugation of4to monoclonal antibody CC49 was effected by two approaches: attachment through the amine (e.g., lysine) residues of the antibody or oxidation of the carbohydrate residues. The extent of conjugations were monitored by MALDI, through evaluation of the increases in molecular weights of the conjugates compared to the unconjugated antibody. The first approach utilizing a ketone linker (6-oxoheptanoic acid, OHA) which served as a hydrazide anchor, is being introduced in this report as a new technique for conjugation of hydrazide derivatives to proteins. The OHA approach proved to be a superior strategy over the aldehyde approach in the ease of the procedure and yield of protein recovery. It also had the advantage of yielding more control in adjusting the ligand-to-protein ratio and was therefore selected for protocol optimization. All conjugates resulting from both approaches were radiolabeled with125I and screened for their immunoreactivity. Furthermore, the conjugates prepared through the optimized OHA protocol were radiolabeled with both99mTc and125I for which the radiolabeling yields and immunoreactivities are reported.