Department of Chemistry, University of Toronto, Toronto, Ontario, Canada.
Biomacromolecules. 2012 Aug 13;13(8):2359-69. doi: 10.1021/bm300613x. Epub 2012 Jul 19.
Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined. Here we describe the synthesis of a new type of MCP based on anionic ring-opening polymerization of an activated cyclopropane (the diallyl ester of 1,1-cyclopropane dicarboxylic acid) using a combination of 2-furanmethanethiol and a phosphazene base as the initiator. This reaction takes place with rigorous control over molecular weight, yielding a polymer with a narrow molecular weight distribution, reactive pendant groups for introducing a metal chelator, and a functional end group with orthogonal reactivity for attaching the polymer to the mAbs. Following the ring-opening polymerization, a two-step transformation introduced diethylenetriaminepentaacetic acid (DTPA) chelating groups on each pendant group. The polymers were characterized by NMR, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The binding properties toward Gd(3+) as a prototypical lanthanide (Ln) ion were also studied by isothermal titration calorimetry (ITC). Attachment to a mAb involves a Diels-Alder reaction of the terminal furan with a bismaleimide, followed by a Michael addition of a thiol on the mAb, generated by mild reduction of a disulfide bond in the hinge region. Polymer samples with a number average degree of polymerization of 35, with a binding capacity of 49.5 ± 6 Ln(3+) ions per chain, were loaded with 10 different types of Ln ions and conjugated to 10 different mAbs. A suite of metal-tagged Abs was tested by mass cytometry in a 10-plex single cell analysis of human adult peripheral blood, allowing us to quantify the antibody binding capacity of 10 different cell surface antigens associated with specific cell types.
金属螯合聚合物（MCP）是基于质谱流式细胞术的多重免疫分析的重要试剂。聚合物的作用是携带多个单个金属同位素，通常为镧系元素离子，并提供方便连接单克隆抗体（mAb）的反应性功能。对于这种应用，还需要确定链长、骨架结构、端基、悬垂基团和合成策略的最佳组合。在这里，我们描述了一种基于环状丙烯（1,1-环丙烷二羧酸的二烯丙酯）的阴离子开环聚合合成新型 MCP 的方法，该聚合反应使用 2-糠硫醇和磷杂环戊二烯碱的组合作为引发剂。该反应对分子量进行严格控制，得到一种分子量分布较窄的聚合物，具有用于引入金属螯合剂的反应性悬垂基团，以及用于将聚合物连接到 mAb 的功能端基。开环聚合后，通过两步转化在每个悬垂基团上引入二乙三胺五乙酸（DTPA）螯合基团。通过 NMR、尺寸排阻色谱（SEC）和热重分析（TGA）对聚合物进行了表征。通过等温滴定量热法（ITC）还研究了聚合物对 Gd（3+）作为典型镧系（Ln）离子的结合特性。与 mAb 的连接涉及末端呋喃与双马来酰亚胺的 Diels-Alder 反应，然后通过在铰链区域中二硫键的轻度还原生成的 mAb 上的硫醇进行迈克尔加成。具有 35 个数均聚合度的聚合物样品，每个链的结合能力为 49.5±6Ln（3+）离子，负载了 10 种不同类型的 Ln 离子并与 10 种不同的 mAb 偶联。通过质谱流式细胞术在人类成人外周血的 10 重单细胞分析中测试了一系列金属标记的 Abs，使我们能够定量测量与特定细胞类型相关的 10 种不同细胞表面抗原的抗体结合能力。
