State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China.
Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China.
Molecules. 2018 Jan 12;23(1):154. doi: 10.3390/molecules23010154.
C5-modified uridines are a valuable class of nucleoside analogues, both as potent chemotherapy agents and through their use as the conjunction site in DNA labeling strategies. As an important C5-modified uridine, BrdU has been used in cell proliferation assays since the 1980s. Currently, the detection of BrdU relies on traditional immunostaining; however, this approach has its limitations. Thus, it is desirable, albeit difficult, to develop chemistry methods to fluorescently label BrdU in a cellular context. In the present study, we report our efforts toward developing a robust chemistry methodology for BrdU fluorescent labeling. The Sonogashira reaction was chosen as the key reaction, and various alkynyl groups (aliphatic or aryl) containing fluorescent dyes were synthesized to cross-couple with BrdU. Various bases and catalyst systems were screened to evaluate the optimum conditions. A mild aqueous Sonogashira reaction (K₂PdCl₄, S-Phos, -Bu₄N⁺OH, Sodium d-isoascorbate, EtOH/H₂O = 1:1, 37 °C, Ar) was obtained to enable high-yielding BrdU fluorescent labeling.
C5-修饰的尿嘧啶核苷是一类很有价值的核苷类似物，可作为有效的化疗药物，也可作为 DNA 标记策略中的连接位点。BrdU 作为一种重要的 C5-修饰尿嘧啶核苷，自 20 世纪 80 年代以来就被用于细胞增殖检测。目前，BrdU 的检测依赖于传统的免疫染色；然而，这种方法有其局限性。因此，开发一种在细胞环境中对 BrdU 进行荧光标记的化学方法是可取的，尽管这很困难。在本研究中，我们报告了开发 BrdU 荧光标记稳健化学方法的努力。选择 Sonogashira 反应作为关键反应，并合成了含有荧光染料的各种炔基（脂肪族或芳基）以与 BrdU 交叉偶联。筛选了各种碱和催化剂体系以评估最佳条件。获得了温和的水相 Sonogashira 反应（K₂PdCl₄、S-Phos、-Bu₄N⁺OH、Sodium d-isoascorbate、EtOH/H₂O = 1:1、37°C、Ar），以实现高产率的 BrdU 荧光标记。
