Maleimide derivatives are privileged reagents for chemically modifying proteins through the Michael addition reaction with cysteine due to their selectivity, operational simplicity, and commercial availability. However, since accessible free cysteine is rarely found in natural proteins, it is highly desirable to find alternative targets to enable direct bioconjugation of proteins with maleimides. In this study, we have developed an operationally simple and straightforward method for the N-terminal modification of proteins without the need for mutagenesis via a copper(II)-mediated [3+2] cycloaddition reaction with maleimides and 2-pyridinecarboxaldehyde (2-PCA) derivatives under non-denaturing conditions at pH 6 and 37 °C in aqueous media. Our method utilizes commercially available maleimides to attach diverse functionalities to various N-terminal amino acids. We demonstrate the preparation of a ternary protein complex cross-linked at the N-termini and dually modified trastuzumab equipped with monomethyl auristatin E (MMAE), a cytotoxic agent, and a Cy5 fluorophore (MMAE–Cy5–trastuzumab). MMAE–Cy5–trastuzumab retained human epidermal growth factor receptor 2 (HER2) recognition activity and exerted cytotoxicity against HER2-positive cells. Furthermore, MMAE–Cy5–trastuzumab allowed successful visualization of HER2-positive cancer cells in mouse tumors. This straightforward method will expand the accessibility of protein conjugates with well-defined structures in a wide range of research fields.

中文翻译：

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基于马来酰亚胺的蛋白质 N 端一步法双重功能化


马来酰亚胺衍生物是通过与半胱氨酸的 Michael 加成反应对蛋白质进行化学修饰的特权试剂，因为它们具有选择性、操作简单性和商业可用性。然而，由于在天然蛋白质中很少发现可接近的游离半胱氨酸，因此非常需要找到替代靶标以实现蛋白质与马来酰亚胺的直接生物偶联。在这项研究中，我们开发了一种操作简单明了的方法，用于蛋白质的 N 端修饰，无需通过铜 （II） 介导的 [3+2] 环加成反应与马来酰亚胺和 2-吡啶甲醛 （2-PCA） 衍生物在 pH 6 和 37 °C 的非变性条件下在水性介质中。我们的方法利用市售的马来酰亚胺将不同的官能团连接到各种 N 端氨基酸上。我们展示了在 N 末端交联的三元蛋白复合物和双重修饰的曲妥珠单抗的制备，该复合物配备单甲基澳瑞他汀 E （MMAE）、细胞毒剂和 Cy5 荧光团 （MMAE-Cy5-曲妥珠单抗）。MMAE-Cy5-曲妥珠单抗保留了人表皮生长因子受体 2 （HER2） 识别活性，并对 HER2 阳性细胞发挥细胞毒性。此外，MMAE-Cy5-曲妥珠单抗允许成功观察小鼠肿瘤中 HER2 阳性癌细胞。这种简单的方法将扩大具有明确结构的蛋白质偶联物在广泛研究领域的可及性。