Efficient and site-specific chemical modification of proteins under physiological conditions remains a challenge. Here we report that 1,4-dinitroimidazoles are highly efficient bifunctional bioconjugation reagents for protein functionalization and peptide macrocyclization. Under acidic to neutral aqueous conditions, 1,4-dinitroimidazoles react specifically with cysteines via a cine-substitution mechanism, providing rapid, stable and chemoselective protein bioconjugation. On the other hand, although unreactive towards amine groups under neutral aqueous conditions, 1,4-dinitroimidazoles react with lysines in organic solvents in the presence of base through a ring-opening & ring-close mechanism. The resulting cysteine- and lysine-(4-nitroimidazole) linkages exhibit stability superior to that of commonly employed maleimide-thiol conjugates. We demonstrate that 1,4-dinitroimidazoles can be applied in site-specific protein bioconjugation with functionalities such as fluorophores and bioactive peptides. Furthermore, a bisfunctional 1,4-dinitroimidazole derivative provides facile access to peptide macrocycles by crosslinking a pair of cysteine or lysine residues, including bicyclic peptides of complex architectures through highly controlled consecutive peptide macrocyclization.

中文翻译：

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二硝基咪唑作为双功能生物偶联试剂，用于蛋白质功能化和肽大环化。

在生理条件下对蛋白质进行有效的位点特异性化学修饰仍然是一个挑战。在这里，我们报告1,4-二硝基咪唑是用于蛋白质功能化和肽大环化的高效双功能生物偶联试剂。在酸性至中性的水性条件下，1,4-二硝基咪唑可通过电影取代机制与半胱氨酸发生特异性反应，从而提供快速，稳定和化学选择性的蛋白质生物结合。另一方面，尽管在中性水溶液条件下不与胺基反应，但是1，4-二硝基咪唑通过开环和闭环机理在碱的存在下与有机溶剂中的赖氨酸反应。所得的半胱氨酸和赖氨酸-（4-硝基咪唑）键表现出优于常用的马来酰亚胺-硫醇缀合物的稳定性。我们证明了1,4-二硝基咪唑类化合物可以用于具有荧光和生物活性肽等功能的位点特异性蛋白质生物缀合。而且，双官能的1，4-二硝基咪唑衍生物通过交联一对半胱氨酸或赖氨酸残基，包括复杂结构的双环肽，通过高度受控的连续肽大环化作用，提供了对肽大环的便捷访问。