Protein poly- and oligophosphorylation are recently discovered post-translational modifications that remain poorly characterized due to (1) the difficulty of extracting endogenously polyphosphorylated species without degradation and (2) the absence of synthetic and analytical tools to prepare and characterize poly- and oligophosphorylated species in biochemical contexts. Herein, we report a methodology for the selective oligophosphorylation of peptides with monodisperse phosphate chain lengths (Pn = 3–6). A library of oligophosphorimidazolide (oligoP-imidazolide) reagents featuring benzyl and o-nitrophenylethyl protecting groups were synthesized in moderate-to-good yields (65–93%). These oligoP-imidazolide reagents enabled the selective and simultaneous conjugation of multiple phosphate units to phosphoryl nucleophiles, circumventing tedious iterative processes. The generalizability of this approach is illustrated by a substrate scope study that includes several biologically relevant phosphopeptide sequences, culminating in the synthesis of >60 examples of peptide oligophosphates (Pn = 2–6). Moreover, we report the preparation of oligoP-diimidazolides (Pn = 3–5) and discuss their application in generating unique condensed phosphate-peptide conjugates. We also demonstrate that human phospho-ubiquitin (pS65-Ub) is amenable to functionalization by our reagents. Overall, we envision the methods described here will enable future studies that characterize these newly discovered but poorly understood phosphorylation modes.

中文翻译：

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通过受保护的寡磷酸咪唑试剂对肽进行单分散化学寡磷酸化


蛋白质多磷酸化和寡磷酸化是最近发现的翻译后修饰，由于 （1） 难以在不降解的情况下提取内源性多磷酸化物质，以及 （2） 在生化环境中缺乏合成和分析工具来制备和表征多磷酸化和寡磷酸化物质，因此其表征仍然很差。在此，我们报道了一种对具有单分散磷酸盐链长度 （Pn = 3-6） 的肽进行选择性寡磷酸化的方法。以中等至良好的收率 （65–93%） 合成了具有苄基和邻硝基苯乙基保护基团的寡磷酸咪唑化物 （oligoP-咪唑化物） 试剂库。这些 oligoP-咪唑试剂能够将多个磷酸盐单元选择性地同时偶联到磷酸基亲核试剂上，从而避免了繁琐的迭代过程。底物范围研究说明了这种方法的普遍性，该研究包括几个生物学相关的磷酸肽序列，最终合成了 >60 肽寡磷酸盐实例 （Pn = 2-6）。此外，我们报道了 oligoP-二咪唑醇 （Pn = 3-5） 的制备，并讨论了它们在生成独特的缩合磷酸肽偶联物中的应用。我们还证明，人磷酸泛素 （pS65-Ub） 适合通过我们的试剂进行官能化。总的来说，我们设想这里描述的方法将使未来的研究能够表征这些新发现但知之甚少的磷酸化模式。