The late-stage site-specific functionalisation of peptides and proteins via polyoxometalate-mediated H-atom abstraction

问题和重要性：在过去的二十年里，基于蛋白质的治疗和诊断试剂的监管批准增加了。蛋白质与其靶标结合的无与伦比的亲和力和选择性确保了这种生物制剂可以有效地用于医疗用途。此外，将天然蛋白质应用于疾病的治疗可以开发出副作用更少的治疗方法，从而减少毒性。特别是在肿瘤学中，基于蛋白质的生物制剂已经走到了癌症诊断和治疗的前沿。然而，尽管在这一领域取得了进展，但仍在积极寻求更安全、更有效的办法。使用显像剂或细胞毒性化合物对临床上相关的蛋白质序列进行功能化进一步增强了它们的治疗应用，并导致了对蛋白质修饰技术的浓厚兴趣。高效、化学选择性和普遍适用的生物偶联化学的发展将使更先进的生物制剂的生产最终可能在临床上产生实质性的影响。与可持续性的关系：尽管开发了几种特定部位的蛋白质修饰技术，但到目前为止使用最多的反应是靶向半胱氨酸残基或赖氨酸，半胱氨酸残基是蛋白质结构和功能所必需的，赖氨酸的丰度导致异质性修饰异构体的产生。不使用天然残基的替代技术可能使用非标准氨基酸，这一策略涉及相关生物机制的定向进化。涉及化学合成肽的半合成技术也得到了广泛的应用，但这种方法使用有毒试剂，使用大量溶剂，限制了这种方法的可持续性。发展一种高效、温和的位点选择性生物偶联反应，可以广泛应用于任何生物表达的蛋白质序列，靶向不是蛋白质结构和功能所必需的天然残基，将是一个非常有影响力的进展。建议的解决方案和方法我们建议开发一种策略，利用自由基化学对肽和蛋白质进行后期特定部位的功能化。多金属氧酸盐(POM)簇能够从未活化的脂肪族三级中心中提取氢原子，从而促进所选氨基酸的氟化。我们希望进一步探索这一POM介导的策略，扩大该方法的范围，使多肽和蛋白质能够以位点选择性的方式实现更广泛的功能。通过将这种H原子提取方法与一个小型的修饰自由基捕捉库相结合，我们的目标是在几个氨基酸残基上的自由基形成位置安装所需的功能(例如放射性核素或细胞毒性化合物)。从一个简单的十钨原子簇开始，我们将探索和利用POM的可调性质来增加控制和位置选择性。该反应将是温和的，在水溶液中快速进行，只需要催化添加POM。我们的战略将是量身定做的，以支持引入一个工具箱的功能，可以应用于任何选定的蛋白质。最终，我们将寻求利用我们的技术使以高侵袭性三阴性乳腺癌表达的受体为靶点的蛋白质具有功能性。

Problems and importance:The last two decades have witnessed an increase in the regulatory approval of protein-based therapeutic and diagnostic agents. The unparalleled affinity and selectivity with which proteins bind to their target, ensures that such 'biologics' can be employed efficaciously for medical use. Additionally, the application of native proteins to the treatment of disease enables the development of therapeutics which may exhibit fewer side effects and thus reduced toxicity. Within oncology especially, protein-based biologics have stepped to the forefront of cancer diagnosis and treatment. However, despite advances within this field, safer and more efficacious approaches are actively sought. Functionalising clinically relevant protein sequences with imaging agents or cytotoxic compounds further enhances their therapeutic application and has led to significant interest in protein modification techniques. The development of efficient, chemoselective and generally applicable bioconjugation chemistry will enable the production of more advanced biologic agents which may ultimately have a substantial impact in the clinic.Relevance to sustainability:Despite the development of several site-specific protein modification techniques, the most utilised reactions to-date target cysteine residues, which are integral to protein structure and function, or lysine, the abundance of which causes the production of heterogeneously modified isoforms. Alternative techniques that do not make use of native residues may employ non-standard amino acids, a strategy that involves directed evolution of the relevant biologically machinery. Semi-synthetic techniques involving chemically synthesised peptides have also been widely utilised, however, such methods employ toxic reagents and use large quantities of solvent which limits the sustainability of this approach. The development of an efficient and mild site-selective bioconjugation reaction that could be broadly applied to any biologically expressed protein sequence, targeting native residues that are not integral to protein structure and function, would be a highly impactful advance. Proposed solution and methodologyWe propose the development of a strategy for the late-stage site-specific functionalisation of peptides and proteins utilising radical chemistry. Polyoxometalate (POM) clusters have previously been reported to enable H-atom abstraction from unactivated aliphatic tertiary centres, facilitating the fluorination of selected amino acids. We wish to further explore this POM-mediated strategy, expanding the scope of the approach to enable the broader functionalisation of peptides and proteins in a site-selective manner. By combining this H-atom abstraction method with a small library of modified radical traps, we aim to install desired functionality (e.g. radioactive nuclides or cytotoxic compounds) at the site of radical formation on several amino acid residues. Starting with a simple decatungstate cluster, we will explore and utilise the tunable properties of the POM to increase control and site-selectivity. The reaction will be mild, proceeding rapidly in an aqueous solution, requiring only catalytic addition of the POM. Our strategy will be tailored to enable the introduction of a toolbox of functionality which could be applied to any chosen protein. Ultimately, we will seek to employ our technique to functionalise proteins that target receptors expressed by highly invasive triple-negative breast cancer.