A Protein Functionalization Platform Based on Selective Modification at Methionine Residues

大自然经常对蛋白质进行位点选择性修饰，使功能多样性显著增加。相比之下，蛋白质的合成操作受到适当化学转化的可用性的限制。然而，获得合成修饰的蛋白质对化学生物学、分子生物学和医学已经变得至关重要。这刺激了对与生物系统相容的化学转化发展的深入研究。理想情况下，一个反应应该在蛋白质上的单个位点具有选择性，其速率与复杂分子的动力学需求相称；应在环境条件下操作，以防止破坏蛋白质结构或功能；在近乎完美的转换中提供同质产品。尽管存在这些挑战，但在过去的20年里，已经出现了许多令人兴奋的方法，用于在体外和体内，在蛋白质中的天然和非天然氨基酸残基上进行转化。虽然大多数化学方法都集中在扩大半胱氨酸（Cys）和赖氨酸（Lys）残基反应的工具箱，但对非天然氨基酸（通过遗传编码）的转化产生了浓厚的兴趣，这些氨基酸显示出与标准残基具有正交反应性的侧链功能。探测生物过程的试剂往往依靠相对简单的反应来规避复杂环境中的化学问题。然而，需要补充工具（对Cys和Lys的反应），通过以前未开发的氨基酸选择性地产生功能性蛋白质偶联物。蛋氨酸（Met）是一种蛋白质原性氨基酸，显示出许多特征，使其成为潜在的生物偶联靶标：例如，蛋氨酸在蛋白质中的丰度<2%，易于编码，在辅助蛋白质功能中的作用有限（主要是防止氧化应激），并通过其弱亲核s原子包含可能的反应处理。直到最近，实际的met选择性生物偶联还是未知的。在我们最初工作的同时，Chang等人报道了一种氧化还原激活的标记策略，该策略将Met转化为亚砜亚胺衍生物，并成功地将其应用于生物学驱动的应用中。我们已经开发了一种基于使用高价碘试剂（我们称之为MetSIS，意思是蛋氨酸选择性碘盐）的蛋氨酸选择性蛋白质功能化策略，该试剂与多肽和蛋白质选择性反应，通常在低于1分钟的时间内，在低浓度的水中将>95%转化为稳定的重氮-磺酸偶联物。我们关于met -生物偶联的初步工作最近发表在Nature 2018, 562, 563-568。任何单一生物体中蛋白质组的结构多样性意味着没有一种蛋白质功能化方法能够为蛋白质结构的制备提供通用的解决方案。半胱氨酸结扎是蛋白质标记的基准，它是快速的，有选择性的，并不断发展强大的方法，可应用于大量的化学生物学和生物医学应用。尽管Cys已经建立了许多独特的应用，但如果另一种氨基酸可以普遍用于蛋白质功能化，与Cys互补呢？在这里，我提出蛋氨酸（Met）的化学靶向可以作为蛋白质修饰的不同策略的基础。通过这项奖学金，我概述了一个合成驱动蛋白质功能化的计划，这些生物大分子在Met标记用于体外和（可能）体内环境，从而扩大了化学生物学家可用的工具。平行研究（阶段a和b）将产生蛋白质功能化工具，并协同进入阶段C，这将寻求将合成进展转化为应用。

Nature routinely carries out site-selective modification of proteins, enabling a dramatic increase in functional diversity. In contrast, synthetic manipulation of proteins is restricted by the availability of suitable chemical transformations. However, access to synthetically modified proteins has become fundamentally important to chemical biology, molecular biology & medicine. This has stimulated intensive research into the development of chemical transformations that are compatible with biological systems. Ideally, a reaction should be selective at a single site on a protein at a rate that is commensurate with the kinetic demands of complex molecules; should operate under ambient conditions to prevent disruption of the protein architecture or function; & provide homogeneous products in near perfect conversion. Despite these challenges, the past 20 years have seen a number of exciting methodologies emerge for executing transformations, both in vitro & in vivo, at natural & non-natural amino-acid residues in proteins. While most chemical methods have focussed on expanding the toolkit for reaction at cysteine (Cys) & lysine (Lys) residues, there has been burgeoning interest in transformations at non-natural amino acids (via genetic encoding) that display side chain functionality with orthogonal reactivity to standard residues. Reagents that probe biological processes tend to rely on relatively simple reactions to circumvent problems with chemistry in complex environments. However, there is a need for complementary tools (to reactions at Cys & Lys) that selectively produce functional protein conjugates via previously unexplored amino acids.Methionine (Met) is a proteogenic amino acids & displays a number of features which make it potentially amenable as a bioconjugation target: For example, Met has a <2% abundance in proteins, is easily encoded, has a limited role in ancillary protein function (mainly protection against oxidative stress) & contains a possible reactive handle via its weakly nucleophilic S-atom. Until recently, practical Met-selective bioconjugation was unknown. Concurrent with our initial work, Chang et al reported a redox-activated tagging strategy that converted Met to sulfoximine-derivatives & successfully applying it in biology-driven applications. We have developed a methionine-selective protein functionalization strategy based on the use of hypervalent iodine reagents (we call these reagents MetSIS, meaning Methionine Selective Iodonium Salts) that react selectively with polypeptides & proteins, often giving >95% conversion to a stable diazo-sulfonium conjugate at low concentration in H2O in <1 minute. Our initial work on Met-bioconjugation was recently published Nature 2018, 562, 563-568.The structural diversity of the proteome in any single organism means that no one protein functionalization method will provide universal solutions to the preparation of protein constructs. Cys-ligation is the benchmark for protein labelling and is is fast, selective and continues to evolve powerful methods that can be applied to a plethora of chemical biology & bio-medical applications. Even though Cys has founded many distinct applications, what if another amino acid can be generally harnessed for protein functionalization that is complementary to Cys? Here, I propose that chemical targeting of methionine (Met) can be the basis of distinct strategies for protein modification. Through this Fellowship, I outline a program for synthesis-driven protein functionalization, where these biomacromolecules are labelled at Met for use in vitro & (possibly) in vivo environments thereby expanding the tools available to chemical biologists. Parallel lines of enquiry (Phases A&B) will generate protein functionalization tools & synergistically feed into a Phase C, which will seek to translate the synthesis advances to application.