New Multifunctional Bioorthogonal Probes

在化学系生命过程化学（CLP）项目的支持下，威斯康星大学麦迪逊分校化学系的Jennifer Schomaker教授旨在开发新的生物相容性化学工具，以研究与正常和功能失调生物学相关的多种相互依赖的过程。所提出的工具的易于合成，加上利用计算研究来调整其互补（生物正交）反应性的能力，是这类新化合物的吸引人的特征。这些工具的实用性将被用来开发选择性的、靶向的分子递送方法，并标记能够穿透血脑屏障的单链抗体片段。所有的发现都将广泛地提供给更广泛的科学界，以促进合作，促进和增加拟议工作的影响。该研究计划的要素将被纳入多课程实验模块，涵盖分析化学、有机化学、计算化学和化学生物学，以提高学生对解决需要多学科方法的科学问题的理解和欣赏。Schomaker实验室将与威斯康星大学麦迪逊分校化学机会（CHOPs）合作，这是一个致力于提高研究生多样性的项目。参与者将参观研究设施，与教师/学生会面，并了解西澳大学跨学科研究的机会。阐明复杂生物过程的功能、动力学和相互依赖细节的重要性，推动了创新工具的设计，以研究细胞系统的行为。设计用于探测生物系统的试剂必须高度稳定，生物相容性，化学选择性和非混杂，即缺乏非特异性标记的倾向。由于这些限制，研究细胞内发生的过程具有挑战性，特别是在同时实时询问多个生物分子时。尽管目前生物正交探针的范围很广，但大多数都是为了检测单一生物事件而设计的，并且通常存在速率慢、化学选择性差/脱靶反应性差、不稳定或无效吸收等问题，这些问题限制了对细胞表面的标记。这项工作介绍了一类新的杂环炔，称为“SNO-OCTs”，其中炔的极化率可预测地调整为与不同的“点击”伙伴互斥的生物正交性。这些强大的工具有可能允许在体外和体内观察多个同时或顺序的信号事件。它们的动力学、生物正交性和物理性质应该是分子可调的，这使得它们有可能适合特定的应用。此外，SNO-OCT支架的多功能性和模块化将被用于开发“点击释放”策略，以将小分子、荧光探针和生物分子递送到特定位点。这些新工具的潜在应用包括控制蛋白质激活，荧光团激活，通过成像和释放气体递质或药物到目标位置来检测RNA和其他生物分子。基于sno - oct的工具，用于制备、生物正交标记和观察单链抗体片段（scFv），这些单链抗体片段能够穿过血脑屏障（BBB）。在生物环境中，SNO-OCTs缺乏与硫亲核试剂的竞争性反应活性，这使得酵母表面的scFv能够有效地同时功能化，避免了传统的可溶性蛋白表达和纯化的需要。在标记/成像事件之后，可以用亲核试剂打开SNO-OCT环，以“重写”scFv以进一步功能化和分析。威斯康辛州的研究小组计划将这些探测器提供给更广泛的科学界，以扩大它们的科学范围和影响。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Professor Jennifer Schomaker in the Department of Chemistry at the University of Wisconsin-Madison aims to develop new biocompatible chemical tools to study diverse and interdependent processes associated with both normal and dysfunctional biology. The ease of synthesis of the proposed tools, coupled with the ability to employ computational studies to tune their complementary (bioorthogonal) reactivity, are attractive features of this new class of compounds. The utility of these tools will be harnessed to develop selective, targeted molecular delivery methods and to tag single-chain antibody fragments capable of permeating the blood-brain barrier. All findings will be made widely available to the broader scientific community to stimulate collaborations that advance and increase the impact of the proposed work. Elements of this research program will be incorporated into multi-course laboratory modules that span analytical, organic and computational chemistry and chemical biology to increase student understanding and appreciation for tackling scientific problems that require multidisciplinary approaches. The Schomaker Lab will partner with UW-Madison Chemistry Opportunities (CHOPs), a program committed to enhancing graduate student diversity. CHOPs participants will tour research facilities, meet with faculty/students and learn about opportunities for interdisciplinary research at UW.The importance of elucidating details of the function, dynamics, and interdependence of complex biological processes drives the design of innovative new tools to study the behavior of cellular systems. Designed reagents used to probe biological systems must be highly stable, biocompatible, chemoselective, and non-promiscuous, i.e. devoid of the propensity for non-specific labeling. Due to these constraints, studying processes occurring inside cells is challenging, especially when interrogating multiple biomolecules simultaneously in real time. Despite the breadth of current bioorthogonal probes, most are designed to examine a single biological event and often suffer from slow rates, poor chemoselectivity/off-target reactivities, instability or ineffective uptake that limits labeling to a cell surface. This work introduces a new class of heterocyclic alkynes, termed ‘SNO-OCTs’, where the polarizability of the alkyne is predictably tuned for mutually exclusive bioorthogonality with diverse ‘click’ partners. These powerful tools have the potential to allow for the observation of multiple simultaneous or sequential signaling events in vitro and in vivo. Their kinetics, bioorthogonality and physical properties should be molecularly tunable, allowing them to be potentially tailored for specific applications. Moreover, the versatility and modularity of SNO-OCT scaffolds is to be exploited to develop ‘click-and-release’ strategies to deliver small molecules, fluorescent probes and biomolecules to specific sites. Potential applications for these new tools include controlled protein activation, fluorophore activation to detect RNA and other biomolecules via imaging and release of gasotransmitters or drugs to targeted locations. SNO-OCT-based tools for the preparation, bioorthogonal labeling and observation of single-chain antibody fragments (scFv) that are able to cross the blood-brain barrier (BBB) are to be developed. The lack of competing reactivity of SNO-OCTs with sulfur nucleophiles in the biological milieu enables efficient simultaneous functionalization of scFv from yeast surfaces and avoids the traditional need for soluble protein expression and purification. The SNO-OCT ring can be opened with nucleophiles subsequent to the labeling/imaging event to ‘rewrite’ the scFv for further functionalization and analysis. The Wisconsin research team plans to make these probes available to the broader scientific community to broaden their scientific reach and impact.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.