New Multifunctional Bioorthogonal Probes

在化学系生命过程化学(CLP)计划的支持下，威斯康星大学麦迪逊分校化学系的Jennifer Schomaker教授致力于开发新的生物兼容化学工具，以研究与正常和功能失调的生物学相关的各种和相互依赖的过程。建议的工具易于合成，再加上使用计算研究来调整其互补(生物正交)反应性的能力，是这类新化合物的诱人特征。这些工具的用途将被利用来开发选择性的、有针对性的分子传递方法，并标记能够渗透血脑屏障的单链抗体片段。所有发现都将广泛提供给更广泛的科学界，以刺激促进和增加拟议工作的影响的合作。这项研究计划的内容将被纳入跨越分析、有机化学和计算化学以及化学生物学的多门课程实验模块，以增加学生对解决需要多学科方法的科学问题的理解和欣赏。Schomaker实验室将与密歇根大学麦迪逊化学机会(CHOPS)合作，这是一个致力于增强研究生多样性的项目。CHOPS的参与者将参观研究设施，与教职员工/学生会面，并了解在威斯康星大学进行跨学科研究的机会。阐明复杂生物过程的功能、动力学和相互依赖的细节的重要性推动了设计创新的新工具来研究细胞系统的行为。设计的用于探测生物系统的试剂必须高度稳定、生物相容、化学选择性和非混杂，即没有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是具有挑战性的，特别是当实时同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，但大多数探针都是设计用于检查单个生物事件，并且往往存在速度慢、化学选择性差/靶外反应、不稳定或无效摄取等问题，从而限制了对细胞表面的标记。这项工作引入了一类新的杂环炔类化合物，称为‘SNO-OCTs’，其中炔的极化率可以预测地调整为与不同的‘点击’伙伴的互斥生物正交性。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连续的信号事件。它们的动力学、生物正交性和物理性质应该是分子可调的，允许它们潜在地为特定的应用量身定做。此外，SNO-OCT支架的多功能性和模块化将被用来开发“点击并释放”策略，将小分子、荧光探针和生物分子输送到特定的位置。这些新工具的潜在应用包括受控的蛋白质激活，通过成像检测RNA和其他生物分子的荧光团激活，以及将气体传递器或药物释放到目标位置。将开发基于SnO-OCT的工具，用于制备、生物正交标记和观察能够穿越血脑屏障(BBB)的单链抗体片段(ScFv)。SNO-OCTs在生物环境中缺乏与硫亲核试剂的竞争反应能力，因此能够有效地同时从酵母表面实现单链抗体的功能化，从而避免了传统上对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’单链抗体以进行进一步的功能化和分析。威斯康星州的研究团队计划将这些探测器提供给更广泛的科学界，以扩大它们的科学覆盖范围和影响。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.