New Multifunctional Bioorthogonal Probes

在化学部生命过程化学（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样化和相互依赖的过程。所提出的工具的合成的容易性，加上采用计算研究来调整其互补（生物正交）反应性的能力，是这类新化合物的有吸引力的特征。这些工具的效用将被利用来开发选择性的、有针对性的分子递送方法，并标记能够渗透血脑屏障的单链抗体片段。所有研究结果将广泛提供给更广泛的科学界，以促进合作，推动和增加拟议工作的影响。该研究计划的元素将被纳入跨分析，有机和计算化学和化学生物学的多课程实验室模块，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（CHOP）合作，该计划致力于提高研究生的多样性。CHOP的参与者将参观研究设施，与教师/学生见面，并了解在UW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，推动了创新的新工具的设计，以研究细胞系统的行为。用于探测生物系统的设计试剂必须是高度稳定的、生物相容的、化学选择性的和非混杂的，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，特别是在真实的时间内同时询问多个生物分子时。尽管目前的生物正交探针的广度，大多数被设计为检查一个单一的生物事件，并经常遭受缓慢的速度，差的化学选择性/脱靶反应性，不稳定性或无效的摄取，限制标记到细胞表面。这项工作介绍了一类新的杂环炔，被称为“SNO-OCT”，其中炔的极化率可预测地调整为与不同的“点击”合作伙伴相互排斥的生物正交性。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传导事件。它们的动力学、生物正交性和物理性质应该是分子可调的，从而使它们能够针对特定应用进行定制。此外，SNO-OCT支架的多功能性和模块化将被开发用于开发“点击和释放”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受控的蛋白质激活，荧光团激活以通过成像检测RNA和其他生物分子，以及将气体递质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过血脑屏障（BBB）的单链抗体片段（scFv）。SNO-OCT与硫亲核试剂在生物环境中的竞争反应性的缺乏使得能够从酵母表面有效地同时官能化scFv，并且避免了对可溶性蛋白质表达和纯化的传统需要。SNO-OCT环可以在标记/成像事件之后用亲核试剂打开，以“重写”scFv用于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，以扩大其科学范围和影响。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.