New Multifunctional Bioorthogonal Probes

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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.