Fluorescent probes that span the spectrum for multi-color imaging of peroxynitrite in living cells

The Colorado School of Mines received a $540,053 Project Grant award, effective September 5, 2025, through the National Institute of General Medical Sciences (NIGMS) Biomedical Research and Research Training program (CFDA 93.859) to develop fluorescent imaging probes for detecting peroxynitrite and other reactive oxygen and nitrogen species in living cells. Over the three-year performance period (through August 31, 2028), the institution will design and synthesize a comprehensive toolkit of optical probes with excitation and emission profiles spanning the visible spectrum from blue to red wavelengths, enabling multi-color, multi-analyte imaging capabilities. The research leverages principles from physical organic chemistry, synthetic chemistry, and molecular probe design to advance understanding of diazaborines—a novel chemical motif for selective metabolite detection—with applications in cell culture models of macrophages to elucidate the roles of reactive oxygen and nitrogen species in inflammation-related diseases.

Embedded within this research initiative is an educational component designed to provide undergraduate students at the Colorado School of Mines with hands-on research training in biomedical chemistry and instrumentation, preparing them for careers in the increasingly collaborative and diverse biomedical research workforce. The deliverables from this award will contribute foundational knowledge and practical tools to advance redox biology research and improve understanding of oxidative stress mechanisms underlying cancer, cardiovascular disease, respiratory diseases, autoimmune diseases, and stroke.

Abstract

Reactive oxygen species and reactive nitrogen species are oxidative metabolites that play important roles in human health and disease. While these oxidative metabolites are required for a diverse array of cellular processes, including cell death, differentiation, and signaling, uncontrolled production of reactive oxygen and nitrogen species can contribute to the underlying inflammation of cancer, heart disease, respiratory diseases, autoimmune diseases, and stroke. We are developing new imaging probes that span the visible spectrum to map reactive oxygen and nitrogen species with improved specificity and versatility to help understand the roles these oxidative metabolites play in inflammation. The scientific premise is that building an imaging toolkit for oxidative metabolites will provide new tools to further our understanding of the dynamics of reactive oxygen and nitrogen species in redox biology. This R15 application uses principles from physical organic chemistry, synthetic chemistry, and molecular probe design to develop new peroxynitrite-specific imaging probes with optical characteristics enable multi-analyte imaging. These tools will be applied in cell culture models of macrophages. Specifically, we will understand the structure-function relationship of diazaborines, a new motif for selective metabolite detection, and pursue diazaborine-based probes with excitation/emission profiles spanning from blue to green to red. Embedded in this work is a research-centric pedagogy to train undergraduates at the colorado school of mines to join an increasingly collaborative and diverse biomedical workforce.