Abstract The overall goal of the Fehl laboratory is to develop chemical biology strategies to determine the functional impact of protein modifications during signaling processes. Specifically, cellular metabolism and stress each lead to diverse protein modifications with O-linked N-acetylglucosamine sugar (O-GlcNAc) but no tools are currently able to capture highly dynamic and transient O-GlcNAc events with defined time and spatial resolution. Lack of “time and space” rigor hinders the scientific community from connecting metabolism with disease physiology, including significantly elevated cancer risk in diabetic patients observed for malignancies like breast cancer. In this MIRA application, we pose our strategies to address this critical gap through the development of real-time and space molecular tools that bridge cell metabolism and cancer processes using O-GlcNAc as the keystone. Excellent NIH-funded research has discovered over 2000 O-GlcNAc on proteins in human cells, but current tools rely on disrupted physiology, leading to artifacts, or miss key GlcNAc-driven signaling events that occur before global metabolic rebalancing occurs in less than an hour. We hypothesize that the key drivers of hyperglycemic metabolism and pathology lie within the first few minutes of nutrient and signaling stimulation, which to date is not possible to observe in living cells. Our published work in photochemistry and systems glycobiology support our unique strategies to trigger O-GlcNAc processes in minutes, before O-GlcNAc rebalancing occurs. Our photocaged sugar tool is able to trigger the oncogenic transcription factor NFkB movement between cytosol or endoplasmic reticulum into the nucleus, simulating physiological events that potentially link aberrant insulin and glucose release in diabetes with breast cancer risk. Our real time system can be used to track O-GlcNAc events during insulin signaling for the first time during the rapid, 15-minute pulses of diseased insulin physiology. Another tool for targeted intracellular O-GlcNAc-targeted proximity labeling is able to track O-GlcNAcylated proteins in subcellular space, which no reported tool has the capability to specifically label in live cells. We propose in the next 5 years to develop our “time and space” molecular tools and apply them for unique mechanistic studies in disease biology through NFkB targeting. We actively collaborate with metabolic disease and cancer specialists to ensure disease relevance, as well as with industrial scientists for technology development to expand industrial awareness of O-GlcNAc biology in metabolism-driven disease pathways. The research outputs of this proposal include molecular probes, spatiotemporal strategies, and targets to connect cellular metabolism with signaling. Our enabling chemical strategies have the potential for broad impact in the scientific community by establishing temporal and spatial methods to study protein modifications. Our platforms can be extende...