UC Berkeley

Formaldehyde is a potent electrophile engaging in reactions at nucleophilic amino acid residues, DNA and RNA bases, and biological metabolites. While traditionally viewed as an environmentally-sourced toxin, recent evidence has emerged indicating formaldehyde is produced endogenously at high micromolar concentrations through cellular processes such as one-carbon metabolism, N-demethylation reactions, and spontaneous folate degradation. Biochemical studies show the intracellular production and metabolism of formaldehyde is directed to preserve the carbon unit on formaldehyde for productive one-carbon metabolism and biosynthetic processes, rather than unspecific release leading to toxic side reactions. This dissertation describes the development of activity-based sensing methods to selectively image formaldehyde in living organisms and proteomics methods to determine proteome-wide reactivity of formaldehyde on nucleophilic amino acid residues. Historical perspective on the development of activity-based sensing methods is provided as context for the development of methods for formaldehyde detection. Development of a general reaction-based trigger utilizing the aza-Cope reaction allowed the functionalization of a palette of fluorescent reporter molecules for formaldehyde detection, and we applied a red-emitting fluorescent probe to verify an endogenous metabolism process for formaldehyde via glutathione and alcohol dehydrogenase 5. We then applied the formaldehyde trigger to develop organelle targetable probes for interrogating formaldehyde fluxes across intracellular compartments. The formaldehyde trigger also enabled functionalization of chemiluminescent dioxetane scaffolds, allowing us to visualize endogenous formaldehyde release from folate degradation in a living mouse model. Finally, activity-based proteomics methods reveal formaldehyde hyper-reactive cysteine residues are specific to enzymes involved in one-carbon metabolism processes, and not ubiquitous across the proteome. We show an isoform specific inhibition of formaldehyde on S-adenosylmethionine synthetases, revealing a feedback loop of formaldehyde to regulate the methylation potential of the cell.