PROJECT ABSTRACT Climate change, agriculture practices, hydrology alterations, and sewage sheds are contributing to an increase in the frequency and intensity of cyanobacteria harmful algal blooms (cHABs) that produce bioactive and toxic secondary peptide metabolites. As a result, public health and drinking water advisories, recreational water closures, and coastal seafood contamination have increased. Cyanobacteria peptide toxins are known causes or suspect in liver, neurological, dermal, gastrointestinal and kidney disease. Although hundreds of potential toxic and beneficial bioactive peptides have been reported, only a handful of reference materials are commercially available. cHAB peptide research, monitoring, and mitigation has been bottlenecked by lack of certified and bulk reference materials. Analytically pure (certified) synthetic standards cyanotoxin and reliable analytical workflows are critically needed to protect public health. Our long-term goal is to create a stable and reliable supply of CHAB cyanopeptide reference materials to enable investigation of cyanopeptides’ occurrence and human health impacts. Our objectives in this proposal are to establish efficient synthetic routes and bioactivity profiles for anabaenopeptins (ABPs) and microcystins (MCs), to create mass spectrometry workflows for their identification and quantification, and to establish the impact of ingestion and inhalation pathways as exposure routes. In particular, we will assess cyanotoxins’ impact on cells from the respiratory tract and on reconstituted human fecal samples. The ABPs and MCs are observed in the Great Lakes region’s freshwater bodies in high concentrations and are known to have hundred(s) of congeners with varying degrees of toxicity and bioactivity. Many of these lakes/rivers contain congeners with tentative or unassigned identifications. Our congener selection is designed to increase the reliability and reproducibility of targeted and untargeted mass spectrometry workflows. Cyanotoxin exposure routes and their health impacts are dependent on the interplay of chemical structure and the environment. The proposed aerosol studies will assist in validating inhalation as an exposure route. There is a critical need for cyanopeptides/congeners to be synthesized, characterized, and then systematically studied to quantify their associated exposure risks in cHAB waters, cHAB-exposed food sources, and aerosols. Our proposed work combines the expertise of several areas of chemistry and pharmacology and is important because it empowers ongoing fundamental cHAB research in the areas of ecology, toxicology, biology, and fate and transport, and provides reliable cyanotoxin quantification through certified standards for water resource managers and public health decision makers.