Synthesis of Dopamine Heptynamide as a Chemical Probe for Catechol Bioactivity

Catechol-containing natural molecules are commonly found in foods, drinks, and drugs that we encounter everyday. Catechol-containing products have been associated with anti-inflammatory activity, and it is known that catechols will bind covalently to specific proteins. Catechols have a broad range of functions in biochemical processes; however their mechanisms of action are not clearly understood. The first objective of this research project is to synthesize a catechol-alkyne chemical probe, named dopamine heptynamide, from dopamine hydrochloride and 6-heptynoic acid. For the synthesis of dopamine heptynamide, 100 mg of 6-heptynoic acid was mixed with with 100 mg dopamine hydrochloride to form an amide bond with the use of EDC and Oxyma Pure as coupling reagents in DMF. The reaction takes place overnight at room temperature. Results obtained from proton NMR as well as thin layer chromatography suggest that formation of dopamine heptynamide was successful. The second objective is to determine how dopamine heptynamide interacts with proteins of E. coli. The interaction of dopamine heptynamide in E. coli will give insight as to how chemical probes affect gut bacteria in humans. The synthesized probe will be introduced to an E. coli culture at a concentration of 4 µM. Once the cells have been lysed, a biotin-streptavidin immunoprecipitation will be used to isolate the protein binding to dopamine heptynamide. Click chemistry will be utilized to attach biotin to the alkyne of dopamine heptynamide, via the copper-catalyzed azide-alkyne cycloaddition reaction. Streptavidin binds to biotin with a high specificity, making it ideal for use in immunoprecipitation to isolate the protein-chemical probe complex. Mass spectrometry will be used to identify the target protein. The results so far demonstrate a greener alternative to amide bond formation to form the catechol-alkyne chemical probe; however, further testing of the chemical probe in E. coli cells is necessary.