UC San Diego

ABSTRACT Cyclic adenosine monophosphate (cAMP) is a second messenger, regulating critical physiological processes. We report the enzymatic synthesis, unique photophysical features, and biological activity of novel emissive cAMP analogs (c th AMP and c tz AMP), comprised of thiopheno‐ and isothiazolo‐based families of isomorphic ribonucleotides. The unexpectedly high brightness of c th AMP compared to th ATP and th AMP, its precursor and hydrolysis products, respectively, driven by an anti ‐to‐ syn conformational shift upon cyclization, is employed for real‐time monitoring of the adenylyl cyclase (AC) mediated synthesis and phosphodiesterase (PDE) mediated hydrolysis. Importantly, these emissive analogs effectively activate protein kinase A (PKA). This study provides critical insights into the biomolecular recognition and catalytic mechanism of cAMP‐regulating enzymes, establishing these isomorphic analogs as potential tools for investigating cAMP‐dependent protein–ligand interaction through fluorescence spectroscopy.