Fluorescent cAMP Analogs: Enzymatic Synthesis, Unexpected Photophysical Behavior and Protein Kinase A Recognition

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^thAMP and c^tzAMP), comprised of thiopheno- and isothiazolo-based families of isomorphic ribonucleotides. The unexpectedly high brightness of c^thAMP compared to ^thATP and ^thAMP, 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.