First, we investigated the alchemical transformation of cyclic adenosine monophosphate (cAMP) to its thio-substituted analog, cyclic adenosine-3 ′ ,5 ′ -monophosphorothioate (Rp-cAMPS). The topology and initial coordinate files for this specific transformation were obtained from the AMBER package, specifically from the directory amber24 src/test/pmemdTI/cAMPTI. This system serves as a well-defined and readily available benchmark for our methodology.