Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

Density functional theory (DFT) was applied to study the thermodynamics and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31G­(d) with the SMD solvation model can reliably predict the Gibbs free energy changes (ΔG) of thiol-Michael addition reactions with an error of less than 1 kcal·mol–1 compared with the experimental benchmarks. Taking advantage of this computational model, the first reversible reaction-based fluorescent probe was developed that can monitor the changes in glutathione levels in single living cells.