In silico screening of photostabilizing reagents for cyanine-based single molecule fluorescence

Fluorophore signal and photostability are desirable when conducting single molecule fluorescence (SMF) studies. For cyanine dyes, this is typically achieved by quenching their excited triplet states via photoinduced electron transfer (PeT). However, to date, only a handful of electron donors or acceptors have been - empirically - identified, most of which do not show diffusion controlled PeT quenching rate constants (k_P). Here we report a screening tool to rapidly explore the potential of thiol-based electron donors to serve as photostabilizers of Cy3, Cy5, and their bridged congeners Cy3B and Cy5B. Based on density functional theory (DFT) and utilizing Marcus theory of electron transfer, our method estimates in silico the activation free energy of PeT. A correlation function is then established between theoretical estimates and empirical values of k_P acquired through transient absorption spectroscopy. The correlation function then enables screening of the photostabilizing potential of untested reagents from a kinetic standpoint. A new compound, glyceryl monothioglycolate (GMTG), was thus identified. SMF studies show the effectiveness of GMTG as a domestic use, non-toxic, non-volatile alternative to the widely utilized β-mercaptoethanol. Altogether, a predictive model is proposed toward discovering photostabilizers to achieve single molecule fluorescence experiments with enhanced photo- and signal stability.