Theoretical investigation and design of two-photon fluorescent probes for visualizing β-galactosidase activity in living cells

As a marker enzyme, the underlying biological mechanisms of β-galactosidase (β-gal) and its role in senescence and aging still remain unknown. Two-photon (TP) fluorescence microscopy (TPFM) is a promising approach for detecting β-gal enzyme activity in living specimens. In the present study, we have firstly designed a series of novel naphthalene-based β-gal ratiometric TP fluorescent chromophores, and carried out quantum-chemical calculations on their structural and linear/nonlinear optical properties. We have thoroughly studied the effects of different biological bridge groups (quinoline, benzo[d]thiophene, thiophene, diazine) and cyano groups on the one-photon absorption (OPA), fluorescence, as well as two-photon absorption (TPA) properties of β-gal probes and the corresponding reaction products. The calculated results show that the product molecules always exhibit longer absorption and emission wavelengths, and possess both stronger TP transition probability and a larger net charge change relative to that of corresponding β-gal probes, due to their better planarity and larger transition dipole moment. Our analysis suggests that the net charge change dominates the TPA cross section (δ_max). There is an optimal match for δ_max and radiation rate (k_r) following the increase of the net charge change to improve nonlinear optical activity in the studied molecules. The substitution of benzo[d]thiazole with benzo[d]thiophene is an optimal approach for β-gal probe design with the largest fluorescence efficiency and δ_max. We hope this contribution can provide detailed theoretical analysis of OPA and TPA properties of β-gal probes and relevant products, and provide insight into the structure-property relationships for guiding experimental design.