Use of Multiple Carboxylates to Increase Intracellular Retention of Fluorescent Probes Following Release From Cell Penetrating Fluorogenic Conjugates

Fluorogenic reporter systems for use inside cells require that the fluorophore be retained inside the cell following activation to ensure accumulation of an observable signal. In the process of developing ester-based nucleic acid-triggered probe activation systems for use in cells, we found that simple O-alkylated fluorescein esters coupled to cell-penetrating peptides led to very poor signals, presumably because the released fluorophore was too membrane permeable and rapidly exited the cell. To circumvent this problem, we have examined the effect of adding one or two carboxylates to the fluorescein to reduce its membrane permeability. N-maleimido d-valine and α-methyl-β-l-alanine esters of fluorescein, in which the second phenolic hydroxyl group was derivatized with a carboxymethyl group and then further conjugated with glutamate, were linked to the cell-penetrating peptide Arg9Cys through conjugate addition of the thiol group to the maleimido group. HeLa cells were incubated with these conjugates, washe...