title: Use of multiple carboxylates to increase intracellular retention of fluorescent probes following release from cell penetrating fluorogenic conjugates.
abstract: 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 alpha-methyl-beta- 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, washed, and then further incubated for various times prior to analysis by flow cytometry. Quantitative analysis of the data by a simplified kinetic scheme showed that the fluorescein with two appended carboxylic acid groups effluxed with a rate constant of about 0.00113 min (-1), corresponding to a half-life of 8.8 h. The dicarboxylated fluorescein effluxed about 6.1 times more slowly than the fluorescein with a single carboxylic acid group and led to a fairly stable signal. The analysis also showed that the d-Val ester was hydrolyzed about 4.6 times more slowly than the beta-alanine ester and had a half-life of about 31 min. These data indicate that the fluorescein with two appended carboxylates may be a useful membrane-impermeant fluorophore for fluorogenic probe applications inside living cells.
authors.affiliation: Chemistry Department, Washington University, St. Louis, Missouri 63130, USA.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Li
authors.forename: Ryuji
authors.initials: R
authors.lastname: Higashikubo
authors.forename: John-Stephen
authors.initials: JS
authors.lastname: Taylor
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry*; D004952:Esters / Q000737:chemistry*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D010455:Peptides / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D002264:Carboxylic Acids; D004952:Esters; D005456:Fluorescent Dyes; D010455:Peptides