Acetoxymethyl esters of phosphates, enhancement of the permeability and potency of cAMP

Acetoxymethyl esters of alkyl or aryl phosphates can be prepared by reacting their trialkylammonium or silver salts with acetoxymethyl bromide. Because acetoxymethyl esters are rapidly cleaved intracellularly, they facilitate the delivery of organophosphates into the cytoplasm without puncturing or disruption of the plasma membrane. In addition, acylation of free hydroxyls, for example with butyryl groups, is useful both for synthetic convenience and increased hydrophobicity of the permeant derivatives. The highly polar intracellular messengers cAMP and cGMP were thus converted into uncharged membrane-permeant derivatives. Extracellularly applied N⁶,2′-O-dibutyryl cAMP acetoxymethyl ester (Bt₂cAMP/AM) is shown to simulate intracellular cAMP in three model systems, namely dissociation of cAMP-dependent protein kinase in fibroblasts, activation of Cl^- secretion of monolayers of the human colon epithelial cell line T₈₄, and dispersion of pigment granules in angel fish melanophores. Bt₂CAMP/AM is effective at concentrations two or three orders of magnitude less than those required for commonly used membrane-permeant cAMP derivatives such as Bt₂CAMP, 8-Br-cAMP, and 8-pCPT-cAMP lacking the acetoxymethyl ester. This methodology should be of general utility for the intracellular delivery of phosphate-containing second messengers.