Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl) methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5' -monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (g-Br-cGMP) are described. The compounds are the axial and equatorial isome rs of the (7-methoxycoumarin-4-yl) methyl (MCM) esters of cyclic nucleotide s. Synthesis is accomplished by treatment of 4-bromomerhyl-7-methoxycoumari n with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nu cleotides or with the fret: acids of 8-Br-cAMP and 8-Br-cGMP in the presenc e of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yiel ds and are resistant to hydrolysis in aqueous buffer solutions. The moderat e fluorescence properties of the caged compounds in comparison with the str ongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct a llow the indirect estimation of the amount of photolytically released cycli c nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olf actory sensory neurons using the patch-clamp technique and confocal laser s canning microscopy. The caged compounds serve as efficient and rapid intrac ellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 ce lls, fluorescence signals cannot be used to monitor the photolysis of MCM-c aged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-O H. (C)1999 Elsevier Science S.A. All rights reserved.