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 (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence 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 yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic 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 olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.
Hagen
V
V
Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany. hagen@fmp-berlin.de
Bendig
J
J
Frings
S
S
Wiesner
B
B
Schade
B
B
Helm
S
S
Lorenz
D
D
Kaupp
U B
UB
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000138:chemical synthesis* / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D006801:Humans; D007473:Ion Channels / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D010777:Photochemistry; D010782:Photolysis; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013997:Time Factors
C406453:(7-methoxycoumarin-4-yl)-methyl-caged 8-bromoguanosine cyclic 3',5'-monophosphate; C406452:(7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate; D003374:Coumarins; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D000242:Cyclic AMP; D006152:Cyclic GMP