Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis

Photochemical release (uncaging) of bioactive messengers with three-dimensi onal spatial resolution in light-scattering media would be greatly facilita ted if the photolysis could be powered by pairs of IR photons rather than t he customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the long er wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitat ion in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmet hyl esters and carbamates efficiently release carboxylates and amines on ph otolysis, with one- and two-photon cross sections up to one or two orders o f magnitude better than previously available. These advantages are demonstr ated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-L- glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less th an one-fifth the intensities needed by one of the best previous caged gluta mates, gamma-(alpha-carboxy-2-nitrobenzyl)-L-glutamate (CNB-glu). Two-photo n photolysis with raster-scanned femtosecond IR pulses gives the first thre e-dimensionally resolved maps of the glutamate sensitivity of neurons in in tact slices. Bhc-glu and analogs should allow more efficient and three-dime nsionally localized uncaging and photocleavage, not only in cell biology an d neurobiology but also in many technological applications.