1.

Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis
1. 2. 3. #. '. +. /. 1. Toshiaki Furuta*, Samuel S.-H. Wang, Jami L. ant!ker", Timoth$ %. ore*&, Wen($ J. )$*ee, ,(-ar( %. .alla-a$", Win0rie( enk, an( 2oger 3. Tsien*&**

4 5uthor 500iliations 1. Departments of *Pharmacology and Chemistry and Biochemistry and Howard Hughes Medical Institute !ni"ersity of California #an Diego C$ %&'%()'*+,. Bell /a0oratories /ucent 1echnologies *'' Mountain $"enue Murray Hill 23 ',%,+- and 4#al5 Institute 1''1' 2orth 1orrey Pines 6oad /a 3olla C$ %&'(, 1. .ontri*ute( *$ 2oger 3. Tsien

Abstract
6hoto7hemi7al release 8un7aging9 o0 *ioa7ti:e messengers -ith three-(imensional s;atial resolution in light-s7attering me(ia -oul( *e greatl$ 0a7ilitate( i0 the ;hotol$sis 7oul( *e ;o-ere( *$ ;airs o0 <2 ;hotons rather than the 7ustomar$ single => ;hotons. The ?ua(rati7 (e;en(en7e on light intensit$ -oul( 7on0ine the ;hotol$sis to the 0o7us ;oint o0 the laser, an( the longer -a:elengths -oul( *e mu7h less a00e7te( *$ s7attering. Ho-e:er, ;re:ious 7age( messengers ha:e ha( :er$ small 7ross se7tions 0or t-o-;hoton e@7itation in the <2 region. We no- sho- that *rominate( /-h$(ro@$7oumarin-#$lmeth$l esters an( 7ar*amates e00i7ientl$ release 7ar*o@$lates an( amines on ;hotol$sis, -ith one- an( t-o-;hoton 7ross se7tions u; to one or t-o or(ers o0 magnitu(e *etter than ;re:iousl$ a:aila*le. These a(:antages are (emonstrate( on neurons in *rain sli7es 0rom rat 7orte@ an( hi;;o7am;us e@7ite( *$ glutamate un7age( 0rom 2-8+-*romo-/h$(ro@$7oumarin-#-$lmetho@$7ar*on$l9-l-glutamate 8)h7-glu9. .on:entional => ;hotol$sis o0 )h7-glu re?uires less than one-0i0th the intensities nee(e( *$ one o0 the *est ;re:ious 7age( glutamates, A-8B-7ar*o@$-2-nitro*en!$l9-l-glutamate 8.C)-glu9. T-o;hoton ;hotol$sis -ith raster-s7anne( 0emtose7on( <2 ;ulses gi:es the 0irst three(imensionall$ resol:e( ma;s o0 the glutamate sensiti:it$ o0 neurons in inta7t sli7es. )h7glu an( analogs shoul( allo- more e00i7ient an( three-(imensionall$ lo7ali!e( un7aging an( ;hoto7lea:age, not onl$ in 7ell *iolog$ an( neuro*iolog$ *ut also in man$ te7hnologi7al a;;li7ations.