Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are on-pathway targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics. INTRODUCTION For 170 years general anesthetics (starting with diethyl ether) have been administered to human patients, who are rendered nonresponsive to painful medical procedures. To this date, surprisingly little is known about the relevant anesthetic targets, which BMS-540215 has compromised the development of not only more potent but also safer anesthetic drugs. Current hypotheses of anesthetic action invoke specific ligandCprotein interactions.1 However, anesthetic drugs display only modest affinity for the putative targets as well as many proteins subserving off-pathway (undesirable) effects.2C4 A wide range of life forms, including plants, are influenced by general anesthetics, which suggests common protein targets that are evolutionarily conserved. The different pharmacology and chemical diversity of anesthetic drugs also suggests that in humans there are multiple routes to achieving the same anesthetic end point. Still lacking are validated, relevant targets, as well as knowledge of their in vivo distribution. The development of anesthetic probe molecules provides a route to address these challenges.5 The ability to identify general anesthetic protein targets and binding sites has been enhanced with anesthetic photolabels containing diazo or azido functional groups.6C11 Carbene or nitrene intermediates can be generated with absorption of long-wavelength ultraviolet light (315C400 nm), enabling ligand attachment onto physiologically relevant sites on cellular macromolecules, when equilibrated prior to photolysis. The covalently linked photolabel provides a tag identifiable with mass spectrometry; however, radiolabeled versions of these BMS-540215 probes have often been essential to novel target discovery and/or binding site identification with Edman degradation.12,13 Previously, our laboratories identified 1-aminoanthracene (1-AMA) as a GABAergic fluorescent general anesthetic that reversibly induces immobility in tadpoles.14 Using confocal laser scanning microscopy, 1-AMA was found to localize to the central nervous system (CNS).14,15 The efficacy of 1-AMA as an anesthetic, combined with optical and biochemical advantages of fluorescent probes over radiolabels, prompted us to investigate the protein targets. To Slc2a4 facilitate BMS-540215 target identification, we synthesized a photoactive analogue, 1-azidoanthracene (1-AZA), by replacement of the amino group with an azide. We exhibited that 1-AZA shares conserved anesthetic targets in tadpoles through targeted in vivo photolabeling, which produces an anesthetic-like state reminiscent of optoanesthesia,16 a light-induced prolongation of the anesthetic state mediated by covalent occupation of relevant ligand BMS-540215 binding sites. The anthracenes 1-AMA and 1-AZA display binding specificity for neuronal tubulin and the disruption of microtubule dynamics in vitro. Finally, we have identified 1-AZA binding sites on tubulin with mass spectrometry and provided in vivo evidence that microtubule destabilization contributes to 1-AMA anesthesia. EXPERIMENTAL SECTION Spectroscopy, Chromatography, and Fluorescence Microscopy 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were obtained at room temperature in CDCl3 on Bruker DMX 500 spectrometers. Spectra were referenced to the central line of the solvent BMS-540215 residual, and the chemical shifts () are reported to a precision of 0.01 and 0.1 ppm for 1H and 13C, respectively. Proton coupling constants (= 8.5 Hz, 1H), 7.44C7.51 (m, 3H), 7.23 (d, = 7.1 Hz, 1H). 13C NMR: 136.9, 132.4, 131.8, 128.9, 128.2, 126.4, 126.3, 125.9, 125.4, 125.2, 124.9, 121.8, 112.9. Scheme 1 Conversion of 1-Aminoanthracene (1-AMA, 1) to 1-Azidoanthracene (1-AZA, 2) Localized in Vivo Photolabeling For 1-AZA labeling studies, stage 40C47 albino tadpoles (Nasco, Fort Atkinson, WI) were incubated for 30 min in 5 mL of pond water (3.15 mM CaCl2, 30.36 mM NaCl, and 0.59 mM NaHCO3 in deionized water) with 0.5% ethanol containing 15 M 1-AZA. Tadpoles were briefly washed with fresh water and directed into a Delta T culture dish (Bioptechs). The Delta T culture dish consisted of a coverslip at the bottom with a tapered agarose channel (solidified 1% w/v in water) to restrict movement. A similar protocol for tadpole incubation and UV laser exposure (except 10 s irradiation at 100% power) was followed for the.