A Protecting Group for Carboxylic Acids That Can Be Photolyzed by Visible Light ² Vishakha R. Shembekar, ‡ Yongli Chen, ‡ Barry K. Carpenter, § and George P. Hess* ,‡ Department of Molecular Biology and Genetics, 216 Biotechnology Building, Cornell UniVersity, Ithaca, New York 14853-2703, and Department of Chemistry and Chemical Biology, Cornell UniVersity, Ithaca, New York 14853-2703 ReceiVed NoVember 2, 2004; ReVised Manuscript ReceiVed December 24, 2004 ABSTRACT: We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the γ-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (ǫ 390 ) 13651 M -1 cm -1 ). Experiments are performed at 400 nm (ǫ 400 ) 12232 M -1 cm -1 ) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 °C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 μs with a quantum yield of 0.11 ( 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between ∼10 12 neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators. Photolabile protecting groups for functional groups com- monly found in biologically important compounds have been developed during the last 40 years (1-4). To solve biological problems, they were first used by Kaplan et al. (5) to rapidly generate significant and known concentrations of biologically active compounds. The rapid release of biologically active compounds from photolabile precursors has become a very useful technique in transient kinetic investigations of neuro- transmitter receptors. These proteins must be studied on cell surfaces, where rapid mixing techniques do not have the time resolution required for the investigation of the fast (micro- second to millisecond) processes involved in activation and inhibition of neurotransmitters receptors (6-9). This problem is overcome by equilibrating the cell surface receptors with a biologically inactive caged neurotransmitter. The reaction to be investigated is initiated in the μs time domain by photolysis of the caged compound (reviewed, 10). The R-carboxy-2-nitrobenzyl photolabile protecting group (11) was successfully used in such investigations to cage the different neurotransmitters that activate different receptors, carbamoylcholine (a stable analogue of acetylcholine) (11), N-methyl-D-aspartic acid (12), kainate (13), γ-aminobutyric acid (14, 15), glutamate (16), glycine (17), and serotonin (18). All these compounds are photolyzed in the μs - ms time region and with adequate quantum yield (reviewed, 19, 20). This and other caging groups for neurotransmitters (21-24) absorb only in the UV wavelength region and require expensive lasers and special rooms in which the instruments must be housed for safety reasons. Because of potential damage to the receptor-containing cells by pro- longed exposure to UV light, only a few measurements can be made with each cell. Compounds that are photolyzed in the UV wavelength region can also be photolyzed by multiphoton excitation in the visible wavelength region (25, 26). Uncaging, however, occurs only at the focus of the laser beam (25, 26). The technique is, therefore, suitable for locating the position of a particular receptor, but not for activating sufficient receptors for transient kinetic measure- ments (19). All these problems can be avoided by use of a photolabile protecting group for neurotransmitters that can be photolyzed in the visible wavelength region. Many organic molecules suitable for caging absorb in the visible wavelength region (27-32). However, they have negligible or no solubility in aqueous medium (29-32). Recently, the substituted 2-nitroveratrole group (33), nitro- indolines (34), the phenacyl group (35), and substituted ² This work was supported by a grant from the National Institutes of Health (GM 04842) awarded to G.P.H. * Author for correspondence. E-mail: gph2@cornell.edu. Tel: 607-255-4809. Fax: 607-255-6249. ‡ Department of Molecular Biology and Genetics. § Department of Chemistry and Chemical Biology. 7107 Biochemistry 2005, 44, 7107-7114 10.1021/bi047665o CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005