Reagents and conditions: i) KNO₃, concentrated H₂SO₄, 0°C, 1 h; ii) NBS, benzoyl peroxide, CCl₄, reflux, 30 h.

Reagents and conditions: i) Et₃N, DMSO, rt, 4.5 h; ii) HBr/HOAC, 0°C, 15 min.

Reagents and conditions: i) Et₃N, DMSO, rt, 5.5 h; ii) TFA, CHCl₃, rt, 21 h.

(A) UV-visible absorption spectra of a 83 μM solution of N-Ncm-Glu in 0.1 M sodium phosphate (pH 7.4) at 22°C. Spectra were recorded at the following times: 0, 20.3, 44.4, 68.2, 91.6, 116.3, 140.6, 165.9, 226.8 hours. Inset shows interconversion of the open and closed forms (H₂O and H⁺ required for balance are omitted for visual clarity). (B) The increase of absorbance at 415 nm is well fit by a single exponential function.

Light absorption leads to the transient aci-nitro intermediate, which decomposes into the free amino acid and the spent cage (7-formyl-6-nitrosocoumarin). The caged molecules are depicted as N-protonated species because the pK_as for the α-amino groups are all estimated to be higher than physiologic pH (details in ).

(A) UV-visible absorption spectra of a solution of N-Ncm-Glu in 0.1 M phosphate buffer (pH 7.4) recorded before (blue solid line, labeled “‒UV”) and after (red solid line, labeled “+UV”) photolysis with 166 mW of UV light from an argon ion laser for 2 min. The blue dashed curve shows the long-wavelength (360–500 nm) region of the ‒UV curve on an expanded scale (refer to the y-axis scale on the right). (B) Transient absorbance change of a solution of N-Ncm-Glu in DMSO following photolysis with an 8.6-nsec pulse of 355-nm light delivered at time zero. In the upper panel, gray points are data and solid curve is a least-squares single-exponential fit to the data. The residuals of the fit are shown in the lower panel. (C) Dependence of photolysis kinetics on mole-fraction of water. The t_1/2 values for transient spectral decay are plotted against mole fraction of water in DMSO for N-Ncm-Glu and N-Ncm-Gly. The t_1/2 for N-Ncm-GABA in DMSO is also shown. Each point is the average of 3 replicate measurements; error bars represent standard deviations (where not seen, error bar is smaller than the symbol).

(A) UV-visible spectrum of a 30 μM solution of 7-formyl-6-nitrosocoumarin (14) in 10 mM sodium phosphate buffer (pH 7). (B) Schematic representation of the reaction of 14 with a thiolate (RS^‒), which adds nucleophilically to the nitroso group, and concomitantly disrupts the chromophore. The bimolecular rate constant is k. (C) Spectrophotometrically monitored time course of the reaction of 14 (25 μM) with glutathione (50 μM) in 10 mM sodium phosphate buffer (pH 7.0, 24°C). Grey circles are experimental data acquired at 20 Hz; red curve is the nonlinear least-squares fit to the function, , where k_app = 0.5[GSH]₀k, (k is the bimolecular rate constant indicated in panel B, and the initial glutathione concentration is [GSH]₀ = 50 μM), and y₀ and S are adjustable parameters (see and for details). For the data set shown, k = 3.74 × 10³ M^-1s^-1.

Bar above the trace marks the 5-ms duration of the UV laser pulse. Caged glutamate concentration was 1 mM. Currents shown are the average from 12 cells for N-Ncm-Glu and 7 cells for N-Nmoc-Glu.

Neurons were loaded with the fluorescent Ca²⁺ indicator, OGB-1, for monitoring intracellular Ca²⁺ signals evoked by glutamate photorelease. In the fluorescence image, red circles mark 21 cells whose responses were analyzed; scale bar represents 100 μm. The center bright area is due to the optical fiber illumination. In the graph, change in OBG-1 fluorescence (ΔF) is plotted as function of time. Individual data are shown as gray points; the solid line is the average response of the 21 cells. A 50-ms laser pulse was used for photolysis. The dotted line indicates the interval during which the photolytic laser pulse precluded fluorescence data acquisition.