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. 2013 Oct 23;135(42):15948-54.
doi: 10.1021/ja408225k. Epub 2013 Oct 11.

Spectral Evolution of a Photochemical Protecting Group for Orthogonal Two-Color Uncaging With Visible Light

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Spectral Evolution of a Photochemical Protecting Group for Orthogonal Two-Color Uncaging With Visible Light

Jeremy P Olson et al. J Am Chem Soc. .
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Abstract

Caged compounds are molecules rendered functionally inert by derivatization with a photochemical protecting group. We describe the design logic behind the development of a diethylaminocoumarin (DEAC) caging chromophore, DEAC450, that absorbs blue light strongly (ε450 = 43,000 M(-1) cm(-1)) and violet light 11-fold more weakly. The absorption minimum is in the wavelength range (340-360 nm) that is traditionally used for photolysis of many widely used nitroaromatic caged compounds (e.g., 4-carboxymethoxy-5,7-dinitroindolinyl(CDNI)-GABA). We used this chromophore to synthesize DEAC450-caged cAMP and found this probe was very stable toward aqueous hydrolysis in the electronic ground state but was photolyzed with a quantum efficiency of 0.78. When DEAC450-cAMP and CDNI-GABA where co-applied to striatal cholinergic interneurons, the caged compounds were photolyzed in an chromatically orthogonal manner using blue and violet light so as to modulate the neuronal firing rate in a bidirectional way.

Figures

Figure 1
Figure 1
Comparison of the absorption spectra of nitroaromatic and aminocoumarin chromophores. (a) Spectra of CDNI-GABA (violet), DEAC (blue), and DEAC450-cAMP (red). (b) Spectra of 3-(p-cyano-phenyl)-DEAC (4, khaki), 3-nitro-4-methyl-DEAC (2, green) and 3-cyano-4-methyl-DEAC (3, orange), and 3-nitro-DEAC (1, dark red).
Figure 2
Figure 2
Bi-directional modulation of neuronal firing rates by optically orthogonal uncaging of cAMP and GABA. Whole-cell current clamp recordings from acute mouse brain slices were used to monitor spontaneous action potential firing in striatal cholinergic interneurons. DEAC450-cAMP (25-75 μM) was included in the intracellular solution, and CDNI-GABA (1 mM) was bath applied. Cell bodies were irradiated at 473 nm (~20 μm spot and 410 mW/cm2 for 100-200 ms) and/or 355 nm (50 μm spot and 16 mW/cm2 for 2 ms). Scale bars: y=10 mV, x=1 sec. a) 473-nm light causes an increase in the spontaneous firing rate of neurons loaded with DEAC450-cAMP (left trace), but 355-nm light does not (right trace). b) 473-nm light does not alter the spontaneous firing of neurons bathed in CDNI-GABA (left trace), but 355-nm light transiently inhibits spontaneous firing (right trace). c) In the presence of both DEAC450-cAMP and CDNI-GABA, sequential illumination with 355-nm and 473-nm light produces bidirectional changes in action potential firing. Bar graphs show summaries of light-induced changes in action potential firing by quantifying the ratiometric change in inter-spike interval (ISI) before (pre) and after (post) each light flash for many cells. The “no flash” condition corresponds to ISI ratios measured from each cell before and after an arbitrary time point. Data are presented as geometric means + the standard deviation upper limit. Asterisks denote a significant difference from the “no flash” condition (p < 0.05) according to paired sample t-test analysis.
Scheme 1
Scheme 1
Synthesis of DEAC450-cAMP. Reagents and conditions: (a) i. TBDMSCl, Et3N (83%). ii. N-Bromosuccinimide, NaOAc (59%). (b) 4-Cyanophenylboronic acid, CsF, Pd(PPh3)4 (69%). (c) i. TBAF (85%). ii. EDC, Boc-Glu-OtBu (76%). iii. TFA, then HPLC purification (71%). (d) ref . (e) TBAF (85%). (f) Methanesulfonyl chloride, TEA (59%). (g) cAMP, tri-n-butylamine (65%). (h) TFA, then HPLC purification (11a, 35%; 11b, 44%).

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