Optically Selective Two-Photon Uncaging of Glutamate at 900 nm

Abstract

We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength selective, two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moeity at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 nm to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.

Cloaked Caged Compounds: Chemical Probes for Two‐Photon Optoneurobiology

“…In contrast we could use G5-DEAC450-GABA at 66 μM or even 45 μM and still reliably evoke currents by 2P excitation at 900 nm (Figure 4c,d). We also confirmed that G5-DEAC450-GABA was, like previous DEAC450-caged transmitters [16,18] , essentially inactive at 720 nm (Fig. S4).…”

“…Thus, we were subsequently surprised to discover that MNI-Glu was not inert towards GABA-A receptors [14,15] . This off-target effect was substantiated by other reports and applies to other caged glutamate probes such CDNI-Glu [16] and RuBi-Glu [16,17] . Much less surprising was that caged GABA probes such as CDNI-GABA [15] , RuBi-GABA [18] and PEG-DEAC450-GABA [18] were also found to be quite antagonistic towards GABA-A receptors.…”

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

“…3-Carboxy derivatives of aminocoumarins are known to have absorption spectra with similar bathochromic shifts to 1-4 , 12 but we found the synthesis of such simple derivatives in a form suitable for elaboration into caged compounds surprisingly problematic (data not shown). In contrast, a functionalized DEAC-based caging chromophore could be created in a facile manner by Heck coupling of methylvinylacrylate 16 or tert -butylacrylate 11 to 5 to give the core DEAC450 chromophore (Supplemental movie). Mesylation of alcohol 8 11 gave 9 and this derivative was used to alkylate the tri-n-butylamine salt of cAMP to give 10 in 35% overall yield.…”

“…In contrast, a functionalized DEAC-based caging chromophore could be created in a facile manner by Heck coupling of methylvinylacrylate 16 or tert -butylacrylate 11 to 5 to give the core DEAC450 chromophore (Supplemental movie). Mesylation of alcohol 8 11 gave 9 and this derivative was used to alkylate the tri-n-butylamine salt of cAMP to give 10 in 35% overall yield. Treatment of 10 with TFA followed by HPLC purification gave equatorial and axial derivatives 11a and 11b , in yields of 35% and 44%, respectively (Scheme 1).…”

“…Importantly, irradiation of DEAC450-Glu at dendritic spine heads with a pulsed laser at short wavelengths (i.e. 720 nm) produced little or no postsynaptic current in neurons when compared to 900 nm 11 . The non-linear nature of two-photon excitation enabled clean chromatically selective uncaging of glutamate and was based on the >60-fold difference in two-photon excitation at the two wavelengths.…”