Caged Neuroactive Amino Acids For Two-Photon Photolysis Or Tp-Photostimulation/Inhibition

Although the widely-used caged L-glutamate MNI-glu has properties well-suited to near-UV photolysis it has a sub-optimal cross-section for two-photon near-IR photolysis (0.05 GM, 730 nm), thus requiring high cage concentrations that interfere with neurotransmitter receptors in network studies. We have developed small hydrophilic cages with large TP uncaging cross-sections specifically for use in photostimulation or photoinhibition. These are based on 8-dimethylamino-quinoline (8-DMAQ) photolysis previously shown to be as efficient in photorelease of L-glutamate as MNI-glu in Purkinje neurons (Petit et al., 2012, Org Lett 14 6366). Systematic substitution on 8-DMAQ identified the importance of electron withdrawing at position 5 in defining TP sensitivity. The 5-benzoyl substitution gave TP uncaging cross-sections 40 fold greater than MNI-glu and an aqueous solubility u003e50 mM (Tran et al. 2015 Org Lett 17, 405). TP uncaging cross-sections were measured from conversion at 730 nm in the focused output of a MaiTai BB (Spectra Physics) in 45 microL quartz cuvettes, using a fluorescein standard.For photostimulation kainate was chosen as the agonist released because of its high potency relative to L-glutamate at native AMPA/K receptors, permitting low cage concentrations to minimise pharmacological interference, and also stability to hydrolysis of the ester when compared with L-glutamate. The uncaging cross-section was 1.0 GM (730 nm, 100 fs pulses).For photoinhibition the 5-benzoyl-8DMAQ was coupled by a carbamate link to the GABA amine function to avoid the hydrolytic instability of the ester bond. The lower photolysis rate of 150 s-1 for release from the carbamate is fast enough for photoinhibition because the delocalisation due to slower GABA uncaging reduces receptor saturation. When evaluated for neurophysiological side-effects these probes should prove useful for precise modulation in network studies.