Optical control of AMPA receptors using a photoswitchable quinoxaline-2,3-dione antagonist† †Electronic supplementary information (ESI) available: Experimental procedures and characterisation data. See DOI: 10.1039/c6sc01621a Click here for additional data file.

We have developed the first photoswitchable AMPA receptor antagonist, termed ShuBQX-3. It permits the precise optical control of AMPA receptors and exhibits a remarkable red-shifting of its photoswitching properties when bound to the receptor.


Index
(quartet) and m (multiplet). Except for multiplets, the chemical shift of all signals is reported as the centre of the resonance range. Additionally to 1 H and 13 C NMR measurements, 2D NMR techniques such as homonuclear correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) were used to assist the compound identification process. Coupling constants J are reported in Hz. All raw fid files were processed and the spectra analysed using the program MestReNova 9.0 from Mestrelab Research S. L.
Infrared Spectroscopy. IR spectra were recorded on a PerkinElmer Spectrum BX II FT-IR instrument equipped with an ATR unit. The measured wave numbers are reported in cm −1 .
Mass Spectrometry. All high-resolution mass spectra (HRMS) were recorded by the LMU Mass Spectrometry Service. HRMS were recorded on a MAT 90 (ESI) spectrometer from Thermo Finnigan GmbH.
UV-Vis Spectroscopy. UV-Vis spectra were recorded on a Varian Cary 50 Scan UV-Vis spectrometer using Helma SUPRASIL precision cuvettes (10 mm light path).

methyl)-3,4-dihydroquinoxalin-1(2H)-yl]methyl}phosphonate (12)
To a solution of compound 9 (932 mg, 1.80 mmol) in THF (90 mL) and Et 3 N (1.28 g, 12.6 mmol, 1.76 mL) at r.t. was added ethyl chlorooxoacetate (11)  was added. The mixture was heated to 120 °C for 2.5 h, cooled to r.t. and the ethanol was removed under reduced pressure. The resulting aqueous layer was washed with CH 2 Cl 2 (2 × 50 mL) and its pH was adjusted to 10-11 using a solution of Na 2 CO 3 (2.0 M in H 2 O). The aqueous layer was then back extracted with CH 2 Cl 2 (2 × 50 mL). The combined organic extracts were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.

methyl)-3,4-dihydroquinoxalin-1(2H)-yl]methyl}phosphonate (13)
To a solution of compound 10 (124 mg, 0.240 mmol) in THF (12 mL) and Et 3 N (170 mg, The mixture was heated to 120 °C for 2.5 h, cooled to r.t. and the ethanol was removed under reduced pressure. The resulting aqueous layer was washed with CH 2 Cl 2 (2 × 20 mL) and its pH was adjusted to 10-11 using a solution of Na 2 CO 3 (2.0 M in H 2 O). The aqueous layer was then back extracted with CH 2 Cl 2 (2 × 20 mL). The combined organic extracts were

HEK293T Cell Electrophysiology
HEK293T cells were incubated in dulbecco ś minimal essential medium + 10% FBS and split at 80 to 90% confluency. For detachment, growth medium was removed, cells were washed with calcium free PBS buffer and cells were treated with trypsin solution at 37 °C for 2 min.
Detached cells were diluted with growth medium and singularised by pipetting. For transfection, acid-etched coverslips were coated with poly-L-lysin and placed in a 24-well plate. 40 000 cells were added to each well in 500 µL standard growth medium. DNA (per coverslip: 350 ng GluA1-L497Y and 50 ng YFP) was mixed with 1 µL polyplus jetprime in 50 µL jetprime buffer. After standing at room temperature for 10-15 min, the DNA-mix was added to the cells shortly after seeding them into the abovementioned 24-well plate. After 3-5 hours the medium was exchanged for standard growth medium. Cells were used for electrophysiological recordings 24 hours post transfection.
Whole-cell patch clamp experiments were performed using a standard electrophysiology setup equipped with a HEKA Patch Clamp EPC10 USB amplifier and PatchMaster software (HEKA Electronik). Micropipettes were generated from a Science Products GB200-F-8P with filament pipettes using a vertical puller. Resistance varied between 3-7 MΩ. The extracellular solution contained in mM: 138 NaCl, 1.5 KCl, 2.5 CaCl 2 , 1.2 MgCl 2 , 10 glucose and 5 HEPES (NaOH to pH 7.4). The intracellular solution contained in mM: 140 K-gluconate, 5 NaCl, 15 KCl, 5.0 MgATP, 0.5 Na 2 ATP and 12.5 HEPES (pH 7.3). The holding potential for voltage clamp experiments was −60 mV. The antagonists ShuBQX-1, ShuBQX-2 and ShuBQX-3 were diluted into the extracellular solution from 100 mM DMSO stock solutions. Glutamate was diluted into the extracellular solution from a 100 mM H 2 O stock solution. Illumination during electrophysiology experiments was provided by a Poly V, FEI monochromator.

Brain Slice Electrophysiology
All animal procedures were performed in accordance with the guidelines of the Regierung Oberbayern. Horizontal slices were prepared from C57Bl6JRj mice (postnatal day 12-14).