Blue and Green Light Responsive Caged Glutamate

Abstract

Glutamate (Glu) is an excitatory neurotransmitter that plays a critical role in memory. Brain mapping activities of such pathways relied heavily on the ability to release Glu with spatiotemporal precision. Several photo-protecting groups (PPGs), referred to as photocages or cages, were designed to accomplish the release of Glu upon irradiation. Previously reported Glu cages responded to UV upon irradiation with single photons, which limited their use in vivo experiments due to cytotoxicity. Other caged designs suffered from lower quantum efficiency (QE) of release necessitating higher concentrations and/or longer photoirradiation times. There have been limited examples of cages that respond to visible light with single photon irradiation. Herein, we report the efficient preparation of 11 caged Glu examples that respond to two visible wavelengths, 467 nm (thiocoumarin based) and 515-540 nm (BODIPY based). The kinetics of photouncaging were studied for all caged designs, and we report all quantum efficiencies, i.e., quantum yields (Φ), that ranged from 0.0001-0.65. Two of the BODIPY cages are reported here for the first time, and one, Me-BODIPY-Br-Glu, shows the most efficient Glu release with a QE of 0.65. Similar caged designs can be extended to the inhibitory neurotransmitter, GABA. This would enable the use of two visible wavelengths to modulate the release of excitatory and inhibitory neurotransmitters upon demand via optical control.

Keywords: BODIPY; Caged Glu; Glutamate; Visible light; photo-protecting group; photoirradiation.

Fig. 1.

a. Previously reported caged Glu molecules that uncage with UV light [, , ,–36] and b. Blue and green light responsive caged Glu molecules presented herein.

Fig. 2.

Designed caged Glu molecules. N-caged Glu (blue light responsive 1 and green light responsive 2–5) and γ-carboxy caged Glu (green light responsive 6–11).

Fig. 3.

UV comparison of a. TC-N-Glu b. BODIPY-N-Glu c. BODIPY-Glu [TC-N-Glu (1), Me-BODIPY-Et-N-Glu (2), Me-BODIPY-Br-N-Glu (3), Me-BODIPY-I-N-Glu (4), F-BODIPY-Et-N-Glu (5), Me-BODIPY-H-Glu (6), Me-BODIPY-Et-Glu (7), Me-BODIPY-Br-Glu (8), F-BODIPY-H-Glu (9), F-BODIPY-Et-Glu (10), F-BODIPY-Br-Glu (11), each caged molecules were prepared with 0.5 mg/mL at 7.6 pH].

Fig 4.

¹H NMR analysis of progressive photolysis of the caged molecules; a. TC-N-Glu (1) was irradiated at 467 nm for 60 s intervals and b. BODIPY-(N)-Glu (6) was irradiated at 520 nm for 120 s intervals.

Scheme 1.

Synthesis of TC-N-Glu Cage, (a) Triphosgene, Pyridine, CH₂Cl₂, 2 h, −10 °C, N₂, 90%; (b) SeO₂, 1 : 0.05 (v/v) dioxane/water, reflux, 14 days; (c) NaBH₄, EtOH, 12 h, rt, 42% (two steps totally, a and b); (d) AcOH, EDC·HCl, DMAP, CH₂Cl₂, 12 h, rt, Ar, 83%; (e) Lawesson’s reagent, toluene, 15 h, reflux, 69%; (f) HCl, EtOH, overnight, reflux, 30%; (g) t-butylisocyanate glutamate (13), Et₃N, Toluene, 72 h, 50 °C, N₂, 76%; (h) TFA, 2 h, rt, N₂, 50% (HPLC).

Scheme 2.

Synthesis of hydroxymethyl-BODIPY compounds, (a) acetoxyacetyl chloride, CH₂Cl₂, 2 h, 50 °C, Ar; (b) BF₃·Et₂O, Toluene, CH₂Cl₂, Et₃N, 1.5 h, 50 °C, Ar, 45% (two steps totally, a and b); (c) 0.1 M NaOH, 2 : 1 (v/v) Methanol/Water, rt, 4 h, N₂, 65% (d) NBS, CH₂Cl₂, overnight, rt, N₂, 76%; (e) CH₃MgBr in Et₂O, Et₂O, Overnight, rt, N₂, 62%; (f) NBS, CH₂Cl₂, overnight, rt, N₂, 76%; (g) NIS, CH₂Cl₂, overnight, rt, N₂, 76%; (h) acetoxyacetyl chloride, CH₂Cl₂, 2 h, 50 °C, Ar; (i) BF₃·Et₂O, Toluene, CH₂Cl₂, Et₃N, 1.5 h, 50 °C, Ar, 60% (two steps totally, g and h); (j) 0.1 M NaOH, 2 : 1 (v/v) Methanol/Water, rt, 4 h, N₂, 65%; (k) CH₃MgBr in Et₂O, Et₂O, Overnight, rt, N₂, 62%.

Scheme 3.

Synthesis of BODIPY-N-Glu caged molecules (a) Isocyanate Glutamate (13), Et₃N, Toluene, 72 h, 50 °C, N₂; (b) 0.05:1 (v/v) TFA/ CH₂Cl₂, 24 h, rt, N₂.

Scheme 4.

Synthesis of BODIPY-Glu caged molecules (a) α-tert-Butyl-N-BOC-glutamate, EDC·HCl, DMAP, CH₂Cl₂, 12 h, rt, N₂; (b) 0.05:1 (v/v) TFA/ CH₂Cl₂, 1 h, rt, N₂; (c) CeCl₃·7H₂O, NaI, Overnight, 50 °C, N₂; (d) TFA, 2 h, rt, N₂.