Improved Synthesis of Caged Glutamate and Caging Each Functional Group

Abstract

Glutamate is an excitatory neurotransmitter that controls numerous pathways in the brain. Neuroscientists make use of photoremovable protecting groups, also known as cages, to release glutamate with precise spatial and temporal control. Various cage designs have been developed and among the most effective has been the nitroindolinyl caging of glutamate. We, hereby, report an improved synthesis of one of the current leading molecules of caged glutamate, 4-carboxymethoxy-5,7-dinitroindolinyl glutamate (CDNI-Glu), which possesses efficiencies with the highest reported quantum yield of at least 0.5. We present the shortest route, to date, for the synthesis of CDNI-Glu in 4 steps, with a total reaction time of 40 h and an overall yield of 20%. We also caged glutamate at the other two functional groups, thereby, introducing two new cage designs: α-CDNI-Glu and N-CDNI-Glu. We included a study of their photocleavage properties using UV-vis, NMR, as well as a physiology experiment of a two-photon uncaging of CDNI-Glu in acute hippocampal brain slices. The newly introduced cage designs may have the potential to minimize the interference that CDNI-Glu has with the GABA receptor. We are broadly disseminating this to enable neuroscientists to use these photoactivatable tools.

Unusual Norrish Type I and Type II Nitro-Acyl Migration Transition State in the Photo-Uncaging of 1-Acyl-7-Nitroindolines Revealed by Computations

“…As a further validation of our methodology, we calculated also the quantum yield of CDNI-Ac. The experimental quantum yield of CDNI is 4 to 5 times higher than the quantum yield of MNI, 38,39,41 and it is about the same magnitude as the quantum yield of MDNI. We are pleased to notice that our calculated results are also in very good agreement with this experimental result.…”

“…37,38 Although the second nitro at the C-5 is not directly participating in the mechanism, as we will show later, its presence results in an improved quantum yield by at least 5 fold according to experimental data. [39][40][41] The mechanisms of uncaging for the 7-indolinyl system was studied by at least three groups using kinetic, fast pulse IR, and computation. 37,42,43 However, the most recently reported computational mechanisms of uncaging do not explain the difference in quantum yield, which is an important parameter especially for consideration in future improved designs.…”

Competition Between Cyclization and Unusual Norrish Type I and Type II Nitro-Acyl Migration Pathways in the Photouncaging of 1-Acyl-7-nitroindoline Revealed by Computations

“…As further validation of our methodology, we also calculated the quantum yield of 4carboxymethoxy-5,7-dinitroindolyl acetate (CDNI-Ac). The experimental quantum yield of CDNI-Ac is 4 to 5 times higher than the quantum yield of MNI, 34,35,37 and it is about the same magnitude as the quantum yield of MDNI. Our calculated relative quantum yield for CDNI is also in excellent agreement with this experimental result.…”

“…33,34 Although the second nitro at the C-5 is not directly participating in the mechanism, as we will show later, its presence results in an improved quantum yield by at least fivefold according to experimental data. [35][36][37] The mechanisms of uncaging for the 7-indolinyl system was studied by at least three groups using kinetic, fast pulse IR, and computation. 33,38,39 However, the most recently reported computational mechanisms of uncaging do not explain the difference in quantum yield, which is an important parameter, especially for consideration in future improved designs.…”

Methods for Three-Dimensional All-Optical Manipulation of Neural Circuits

“…Controlling and monitoring neuronal activity with a light has become a common practice in many neurobiological studies, throughout the last decade. The continuously expanding toolbox of molecular probes that activate/inhibit (Herlitze and Landmesser, 2007; Airan et al, 2009; Levitz et al, 2013; Klapoetke et al, 2014; Shemesh et al, 2017; Becker et al, 2018; Guruge et al, 2018; Mardinly et al, 2018) or image (Emiliani et al, 2015; Kim et al, 2018) neuronal activity as well as the development of original light-microscopy methods for stimulating these tools (Ronzitti et al, 2017b; Chen et al, 2018c; Yang and Yuste, 2018), has tremendously contributed to the direction of research and has led to innovative experimental concepts (Rickgauer et al, 2014; Carrillo-reid et al, 2016). Photostimulation via optogenetics and/or uncaging (Kwon et al, 2017) is suitable for single cell and, most importantly, circuit studies, since light gives access to a large number of targets simultaneously, at high spatial precision via parallel illumination methods (Papagiakoumou et al, 2010; Packer et al, 2015; Forli et al, 2018; Yang et al, 2018).…”