A new synthesis of caged GABA compounds for studying GABAA receptors

Article abstract of DOI:10.1016/j.bmcl.2009.03.065

A short and convergent synthetic approach to new photoactivatable precursors of γ-aminobutyric acid (GABA) is described. When photolyzed, the 'caged' GABA precursor efficiently releases GABA, as judged by depolarization measurements on the mammalian GABA_A receptor.

Full text of DOI:10.1016/j.bmcl.2009.03.065

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3932–3933
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new synthesis of caged GABA compounds for studying GABAA receptors
Lijun Fan a, Ryan W. Lewis b, George P. Hess b, Bruce Ganem a,
*
a Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301, USA
b Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A short and convergent synthetic approach to new photoactivatable precursors of c-aminobutyric acid
Received 17 February 2009
Accepted 17 March 2009
Available online 21 March 2009
(GABA) is described. When photolyzed, the ‘caged’ GABA precursor efficiently releases GABA, as judged
by depolarization measurements on the mammalian GABAA receptor.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
GABA
Neurotransmitter
Caged compound
Synthesis
The primary inhibitory neurotransmitter receptors in the mam-
malian central nervous system (CNS) are -aminobutyric acid type
To circumvent this unwanted activity, we devised an alternative
and convergent route to a family of carboxy-substituted DECM–
GABA derivatives, exemplified by structure 2. This caged GABA
derivative exhibits all the requisite properties of a desirable GABA
precursor (decaging at 400 nm with good quantum yield), but with
the particular advantage that both 2 and its photolysis byproducts
are inert towards the GABA receptor tested.
c
A (GABAA) receptors.1 These receptors exist as heteropentameric,
transmembrane, ligand-gated ion channels composed of diverse
subtypes that exhibit distinct kinetic and pharmacological pro-
files.2 Mutations in the various GABAA subunits are associated with
dysfunctional neuronal transmission.3 Mutations of GABAA recep-
tors have been linked to several genetically heritable forms of epi-
lepsy, a disease that in the US affects approximately 2.5 million
people at an estimated annual cost of $12.5 billion.4
The synthetic approach we developed took advantage of the
well-known Passerini 3-component condensation, which produces
a-acyloxyesters from the reaction of simple carbonyl compounds
To investigate the kinetics and mechanism of wild-type GABAA
receptor function, and how mutations affect these properties, we
required a biologically inert (‘caged’) carboxyl-linked GABA precur-
sor that can release free GABA by photoactivation using visible
light, which causes less damage to biological systems than UV
light. Such caged neurotransmitters have proven to be powerful
tools in probing signal transduction within the CNS.5
Of the various caging groups for carboxylic acids, the 7-(N,N-
diethylamino)-4-(hydroxymethyl)coumarin (DECM) moiety is par-
ticularly effective, since it undergoes rapid and efficient photolytic
ester cleavage to release the carboxylic acid using visible light. The
with carboxylic acids and isonitriles.8 In this instance, the known9
aldehyde 3, prepared by selenium dioxide oxidation of 7-(N,N-
diethylamino)-4-methylcoumarin, underwent a smooth 3-compo-
nent condensation with ethyl isocyanoacetate and BOC-protected
GABA using concentrated reaction conditions to afford the acy-
loxydiester 4 (Scheme 1) in 96% yield. Similar condensations of 3
and BOC–GABA with t-butyl isonitrile and o-tolylisonitrile afforded
caged GABA derivatives 5 and 6, respectively. Although sensitive to
light, compounds 4–6 could be purified by flash chromatography
(darkroom) for full structural characterization.
DECM group has recently been developed to protect the c-carboxyl
group of the neurotransmitter glutamic acid.6 Besides releasing
glutamate efficiently at 400 nm, both DECM–glutamate and its
photolytic byproducts were shown to be biologically inert. The cor-
responding caged GABA precursor 1 (Fig. 1), which was recently
synthesized,7 also released GABA upon photolysis. Unfortunately,
however, 1 itself inhibited various GABAA receptors.
R
O
NH2 • TFA
O
O
Et2N
O
1 R= H
2 R= CONHCH2CO2Et
* Corresponding author. Tel.: +1 607 255 6360; fax: +1 607 255 6318.
E-mail address: bg18@cornell.edu (B. Ganem).
Figure 1. DECM-caged GABA compounds of interest.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.065

L. Fan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3932–3933
3933
CHO
O
Et2N
O
3
a
O
O
O
R-NH
NHBOC
Et2N
O
O
4 R= CH2CO2Et
5 R= t-butyl
6 R= o-tolyl
b
O
O
R-NH
NH2 • TFA
O
Et2N
O
O
2 R= CH2CO2Et
7 R= t-butyl
Figure 2. Electrophysiological measurements using caged GABA 2. (A) Diagram of
the system used to measure GABAA receptor activity; (B) a representative current
trace in which the flash lamp was pulsed at the zero time point to induce photolysis.
8 R= o-tolyl
Scheme 1. Passerini route to DECM-caged GABA compounds. Reagents and
conditions: (a) BOCNH(CH2)3CO2H (1 equiv), CNCH2CO2Et (1 equiv), CH2Cl2 rt,
12 h, dark (96%); (b) CF3CO2H (5 equiv), CH2Cl2, rt, 18 h, dark (90%).
measurements of GABAA neurotransmitter receptors on cell sur-
faces without causing damage to key cellular constituents.
To remove the BOC group, freshly prepared samples of 4 were
exposed to TFA at rt, affording the desired caged DECM–GABA 2.
In like fashion, BOC–GABA compounds 5 and 6 were also deprotec-
ted to afford caged GABA salts 7 and 8, respectively.
Electrophysiological measurements with caged GABA 2 were
conducted as shown in Figure 2. A borosilicate pipette fitted with
an electrode (Fig. 2A) was attached to an HEK 293T cell transiently
expressing a GABAA receptor (alpha1, beta2, delta subunits). The
cell membrane was ruptured, exposing the cytosol to the pipette
buffer. The cell was lifted from the substratum and suspended in
Acknowledgments
Support of the Cornell NMR Facility has been provided by NSF
(CHE 7904825; PGM 8018643) and NIH (RR02002). This research
was supported in part byNIH (GM 04842).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.065.
front of a U-tube having a small (150 lm) opening for flowing solu-
tions of 2 over the cell surface. Solutions of 2 were kept on ice;
photolysis measurements were made at rt. An optical fiber posi-
tioned perpendicular to the U-tube directed light from a Rapp
SP-20 Xe flash-lamp onto the cell and surrounding buffer. A pulse
of 385–450 nm light released free GABA, activating the opening of
GABAA receptor-channels in the cell membrane and increasing the
permeability of the cell membrane to chloride ions. This resulted in
a rapid change in the current amplitude across the membrane as
measured by the pipette electrode (Fig. 2B).
References and notes
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Like other coumarin-based caged compounds, GABA precursor 2
proved resistant to hydrolysis in the dark. Moreover, initial biolog-
ical experiments demonstrated that 2 was sufficiently soluble in
the assay buffer10 and was stable to hydrolysis. Upon photolysis
at 400 nm (Fig. 2B), 2 smoothly released GABA with a quantum
yield of 0.1. Moreover, no current was observed in the presence
of 2 or its photolysis byproduct(s). These characteristics suggest
that 2 and its congeners may prove useful in transient kinetic
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