Reversibly caged glutamate: A photochromic agonist of ionotropic glutamate receptors

Article abstract of DOI:10.1021/ja067269o

The design, synthesis, and biological evaluation of a photochromic glutamate analogue is described. The molecule functions as a reversibly caged neurotransmitter and can be used to influence neural activity with light. Copyright

Full text of DOI:10.1021/ja067269o

Published on Web 12/21/2006
Reversibly Caged Glutamate: A Photochromic Agonist of Ionotropic
Glutamate Receptors
Matthew Volgraf,† Pau Gorostiza,‡,£ Stephanie Szobota,‡,§ Max R. Helix,† Ehud Y. Isacoff,*,‡,# and
Dirk Trauner*,†,#
Department of Chemistry, UniVersity of California, Berkeley, California 94720, Department of Molecular and Cell
Biology, UniVersity of California, Berkeley, California 94720, and Material Science DiVision and Physical
Bioscience DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received October 10, 2006; E-mail: ehud@berkeley.edu; trauner@berkeley.edu
Scheme 1. Structures of (2S,4R)-4-Substituted Glutamate
Analogues
The ability to control the active concentration of neurotransmitters
in a spatially and temporally precise manner has revolutionized the
study of the central nervous system (CNS). In particular, caged
glutamate has emerged as a tool for the dissection of both neural
circuitry1,2 and the fast kinetic events of channel activation.3 Upon
the irreversible photochemical cleavage of a protecting group,2
released glutamate is free to bind ionotropic glutamate receptors
(iGluRs), the major mediators of excitatory information transfer in
the CNS.4
Scheme 2. Synthesis of Photochromic Agonist 2
Photochromic ligands provide another opportunity for the control
of neural excitability. First reported in the late 1960s in the form
of a photoisomerizable inactivator of chymotrypsin,5 photochromic
ligands control the function of proteins through a reversible change
in shape and/or polarity of an integral bistable photoswitch. In the
case of photochromic agonists, one configuration functions as an
activating ligand, while the other configuration is, ideally, inert
toward the system of study.1 While photochromic nicotinic
acetylcholine receptor agonists,6 enzyme inhibitors,7 and regulatory
peptides8 have been reported, their benefits have been tempered
by the difficulty of achieving perfect on/off activity between states.9
Their successful implementation, however, avoids many disadvan-
tages of caged systems, including precursor instability and photo-
product toxicity.10
We recently developed a light-activated iGluR6 that is based on
a photoisomerizable agonist tethered to an engineered cysteine.11
Encouraged by this success, we investigated the possibility of a
nontethered photochromic agonist that could function at wild-type
receptors. Our design was based upon the report of a series of
(2S,4R)-4-substituted glutamate analogues that are potent and
selective iGluR5 and 6 kainate receptor (KAR) agonists.12 In
particular, we decided to replace the napthyl group of LY339434
(1) with an azobenzene to generate compound 2 (Scheme 1). We
envisioned that the change in shape and polarity between trans-2
and cis-2 would generate differences in affinity to the receptor
ligand binding domains.
NMR experiments with in situ illumination of the sample
revealed photostationary states containing 76.3 ( 0.3% cis-2 at 380
nm and 89 ( 1% trans-2 at 500 nm (mean ( SEM, n ) 3).13
Furthermore, the half-life of thermal relaxation from cis- to trans-2
in the dark was measured at 18 ( 3 h (mean ( SEM, n ) 3).
Compound 2 was assayed with whole-cell voltage clamp
recordings in HEK293 cells transiently expressing wild-type
iGluR6(Q)14 and pretreated with 0.3 mg/mL concanavalin A (ConA)
to block desensitization.15 Channel activation depended on the
wavelength of irradiation, displaying increased inward currents in
the trans relative to the cis state (Figure 1). Activity was
competitively blocked by the non-NMDA receptor antagonist
DNQX (Supporting Information).16
To investigate the cis/trans agonist profile of 2, dose-response
curves were generated with iGluR5(Q) and iGluR6(Q) under both
380 and 500 nm light (Figure 2). In agreement with previous reports
of (2S,4R)-4-glutamic acid analogues,12 2 demonstrated high
selectivity at iGluR5 over iGluR6 receptors, with approximately
half-maximal efficacy with respect to a 300 µM glutamate evoked
response. The apparent agonist affinity (EC50) of iGluR5 was 9
µM under 500 nm illumination and reduced ∼10-fold under 380
nm light. Due to the solubility constraints of 2, we were unable to
generate full titration curves at iGluR6. The AMPA receptor iGluR2
failed to produce inward currents in the presence of 250 µM 2,
demonstrating the subtype selectivity of this agonist.
The stereoselective synthesis of 2 was achieved starting from
N-Boc protected ethyl pyroglutamate 3 (Scheme 2). Diastereo-
selective alkylation of 3 with propargyl bromide gave alkyne 4,
which underwent hydrostannation to afford vinyl stannane 5.
Palladium-catalyzed Stille coupling with iodoazobenzene 6, ob-
tained by the condensation of 4-iodoaniline with nitrosobenzene,
then gave the N-Boc protected azobenzene pyroglutamate 7. Finally,
deprotection of 7 yielded 2 as the dihydrate, monosodium salt.
† Department of Chemistry.
Activation and deactivation proceeded at rates (τon-500nm ) 310
( 15 ms; τoff-380nm ) 250 ( 14 ms; mean ( SEM, n ) 3) slower
than the microsecond time scale of traditional uncaging experi-
ments.17 However, our studies were conducted at light intensities
‡ Department of Molecular and Cell Biology.
£ Current address: Centre de Recerca en Bioenginyeria de Catalunya, Barcelona,
Spain 08028.
§ Biophysics Graduate Program.
# Materials Science Division and Physical Bioscience Division.
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C O M M U N I C A T I O N S
controlled by the wavelength of light used. Future work will concern
the synthesis of substituted analogues of 2 and in-depth studies
profiling their selectivity, receptor desensitization, and utility in
neuronal excitation. Ideally, these photochromic agonists will
become a valuable complement to irreversibly caged neurotrans-
mitters as well as other methods of remote neuronal control.20-22
Acknowledgment. This work was supported by grants from
the National Institutes of Health, the Human Frontier Science
Program, Lawrence Berkeley National Laboratory, the Coates
Microscopy fund, predoctoral fellowships from Wyeth Research
and the ACS Medicinal Chemistry Division (M.V.) and the National
Science Foundation (S.S.), and postdoctoral fellowships from the
Human Frontier Science Program and the Nanotechnology Program
of the Generalitat de Catalunya (P.G.). The iGluR2(Q)flip-L483Y,
iGluR5(Q), and iGluR6(Q) cDNAs were kind gifts of Kathy Partin.
Figure 1. Whole-cell voltage clamp recording of iGluR6(Q) currents in
HEK293 cells under UV-visible illumination.
Supporting Information Available: Detailed synthetic protocols,
relevant spectroscopic data, methods of receptor transfection, electro-
physiological recordings, and illumination protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Gillespie, D. C.; Kim, G.; Kandler, K. In Dynamic Studies in Biology,
1st ed.; Goeldner, M., Givens, R., Eds.; Wiley-VCH: Weinheim, Germany,
2005; pp 232-251.
Figure 2. Dose-response curves for inward currents evoked by 2 at iGluR5
and iGluR6 expressing HEK293 cells under 380 and 500 nm illumination.
(2) Callaway, E. M.; Yuste, R. Curr. Opin. Neurobiol. 2002, 12 (5), 587-
592.
(3) Hess, G. P. In Dynamic Studies in Biology, 1st ed.; Goeldner, M., Givens,
R., Eds.; Wiley-VCH: Weinheim, Germany, 2005; pp 232-251.
(4) Mayer, M. L. Nature 2006, 440 (7083), 456-462.
(5) Kaufman, H.; Vratsanos, S. M.; Erlanger, B. F. Science 1968, 162 (861),
1487-1489.
(6) Bartels, E.; Wasserman, N. H.; Erlanger, B. F. Proc. Natl. Acad. Sci. U.S.A.
1971, 68 (8), 1820-1823.
(7) Fujita, D.; Murai, M.; Nishioka, T.; Miyoshi, H. Biochemistry 2006, 45
(21), 6581-6586.
(8) Caamano, A. M.; Vazquez, M. E.; Martinez-Costas, J.; Castedo, L.;
Mascarenas, J. L. Angew. Chem., Int. Ed. 2000, 39 (17), 3104-3107.
(9) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45 (30), 4900-
4921.
Figure 3. Whole-cell current clamp recording of cultured rat hippocampal
neurons in the presence of 25 µM 2 under 380 and 500 nm illumination.
(10) Givens, R. S.; Weber, J. F. W.; Jung, A. H.; Park, C. In Methods in
Enzymology; Marriott, G., Ed.; Academic Press: New York, 1998; Vol.
291, pp 1-29.
(4.8 mW/mm2 at 500 nm and 1.8 mW/mm2 at 380 nm) several
orders of magnitude weaker than laser pulse-photolysis techniques.
Much faster rates of isomerization are expected at comparable light
intensities.18
(11) Volgraf, M.; Gorostiza, P.; Numano, R.; Kramer, R. H.; Isacoff, E. Y.;
Trauner, D. Nat. Chem. Biol. 2006, 2 (1), 47-52.
(12) Pedregal, C.; Collado, I.; Escribano, A.; Ezquerra, J.; Dominguez, C.;
Mateo, A. I.; Rubio, A.; Baker, S. R.; Goldsworthy, J.; Kamboj, R. K.;
Ballyk, B. A.; Hoo, K.; Bleakman, D. J. Med. Chem. 2000, 43 (10), 1958-
1968.
We tested the ability of 2 to depolarize cultured rat hippocampal
neurons, which are known to express the KAR subunits iGluR6
and KA2, but not iGluR5.19 Neurons were exposed to a 25 µM
concentration of 2 under 380 nm light and current clamped at -65
mV, without pretreatment with ConA. Switching wavelengths
between 380 and 500 nm light was then sufficient to trigger, and
extinguish, sustained trains of action potentials (Figure 3). While
iGluR6 channels are only activated to a small extent at 25 µM
(Figure 2), this can still produce significant depolarization. Given
the large number of receptors in the cell, only a small fraction needs
to be concurrently activated by glutamate release to trigger neuronal
firing.
(13) Tait, K. M.; Parkinson, J. A.; Bates, S. P.; Ebenezer, W. J.; Jones, A. C.
J. Photochem. Photobiol. A 2003, 154 (2-3), 179-188.
(14) Kohler, M.; Burnashev, N.; Sakmann, B.; Seeburg, P. H. Neuron 1993,
10 (3), 491-500.
(15) Partin, K. M.; Patneau, D. K.; Winters, C. A.; Mayer, M. L.; Buonanno,
A. Neuron 1993, 11 (6), 1069-1082.
(16) Honore, T.; Davies, S. N.; Drejer, J.; Fletcher, E. J.; Jacobsen, P.; Lodge,
D.; Nielsen, F. E. Science 1988, 241 (4866), 701-703.
(17) Ramesh, D.; Wieboldt, L. N.; Carpenter, B. K.; Hess, G. P. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90 (23), 11074-11078.
(18) Chen, E.; Kumita, J. R.; Woolley, G. A.; Kliger, D. S. J. Am. Chem. Soc.
2003, 125 (41), 12443-12449.
(19) Janssens, N.; Lesage, A. S. J. J. Neurochem. 2001, 77 (6), 1457-1474.
(20) Banghart, M.; Borges, K.; Isacoff, E. Y.; Trauner, D.; Kramer, R. H. Nat.
Neurosci. 2004, 7 (12), 1381-1386.
Taken together, these results describe a simple approach for
obtaining remote control of iGluR activity and neuronal firing with
a photochromic agonist. The active agonist is subtype-specific,
possesses good efficacy and affinity, and can be conveniently
(21) Boyden, E. S.; Zhang, F.; Bamberg, E.; Nagel, G.; Deisseroth, K. Nat.
Neurosci. 2005, 8 (9), 1263-1268.
(22) Lima, S. Q.; Miesenbock, G. Cell 2005, 121 (1), 141-152.
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