Synthesis, photophysical, photochemical and biological properties of caged GABA, 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy) carbonyl] amino] butanoic acid

Article abstract of DOI:10.1562/2004-07-08-RA-226.1

The photorelease of a caged neurotransmitter can be used to investigate the function of neuronal circuits in tissues. We have designed and synthesized a stable, caged γ-aminobutyric acid (GABA) derivative, 4-[[(2H-l-benzopyran- 2-one-7-amino-4-methoxy)carbonyl]amino] butanoic acid (BC204), that releases the neurotransmitter in physiological medium when irradiated with UV light at 300-400 nm in PBS at pH 7.4. The release of GABA occurs with the formation of the major photoproduct, 7-amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one, via a solvolytic photodegradation mechanism of the coumarin moiety and was confirmed by electrospray mass spectrometry/mass spectrometry (ESI MS/MS). BC204 is chemically stable and shows no intrinsic activity after many hours under physiological dark conditions. These properties suggest that BC204 is an excellent form of caged GABA that is well suited for long-term biological studies.

Full text of DOI:10.1562/2004-07-08-RA-226.1

Synthesis, Photophysical, Photochemical and Biological Properties of Caged
GABA, 4-[[(2H-1-Benzopyran-2-one-7-amino-4-methoxy) carbonyl] amino]
Butanoic Acid¶
Author(s): Beate Cürten, Paul H. M. Kullmann, Mark E. Bier, Karl Kandler, and Brigitte F. Schmidt
Source: Photochemistry and Photobiology, 81(3):641-648.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/2004-07-08-RA-226.1
URL: http://www.bioone.org/doi/full/10.1562/2004-07-08-RA-226.1
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Photochemistry and Photobiology, 2005, 81: 641–648
Synthesis, Photophysical, Photochemical and Biological Properties of
Caged GABA, 4-[[(2H-1-Benzopyran-2-one-7-amino-4-methoxy)
carbonyl] amino] Butanoic Acid{
Beate Cu¨rten1, Paul H. M. Kullmann2, Mark E. Bier3, Karl Kandler2 and Brigitte F. Schmidt*1
1Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213
2Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
3Center for Molecular Analysis and Department of Chemistry, Carnegie Mellon University,
4400 Fifth Avenue, Pittsburgh, PA 15213
Received 7 July 2004; accepted 12 December 2004
glutamate, only a few caged forms of the fast inhibitory neuro-
transmitters c-aminobutyric acid (GABA) and glycine are currently
ABSTRACT
The photorelease of a caged neurotransmitter can be used to
investigate the function of neuronal circuits in tissues. We have
designed and synthesized a stable, caged c-aminobutyric acid
(GABA) derivative, 4-[[(2H-1-benzopyran-2-one-7-amino-4-
methoxy)carbonyl]amino] butanoic acid (BC204), that releases
the neurotransmitter in physiological medium when irradiated
with UV light at 300–400 nm in PBS at pH 7.4. The release of
GABA occurs with the formation of the major photoproduct,
7-amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one, via a sol-
volytic photodegradation mechanism of the coumarin moiety
and was confirmed by electrospray mass spectrometry/mass
spectrometry (ESI MS/MS). BC204 is chemically stable and
shows no intrinsic activity after many hours under physiolog-
ical dark conditions. These properties suggest that BC204 is an
excellent form of caged GABA that is well suited for long-term
biological studies.
available (5–7). These forms have several drawbacks, such as low
stability under physiological dark condition (8), noninert photolysis
intermediates or byproducts (7) and the need for high laser power (9–
11), all of which limit a more widespread use of these compounds
in neurobiological research. In order to develop an improved form
of caged GABA, which would overcome some of these limitations,
we predicted new structures based on molecular modeling studies.
Caging reagents based on the coumarin structure originated in
4-bromomethyl-7-methoxycoumarin that was used as a phosphate
protecting group (12). These substances were reported to have good
quantum yields and sufficient stability in aqueous medium (13).
Brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates
of glutamate where shown to release the neurotransmitter under
one- and two-photon photolysis (14,15). Coumarins show a large
variability in their absorption properties, dependent on the substi-
tution pattern, and are readily synthesized by employing a modified
Pechmann cyclization (16). Therefore, this substance class appeared
to be a suitable candidate for further development to improve its
quality as a photosensitive protecting group. In this article, we re-
port a facile synthesis for an amino-substituted coumarin-caged
GABA (BC204), the investigation of its photo physical and
photochemical properties, and its biological activity.
INTRODUCTION
Caged neurotransmitters, which are photolabile, biologically inert
prodrugs, have been increasingly used as a tool in cell biology,
particularly in studying neuronal processes (1–4). In the past, many
caged forms of the major fast excitatory neurotransmitter glutamate
have been produced. Homologous forms of caged glutamate have
different properties regarding their kinetics, quantum yield of photol-
ysis, stability or release probability upon two-photon excitation. The
differences between these molecules give researchers the opportu-
nity to select the compound that best accommodates their specific
experimental needs. In contrast with the wide variety of caged
MATERIALS AND METHODS
Anhydrous THF, DMF, DMSO (Aldrich Chem. Co., Milwaukee, WI) and
all reagents were used as received. Other solvents were reagent grade and
used without further purification if not stated otherwise. Tetrodotoxin (TTX)
was purchased from Alomone Labs (Jerusalem, Israel), bicuculline
methiodide from Tocris (Ballwin, MO), 2-OH-saclofen from RBI (Natick,
MA). If not stated otherwise, all other chemicals used for electrophysiolog-
ical recordings were from Sigma (St. Louis, MO).
{Posted on the website on 28 December 2004
Analytical high-performance liquid chromatography (HPLC) was per-
formed on a Waters Delta 600 system with UV-VIS detection. A C18 column
(Nova-Pak 4 l, 3.93300 mm, Waters) was used for HPLC analysis at a flow
rate of 1 mL/min at 208C with an injection volume of 10 lL. The solvent
system comprises eluent A acetonitrile/water/TFA 5 80/20/0.07%, and
eluent B water/0.1% TFA. 1H-NMR spectra were recorded at 300 MHz with
the residue solvent peak as reference relative to TMS. 13C-NMR spectra
were recorded at 75.4 MHz and calibrated to the solvent peak. The spectra
recorded in D2O were calibrated using MeOH as an internal standard. Flash
chromatography was performed on silica gel (J. T. Baker, 40 mm particle
size). Melting points are not corrected. UV/VIS spectra were recorded on
*To whom correspondence should be addressed: Molecular Biosensor and
Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue,
Pittsburgh, PA 15213. Fax: 412-268-7548; e-mail: bschmidt@cmu.edu
Abbreviations: ACSF, artificial cerebrospinal fluid; BC204, 4-[[(2H-1-
benzopyran-2-one-7-amino-4-methoxy)carbonyl]amino] butanoic acid;
ESI MS, electrospray ionization mass spectrometry; GABA, c-amino-
butyric acid; HPLC, high-performance liquid chromatography; LC, liquid
chromatography; MS/MS, mass spectrometry/mass spectrometry; TTX,
tetrodotoxin.
Ó 2005 American Society for Photobiology 0031-8655/05
641

642 Beate Cu¨rten et al.
a Hewlett-Packard spectrophotometer. Elemental analyses were performed
by Atlantic Microlab, Inc. (Norcross, GA).
6.63–6.59 (m, 1 H), 6.52–6.51 (m, 1 H), 6.16 (s, 1 H), 5.63 (s, br, 2 H), 5.10
(s, br, 1 H), 4.72 (s, br); 13C-NMR (DMSO-d6) d 161.2 157.1, 155.5, 152.9,
125.0, 111.2, 106.4, 103.8, 98.7, 59.1; analytically calculated for
C10H9NO3: C, 62.82, H, 4.74, N, 7.25; found: C, 62.62, H, 4.78, N, 1.25.
4-[[(2H-1-Benzopyran-2-one-7-amino-4-methoxy)carbonyl] imidazolide
7. Crude 7-Amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one 6 (191 mg,
1 mmol) and carbonyldiimidazole (194 mg, 1.2 equivalents) were dissolved
in 2 mL dry DMF. The product 7 precipitates within 1 h. The yellow solid is
filtered off, washed with 2 mL dry DMF, followed by 4 mL dry acetonitrile
and dried under vacuum. The product can be stored at room temperature
as a dry powder in a dark glass bottle in a desiccator over a prolonged period
of time. 1H-NMR (DMSO-d6) d 8.32 (s 1H), 7.64 (s, br 1H), 7.4 (d 1 H),
7.06 (s, br 1 H), 6.54 (dd, 1 H), 6.42 (d, 1 H), 6.15 (s, br, 2 H), 6.12 (s, 1H),
5.55 (s, 2H).
Syntheses: (3-Hydroxyphenyl)-carbamic acid ethyl ester 2. Commercially
available 3-aminophenol 1 (10.0 g, 92 mmol) was dissolved in a mixture of
10 mL dry THF and 9.0 mL (113.4 mmol, 1.2 equivalents) pyridine under
argon atmosphere. The reaction mixture was cooled to þ38C and 8.8 mL
(10.0 g, 92 mmol) of ethyl chloroformate were added slowly, so that the
temperature remained below þ108C. Stirring was continued at room
temperature for 14 h. Then 50 mL of water were added and the mixture
was extracted with 30 mL of CH2Cl2. The organic layer was separated,
washed with 3 3 30 mL water, dried over Na2SO4, filtered and the solvent
was evaporated. The greenish, oily residue was treated with 5 mL of toluene
and cooled to þ48C for 1 h yielding 10.26 g (62%) of colorless crystals: mp
958C; IR (KBr) 3301 (s, br, OHþNH), 1693 (s, CO); 1H-NMR (acetone-d6)
d 8.39 (s, br, 1 H), 8.17 (s, 1 H), 7.20–6.93 (m, 3 H), 6.50–6.46 (m, 1 H),
4.12 (q, 2 H, 3J57.1 Hz), 1.23 (t, 3 H, 3J57.1 Hz); 13C-NMR (acetone-d6)
d 159.7, 155.3, 142.4, 131.1, 111.3, 111.2, 107.2, 61.9, 15.8; analytically
calculated for C9H11NO3: C, 59.66; H, 6.12; N, 7.73; found C, 59.69;
H, 6.20; N, 7.76.
[4-(Chloromethyl)-2-oxo-2H-1-benzopyran-7-yl]-carbamic acid ethyl
ester 3. Sulfuric acid (80%, 50 mL) was precooled with an ice bath.
Then 1.81 g (10 mmol) of the protected aminophenol and 1.81 mL (2.20 g,
13 mmol) ethyl 4-chloroacetoacetate were added. The mixture was stirred at
room temperature for 4 h, then poured into 50 mL ice water, and the grayish
precipitate was filtered and dried in the air. The semisolid was treated with
50 mL MeOH at 408C for 20 min. Filtration of this slurry yielded 1.89 g
(67%) of a white solid: mp 2478C (decomp.); IR (KBr) 3284 (m, br, NH),
1702 (s, CO), 1H-NMR (DMSO-d6) d 10.15 (s, 1 H), 7.77-7.74 (m, 1 H),
7.59-7.58 (m, 1 H), 7.45-7.40 (m, 1 H), 6.51 (s, 1 H), 4.97 (s, 2 H), 4.18 (q,
2 H, 3J 5 7.0 Hz), 1.27 (t, 3 H, 3J 5 7.35 Hz); 13C-NMR (DMSO-d6) d
198.0, 168.5, 159.9, 153.3, 150.6, 143.1, 125.7, 114.3, 112.6, 104.6, 60.7,
41.2, 14.3; UV (MeOH) k 5 332 nm, e 5 13976; analytically calculated for
C13H12ClNO4: C, 55.43; H, 4.29; N, 4.97; found C, 55.81; H, 4.35; N, 5.22.
7-Amino-4-(chloromethyl)-2H-1-benzopyran-2-one 4.[4-(Chloromethyl)-
2-oxo-2H-1-benzopyran-7-yl]-carbamic acid ethyl ester 3 (1.0 g, 3.55
mmol) was suspended in a mixture of 3 mL concentrated H2SO4 and 3 mL
glacial acetic acid. The suspension was heated to 1258C for 2 h. After
cooling to room temperature, the brown solution was poured on 100 mL
crushed ice and brought to pH 9 with 1 N NaOH solution. The light-yellow
solid was filtered and dried in vacuo (yield 63%). The product was used for
synthesis without further purification. A sample amount was purified for
analysis purposes as follows: The product was dissolved in THF and
acidified with diluted HCl. The precipitate was extracted with water. The
aqueous layer was separated and basified with concentrated NaOH solution
until precipitation occurred. The purified product was filtered and dried in
vacuo, yielding 64% of a yellowish solid, mp 1878C (decomp.); IR (KBr)
3450 (s, NH2), 3351 (s, NH2), 1689 (ss, CO), 1622 (s, NH2); 1H-NMR
(DMSO-d6) d 7.53–7.49 (m, 1 H), 6.65–6.61 (m, 1 H), 6.49–6.48 (m, 1 H),
6.23–6.21 (sþs, nonres. 3 H), 4.90 (s, 2 H); 13C-NMR (DMSO-d6) d 160.5,
1559, 153.3, 151.1, 126.0, 111.2, 107.8, 106.1, 98.6, 41.3; UV (MeOH)
kmax 366 nm, e 5 15 066; analytically calculated for C10H8ClNO2: C,
57.30; H, 3.85; N, 6.68; found C, 57.31; H, 3.84; N, 6.79.
4-[7-Amino-2H-1-benzopyran-2-one)-methyl acetate 5. 7-Amino-4-
(chloromethyl)-2H-1-benzopyran-2-one 4 (500 mg, 2.39 mmol) and KOAc
(1.2 equivalents) were dissolved in 2 mL dry DMF and a small amount of
tetrabutylammonium bromide was added. The mixture was warmed up to
508C and stirred at room temperature for 5 h. The reaction mixture was poured
into 15 mL water, the solid was suction filtered and dried. The raw product
was crystallized from ether affording 445 mg of 5 as a yellowish solid (80%):
mp 218 8C; IR (KBr): 3447 (s, NH), 3355 (s, NH), 1750 (s, C5O ester), 1688
(s, br, C5O, lactone); 1H-NMR (DMSO-d6) d 7.42–7.39 (m, 1 H), 6.62–6.59
(m, 1 H), 6.48–6.47 (m, 1 H), 6.20 (s, br, 2 H), 6.00 (s, 1 H), 5.27 (d, 2 H, J5
1.5 Hz), 2.20 (s, 3 H); 13C-NMR (DMSO-d6) d 170.0, 160.6, 155.7, 153.3,
150.8, 125.5, 111.4, 105.9, 104.9, 98.7, 61.1, 20.5; analytically calculated for
C12H11NO4: C, 61.80, H, 4.75, N, 6.01; found: C, 61.57, H, 4.85, N, 5.84.
7-Amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one 6. 4-[7-Amino-2H-
1-benzopyran-2-one)-methyl acetate 5 (100 mg, 0.43 mmol) and K2CO3
(60 mg, 1.1 equivalents) were suspended in 5 mL MeOH and stirred at
room temperature for 18 h. Then 20 mL THF and Na2SO4 were added and
the solution was filtered to remove all inorganic salts. The solvents were
evaporated and the residue was treated with MeOH to afford the product 6
as a light-yellow solid (yield: 45 mg, 55%): mp 1878C (decay); IR (KBr)
3445 (s, br, NH2), 3350 (ss, br, NH2-þ OH), 1674 (ss, br, CO-valence,
lactone); 1H-NMR (acetone-d6 þ 2 dr. DMSO-d6) d 7.35–7.32 (m 1 H),
4-[[(2H-1-Benzopyran-2-one-7-amino-4-methoxy)carbonyl]amino] bu-
tanoic acid 8. 7-Amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one
6
(100 mg, 0.52 mmol) and carbonyldiimidazole (104 mg, 1.2 equivalents)
were dissolved in 1 mL dry DMSO. The solution was stirred at room tem-
perature for 3 h, then GABA (65 mg, 1.2 equivalents) was added and the
mixture was heated to 808C for 1 h. After cooling to room temperature, 50
mL of water were added and the mixture was acidified to pH 3–4 with 1 M
HCl. The precipitate was extracted with 3340 mL ethyl acetate. The organic
layers were combined, washed with 3330 mL water, dried (Na2SO4) and the
solvent evaporated. The residue was crystallized by treatment with a mixture
of 1 mL acetone and 1 mL ether yielding 88 mg (53%) of 8 (yellowish solid),
mp 194 8C; IR (KBr) 3464 (s, br, NH), 3363 (s, br, NH), 3093 (s, br, OH
carboxylic acid), 1724 (ss, CO carboxylic acid), 1695 (ss, CO urethaneþCO
lactone); 1H-NMR (DMSO-d6) d 7.53 (t, 1 H, 3J 5 6.0 Hz), 7.40–7.37
(m, 1 H), 6.62–6.58 (m, 1 H), 6.48–6.47 (m, 1 H), 5.97 (s 1 H), 5.22 (s, 2 H),
3.08 (m, 2 H), 2.28 (t, 2 H, 3J57.4 Hz), 1.70 (mc, 2 H); 13C-NMR (DMSO-
d6) d 174.1, 160.7, 155.6, 155.5, 153.2, 152.2, 125.3, 111.3, 105.8, 104.2,
98.6, 60.8, 39.8, 30.8, 24.7; analytically calculated for C15H16N2O6: C,
56.25, H, 5.03, N, 8.75; found: C, 56.02, H, 4.90, N, 8.59.
4-[[(2H-1-Benzopyran-2-one-7-amino-4-methyl)amino] butanoic acid
11. 7-Amino-4-(chloromethyl)-2H-1-benzopyran-2-one
4 (418 mg, 2
mmol) and the tetrabutylammonium salt of GABA (1.2 equivalents) were
dissolved in 2 mL dry DMF and a small amount of potassium iodide was
added. The mixture was warmed to 608C and stirred for 5 h. The solvent
was removed under high vacuum and the residue was dissolved in water.
The pH was adjusted to 3–4 with 1 N HCl. The reaction mixture was
concentrated and purified on RP-18 (eluent: water; water/25% methanol).
Yield: 40 mg: 1H-NMR (DMSO-d6) d 7.45 (t, 1 H, 3J 5 6.0 Hz); 6.64 (dd,
1H); 6.41 (d, 1H); 6.09 (s, 1 H); 3.89 (s, br, 2 H), 2.58 (t, 2 H), 2.28 (t, 2 H),
1.68 (m, 2 H). The molecular weight was determined by electrospray mass
spectrometry/mass spectrometry (ESI MS) to be 275.9 Da (calc. 276.1Da)
and the mass spectrometry/mass spectrometry (MS/MS) product ions at m/z
259.0 and 174.0 agree with a loss of water and H2NCH2CH2CH2COOH.
Decay of 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)carbonyl]a-
mino] butanoic acid 8 and quantification of GABA release upon irradiation
with UV light. Photolysis was carried out using a high-pressure mercury
lamp (HBO 200, Oriel) with a filter allowing 240–400 nm light trans-
mission. For the quantification of the GABA release, the irradiated solutions
were postderivatized and analyzed by HPLC. Six hundred microliters of
a 1.25 3 10ꢀ4 M solution of, in 0.1 M PBS, pH 7.4 were placed in a quartz
corvette with a path length of 0.25 cm and irradiated for time periods
ranging from 5 to 90 s. Time points were taken every 5 s. The amount of
remaining caged compound 8 was determined by direct injection of 10 lL
of the irradiated sample. For the determination of GABA, a 100 lL aliquot
was removed and added to 100 lL of sodium bicarbonate buffer (0.2 M, pH
9) containing the valine standard (1 3 10ꢀ4 M). To this solution, 200 lL of
dabsyl chloride (2 nmol/lL in acetone) were added, the mixture was
vortexed and incubated at 608C for 15 min. Under these dabsylation
conditions, 8 is stable and does not undergo degradation. After cooling,
samples of 10 lL were analyzed by HPLC. The gradient is 10–20% A from
0 to 4 min; 20–60% A from 4 to 8 min; 60–10% A 8–8.5min and 100%A
from 8.5 to 16 min. The dabsylated derivatives were detected at k 5 436
nm. Coumarin derivatives were detected at k 5 345 nm.
Quantum yield of photodegradation. To provide an average quantum
yield for the decaging reaction, we calibrated the irradiation system in the
300–400 nm range using ferrioxalate actinometry (17). The quantum yield
of decaging was calculated for each Hg spectral line according to the
equation udec 5 (dc/dt)obs abs
I
/(1 ꢀ V). The value of the reaction rate, dc/dt
(mol Lꢀ1 sꢀ1), was calculated from the initial slope of the degradation curve
of 8 (Fig. 2B). The value of Iabs (absorbed light intensity in moles of

Photochemistry and Photobiology, 2005, 81 643
Figure 1. Synthesis of 4-[[(2H-1-benzo-
pyran-2-one-7-amino-4-methoxy)carbo-
nyl]amino] butanoic acid (BC204)
prepared in seven steps from 3-amino-
phenol.
photons per second at t 5 0 and k 5 313, 333 or 365 nm) was calculated by
multiplying the absorption factor (taken from the UV spectrum of 8) by the
light intensity of the HBO 200 Oriel lamp (determined using the
actinometer compound.). The solution volume was 3 mL.
Membrane resistance was determined by applying 50 ms long, 5–10 mV
voltage steps. Series resistance (less than 50 MX) was compensated 65–
70%. Data were filtered at 3 KHz, digitized at a rate of 10 KHz and stored
on hard disk for off-line analysis using custom-written software (P.H.M.K.)
in the LabVIEW environment (National Instruments, Austin, TX).
Focal photolysis of BC204. For photolytic cleavage, BC204 stock
solution (0.1 M in 0.1 M NaOH) was added to the ACSF to result in a final
concentration of 200 lM. Focal photolysis was achieved by guiding the
light from a 100 Watt mercury arc lamp (Oriel, Stratford, CT) to the re-
corded cell using small diameter fused silica optical fibers (core diameter of
50 lm; Polymicro Technologies, Phoenix, AZ). Duration of the light pulses
was regulated by an electronically controlled shutter (Vincent Associates,
Rochester, NY) located between the arc lamp and the optical fiber. For more
details, see (20).
Liquid chromatography/mass spectrometry/mass spectrometry. Liquid
chromatography MS/MS analysis was done using a Magic 2002 liquid
chromatograph (Michrom BioResources, Inc. Auburn, CA) operated with
EZChrom software (Scientific Software, Inc.). The 1-mm column (Michrom
Bioresources, Inc. Auburn, CA) was packed with C18 particles with 200
angstrom pores to a length of 15 cm. The mobile phase A was 99.9% water
and 0.1% acetic acid (v/v) and mobile phase B was 99.9% acetonitrile and
0.1% acetic acid (v/v). The chromatographic runs were performed at a flow
rate of 50 lL/min and used a gradient from 0% B to 100% B in 15 min. The
column effluent was electrosprayed directly into the LCQ ion trap mass
spectrometer (Thermo Electron Corporation, San Jose, CA) operated in
positive ion mode for either full scan MS or MS/MS mode. The LCQ was
operated with Xcalibur software version 1.2.
Animals and brain slice preparation. Experiments were performed in
living brain slices prepared from 5–7 day old mice (C57Bl/6J, Jackson Lab-
oratory, Bar Harbor, ME). Experimental procedures were in accordance with
National Institutes of Health guidelines and were approved by the IACUC at
the University of Pittsburgh. Preparation and maintenance of slices was per-
formed as described in detail elsewhere (18). In short, animals were deeply
anesthetized by hypothermia and decapitated. The brain was quickly re-
moved and transferred to cold (4–88C) artificial cerebrospinal fluid (ACSF;
composition in mM: NaCl 124, NaHCO3 26, Glucose 10, KCl 5, KH2PO4
1.25, MgSO4 1.3, CaCl2 2, pH 5 7.4, when aerated with 95% O2/5% CO2).
ACSF used for preparation also contained 1 mM kynurenic acid. Coronal
slices of the brainstem (250 lm thick) were cut on a vibrating microtome and
kept at room temperature in an interface chamber until recordings.
Electrophysiological recordings. All recordings were made from neurons
in the lateral superior olive, an auditory brainstem nucleus that receives
GABAergic input (19). Slices were placed in a submerged-type recording
chamber and continuously superfused with freshly oxygenated ACSF cir-
culating at a rate of 2 mL/min (total ACSF volume 15 mL). Standard whole-
cell patch clamp recordings were obtained with pipettes filled with solution
containing (in mM) K-gluconate 84, KCl 38, MgCl2 2, HEPES 10, Na2-
phosphocreatine 5, Mg-ATP 3, Na2-GTP 0.3, adjusted with KOH to pH 7.2.
Whole-cell currents were recorded in voltage-clamp using an Axopatch 1D
amplifier (Axon Instruments). All recordings were made in the presence
of the sodium channel antagonist tetrodotoxin (1 lM, Alomone Labs,
Jerusalem, Israel) to prevent action potentials and synaptic transmission.
RESULTS AND DISCUSSION
Design approach and theoretical calculations
Our design criteria called for a caged GABA derivative that was
stable under physiological dark conditions, but would rapidly re-
lease GABA when illuminated with a mercury arc lamp as used in
a previously described one-photon uncaging setup (21). Thus we
focused on a coumarin-caging reagent that was linked to GABA via
a carbamate function, which combines a high chemical stability
with useful properties for photodeprotection (as proven for
nitrobenzyl systems). In order to tune the absorption properties of
caging reagent to an optimal irradiation wavelength range, we
investigated the influence of the substitution pattern on the 2H-1-
benzopyran system on its long wavelength absorption maximum. In
aqueous media under physiological pH, the substituent in the 7-
position of the 2H-1-benzopyran-2-one derivatives had the most
significant influence on the absorption. This effect was simulated by
theoretical calculations using CAChe 3.0 (Oxford Molecular).
Geometry optimization of the structures was carried out with Mo-
lecular Mechanics (MM2) (22), followed by a MOPAC (Molecular
Orbital Package) calculation using the PM3 Hamiltonian (23). From

644 Beate Cu¨rten et al.
Table 1. UV and fluorescence data for coumarin caged GABA 8–10
(5 3 10ꢀ6 M solutions in PBS buffer solution, pH 7.4)
kmax,abs nm kmax,abs nm kmax,em nm e 1/mol
Substituent-R (theoretical) (experimental) (experimental) cm
/
fi
9, -O-CH3
10, 7-OH
8, 7-NH2
308
310
320
324
326
348
418
483
477
13 000 0.41
10 000 0.21
14 000 0.40
the optimized structures, UV spectra were calculated with ZINDO3
(Professor M. C. Zerner’s Intermediate Neglect of Differential
Overlap program). We calculated 7-methoxy-, 7-hydroxy- and 7-
amino-substituted coumarin-caged GABA derivatives and obtained
theoretical UV absorption wavelengths that showed a bathochromic
shift of 10 nm in the absorption maximum of the 7-amino-
substituted coumarin derivatives versus the 7-hydroxy- and 7-
methoxy-substituted compounds. These calculations encouraged
our efforts to synthesize and study the photochemical and photo-
physical properties of 4-[[(2H-1-benzopyran-2-one-7-amino-4-
methoxy)carbonyl]amino] butanoic acid 8.
Synthesis and photophysical properties
To synthesize BC204, 8, 7-Amino-4-(chloromethyl)-2H-1-benzo-
pyran-2-one, 4 was prepared via Pechmann cyclization of (3-
hydroxyphenyl)-carbamic acid ethyl ester 2 (24) followed by
deprotection of the amino group (25). The protection of the amino
group is required to avoid excessive oxidation during the cyclization
reaction. To allow the introduction of a carbamate linker, 7-amino-4-
(chloromethyl)-2H-1-benzopyran-2-one 4 had to be converted into
the 4-hydroxymethyl derivative. Simple heating in water for 48 h, as
reported for 6-bromo-4-chloromethyl-7-hydroxy coumarin (15), did
not result in the desired product. Thus, 7-amino-4-(chloromethyl)-
2H-1-benzopyran-2-one 4 was converted into 7-amino-4-(hydrox-
ymethyl)-2H-1-benzopyran-2-one 6 as shown in Fig. 1 by a two-step
reaction that involved the formation of 4-[7-amino-2H-1-benzo-
pyran-2-one)-methyl acetate 5 under SN2 conditions and its sub-
sequent methanolysis. This reaction sequence could be combined
in a two-step-one-pot synthesis. Coupling of 6 with GABA via a
carbamate linker was achieved in another multistep-one-pot pro-
cedure. In the first step, 7-amino-4-(hydroxymethyl)-2H-1-benzo-
pyran-2-one 6 was reacted with carbonyl-diimidazol to give the
imidazolide 7 and without isolation directly reacted with GABA to
afford 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)carbony-
l]amino] butanoic acid 8 in good yield. For this reaction, unprotected
GABA could be used due to the higher reactivity of the amino group.
When DMF is used as a solvent in the reaction of 7-amino-4-
(hydroxymethyl)-2H-1-benzopyran-2-one 6 with carbonyl diimi-
dazol, the imidazolide 7 precipitates from the reaction mixture and
can be isolated by simple filtration. The solid can be washed and
stored as a stable dry powder. This variant allows the use of crude
7-amino-4-(hydroxymethyl)-2H-1-benzopyran-2-one 6 and is par-
ticularly valuable for obtaining a clean active caging reagent 7 with
out a sophisticated purification step. We have successfully used 7
to synthesize caged glycine and caged glutamate (results will be
published elsewhere).
Figure 2. A: Absorption spectrum of BC204; wavelength range of
irradiation; lines and spectral energy distribution of a medium-pressure
mercury arc lamp. B: BC204 photodegradation where 50% of the GABA is
release within the first 10 s.
Dk 5 22–24 nm as compared with the 7-hydroxy- and 7-methoxy-
substituted compounds 9 and 10, respectively (structures not
shown). The experimental data confirmed the trend that we had seen
in our theoretical calculations, although our absolute values for the
theoretical maximum absorption wavelengths are low. The bath-
ochromic shift in 8 can be attributed to the enhanced donor effect of
the amino group. At pH 7.4, the amino group on the 7 position is not
protonated and thus can display its donor effect in contrast with
7-hydroxy–substituted coumarin derivative 10.
Photolysis
We studied the photochemical degradation under similar conditions
that are employed in the biological decaging studies. Thus we used
a 1.25310ꢀ4M solution of 8 in PBS and irradiated a 600 lL aliquot
with UV light from a medium-pressure mercury arc lamp used with
a bandpass filter between 240 and 400 nm. Figure 2A shows the
Spectral properties
As shown in Table 1, the absorption maximum of the 7-amino
substituted coumarin 8 exhibits a large bathochromic shift of

Photochemistry and Photobiology, 2005, 81 645
Figure 3. A: Summed selected mass
chromatogram for each [MþH]þ ion at
m/z 192.1, 277.1 and 321.1 of peaks 1, 2
and 3, respectively. B: ESI mass spectrum
of peak 1 with assigned structure. C: ESI
mass spectrum of peak 2 with assigned
structure. D: ESI mass spectrum of peak 3
with assigned structure. E: ESI MS-MS of
peak 1, m/z 277.1, with a precursor
isolation window of ca 4 amu, showing
fragment ions from the loss of water and
absorption spectrum of 8, the range of irradiation and the spectral
lines of the mercury arc lamp. Under these irradiation conditions,
4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)-carbonyl]amino]
butanoic acid 8 degrades rapidly, as shown qualitatively by NMR-
spectroscopy and quantitatively by HPLC. The fluorescamine assay
(26) as a quantification method for the GABA release upon irra-
diation can not be applied for 7-amino-substituted coumarin deriv-
atives because fluorescamine forms a chromophore with the 7-amino
group that strongly interferes with the detection of the GABA-
fluorescamine adduct. Thus, we postderivatized the released GABA
with dabsyl chloride and quantified the dabsylated GABA via HPLC
(27). Valine was added after the irradiation as an internal standard.
Fifty percent of the GABA is released within the first 10 s from 8, as
shown in Fig. 2B. The quantum yield of photodegradation was
determined to be 0.04 and is within the range of quantum yields
reported for various (coumarin-4-yl)methyl esters of cAMP (28).
After complete photodegradation of 8, only 85% of free GABA was
determined. To investigate the fate of the residual GABA, we sepa-
rated the photoproducts byHPLC and analyzedthe peaksbyESI MS.
weight of reaction products with little to no fragmentation due to
the soft ionization process. The summed ion mass chromatogram
from LC ESI MS for a reaction mixture that was irradiated for 20 s
shows the parental compound 8 and two photoproducts in Fig. 3A.
As expected, one of these photoproducts was identified as 7-amino-
4-(hydroxymethyl)-2H-1-benzopyran-2-one 6 m/z 192.2 (Fig. 3C).
In addition, a second photoproduct with a molecular mass [MþH]þ
of m/z 277.1 is formed (Fig. 3B) and the ESI mass spectrum for the
parent compound 8 is shown in Fig. 3D. The ion signals in the
mass chromatogram are not quantitative because of the differences
in proton affinity among the photoproducts. ESI mass spectrom-
etry/mass spectrometry (MS/MS) was performed on the precursor
[MþH]þ at m/z 277.1 as shown in Fig. 3E and the mass
assignments support the structure of 4-[(2H-1-benzopyran-2-one-
7-amino-4-methyl]- amino butanoic acid 11 showing loss of water
and GABA. Isolation and characterization of 11 for NMR analysis
proved to be too difficult because the compound forms only in small
amounts. Thus, we synthesized the predicted compound 4-[(2H-1-
benzopyran-2-one-7-amino-4-methyl]- amino butanoic acid, and
performed LC, MS and MS/MS to further confirm its structure. The
LC retention time, molecular weight and product ions formed from
MS/MS agreed with the assigned structure 11.
Liquid chromatography electrospray mass spectrometry
Liquid chromatography (LC) electrospray ionization (ESI) mass
spectrometry (MS) was used to ionize and determine the molecular
In order to understand the formation of 11 from 8, we added
valine as a competing nucleophile to the PBS solution prior to the

646 Beate Cu¨rten et al.
Figure 4. The formation of 6 and GABA is the result of photosolvolysis of
8. For the formation of 11, two mechanisms are proposed: an intramolecular
1–3 rearrangement under extrusion of carbon dioxide C or a nucleophilic
competition reaction B of the photoreleased GABA with the water
photosolvolysis step A.
irradiation (nucleophiles such as cysteine and isoleucine work
equally well). Under dark conditions, the valine did not cause
a release of GABA, however, the concentration of photoreleased
GABA increased when valine competitively inhibited the formation
of 11. Thus, we conclude that 11 most likely forms through an ion-
pair return of the photoreleased GABA after the loss of CO2. This
pathway is supported by the fact that the ratio of GABA to 11
remained fairly constant during photolysis. This process is relevant
in a closed system where the released GABA stays present. In
a biological open system, GABA will be immediately taken up by
receptors or can diffuse away and thus minimize the formation of
the by-product 11.
Biological Activity of BC204
To determine the biological properties of BC204 and to estimate its
potential as a tool in neurobiological research, BC204 was tested in
auditory neurons in the lateral superior olive in brainstem slices of
mice. In these experiments, we focused on those basic properties
that are most important for the general use of caged neurotrans-
mitters (3,29). First, the caged neurotransmitter should be biologi-
cally inactive and in particular should not act as an agonist or
antagonist on the receptors under study. Second, under physiolog-
ical darkness conditions, the compound should be stable so that
biological specimens can be exposed to the caged compound over
longer time periods. Third, the compound should be readily cleav-
able with light a wavelength .300 nm and with moderate inten-
sity to minimize UV-induced physiological changes (30) or cell
damage and to avoid the necessity of using expensive, high-power
light sources.
Figure 5. A: Photolysis of BC204 elicits whole-cell currents in auditory
brainstem neurons. The slice was bathed in ACSF containing 200 lM
BC204. The inward currents evoked by illuminating the area of the slice
around the recorded neuron increase in amplitude and duration with
increasing illumination times (opening of light shutter indicated by arrow).
For a given flash duration, repeated photolysis of BC204 results in repro-
ducible current responses. No responses are observed when the UV com-
ponent of the illuminating light is removed by placing a filter in the light
path. B: Currents elicited by photolysis of BC204 are due to activation of
GABAA receptors. The specific GABAA receptor antagonist bicuculline (20
lM) eliminates the current response evoked by photolysis (15 ms flash
duration) of BC204 (200 lM). Wash-in of bicuculline starts at time 0,
reaching the slice after about 3 min. Current trace insets show single
responses at the times indicated in the graph. C: The reversal potential of
BC204-evoked currents is consistent with the activation of a ligand-gated
chloride conductance. Currents elicited by photolysis of BC204 were
measured while holding the cell membrane at various potentials. Currents
reverse polarity at ꢀ29 mV in this cell, close to the theoretical chloride
reversal potential of ꢀ30 mV. Current trace inset shows averaged responses
at the holding potential indicated to the left (200 lM BC204, flash duration
3 ms). The arrows above the traces indicate the time of UV flash.
The effects of photolyzed BC204 on membrane currents was
tested using whole-cell patch clamp recordings from individual
neurons. For uncaging, the output of a mercury arc lamp was aimed
to a small area around the recorded cell using a 50 lm diameter
quartz fiber as previously described (18). Action potentials and
synaptic transmission were blocked with tetrodotoxin (1 lM) and
metabotropic GABAB receptors were blocked by 2-OH-saclofen
(10 lM). Under these conditions, photolysis of BC204 (200 lM)
with short light pulses (5–40 ms) produced transient whole-cell
inward currents. Increasing the duration of the pulses generated
currents of larger amplitude and longer duration. For a given pulse
length, response amplitudes and durations were remarkably repro-
ducible (Fig. 5A), which indicated that hydrolysis products or the
amount of the UV component delivered during repetitive illumi-
nation had no obvious detrimental effects on the recorded neuron.
As expected from the absorption spectrum of BC204 (Fig. 2A),
blockade of the UV light component (filter with center frequency
540 nm, bandpass 25 nm) completely prevented these responses.
Two lines of evidence indicate that photolysis-evoked membrane
currents were mediated by specific activation of GABAA receptors, a
ligand-gated chloride channel (31). First, responses were abolished
when GABAA receptors were blocked by the specific antagonist
bicuculline (20 lM) (Fig. 5B). Second, when responses were elicited

Photochemistry and Photobiology, 2005, 81 647
readily to photolyzed BC204. Furthermore, bath application of 20
lM GABA to these cells induced a steep drop in the membrane
resistance to 25 6 3.6% of its initial value (n 5 2). This indicates
that the absence of any noticeable membrane resistance changes
upon BC204 application cannot be attributed to desensitization of
GABAA receptors due to slow buildup of GABA in the slice.
GABA a-carboxy-2-nitrobenzyl ester [CNB-caged GABA] at
more than 180 lM depresses presynaptic calcium influx at granule
cell-Purkinje cell synapses (32), consistent with the original report
of the synthesis of this substance by Gee et al. (33) (also: absorption
maximum of CNB-caged GABA at 262 nm). Use of a 1 day old
stock solution kept at room temperature also had no effect on the
membrane resistance (n53 cells). Storage of stock solution did not
seem to affect the ability to produce responses on uncaging, as we
observed no obvious differences in response amplitudes between
using fresh and stored stock solutions (fresh, 125–160 pA; 1 day
old, 100–450 pA; 11 day old, 40–700 pA). Finally, our recordings
also indicate that BC204 is also stable at physiological pH, a basic
requirement for using caged compounds in longer experiments (34).
In our recordings lasting up to 1 h, we found no indication for
spontaneous degradation of BC204 in ACSF at pH 7.4, allowing us
to reuse BC204-containing ACSF in another slice or to keep a slice
in BC204-containing ACSF for 2 h before starting another
recording. In summary, these results indicate that BC204 does not
spontaneously degrade over the period tested at a rate detectable
with our biological system while it still maintains its ability to
undergo fast photolysis.
Figure 6. BC204 in solution is stable and can be kept for several days
without showing signs of spontaneous degradation. A: Wash-in of BC204
taken from freshly prepared stock solution (0.1 M in 0.1 M NaOH) does not
result in a change of neuronal membrane resistance (r peak current to
10 ms light pulse, u membrane input resistance). B: For another neuron,
wash-in of BC204 from a stock solution prepared 11 days before and kept
at 48C also has no effect on membrane resistance, indicating the absence of
free GABA (both cells: 200 lM BC204; flash duration 10 ms).
The 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)carbonyl]
amino] butanoic acid (BC204) is a stable form of caged GABA
that can be easily synthesized. This compound is chemically stable
under physiological dark conditions and shows no intrinsic
biological activity. ESI MS supports the stability of the structure
as little to no fragmentation was observed. GABA is readily
released from BC204 with an inexpensive one-photon irradiation
system that uses light at 300–400 nm from a mercury arc lamp.
The photo products were identified by LC ESI MS and the
structure of the by-product 4-[(2H-1-benzopyran-2-one-7-amino-4-
methyl]-amino butanoic acid was confirmed by LC ESI MS/MS
and comparison to synthetically derived compound. These
properties suggest that BC204 is an excellent form of caged
GABA that is particularly suited for the use in long-term
neurobiological studies.
at depolarized membrane potentials, current amplitudes decreased or
reversed in polarity (Fig. 5C), with a reversal potential of 32.8 6 4.3
mV (n 5 8 cells). This reversal potential is close to the theoretical
value of ꢀ30 mV as calculated with the Nernst equation for the
chloride concentration in our pipette (42 mM) and bath solution (133
mM), indicating that photolysis of BC204 activated chloride mem-
brane currents. Together, these results provide strong evidence that
responses elicited by photolysis of BC204 are not caused by
unspecific effects of hydrolysis byproducts, but rather are generated
by free GABA acting on GABAA.
Acknowledgements—We would like to thank the National Science Foun-
dation (NSF) for the grant MCB-8920118. M.E.B. would like to thank
the NSF for grant DBI-9729351 and Carnegie Mellon University for sup-
port in the purchase of the LC MS.
To address the stability of BC204 in solution, prephotolytic
activity and photolysis responses were tested in freshly prepared
and stored stock solutions. Prephotolytic activity due to the pres-
ence of free GABA was assessed by monitoring the membrane
input resistance with short voltage command steps during wash-in
of diluted stock solutions (200 lM). Under these conditions, the
presence of free GABA would become apparent as a reduction in
the membrane resistance due to activation of GABAA-activated
chloride channels. As shown in Fig. 6, wash-in of freshly dissolved
BC204 had no effect on the membrane resistance, indicating the
absence of detectable free GABA. More importantly, wash-in of
BC204 (200 lM) taken from stock solution, which was prepared 11
days before and stored in the dark at 48C, also did not change the
membrane resistance (n 5 3 cells). The absence of any detectable
membrane resistance change was not due to an unresponsiveness of
the recorded cells to GABA, as all of the tested cells responded
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