Synthesis and photocleavage of 7-[{Bis(carboxymethyl)amino}-coumarin-4-yl] methyl-caged neurotransmitters

Article abstract of DOI:10.1246/bcsj.80.2384

7-[{Bis(carboxymethyl)amino}coumarin-4-yl]methyl-caged neurotransmitters (glutamate and GABA) were synthesized. Both caged compounds showed sufficient stability in the dark, were water-soluble at pH 7.2 without using organic solvents, and exhibited relati

Full text of DOI:10.1246/bcsj.80.2384

2384 Bull. Chem. Soc. Jpn. Vol. 80, No. 12, 2384–2388 (2007)
ꢀ 2007 The Chemical Society of Japan
Synthesis and Photocleavage of 7-[{Bis(carboxymethyl)amino}-
coumarin-4-yl]methyl-Caged Neurotransmitters
ꢀ
Naoko Senda, Atsuya Momotake, and Tatsuo Arai
Graduate School of Pure and Applied Sciences, University of Tsukuba, Ibaraki 305-8571
Received May 18, 2007; E-mail: arai@chem.tsukuba.ac.jp
7-[{Bis(carboxymethyl)amino}coumarin-4-yl]methyl-caged neurotransmitters (glutamate and GABA) were syn-
thesized. Both caged compounds showed sufficient stability in the dark, were water-soluble at pH 7.2 without using
organic solvents, and exhibited relatively high quantum yield for photolysis upon irradiation with 390 nm light.
Photolabile precursors of bioactive effecter molecules (cag-
ed compounds) are powerful tools for understanding, control-
ling, and manipulating cell physiology.1–6 In many caged com-
pounds, covalent bond formation with a photoremovable pro-
tecting group masks important features for biological recog-
nition, and photocleavage (un-cage) of the caged compound
produces rapid jumps in the concentration of the bioactive
molecules. For biological use, photolabile protecting groups
must undergo rapid and efficient photolysis upon photo excita-
tion at wavelengths that are not harmful to the cells. In addi-
tion, enhancement of the hydrophilicity of the photolabile
protecting group makes the caged compound more soluble in
aqueous buffers without using unnecessary organic solvents.
Functionalized coumarins have been recognized and wide-
ly used as suitable chromophores caging for chemical mes-
senger molecules, such as cyclic nucleotide monophosphates
(cNMPs),7–12 cytidine diphosphate,13 and glutamic acid.14,15
Furuta et al. first reported that (7-methoxycoumarin-4-yl)-
methyl-caging group (MCM-caging group, Scheme 1) has
certain advantages, such as stability in the dark in physio-
logical buffer, high extinction coefficient at longer wavelength
region, and relatively high quantum yield of photorelease,
over other photolabile protecting groups.7 Hagen et al. have
introduced both axial and equatorial diastereomers of caged-
cNMPs using a (7-diethylaminocoumarin-4-yl)methyl-caging
group (DEACM-caging group).16,17 The DEACM-caged
cNMPs photorelease free cNMPs by photoirradiating at longer
wavelength more efficiently than the other coumarinylmethyl-
caged cNMPs. DEACM-caging group, itself, is however
hydrophobic, and therefore, an organic solvent must be
used to dissolve DEACM-caged cNMPs. Thus, they have
developed 7-[bis(carboxymethyl)amino]-4-methylcoumarin-
caged cyclic nucleotides (BCMACM-caged cNMPs), in which
the BCMACM-caging group maintains its positive photo-
chemical properties and there is an enhancement in the aque-
ous solubility due to the carboxylate groups in its structure.18
As a parent chromophore for BCMACM-caging group, we
have studied on the photochemical properties of 7-[bis(car-
boxymethyl)amino]-4-methylcoumarin (1).19 This molecule
is soluble in aqueous buffers at physiological pH without
using organic solvents, absorbs in the visible region, and
shows an excited singlet lifetime of 2.0 ns, which is considered
to be sufficiently long to undergo photolysis efficiently. Thus,
it should be possible to use 1 as the light-absorbing part
of the water soluble caged compounds not only for cNMPs
but also for other biologically active substances. Here, we de-
scribe the photocleavage of BCMACM-caged glutamate 6 and
BCMACM-caged GABA 7. Both compounds showed suffi-
cient solubility in aqueous buffer at pH 7.2, were stable in
the dark and had a reasonable quantum yield on photolysis.
Because glutamate and GABA are the major excitatory and in-
hibitory neurotransmitters, respectively, in the central nervous
system, 6 and 7 should facilitate research on the mechanism of
the neurotransmitter receptor.
Results and Discussion
Caged compounds 6 and 7 were prepared as shown in
Scheme 1 by condensing alcohol 2 with N-Boc-protected
glutamic acid and N-Boc-protected GABA to yield 4 and 5,
respectively, followed removal of tert-butyl ester groups with
trifluoroacetic acid (TFA). The TFA deprotection resulted in
the pure caged glutamic acid 6 and caged GABA 7, respective-
ly, which was confirmed by using 1H NMR, UV, and fluo-
rescence spectroscopy, and HPLC analysis. 7-[Bis(carboxy-
methyl)amino]-4-(hydroxymethyl)coumarin (3) was prepared
and used as an authentic photoproduct.
The thermal stability of the caged compounds 6 and 7 was
measured in HEPES buffer solution at pH 7.2. Caged com-
pounds 6 and 7 were pratically stable for 1 h in the dark at
room temperature but were gradually hydrolyzed to give the
half-lives of longer than 200 h for both 6 and 7. Caged com-
pounds 6 and 7 showed very little hydrolysis in the dark at
room temperature during 2 h of measurement. Hydrolysis
was not detected after 2 days, when the caged compounds were
dissolved in HEPES buffer at pH 7.2 and were stored at ꢁ4 ꢂC
in the dark.
The photochemical properties of the 1, 3, 6, and 7 in HEPES
buffer (pH 7.2) are summarized in Table 1. The absorption
and emission bands in both Figs. 1a and 1b originate from
the coumarin chromophore. The band properties of the caged
compounds 6 and 7 are very similar to each other, and ap-
peared at longer wavelengths than those of model compounds
1 and 3. The fluorescence quantum yields (ꢀf) of 6 and 7 are
lower than those of model compounds 1 and 3 (Table 1) prob-
Published on the web December 13, 2007; doi:10.1246/bcsj.80.2384

N. Senda et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 12 (2007) 2385
Scheme 1. (a) TFA, CH2Cl2, H2O, rt, 20 min; (b) EDC, DMAP, CH2Cl2, N-Boc-Glu-O-t-Bu for 4, or N-Boc-GABA for 5, rt, 40 min.
Table 1. Absorption Maxima (ꢀabsmax), Extinction Coefficients ("max), Fluorescence Maxima (ꢀfmax),
Fluorescence Quantum Yields (ꢀf), Stokes Shifts, Quantum Yields for Photolysis (ꢀp), and Solubility
of Compounds 1, 3, 6, and 7 in HEPES Buffer (pH 7.2) at Room Temperature
Solubility
/mM
max
abs
Compounds
ꢀ
/nm ("max/Mꢁ1 cmꢁ1
)
ꢀmf ax/nm (ꢀf) Stokes shifts/cmꢁ1
ꢀp
1
3
6
7
371 (17600)
375 (16900)
379 (13100)
381 (15000)
472 (0.32)
489 (0.22)
498 (0.10)
498 (0.10)
5800
6200
6300
6200
—
—
0.10
0.20
>5
>5
>1
>1
ably due to the existence of competitive decay pathway involv-
ing the photocleavage from the excited singlet state. The large
values of Stokes shifts for all compounds are characteristic for
7-aminocoumarin derivatives, which undergo photoinduced
intramolecular charge transfer in their excited singlet state.19
Efficient release of the neurotransmitters upon photoirradia-
tion of caged compounds is needed for biological applications.
Upon irradiation at 380 nm in aqueous buffer, the absorption
spectra of caged glutamate 6 shifted to shorter wavelength
region (Fig. 2a). The blue shift of the absorption spectrum is
due to the production of alcohol 3 by photolysis, which was
detected by using HPLC as a major photoproduct and con-
firmed by using an authentic sample of 3 (Fig. 2b). A similar
spectral change during the irradiation was observed by photol-
ysis of caged GABA 7. As depicted in Scheme 2, formation of
alcohol 3 during irradiation should indicate the production of
‘‘free’’ neurotransmitters. The quantum yield of photolysis
(ꢀp) was determined by HPLC analysis to be 0.10 for 6 and
0.20 for 7. The quantum yield of 6 is similar to that of previ-
ously reported DECM-caged glutamate (ꢀp 0.11),15 whereas
the efficiency for photolysis of caged GABA 7 is twice as high
as those of caged glutamates (Table 1). The fluorescence life-
times of 6 and 7 were 700 and 760 ps, respectively, and are
shorter than 1 (2.0 ns). The rate constant of the cleavage reac-
tion was estimated to be 1:4 ꢃ 108 and 2:6 ꢃ 108 sꢁ1 for 6 and
7, respectively, from the fluorescence lifetime and the quantum
yield of photolysis. According to the reported mechanism
for the photocleavage of MCM-caged acids,9 an ion pair of

2386 Bull. Chem. Soc. Jpn. Vol. 80, No. 12 (2007)
Synthesis and Photocleavage of Caged Neurotransmitters
Scheme 2.
Fig. 1. (a) UV–vis absorption spectra for 1, 3, caged gluta-
mate 6, and caged GABA 7 in HEPES buffer at pH 7.2.
Fig. 2. (a) Change in the absorption spectra of 6 in HEPES
buffer (1:0 ꢃ 10ꢁ5 M) at pH 7.2 on photoirradiation with
380 nm light. (b) HPLC chromatograms of 3 as an authen-
tic sample, 6 before irradiation, 6 after irradiation, 7 before
irradiation, and 7 after irradiation from the top to the
bottom, respectively.
coumarinylmethyl cation and the anionic product are produced
by photoexcitation, and the following escape process to pro-
duce the free acids might compete with the recombination
reaction to give starting caged compound. In this mechanism,
the quantum yield of photocleavage can be affected by the
ability of the cleaved group to escape from the solvent cage,
which should depend on the molecular structure. The quantum
yields (ꢀp) and the extinction coefficients (") are high for
caged glutamates, resulting in good photosensitivity at longer
wavelength.
ation with 390 nm light. These favorable properties will make
it possible to obtain high concentrations of free neurotransmit-
ters by flash photolysis.
Experimental
The coumarin chromophore and glutamate moiety in 6 or
GABA moiety in 7 is linked by ester bond, which should be
hydrolyzed by intracellular esterase. Such caged compounds,
therefore, can be used extracellularly, where 6 and 7 were con-
firmed to be stable in the dark in physiological buffer. Thus, it
is expected that 6 and 7 can activate the glutamate or GABA
receptors, respectively, on the cell surface by flash photolysis.
In summary, BCMACM-caged glutamate 6 and BCMACM-
caged GABA 7 were prepared from readily available starting
materials, and their photochemistry was investigated. Both
compounds 6 and 7 showed sufficient stability in the dark,
were water-soluble at pH 7.2 without using organic solvents,
and had relatively high quantum yield for photolysis on irradi-
Compound 3. Compound 2 (43 mg) was dissolved in di-
chloromethane (3 mL), and the reaction mixture was cooled to
0 ꢂC. Trifluoroacetic acid (1 mL) and water (50 mL) was added,
and the solution was stirred for 20 min at room temperature.
The solvent was evaporated under reduce pressure, and the
residue was washed three times with 1 mL of diethyl ether and
twice with 1 mL of acetonitrile to give 26 mg (84% yield) of 3
as a pure product.
1H NMR (CD3OD) ꢁ 7.51 (d, J ¼ 8:8 Hz, 1H), 6.65 (dd, J ¼ 8:8,
2.4 Hz, 1H), 6.51 (d, J ¼ 2:4 Hz, 1H), 6.30 (s, 1H), 4.80 (s, 2H),
4.27 (s, 4H).
Compound 4. A mixture of Boc-Glu-O-t-Bu (146 mg, 0.48
mmol), DMAP (7.8 mg, 0.064 mmol), and EDC (91 mg, 0.48

N. Senda et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 12 (2007) 2387
mmol) in dichloromethane (20 mL) was stirred for 10 min at
room temperature. Compound 2 (102 mg, 0.24 mmol) in dichloro-
methane was then added to the reaction mixture, and the reaction
mixture was stirred in the dark for 40 min at room temperature.
The reaction mixture was poured into water, and the separated
organic layer was washed with NaHCO3 aq and brine, dried over
MgSO4, and filtered. Then, the solvent was evaporated under re-
duce pressure. The residue was purified by flash chromatography
(SiO2, hexane–AcOEt 3:1) to give 0.151 g of 4 in 89% yield.
1H NMR (CDCl3) ꢁ 7.34 (d, J ¼ 8:8 Hz, 1H), 6.52 (dd, J ¼ 8:8,
2.8 Hz, 1H), 6.47 (d, J ¼ 2:8 Hz, 1H), 6.21 (s, 1H), 5.23 (s, 2H),
5.23–5.12 (m, 1H), 4.28–4.20 (m, 1H), 4.06 (s, 4H), 2.61–2.45 (m,
1H), 2.27–2.20 (m, 1H), 1.96–1.93 (m, 1H), 1.48 (s, 18H), 1.44 (s,
18H). 13C NMR (CDCl3) ꢁ 172.1, 171.2, 168.8, 161.3, 155.7,
155.4, 151.3, 149.1, 128.3, 124.5, 109.2, 108.4, 108.2, 99.3, 82.5,
82.4, 79.9, 61.4, 54.3, 53.2, 30.1, 28.3, 28.1, 28.0. Found: C,
61.13; H, 7.51; N, 3.72%. Calcd for C36H52N2O12: C, 61.35; H,
7.44; N, 3.97%.
Compound 5. A mixture of Boc-GABA (98 mg, 0.48 mmol),
DMAP (7.8 mg, 0.064 mmol), and EDC (91 mg, 0.48 mmol) in di-
chloromethane (20 mL) was stirred for 10 min at room tempera-
ture. Compound 2 (101 mg, 0.24 mmol) in dichloromethane (30
mL) was added to the reaction mixture, and the reaction mixture
was stirred in the dark for 40 min at room temperature. The reac-
tion mixture was poured into water, and the separated organic
layer was washed with NaHCO3 aq and brine, dried over MgSO4
and filtered. The solvent was then evaporated under reduce pres-
sure. The residue was purified by flash chromatography (SiO2,
hexane–AcOEt 3:1) to give 0.134 g of 5 in 92% yield.
(m, 2H), 2.61–2.58 (m, 2H), 1.85–1.80 (m, 2H). 13C NMR
(DMSO) 172.1, 171.6, 160.2, 155.1, 150.9, 150.3, 125.5, 108.8,
106.7, 106.7, 97.6, 61.3, 40.0, 38.1, 30.0, 22.3. ESI-MS observed
m=z 393.12 (½M þ Hꢄþ).
Measurements. The 1H and 13C NMR spectra were measured
on a Bruker ARX-400 (400 MHz for 1H NMR) and Bruker
AVANCE 600 (600 MHz for 1H NMR, 150 MHz for 13C NMR)
spectrometer. FAB mass spectra were recorded on a JEOL MS-
600H mass spectrometer. ESI mass spectra were recorded on an
Applied Biosystems Qstar/Pulsar i spectrometer. The UV absorp-
tion and fluorescence spectra were recorded on a Shimadzu UV-
1600 UV–visible spectrophotometer and on a Hitachi F-4500 fluo-
rescence spectrometer, respectively. Fluorescence lifetimes were
determined on a Horiba NAES-1100 time resolved spectrofluoro-
meter.
Solution of 6 and 7 were photolyzed with UV light (365 nm, slit
5 nm) from 150 W xenon lamp from a JASCO FP777 fluorescence
spectrometer, and analytical HPLC (ODS100V) was performed to
determine the decrease in the amount of 6 and 7 in the photolysis.
Quantum efficiencies were calculated as ðI"t10%Þꢁ1, where I is the
irradiation intensity in einsteins cmꢁ2 sꢁ1, " is the molar extinc-
tion coefficient in cm2 (mol substrate)ꢁ1 (103 times the conven-
tional extinction coefficient in Mꢁ1 cmꢁ1), and t10% is the irradia-
tion time in seconds for 10% conversion to product. Total UV
intensity I was measured by using chemical actinometry with
potassium ferrioxalate in the same setup. The intensities were
2:8 ꢃ 10ꢁ8 einsteins cmꢁ2 sꢁ1
.
This work was supported by a Grant-in-Aid for Scientific
Research on Priority Areas (417), a Grant-in-Aid for Scientific
Research (No. 16350005) and the 21st Century COE Program
from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of the Japanese Government, by Univer-
sity of Tsukuba Research Projects, Asahi Glass Foundation
and JSR Corporation.
1H NMR (CDCl3) ꢁ 7.34 (d, J ¼ 8:8 Hz, 1H), 6.52 (dd, J ¼ 8:8,
2.8 Hz, 1H), 6.47 (d, J ¼ 2:8 Hz, 1H), 6.21 (s, 1H), 5.23 (s, 1H),
4.62 (s, 1H), 4.06 (s, 4H), 3.22–3.178 (m, 2H), 2.49 (t, J ¼ 7:2
Hz, 2H), 1.91–1.83 (m, 2H), 1.48 (s, 18H), 1.44 (s, 9H). 13C NMR
(CDCl3) 172.4, 168.8, 161.4, 156.0, 155.7, 151.3, 149.2, 143.6,
129.6, 129.1, 127.5, 124.5, 109.2, 108.4, 108.2, 99.3, 82.6, 79.4,
61.3, 54.3, 51.9, 39.8, 31.3, 28.4, 28.1, 25.3. MALDI-TOF-MS:
Found: m=z 604.30. Calcd for C31H44N2O10: M, 604.30.
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General Procedures for the Preparation of the Caged Glu-
tamate 6 and 7. Compound 4 or 5 (40 mg) was dissolved in di-
chloromethane (3 mL) and the reaction mixture was cooled to
0 ꢂC. Trifluoroacetic acid (1 mL) and water (50 mL) were added,
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