Synthesis and photoreactivity of caged blockers for glutamate transporters

Article abstract of DOI:10.1016/S0960-894X(02)01042-9

L-TBOA (L-threo-β-benzyloxyaspartate) is, so far, the most potent non-transportable blocker for glutamate transporters. We synthesized α-CMCM-L-TBOA (1a) possessing [7-(carboxymethoxy)coumarin-4-yl]methyl ester as a caging group. α-CMCM-L-TBOA (1a) is biologically inactive until UV irradiation and the photolysis of 1a immediately released L-TBOA to show glutamate uptake inhibition. The photoreactivity of the coumarin-type caging group was superior to that of the o-nitrobenzyl-type caging group.

Full text of DOI:10.1016/S0960-894X(02)01042-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 965–970
Synthesis and Photoreactivity of Caged Blockers for
Glutamate Transporters
Kiyo Takaoka,a,b Yoshiro Tatsu,c Noboru Yumoto,c Terumi Nakajimaa
and Keiko Shimamotoa,*
aSuntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan
bJapan Society for the Promotion of Science, 1-1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan
cNational Institute of Advanced Industrial Science and Technology, Midorigaoka, Ikeda 563-8577, Japan
Received 29 October 2002; accepted 2 December 2002
Abstract—l-TBOA (l-threo-b-benzyloxyaspartate) is, so far, the most potent non-transportable blocker for glutamate transporters.
We synthesized a-CMCM-l-TBOA (1a) possessing [7-(carboxymethoxy)coumarin-4-yl]methyl ester as a caging group. a-CMCM-l-
TBOA (1a) is biologically inactive until UV irradiation and the photolysis of 1a immediately released l-TBOA to show glutamate
uptake inhibition. The photoreactivity of the coumarin-type caging group was superior to that of the o-nitrobenzyl-type caging
group.
# 2003 Elsevier Science Ltd. All rights reserved.
l-Glutamate plays major roles in the excitatory neuro-
transmission in mammalian central nervous systems
(CNS). Glutamate transporters (excitatory amino acid
transporters: EAATs) maintain the extracellular gluta-
mate concentrations at low level to limit the receptor
activation and to protect neurons from the excitotoxi-
city.1 Recent studies have revealed that EAATs mod-
ulate synaptic transmission.2 Signal transmission can
occur on a milisecond order and, thus, methods for the
activation and/or inactivation of EAATs with good
time resolution are strongly required.
Although o-nitorobenzyl-type groups have been most
widely used as photocleavable protective groups,
recently coumarin derivatives were reported as more
sensitive caged compounds.5,6 Therefore, we synthesized
the [7-(carboxymethoxy)coumarin-4-yl]methyl (CMCM)
ester of l-TBOA (1a: a-CMCM-l-TBOA) and the (7-
methoxycoumarin-4-yl)methyl (MCM) ester (1b: a-
MCM-l-TBOA). We also synthesized the 2-(4,5-dime-
thoxy-2-nitrophenyl)ethyl (DMNPE) esters (3a, b) to
compare the photosensitivity.7 By photolysis, coumarin-
esters (1a, b) generate l-TBOA and the corresponding
alcohols (2a, b) (eq 1) whereas DMNPE-esters (3a, b)
provide l-TBOA and 40,50-dimethoxy-2-nitrosoaceto-
phenone (4) (eq 2).
Caged compounds whose activities are masked by a
photocleavable group are a useful tool to overcome the
limitations in time resolution.3 An active compound can
be generated at the desired time and position by a
photochemical reaction using pulsed UV-light irradia-
tion. In order to elucidate the regulation of EAATs at
excitatory synapses, we synthesized caged derivatives of
l-TBOA (l-threo-b-benzyloxyaspartate), which is, so
far, the most potent non-transportable blocker for all
subtypes of glutamate transporters (EAAT1-5).4
The preparation of esters from carboxylic acid (5) and
alkyl halide or alcohol under basic conditions8 resulted
in significant epimerization of the amino group or the
benzyloxy groupand low yield. Therefore, we used
diazoalkane under neutral conditions.5b,9a Treatment of
5 with the corresponding diazoalkane 6a9a in benzene
provided the protected ester (7a) in 69% yield (Scheme
1). No epimerization was detected by 400 MHz 1H
NMR. Deprotection of 7a with TFA followed by HPLC
purification gave a-CMCM-l-TBOA (1a).10 MCM
analogue 1b was synthesized in the same manner.10
*Corresponding author. Tel.: +81-75-962-6114; fax: +81-75-962-
2115; e-mail: shimamot@sunbor.or.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01042-9

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K. Takaoka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 965–970
Similarly, a-DMNPE-l-TBOA (3a, b) were synthesized
from 5 and diazoalkane 8.9bÀd Although the diaster-
eomers (3a and 3b) were separated by HPLC (Rt: 17.1
and 20.1 min), the configuration of the DMNPE group
could not be determined (Scheme 1).11
was analyzed by HPLC.12,13 HPLC analyses indicated
that the peak of 1a (Rt: 5.3 min) decreased during UV-
irradiation while that of 2a (Rt: 4.5 min) increased (Fig.
1a–d). CMCM-ester (1a) completely disappeared within
20 min. We found that a part of CMCM-OH (2a) was
further converted to the decarboxylated alcohol MCM-
OH (2b: Rt: 8.0 min)14 but decarboxylation from 1a to
1b (Rt: 10.7 min) was not observed. The rate of dis-
appearance of a-MCM-l-TBOA (1b) was almost the
same as that of 1a (data not shown). On the other hand,
DMNPE-ester (3a) hardly decomposed after 30 min
(Fig. 2a and b). The peaks of 3a (Rt: 17.1 min) and 3b
(Rt: 20.1 min) were still detected after 180 min-irradia-
tion (Fig. 2c and d). Although the photolysis products were
not identified, 3a and 3b provided the same peaks (Rt: 15.0,
18.9 and 23.2 min). Quantitative analyses revealed the half-
life of 1a and 1b was 3.7 and 3.9 min, respectively, while
that of 3a and 3b was 57.2 and 45.2 min, respectively.
Therefore, it is found that coumarin-esters are much more
photosensitive than nitrobenzyl-esters.5,6 Although the
irradiated caged TBOA solution contained byproducts,
the disappearance of the starting materials can be fitted
to single exponential decay, suggesting the photolysis
proceeds without any interference of the photolysis
products.
Prior to the physiological application, we checked the
photosensitivity of the caged compounds. A solution of
caged TBOAs during steady-state irradiation with UV
lamp (max power at 10 cm from the lamp: 2.3 mW/cm2)
Restoring of l-TBOA from 1a by photolysis was
directly confirmed by the glutamate uptake inhibition
assay on EAAT2 stably expressed on MDCK cells,15
since it was difficult to quantitate l-TBOA by HPLC.
The activity of the caged compound should be masked
until the photoirradiation. Before UV irradiation, the
inhibitory activity of 1a (IC50: 124 mM) was 100-fold less
potent than that of l-TBOA (IC50: 1.3 mM) and 1a was
practically inactive at the effective concentration range
of l-TBOA. Its activity was restored time-dependently
as shown in Figure 3, as we expected. The IC50 values of
1a with UV irradiation (365 nm) were as follows: 24 mM
(1 min), 8.6 mM (5 min), 7.0 mM (10 min), 5.6 mM
(20 min). However, the activity was not completely
restored after 20-min irradiation, in which the peak of
1a disappeared in HPLC, and even if 1a was irradiated
for more than 20 min, the inhibitory activity was not
Scheme 1. (a) TFA, 50%; (b) TFA, 22%.

K. Takaoka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 965–970
967
Figure 1. HPLC analysis of a-CMCM-l-TBOA [1a: 100 mM solution in PBS(+) buffer] and the products with UV irradiation (365 nm) monitored at
320 nm. Condition of HPLC: 32% CH3CN/H2O/0.1% TFA. Irradiation time of sample: (a) 0 min, (b) 1 min, (c) 5 min, (d) 20 min.
Figure 2. HPLC analyses of a-DMNPE-l-TBOA [3a and 3b: 100 mM solution in PBS(+) buffer] and the products with UV irradiation (365 nm)
monitored at 254 nm. Condition of HPLC: Dashed lines indicate the acetonitrile gradient (32–77% CH3CN/H2O/0.1% TFA). Irradiation time of
sample: (a) 0 min, (b) 30 min, (c) 180 min for a-DMNPE-l-TBOA (3a), (d) 180 min for a-DMNPE-l-TBOA (3b). Photolysis of 3a and 3b gave the
peaks of the same retention time (A: 15.0, B: 18.9 and C: 23.1 min).
improved. The activity of l-TBOA was not disturbed
after 120-min-irradiation and CMCM-OH (2a) or
MCM–OH (2b) produced by photolysis did not affect
the inhibitory activity (data not shown). These results
indicate that an unknown byproduct(s) was produced
by the photoreaction. The byproduct(s) could not be
specified by HPLC in both 320 and 254 nm detection,
suggesting the byproduct is l-TBOA possessing a
decomposed coumarin ring.16 There was no obvious
difference in IC50 values among the other caged TBOAs
(1b: 124 mM, 3a: 103 mM and 3b: 111 mM), showing that
type of ester group is not important for the caging
effect.
The photosensitivity of the caged TBOAs and the gen-
eration of l-TBOA were confirmed by the UV lamp
irradiation. Using laser-pulse, which is higher energy
than UV light, in a sample specimen would be more
useful for physiological applications because the active
compound can be generated at the desired time and
position in a moment. Therefore we next examined
photolysis of these compounds by using YAG Laser
(SureLite II, Continuum, Santa Clara, CA, USA,
355 nm, 8 ns pulse, 40 mJ).17 The photochemical quan-
tum yields (fq) were determined from the disappeared
amount of the caged compounds and the absorbed light
intensity (Table 1). The concentrations of the remaining
caged compounds after one shot irradiation were mea-
sured from HPLC and the chemical reaction yields (fc)
Table 1. Spectroscopic and photolytic characteristics of caged
compounds
Compd
UV absorption
lmax (nm)a
Laser photolysis
1 shot (40 mJ) 355 nm
(e: M-1 cmÀ1
)
b
b
fc
fq
e355fq
(e355: MÀ1 cmÀ1 c
)
a-CMCM-l-TBOA (1a)
a-MCM-l-TBOA (1b)
a-DMNPE-l-TBOA (3a)
a-DMNPE-l-TBOA (3b)
325 (4600)
325 (4300)
352 (4000)
352 (3300)
8Æ2.3 0.04
13Æ0.6 0.06
3Æ2.1 0.005
44 (1100)
66 (1100)
20 (3900)
n.d.d
n.d.d
n.d.d
Figure 3. Release of l-TBOA from a-CMCM-l-TBOA (1a) with UV
lampirradiation (365 nm). Inhibition of [ 14C]Glu uptake in MDCK
cells expressing EAAT2 was measured as previously reported.15 Non-
irradiated a-CMCM-l-TBOA (1a) (&) was much less potent than l-
TBOA (!). Irradiated samples of 1a restored the activity according to
the irradiation time (1–20 min). Values are presented as meanÆSEM
of at least three determinations.
ae, wavelength of absorbance maximum (lmax) and extinction coefficient in
100 mM solution in PBS(+) solution.
bfc, chemical yield; fq, quantum yield.
ce355fq, product of the quantum yield and extinction coefficient (indication of
the efficiency of photolysis).
dNot determined.

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K. Takaoka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 965–970
were obtained by the subtracted concentrations.18 The
light intensity was determined by using potassium fer-
rioxalate actinometry, and the absorption factor at 355
nm (e355) of each compound was obtained from the UV
spectra. Chemical yields (fc), quantum yields (fq) and
e355fq values (indication of the efficiency of photolysis:
high value reflects high efficiency)19 of coumarin-esters
(1a, b) were better than those of DMNPE-ester (3a). As
the e355 values of 1a, b were relatively small, laser irra-
diation with closer wavelength to lmax (325 nm) would
more efficiently provide l-TBOA. Alternatively,
introduction of substituents on the coumarin ring to
shift lmax to longer wavelength would be useful.5,10
because such peak was also detected in the transient
absorption of CMCM-OH (2a: 540 nm) but not in those
of a-MCM-l-TBOA (1b) and MCM-OH (2b). Never-
theless, the change in the transient spectroscopy was
completed within 1.5 ms and the reaction pathway of the
photolytic cleavage of 1a and 1b is thought to be the
same as that of the coumarin caged compounds reported
previously.5,6 Therefore, the liberation of l-TBOA from
1a and 1b is much faster than that from 3a and 3b, in
agreement with the photosensitivity of these compounds
using UV lamp, and the rapid generation of the blocker
would enable the precise kinetic analysis of EAATs.
The HPLC analysis of the non-irradiated sample 1a
revealed a small contamination of 2a, suggesting
decomposition of the caged compound in a solution.
Therefore, we examined the chemical stability of 1a
both in aqueous solution and in DMSO (Fig. 4) In the
aqueous buffer [PBS(+), pH 7.4], 1a was stable at
À20 ꢀC but gradually decomposed at 0 ꢀC and it was
rather unstable at room temperature. Hydrolysis of the
coumarin-ester occurred even in the dark condition.21
In contrast, DMSO solution was extremely stable and
only a little decomposition was observed after more
than 30 days (0 ꢀC, dark). As far as a stock solution of
1a is prepared in DMSO and is added to the buffer just
prior to the physiological experiments, it would be
stable enough during the experiments. Similarly, we also
confirmed that 1b is stable in DMSO for more than 30
days (data not shown). Both DMNPE-esters (3a, b)
were more stable in the aqueous buffer [PBS(+), pH
7.4] than the coumarin-esters at 0 ꢀC, but the benzyl-
esters were also hydrolyzed. However, the chemical sta-
bility of diastereomers (3a, b) at room temperature was
different. Only 3b unexpectedly decreased by 50% for a
month even in DMSO at room temperature. The reason
for the difference in diastereomers (3a and 3b) is unclear.
It is well-known that decomposition of nitrobenzyl
compounds can be monitored by the transient absorp-
tion at 420 nm of its aci-nitro intermediate.20 The
transient absorption during the laser-irradiation was
measured using a photomultiplier tube and monochro-
matic light from a 150-W xenon lamp. The photocurrent
was fed into an oscilloscope (TDS340AP, Sony-Tek-
tronics, Tokyo, Japan). The half-life of aci-nitro inter-
mediates for 3a and 3b was 445 and 507 ms, respectively.
On the other hand, the accurate rate of the photolysis of
coumarin-esters (1a, b) could not be obtained from the
transient absorption spectra at intervals of 20 nm from
380 to 600 nm with or without O2, because these cou-
marin-esters showed very strong fluorescence at
400 nm,10 of which the decay curves were very complex
(data not shown).5d In the transient absorption of
a-CMCM-l-TBOA (1a) at 500 nm, a very sharppeak
was detected at 0.15 ms. It was reported that the tran-
sient absorption at 480 nm in degassed acetonitrile is
assigned to the triplet state of N,N,N-tributyl-N-(4-
methylene-7-methoxycoumarin) ammonium borates.5d
However, we could not conclude whether the observed
peak of 1a reflects its triplet state in aqueous solution,
Figure 4. Stability of caged compounds (1a, 3a, and 3b) in PBS(+)(pH 7.4) or DMSO. The concentrations of the caged compounds were quantified
by HPLC.

K. Takaoka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 965–970
969
In summary, we have synthesized caged blockers for
glutamate transporters, a-CMCM-l-TBOA (1a) and a-
MCM-l-TBOA (1b), which are practically inactive
around the effective concentration of l-TBOA while
rapidly generating l-TBOA by UV lampirradiation or
laser-pulse irradiation. They are stable in DMSO solu-
tion and can be stocked for several weeks. Therefore,
these compounds would be useful tools for elucidating
the physiological roles of transporters. Applications of
these samples to electrophysiological studies are now in
progress.
2516) is commercially available from Molecular probes (OR,
USA).
10. a-CMCM-L-TBOA (1a): 1H NMR (DMSO-d6) d 4.4 (d,
1H, J=11.6 Hz), 4.5 (d, 1H, J=3.2 Hz), 4.7 (br, 1H), 4.7 (d,
1H, J=11.6 Hz), 4.8 (s, 2H), 5.3 (d, 1H, J=15.6 Hz), 5.5 (d,
1H, J=15.6 Hz), 6.4 (s, 1H), 6.9 (dd, 1H, J=2.4, 8.8 Hz), 7.0
(d, 1H, J=2.4 Hz), 7.2–7.3 (m, 5H), 7.6 (d, 1H, J=8.8 Hz).
HRMS (FAB) m/z calcd for C23H22NO10 (M+H)+ 472.1244,
28
Found 472.1244. ½aꢁD +225.2ꢀ (c 0.46, DMSO), mp148–
150 ꢀC. Fluorescence lex/lem (1 mM): 320 nm/401 nm. a-
1
MCM-L-TBOA (1b): H NMR (DMSO-d6) d 3.9 (s, 3H), 4.5
(d, 1H, J=12 Hz), 4.6 (d, 1H, J=4.6 Hz), 4.7–4.8 (m, 2H), 5.3
(d, 1H, J=15.6 Hz), 5.5 (d, 1H, J=15.6 Hz), 6.4 (s, 1H), 6.9
(dd, 1H, J=2.4, 8.8 Hz), 7.0 (d, 1H J=2.4 Hz), 7.2–7.3 (m,
5H), 7.6 (d, 1H, J=8.8 Hz). HRMS (FAB) m/z calcd for
Acknowledgements
28
C22H22NO8 (M+H)+ 429.1345, Found 428.1361. ½aꢁD À13.1ꢀ
(c 0.35, DMSO). Fluorescence lex/lem (1 mM): 320 nm/407 nm.
This work was financially supported by Grant-in-Aid
for Creative Scientific Research (#13NP0401) and
Grant-in-Aid for Scientific Research (#13680681) from
the Ministry of Education, Culture, Sports, Science and
Technology. We wish to thank Prof. Susan. G. Amara
at Oregon Health and Sciences University for providing
the MDCK cells expressing EAAT2 and Dr. Yasushi
Shigeri at National Institute of Advanced Industrial
Science and Technology for critical reading of this
manuscript.
1
11. a-DMNPE-L-TBOA (3a): H NMR (DMSO-d6) d 1.6 (d,
3H, J=6.4 Hz), 3.8 (s, 3H), 3.9 (s, 3H), 4.2 (d, 1H,
J=11.2 Hz), 4.4 (d, 1H, J=3.2 Hz), 4.5 (d, 1H,J=4.6 Hz), 4.7
(d, 1H, J=11.2 Hz), 6.4 (q, 1H, J=6.4 Hz), 7.04–7.06 (m, 2H),
7.20–7.22 (m, 4H), 7.51 (s, 1H). HRMS (FAB) m/z calcd for
25
C21H25N2O9 (M+H)+ 449.1560, Found 449.1563. ½aꢁD
À749.1ꢀ (c 0.50, DMSO). a-DMNPE-L-TBOA (3b): H NMR
1
(DMSO-d6) d 1.44 (d, 3H, J=6 Hz), 3.84 (s, 3H), 3.90 (s, 3H),
4.4 (d, 1H, J=2.8 Hz), 4.51–4.54 (m, 2H), 4.8 (d, 1H,
J=11.6 Hz), 6.1 (q, 1H, J=6.4 Hz), 7.1 (s, 1H), 7.6–7.35 (m,
5H), 7.57 (s, 1H). HRMS (FAB) m/z calcd for C21H25N2O9
26
(M+H)+ 449.1560, Found 449.1578. ½aꢁD À65.7ꢀ (c 0.52,
DMSO).
References and Notes
12. Condition of analytical HPLC: Column; SP-120-5-ODS-
AP (Daiso Co. Ltd. Japan), 150 mm ꢂ 6 mm I.D.: Eluent;
CH3CN/H2O/TFA=32:68:0.1. Flow rate; 1 mL/min; Detec-
tion; 320 nm and 254 nm. As an internal standard for the
quantitative analyses, 10 mM of 30,40-dimethoxyacetophenone
(Rt: 11.1 min) for 1a, 3a and 3b or 40 mM of 2-(3,4-
dimethoxyphenyl)ethanol (Rt: 6.7 min) for 2a was included in
the sample solution.
13. Irradiation condition: UV handy-lamp; Spectroline ENF-
260C/J (Spectronics Co. USA), 365 nm: Sample solution;
100 mM in PBS(+) buffer (phosphate buffered saline including
1 mM CaCl2 and 1 mM MgCl2, pH 7.4). A solution (300 mL)
of the caged compounds was placed in quartz cuvettes with a
path length of 1 mm in a dark box and irradiated at room
temperature for 1–240 min.
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14. We confirmed that CMCM-OH (2a) was gradually
converted to MCM-OH (2b) with UV irradiation. The struc-
ture of the product was determined by 1H NMR. CMCM-OH
(2a): 1H NMR (DMSO-d6) d 4.7 (s, 2H), 4.8 (s, 2H), 5.6
(br, 1H), 6.2 (s, 1H), 6.93–6.97 (m, 2H), 7.61 (d, 1H,
J=8.8 Hz). Mp >260 ꢀC. Fluorescence lex/lem (1 mM):
1
320 nm/400 nm. MCM-OH (2b): H NMR (DMSO-d6) d 3.8
(s, 3H), 4.7 (dd, 2H, J=1.6, 5.6 Hz), 5.5 (t, 1H, J=5.6 Hz), 6.2
(s, 1H), 6.9 (dd, 1H, J=2.4, 9.2 Hz), 7.0 (d, 1H, J=2.4 Hz),
7.6 (d, 1H, J=9.2 Hz). Fluorescence lex/lem (1 mM): 320 nm/
402 nm.
15. The uptake assay was performed as previously described4a
except that MDCK cells stably expressing EAAT2 were used
instead of the transiently transfected COS-1 cells. The relative
specific uptake of [14C]glutamate (1 mM) was determined from
three different experiments. The Michaelis constant (Km) and
IC50 values of l-glutamate for EAAT2 were 63Æ3.8 and
41Æ1.9 mM, respectively.
16. MCM-OH (2b) was decomposed 20% for 30 min and 50%
for 120 min with UV lampirradiation (365 nm).
17. A sample [100 mM in PBS(+) solution, 100 mL] placed in a
quartz cuvette (path length 1 mm) was irradiated with laser
pulse.
18. The chemical yields of the photolysis by repeated irradi-

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K. Takaoka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 965–970
ation (8 shots) were 38Æ1.6% (1a), 34Æ5.6% (1b), and
21Æ2.9% (3a), respectively.
21. For the purification of the caged compounds, column of
SP-120-5-ODS-AP (Daiso Co. Ltd. Japan), 250 mm ꢂ 20 mm
I.D. was used and peaks were monitored at only 254 nm to
avoid decomposition. Because 1a was hydrolyzed during the
lyophilization after HPLC purification, we could not remove a
small contamination of l-TBOA from 1a. It may contribute to
the activity at the higher concentrations.
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