Development of the 8-aza-3-bromo-7-hydroxycoumarin-4-ylmethyl group as a new entry of photolabile protecting groups

Article abstract of DOI:10.1016/j.tet.2014.04.063

A significant substitution effect of the position of the bromo group on the photosensitivity of the 8-azacoumarin chromophore leads to the development of a highly photosensitive 8-aza-3-bromo-7-hydroxycoumarin-4-ylmethyl (aza-3-Bhc) group that shows excellent photolytic efficiency and hydrophilicity with long-wavelength absorption maxima. The newly identified aza-3-Bhc group can be applied to caged glutamates for ester-type and carbamate-type protections of carboxyl and amino functionalities.

Full text of DOI:10.1016/j.tet.2014.04.063

Tetrahedron xxx (2014) 1e5
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Tetrahedron
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Development of the 8-aza-3-bromo-7-hydroxycoumarin-4-ylmethyl
group as a new entry of photolabile protecting groups
, ,
Hikaru Takano a, Tetsuo Narumi a y, Nami Ohashi a, Akinobu Suzuki b, Toshiaki Furuta b,
*
,
Wataru Nomura a, Hirokazu Tamamura a
*
a Institution of Biomaterial and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A significant substitution effect of the position of the bromo group on the photosensitivity of the 8-
azacoumarin chromophore leads to the development of a highly photosensitive 8-aza-3-bromo-7-
hydroxycoumarin-4-ylmethyl (aza-3-Bhc) group that shows excellent photolytic efficiency and hydro-
philicity with long-wavelength absorption maxima. The newly identified aza-3-Bhc group can be applied
to caged glutamates for ester-type and carbamate-type protections of carboxyl and amino functionalities.
ꢀ 2014 Published by Elsevier Ltd.
Received 5 March 2014
Received in revised form 22 April 2014
Accepted 22 April 2014
Available online xxx
Keywords:
Azacoumarin
Photolabile protecting groups
Caged compounds
Photolytic efficiency
Hydrophilicity
1. Introduction
whose features are superior to those of the 6-bromo derivatives 2
(Fig. 1).
Photosensitive biologically active compounds (referred to as
caged compounds) have attracted considerable attention due to
their practical potentials as phototriggers of biological functions.1
Progress in the field has been driven by the development of new
photolabile protecting group types,2 such as nitrobenzyl,3 benzoin,4
phenacyl,5 and coumarin.6 Although a number of chromophores
have been applied to photolabile protecting groups, 8-azacoumarin
derivatives have not been applied to the caged compounds until our
identification of several advantages as an attractive chromophore
for caging chemistry, such as excellent water solubility, high molar
absorptivity, and efficient photorelease at low pH as described in
our previous report.7 This report describes the unexpected sub-
stitution effects on the photosensitivity of 8-azacoumarin chro-
mophore, leading to the development of a novel 8-aza-3-bromo-7-
hydroxycoumarin-4-ylmethyl caging group (aza-3-Bhc 1) that
shows excellent photolytic efficiency and hydrophilicity with long-
wavelength absorption maxima and high molar absorptivity,
Fig. 1. Structures of azacoumarin- and coumarin-based Bhc groups.
2. Results and discussion
Our study on the development of the aza-3-Bhc group emerged
from the bromination of azacoumarin derivative 3 that provided
the 6-brominated compound 4a in a 67% yield as a major product,7
accompanied with a 16% yield of the 3-brominated compound 4b as
a minor product (Scheme 1a, Supplementary data). Bromination of
the coumarin chromophore provides several advantages for pho-
tolysis reactions including lowering the pKa of the adjacent hy-
droxyl group accelerating the formation of the strongly absorbing
anion and the promotion of intersystem crossing to the triplet,
which is considered to be the photochemically reactive state.6d
* Corresponding authors. Tel./fax: þ81 53 478 1198 (T.N.); tel.: þ81 3 5280
8036; fax: þ81 3 5280 8039 (H.T.); e-mail addresses: ttnarum@ipc.shizuoka.ac.jp
(T. Narumi), tamamura.mr@tmd.ac.jp (H. Tamamura).
y
Present address. Department of Applied Chemistry and Biochemical Engineer-
ing, Faculty of Engineering, Shizuoka University, Hamamatsu, Shizuoka 432-8561,
Japan.
0040-4020/$ e see front matter ꢀ 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tet.2014.04.063
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H. Takano et al. / Tetrahedron xxx (2014) 1e5
Table 1
Photophysical and hydrophilic properties of aza-3-Bhc-CH2OAc 7, aza-Bhc-CH2OAc
10, and Bhc-CH2OAc 11
b (Mꢀ1 cmꢀ1
)
Cs
(
mM)
pKa
a
c
d
Compd
lmax (nm)
3
max
Aza-3-Bhc-CH2OAc (7)
Aza-Bhc-CH2OAc (10)
Bhc-CH2OAc (11)
378
362
370
27,086
21,107
16,584
3260
10,832
602
5.08
4.22
5.88
a
Long-wavelength absorption maxima in PBS (0.1% DMSO).
Molar absorptivity at the absorption maxima.
Concentration at saturation in PBS (0.1% DMSO).
b
c
d
Determined using citric/phosphate buffer in the pH range 2.6e7.0.
original Bhc-CH2OAc 11, the absorption maximum of 7 shifted to
longer wavelength from 370 nm for 11 to 378 nm for 7, whereas
that of 10 shifted to shorter wavelength (lmax¼362 nm). The molar
Scheme 1. Synthesis of brominated 8-azacoumarin esters 4 and aza-3-Bhc-CH2OAc 7.
absorptivity at the maximum wavelength
(
3
) of 7 is
max
27,086 Mꢀ1 cmꢀ1
, which is also higher than those of 10
Preliminary studies revealed that the 8-aza-7-hydroxycoumarin
chromophore as well as the 6-bromo-7-hydroxycoumarin has
a pKa value, which is lower than 7.4, and mostly assumes the
deprotonated (ionic) form under physiological conditions without
additional electron-withdrawing groups. Therefore, the absorption
spectrum of 9 has a single peak as the spectrum of 8 (Fig. 2). These
features imply the possibility that the regioisomeric 3-brominated
8-azacoumarin chromophore would also work as a hydrophilic
caging group. Furthermore, several papers8 that identified strong
substitution effects on quinolone chromophores prompted us to
invoke that the substitution pattern could positively affect photo-
physical and photochemical properties of 8-azacoumarin
chromophore.
(
3
¼21,107 Mꢀ1 cmꢀ1) and 11 (
3
¼16,584 Mꢀ1 cmꢀ1). These
max
max
results indicated that the 3-brominated 8-azacoumarin would be
a superior chromophore to the 6-brominated coumarins. As ex-
pected, aza-3-Bhc-CH2OAc 7 has a pKa value below the physiolog-
ical pH and also shows high aqueous solubility, which is important
for the photorelease of high concentrations of the caged com-
pounds under physiological conditions. HPLC monitoring of the
hydrolysis stability revealed that while aza-3-Bhc-CH2OAc 7 was
more sensitive to hydrolysis in PBS (pH 7.4) than aza-Bhc-CH2OAc
10, the hydrolysis stability of 7 in KMOPS buffer (10 mM MOPS; 3-
morpholinepropane-1-sulfonic acid, and 100 mM KCl, pH 7.1) is
comparable to that of 10 (Fig. 3).
Fig. 3. Hydrolysis stability of compounds 7 and 10 at room temperature in dark. Time
course of compounds 7 and 10 in PBS or KMOPS was analyzed by reversed-phase HPLC.
Fig. 2. Absorption spectra of Bhc derivative 8 (dash line) and 8-aza-hc 9 (solid line).
The 3-brominated acetate aza-3-Bhc-CH2OAc 7 was synthesized
from 3 in four steps (Scheme 1b). Briefly, reduction of 3 with LiBH4
gave the corresponding alcohol 5 in moderate yield. In contrast
with the bromination of 3 that gave a mixture of regioisomers, the
bromination of alcohol 5 proceeded regioselectively to provide the
3-brominated alcohol 6 in 80% yield, which was subjected to
acetylation followed by TFA treatment to give the desired aza-3-
Bhc-CH2OAc 7.
Having recognized promising photophysical and hydrophilic
behaviors of the 3-brominated 8-azacoumarin chromophore, we
evaluated the photochemical properties of aza-3-Bhc-CH2OAc 7
under the photolysis in 5 mM KMOPS buffer solution at pH 7.2 at
350 nm. The time course of photolysis reaction of 7 was monitored
by HPLC in terms of the consumption of the starting materials
(Table 2), and indicates that the photolytic reaction at 350 nm of 7
follows a single-exponential decay as in the result of the original
Bhc compound 11. The time to reach 90% conversion (t90) for
photolysis of 7 is 19 s, which is shorter than those of the 6-
Initially, we investigated the photophysical and hydrophilic
behaviors of aza-3-Bhc-CH2OAc 7 (Table 1). Compared with the
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H. Takano et al. / Tetrahedron xxx (2014) 1e5
3
Table 2
Time course for photolysis reactions of aza-3-Bhc-CH2OAc 7, aza-Bhc-CH2OAc 10,
and Bhc-CH2OAc 11 and selected photochemical properties
Scheme 2. Synthesis of aza-3-Bhc-caged glutamates.
a (Mꢀ1 cmꢀ1
)
Fchem
3
350
$Fchem
b
c
Compd
t90 (s)
3
350
Aza-3-Bhc-CH2OAc (7)
Aza-Bhc-CH2OAc (10)
Bhc-CH2OAc (11)
19
42
28
20,175
20,583
13,774
0.17
0.059
0.13
2667
1211
1806
value of
3
$
F
of 12/14¼1.19 and of 13/15¼1.27). These results in-
dicate that the newly identified aza-3-Bhc chromophore can serve
as a variant of Bhc chromophore in biologically relevant com-
pounds. Further studies for the application of the 8-aza-3-Bhc
group to the protection of other functionalities, such as alcohols,
phosphoric acids, and thiols are in progress.
a
Molar absorptivity at 350 nm.
b
Quantum yields for the disappearance of starting materials upon irradiation at
350 nm.
c
Product of the photolysis quantum yield and molar absorptivity.
3. Conclusion
brominated coumarins 10 and 11 (t90¼42 s for 10 and 28 s for 11,
respectively). The photolysis quantum yields of disappearance of
starting materials were calculated from the single decay curves
We have reported the development of the aza-3-Bhc group as
a new entry of photolabile protecting groups for caging chemistry
through the strong influence of the position of a bromo substituent
on the photosensitivity of the 8-azacoumarin chromophore. The 3-
brominated 8-azacoumarin 7 is considerably more efficient than
the 6-brominated regioisomer 10. Aza-3-Bhc-CH2OAc 7 shows ex-
cellent photolytic efficiency with a bathochromic shift of the ab-
sorption maximum of 8-azacoumarin from 362 to 378 nm.
Moreover, we have disclosed the potentials of the aza-3-Bhc group
as protecting groups of carboxyl and amino functionalities for
caged glutamates. A key to the development of the aza-3-Bhc group
is the acidic azacoumarin chromophore, whose feature can provide
a new opportunity to improve the photosensitivity by chemical
modifications that are distinct from those in previous approaches.
Efforts to elucidate the reason for the markedly enhanced photo-
sensitivity by the 3-bromo substituent and studies on the two-
photon sensitivity of the 8-azacoumarin chromophore are cur-
rently in progress.
using the equation
F
¼1/(Iꢁ103
3
t90) as reported by Tsien.9 Notably,
the quantum yield of disappearance of 7 (Fchem¼0.17) is approxi-
mately three times higher than that of 10 (Fchem¼0.059) and sig-
nificantly higher than that of 11 (Fchem¼0.13). In addition, the
photolytic efficiency,10 the product of the photolysis quantum yield
(
Fchem) and molar absorptivity (
3
) of 7 (
3
$
Fchem¼2667), is ap-
350
proximately 1.5e2.2-fold higher than that of 10 (
and 11 (
3
350
$
Fchem¼1211)
3
$
Fchem¼1806). These results indicate that the bromo
350
substitution on position 3 of the 8-azacoumarin chromophore leads
not only to improve the photophysical behavior but also to increase
the photosensitivity. Although the reason for the significant en-
hancement of photochemical reactivity of 7 is not fully understood
at this stage, these observations suggest that the aza-3-Bhc group
has a powerful set of photophysical, photochemical, and hydro-
philic properties for caging chemistry.
Next, we examined the synthetic methods for the introduction
of the aza-3-Bhc group to biologically relevant compounds
(Scheme 2). The aza-3-Bhc group is not limited to the caging group
for ester-types. In addition to the
a
-Glu ester 12, the aza-3-Bhc
4. Experimental section
4.1. General methods
group can be applied to the -Glu carbamate 13 for the pro-
a
tection of an amino functionality, which can be removed efficiently
with light of wavelength of 365 nm to produce the corresponding
alcohol.11 Compared to the corresponding Bhc-protected com-
pounds 14 and 15,6d both aza-3-Bhc-protected compounds 12 and
13 showed improved photosensitivity (Table 3). The time to reach
90% conversion (t90) for photolysis of 12 is 32 s, which corresponds
to be approximately 20% faster than that of the corresponding Bhc-
All reactions utilizing air- or moisture-sensitive reagents were
performed in dried glassware under an atmosphere of nitrogen,
using commercially supplied solvents and reagents unless other-
wise noted. CH2Cl2 was distilled from CaH2 and stored over mo-
lecular sieves. Thin-layer chromatography (TLC) was performed on
Merck 60F254 precoated silica gel plates and were visualized by
fluorescence quenching under UV light and by staining with
phosphomolybdic acid, p-anisaldehyde, or ninhydrin, respectively.
Flash column chromatography was carried out using silica gel 60 N
(Kanto Chemical Co., Inc.).
protected
a
-Glu ester 14 (t90 of 14¼38 s). A similar superiority of the
aza-3-Bhc group was observed with the carbamate substrates 13
and 15. In accordance with the greater quantum yields of disap-
pearance and comparable molar absorptivities, the photolytic effi-
ciency of both 12 and 13 is higher than those of 14 and 15 (relative
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4
H. Takano et al. / Tetrahedron xxx (2014) 1e5
Table 3
Selected photophysical and photochemical properties of aza-3-Bhc-caged glutamates 12 and 13, and Bhc-caged glutamates 14 and 15
c (Mꢀ1 cmꢀ1
)
t90d (s)
Fchem
3 $Fchem
365
rel. 3 $
Fg
a
b
e
f
Compd
lmax (nm)
3
3
365
max
(Mꢀ1 cmꢀ1
)
Aza-3-Bhc-Glu-ester (12)
Aza-3-Bhc-Glu-carbamate (13)
Bhc-Glu-ester (14)
378
376
370
370
13,648
17,068
16,912
17,822
12,153
15,623
16,466
17,636
32
10
38
13
0.17
0.43
0.11
0.30
2063
6717
1740
5280
1.19
1.27
d
Bhc-Glu-carbamate (15)
d
a
Long-wavelength absorption maxima in PBS (0.1% DMSO).
Molar absorptivity at the absorption maxima.
Molar absorptivity at 365 nm.
Time to reach 90% conversion.
Quantum yields for the disappearance of starting materials upon irradiation at 365 nm.
b
c
d
e
f
Photolytic efficiency: product of the photolysis quantum yield and molar absorptivity.
Relative value of photolytic efficiency [aza-3-Bhc/Bhc]. For full experimental protocol, see Supplementary data.
g
4.2. Characterization data
254e257 ꢂC (dec); 1H NMR (500 MHz, CDCl3)
2H), 6.67 (m, 1H), 8.13 (m, 1H); 13C NMR (125 MHz, CDCl3)
61.9, 82.9, 106.1, 109.3, 111.3, 136.8, 150.3, 156.23, 157.7, 164.7; IR
d
1.64 (s, 9H), 5.02 (s,
28.7,
d
1H NMR (400 or 500 MHz) and 13C NMR (125 MHz) spectra were
recorded using a Bruker Avance II spectrometer with a CryoProbe.
Chemical shifts are reported in
as internal standard. Infrared (IR) spectra were recorded on a JASCO
FT/IR 4100, and are reported as wavenumber (cmꢀ1). Low- and
high-resolution mass spectra were recorded on a Bruker Daltonics
micrOTOF (ESI-MS) spectrometers in the positive and negative
detection modes.
(ATR) n 3465 (OH) 2977 (CH), 2929 (CH), 1707 (CO), HRMS (ESI), m/z
d
(ppm) relative to Me4Si (in CDCl3)
calcd for C13H14BrNNaO4 [MþNa]þ 350.0004, found 350.0000.
4.4.3. (3-Bromo-7-hydroxy-2-oxo-2H-pyrano[2,3-b]pyridin-4-yl)
methylacetate (7). To
0.389 mmol) and DMAP (6.20 mg, 0.0507 mmol) in CH2Cl2 (5.6 mL)
were added sequentially pyridine (313.1 L, 3.89 mmol) and acetic
anhydride (183.7 L, 1.94 mmol), and the mixture was stirred at
a solution of compound 6 (127.1 mg,
m
m
4.3. HPLC condition
room temperature for 1 h. The reaction mixture was diluted with
CH2Cl2, washed with NaHCO3 aq, and dried over Na2SO4. Concen-
tration under reduced pressure gave the corresponding acetate
(138.1 mg, 96% yield). To a solution of the corresponding acetate
(138.1 mg, 0.374 mmol) in CH2Cl2 (1 mL) was added trifluoroacetic
acid (1 mL), and the mixture was stirred at room temperature for
30 min. Concentration under reduced pressure followed by flash
column chromatography over silica gel with CHCl3/MeOH (5:1) to
give the title compound 7 (80.1 mg, 68% yield) as a white solid. Mp:
For analytical HPLC, a Cosmosil C18-ARII column (4.6ꢁ250 mm,
Nacalai Tesque, Inc., Kyoto, Japan) was employed with a linear
gradient of MeCN containing 0.1% (v/v) TFA at a flow rate of
1 cm3 minꢀ1 an Agilent HP 1100 system with DAD detection (Agi-
lent Technologies JAPAN Ltd., Tokyo, Japan) and JASCO PU-2086
plus (JASCO corporation, Ltd., Tokyo, Japan), and eluting products
were detected by UV at 340 nm.
273e276 ꢂC (dec); 1H NMR (500 MHz, CD3OD)
d 1.99 (s, 3H), 5.35 (s,
4.4. Experimental procedures of 8-azacoumarin derivatives 5,
6, 7, 12, and 13
2H), 6.53 (d, J¼9.0 Hz, 1H), 7.97 (d, J¼9.0 Hz, 1H); 13C NMR
(125 MHz, CD3OD) d 20.5, 61.1, 102.0,106.1,109.4,136.2,150.9,158.4,
160.4, 166.8, 170.0; IR (ATR) n 2923 (OH) 1736 (CO), HRMS (ESI), m/z
4.4.1. 7-(tert-Butoxy)-4-(hydroxymethyl)-2H-pyrano[2,3-b]pyridin-
2-one (5). A suspension of LiBr (1.09 g, 12.6 mmol) and NaBH4
(410.2 mg, 10.8 mmol) in THF (36.0 mL) was stirred under nitrogen
at 50 ꢂC for 2 h, producing the solution of LiBH4 (ca. 0.3 M). To the
solution was added compound 3 (1.03 g, 3.72 mmol) in THF
(36.0 mL), and the mixture was stirred at ꢀ20 ꢂC for 2 h. The re-
action mixture was quenched by 1 M HCl aq, and the organic layer
was removed under reduced pressure. The residue was extracted
with EtOAc, washed with brine, and dried over Na2SO4. Concen-
tration under reduced pressure followed by flash column chro-
matography over silica gel with n-hexane/EtOAc (1:1) gave the title
calcd for C11H8BrNNaO5 [MþNa]þ 335.9484, found 335.9485.
4.4.4. (S)-4-Amino-5-((3-bromo-7-hydroxy-2-oxo-2H-pyrano[2,3-
b]pyridin-4-yl)methoxy)-5-oxopentanoic acid (12). To a solution of
Boc-Glu(OBn)-OH (158.0 mg, 0.468 mmol), EDCI$HCl (541.2 mg,
2.82 mmol), and DMAP (13.8 mg, 0.113 mmol) in CH2Cl2 (10.2 mL)
was added compound 6 (100.7 mg, 0.308 mmol), and the mixture
was stirred at room temperature for 24 h. The mixture was poured
into water and extracted with EtOAc, and dried over Na2SO4.
Concentration under reduced pressure gave the crude compound
(321.9 mg), which was used in the next step without further pu-
compound
236e244 ꢂC (dec); 1H NMR (500 MHz, CDCl3)
2H), 6.45 (m, 1H), 6.60 (m, 1H), 7.74 (m, 1H); 13C NMR (125 MHz,
CDCl3) 28.3, 60.9, 82.5, 104.9, 109.1, 110.5, 134.6, 153.9, 157.4, 161.5,
164.6; IR (ATR)
5
(411.8 mg, 45% yield) as white powder. Mp:
rification. A solution of the crude compound (321.9 mg,
d
1.64 (s, 9H), 4.85 (m,
0.498 mmol), 1 M TMS-Br/TFA (3.32 mL), and 1 M thioanisole/TFA
(3.32 mL) was stirred at room temperature for 3 h. Purification by
preparative HPLC (Gradient: 0 min, 0% CH3CN in H2O; 90 min, 40%
CH3CN in H2O) followed by lyophilization to give a title compound
12 (23.7 mg, 16% yield) as a pale purple solid. Mp: 149e154 ꢂC
d
n
3395 (OH), 1704 (CO); HRMS (ESI), m/z calcd for
C9H8NO4 [Mꢀtert-Buþ2H]þ 194.0453, found 194.0450.
(dec); 1H NMR (500 MHz, CD3OD)
d
2.06 (m, 2H), 2.39 (m, 2H), 4.10
(m, 1H), 5.56 (m, 2H), 6.60 (d, J¼8.5 Hz, 1H), 8.06 (d, J¼8.5 Hz, 1H);
13C NMR (125 MHz, CD3OD)
26.6, 30.2, 53.2, 64.7, 106.3, 110.4,
112.0, 139.1, 147.5, 157.9, 158.3, 166.6, 169.9, 175.4; IR (ATR) 3505
4.4.2. 3-Bromo-7-(tert-butoxy)-4-(hydroxymethyl)-2H-pyrano[2,3-
b]pyridin-2-one (6). To a solution of compound 5 (121.4 mg,
0.487 mmol) in CH3CN (1.37 mL) was added NBS (428.6 mg,
125 mmol), and the mixture was stirred at room temperature for
4 h. After being concentrated under reduced pressure, the residue
was dissolved in EtOAc, washed with H2O, and dried over Na2SO4.
Concentration under reduced pressure followed by flash column
chromatography over silica gel with n-hexane/EtOAc (1:1) gave the
title compound 6 (127.1 mg, 80% yield) as a dark green solid. Mp:
d
n
(NH), 2981 (OH), 2865 (CH), 1737 (CO), 1690 (CO) 1682 (CO), HRMS
(ESI), m/z calcd for
400.9981.
C
14H14BrN2O7 [MþH]þ 400.9984, found
4.4.5. (S)-2-((((3-Bromo-7-hydroxy-2-oxo-2H-pyrano[2,3-b]pyridin-
4-yl)methoxy)carbonyl)amino)-pentanedioic acid (13). To a solution
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H. Takano et al. / Tetrahedron xxx (2014) 1e5
5
of CDI (25.4 mg, 0.157 mmol) of CH2Cl2 was added 6 (48.5 mg,
0.148 mmol), and the mixture stirred at 0 ꢂC for 1 h. After H-
Glu(OtBu)-OtBu (90.4 mg, 0.306 mmol) and Et3N (52.3
L,
Acknowledgements
m
This research was supported by the Naito Foundation,
KEN10000322 (Natural Science Scholarship) and in part by a Grant-
in-Aid for Young Scientist (B) from the Ministry of Education, Cul-
ture, Sports, Science, and Technology.
0.375 mmol) were added, the mixture was stirred at room tem-
perature for additional 12 h. The reaction mixture was concentrated
with reduced pressure. The residue was diluted with Et2O, and
washed with water and brine, dried over Na2SO4. Concentration
under reduced pressure gave the crude compound (168.1 mg),
which was used in the next step without further purification. To
a solution of the crude compound (168.1 mg, 0.275 mmol) in CH2Cl2
(0.90 mL) was added TFA (2.7 mL), and the mixture was stirred at
room temperature for 1 h. Purification by preparative HPLC (Gra-
dient: 0 min, 5% CH3CN in H2O; 90 min, 40% CH3CN in H2O) fol-
lowed by lyophilization to give a title compound 13 (17.6 mg, 22%
yield) as a pale green solid. Mp: 196e198 ꢂC (dec); 1H NMR
Supplementary data
This section presents the experimental details, and 1H and 13C
NMR spectra. Supplementary data related to this article can be
found at http://dx.doi.org/10.1016/j.tet.2014.04.063.
References and notes
(500 MHz, CD3OD)
(m, 1H), 5.46 (s, 2H), 6.68 (m, 1H), 8.19 (m, 1H); 13C NMR (125 MHz,
CD3OD) 27.8, 31.1, 54.8, 64.0, 106.3, 110.4, 110.8, 139.6, 149.4, 157.6
(2C), 158.4, 166.3, 175.2, 176.3; IR (ATR) 3306 (NH), 2981 (OH),
d 1.88 (m, 1H), 2.16 (m, 1H), 2.38 (m, 2H), 4.19
1. (a) Caged Compounds; Marriot, G., Ed.Methods in Enzymology; Academic: New
York, NY, 1998; Vol. 291; (b) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006,
45, 4900e4921; (c) Ellis-Davies, G. C. R. Nat. Methods 2007, 4, 619e628; (d) Lee,
H. M.; Larson, D. R.; Lawrence, D. S. ACS Chem. Biol. 2009, 4, 409e427.
d
n
2. (a) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A. Angew. Chem., Int.
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Ed. 2012, 51, 8446e8476; (b) Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens,
1742 (CO), 1698 (CO), HRMS (ESI), m/z calcd for C15H13BrN2NaO9
R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Chem. Rev. 2013, 113, 119e191.
3. Nitrobenzyl-type: (a) Engels, J.; Schlaeger, E.-J. J. Med. Chem. 1977, 20, 907e911;
(b) Kaplan, J. H.; Forbush, B., III; Hoffman, J. F. Biochemistry 1978, 17, 1929e1935.
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5277e5281; (b) Givens, R. S.; Athey, P. S.; Kueper, L. W., III; Matuszewski, B.;
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Larsen, R. W.; Chan, S. I. J. Am. Chem. Soc. 2000, 122, 11567e11568.
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3771e3774; (b) Givens, R. S.; Weber, J. F. W.; Jung, A. H.; Park, C.-H. Methods
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[MþNa]þ 466.9702, found 466.9701.
4.5. Determination of saturated concentrations
Saturated concentrations of compounds were calculated from
the standard curves that related peak area (340 nm) against known
concentration of compounds in PBS.
6. Coumarin-type: (a) Givens, R. S.; Matuszewski, B. J. Am. Chem. Soc. 1984, 106,
6860e6861;(b)Furuta, T.;Torigai, H.; Sugimoto, M.; Iwamura, M.J. Org. Chem.1995,
60, 3953e3956; (c) Furuta, T.; Iwamura, M. Methods Enzymol.1998, 291, 50e63; (d)
Furuta, T.; Wang, S. S. H.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.;
Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A.1999, 96, 1193e1200; (e) Hagen, V.;
Bendig, J.; Fring, S.; Eckardt, T.; Helm, S.; Reuter, D.; Kaupp, U. B. Angew. Chem., Int.
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Schweitzer, C.; Bendig, J. J. Org. Chem. 2002, 67, 703e710; (g) Geisler, D.; Kresse, W.;
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4.6. Determination of the pKa values
The pKa values of compounds were estimated from the ti-
tration curves of absorbance against pH using 10
solution in citric/phosphate buffer solution in the pH range
2.6e7.0 by adjusting the acidity with 10 L of 2 M NaOH se-
quentially. The pH values were analyzed by a sensitive pH meter
(HORIBA, F51).
mM substrate
m
7. Narumi, T.; Takano, H.; Ohashi, N.; Suzuki, A.; Furuta, T.; Tamamura, H. Org. Lett.
2014, 16, 1184e1187.
4.7. Photolysis and quantum efficiency measurement
8. (a) Petit, M.; Tran, C.; Roger, T.; Gallavardin, T.; Dhimane, H.; Palma-Cerda, F.;
Blanchard-Desce, M.; Acher, F. C.; Ogden, D.; Dalko, P. I. Org. Lett. 2012, 14,
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6366e6369; (b) Zhu, Y.; Pavlos, C. M.; Toscano, J. P.; Dore, T. M. J. Am. Chem. Soc.
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Zhu, Y.; Dore, T. M. J. Org. Chem. 2009, 74, 1721e1729; (d) Laras, Y.; Hugues, V.;
Chandrasekaran, Y.; Blanchard-Desce, M.; Acher, F. C.; Pietrancosta, N. J. Org.
Chem. 2012, 77, 8294e8302.
Into a Pyrex test tube of 12 mm diameter was placed 2 mL of
10
DMSO. The solution was irradiated at 350 nm using four RPR
350 nm lamps (10 mJ sꢀ1). Aliquots of 10
M were removed peri-
mM substrate solution in KMOP solution (pH 7.2) containing 0.1%
m
9. Tsien, R. Y.; Zuker, R. S. Biophys. J. 1986, 50, 843e853.
10. Brown, E. B.; Shear, J. B.; Adams, S. R.; Tsien, R. Y.; Webb, W. W. Biophys. J. 1999,
76, 489e499.
odically and analyzed by HPLC. The light output for the quantum
efficiencies measurement was performed using ferrioxalate
actinometry.
11. See Supplementary data for details.
Please cite this article in press as: Takano, H.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.04.063