Synthesis and photochemical properties of a new water-soluble coumarin, designed as a chromophore for highly water-soluble and photolabile protecting group

Article abstract of DOI:10.1246/bcsj.79.1753

The absorption spectra and fluorescence spectra of a coumarin derivative (1), designed to be a water-soluble photolabile protecting group for caged compounds, were successfully observed in aqueous buffer solutions at pH 2.0-12.5 for the first time. Coumar

Full text of DOI:10.1246/bcsj.79.1753

ꢀ 2006 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 79, No. 11, 1753–1757 (2006) 1753
Synthesis and Photochemical Properties of a New Water-Soluble
Coumarin, Designed as a Chromophore for Highly
Water-Soluble and Photolabile Protecting Group
ꢀ
Naoko Senda, Atsuya Momotake, Yoshinobu Nishimura, and Tatsuo Arai
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571
Received March 17, 2006; E-mail: arai@chem.tsukuba.ac.jp
The absorption spectra and fluorescence spectra of a coumarin derivative (1), designed to be a water-soluble photo-
labile protecting group for caged compounds, were successfully observed in aqueous buffer solutions at pH 2.0–12.5 for
the first time. Coumarin 1 is highly soluble (>5 mM) in aqueous buffer at pH 7.2 and is stable in the dark at room tem-
perature for up to a week. The pKa value for the protonation of anilino nitrogen in coumarin 1 was determined to be 4.5.
Coumarin 1 has a fluorescence lifetime of 2.0 ns and undergoes intersystem crossing to the triplet state. Therefore, com-
pound 1 can be used for the light absorbing part of the new water-soluble caged compounds, such as caged amino acids
or caged nucleotides, which can release free amino acids or nucleotides, respectively, by excitation in the visible region.
Photochemical properties of conjugated molecules have
been extensively studied in organic solvents. Solvent proper-
ties, such as polarity and hydrogen-bonding ability, sometimes
affect the properties of molecules both in the ground state and
excited state. However, photochemistry in water has hardly
been studied because many organic molecules do not dissolve
in water.
In the course of our study on the photochemistry in water,1–5
we have come to study caged compounds soluble in water.
Although a lot of studies concerning caged compound have
been done, quite a few have been reported on a water-soluble
photolabile protecting group. In this respect, we are synthesiz-
ing a new chromophore for caged compounds that is capable
of uncaging carboxylic acid and phosphoric acid in water.
The points for the development of a new chromophore for
photolabile protecting group in caged compound are: 1) suffi-
cient solubility and stability in water at neutral pH and 2) high
quantum yield for photolysis by excitation in the visible wave-
group at the 7-position and two carboxyl groups to make it wa-
length region. The latter is closely related to the lifetime for
Scheme 1.
a water-soluble version of DECM, which has a dialkylamino
ter soluble. When our studies have almost finished, we come to
the singlet excited state. It is also advantageous if the photoly-
realize the report of the use of coumarinylmethyl esters for the
sis proceeds in the triplet excited state, because the radical re-
caging of phosphate and other groups.15 However, there is no
combination is not efficient to result in enhancing the quantum
yield for photolysis.
report on the photochemical properties of coumarin derivative
in pure water. Therefore, it is worthwhile to report the photo-
physical features of coumarin derivatives in pure water. We
now wish to report the synthesis of coumarin 1 and the studies
of photochemical and photophysical properties in water.
Coumarin derivatives have been successfully employed as a
photolabile protecting group in caged compounds because of
their favorable properties such as rapid photolytic release with
high efficiency, stability under physiological conditions, and
large extinction coefficients at longer wavelength.6–11 Among
coumarin-based caged compounds, 7-diethylamino-4-methyl-
coumarin (DECM) caging group showed significant properties
such as high efficiency of photolysis in the visible wavelength
region.12–14 However, DECM itself is not soluble in water. In-
Experimental
Materials. Coumarin 1 was synthesized by the four steps as
shown in Scheme 1.
Compound 2: A mixture of 3-aminophenol (6.98 g, 64 mmol),
imidazole (10.9 g, 160 mmol) and tert-butyldimethylchlorosilane
solubility in water is an obvious problem for caging group, es-
pecially when the biologically active molecule does not have
strong hydrophilic groups either. In this respect, we designed
a new coumarin derivative 1 (Scheme 1), being designed as
(TBDMS) (11.6 g, 77 mmol) in DMF (56 mL) was stirred for 2 h
at 0 ꢁC under nitrogen. A saturated aqueous solution of NaHCO3
(100 mL) was then added and the solution was extracted three
times with ether. The organic layer was collected, washed with
Published on the web November 9, 2006; doi:10.1246/bcsj.79.1753

1754 Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006)
Properties of a New Water-Soluble Coumarin
brine, and dried over MgSO4. After evaporation of the solvent, the
residue was purified by flash chromatography (SiO2, hexane–
1
AcOEt 5:1) to give 8.4 g of 2 in 66% yield. H NMR (CDCl3) ꢀ
6.98 (t, J ¼ 8:0 Hz, 1H), 6.30–6.18 (m, 3H), 3.58 (s, 2H), 0.97
(s, 9H), 0.18 (s, 6H).
Compound 3: A mixture of 2 (4.13 g, 19 mmol), ethyldiisopro-
pylamine (9.56 mL, 56 mmol), tert-butyl bromoacetate (6.14 mL,
42 mmol), and sodium iodide (8.32 g, 56 mmol) in acetonitrile
was refluxed for 18 h under nitrogen. The solution was poured into
water and was extracted with dichloromethane. The organic layer
was dried over MgSO4 and filtered, and the solvent was evapo-
rated. The residue was purified by flash chromatography (SiO2,
hexane–AcOEt 7:1) to give 6.82 g of 3 in 80% yield; 1H NMR
(CDCl3) ꢀ 7.03 (t, J ¼ 8:0 Hz, 1H), 6.25–6.07 (m, 3H), 3.97 (s,
4H), 1.46 (s, 18H), 0.97 (s, 9H), 0.17 (s, 6H).
Fig. 1. Absorption and fluorescence spectra of coumarin 1
(1:02 ꢃ 10ꢂ5 M) in HEPES buffer at pH 7.2.
coumarin synthesis,18 proceeded smoothly to give coumarin
1 in 18% overall yield, as described in Scheme 1.
Compound 4: To a solution of 3 (5.35 g, 12 mmol) in THF–
ethanol (10:1, 110 mL) was added 1.0 M THF solution of tetra-
butylammonium fluoride (17.7 mL). After stirring for 2 h under
nitrogen, water was added, and the solution was extracted with
dichloromethane. The organic layer was dried over MgSO4 and
filtered, and the solvent was evaporated, followed by purification
by flash chromatography (SiO2, hexane–AcOEt 5:1) to give 3.45 g
Steady State Absorption and Fluorescence Spectra.
For the absorption and the fluorescence measurements, a
McIlvaine buffer solution19 was used in the range of pH 2.0 to
6.4, a HEPES buffer solution (HEPES 40 mM, KCl 100 mM)
was used for pH at 7.2, and a KCl buffer solution (KCl 100
mM) was used in the range of pH 6.4 to 12.5. Coumarin 1 is
soluble (>5 mM) in HEPES buffer at pH 7.2. Figure 1 shows
the UV absorption and fluorescence spectra of coumarin 1 in
HEPES buffer solution at pH 7.2. The absorption maximum
of coumarin 1 was at 371 nm with the extinction coefficient
of 17600 Mꢂ1 cmꢂ1. The fluorescence spectrum was observed
in the same buffer with the peak at 472 nm. The value of the
Stokes shift of coumarin 1 was 5800 cmꢂ1. The fluorescence
band did not depend on the excitation wavelength at pH 7.2.
The stability of coumarin 1 in the neutral buffer was tested
by monitoring the absorption spectra. The HEPES buffer solu-
tion of coumarin 1 (0:5 ꢃ 10ꢂ6 M) at pH 7.2 was kept in the
dark at room temperature under air. After a week, the solution
gave the identical absorption spectra to that of a freshly pre-
pared solution, indicating that coumarin 1 is stable in aqueous
neutral buffer in the dark at room temperature for a week.
The pKa value for the protonation of the anilino nitrogen in
coumarin 1 was determined by monitoring the absorption spec-
tra (Fig. 2) in a McIlvaine buffer solution19 at various pH
(pH 2.0–6.4). The UV absorption spectra of coumarin 1 large-
ly changed between pH at 6.0 and 3.0, where the intensity of
the absorption band at 353 nm decreased and that at 370 nm
increased with increasing pH. The pKa value was calculated
to be 4.5. The absorption spectra slightly changed in the acidic
solution between pH 3.0 and 2.0, probably because the proton-
ation of the two carboxylic acid moieties in coumarin 1 affects
its absorption spectra. However, the exact pKa values for the
two carboxylic acids could not be determined. Coumarin 1
shows stability and solubility in acidic conditions even at
pH 2.0. The absorption spectra of coumarin 1 did not change
in the range of pH 6.4 to 12.5 in KCl buffer, indicating that
coumarin 1 takes a single form in the ground state in aqueous
buffer between pH 6.4 and 12.5.
1
of 4 in 87% yield; H NMR (CDCl3) ꢀ 7.00 (t, J ¼ 8:0 Hz, 1H),
6.20–6.06 (m, 3H), 3.97 (s, 4H), 1.45 (s, 18H).
Coumarin 1: To a mixture of 4 (0.74 g, 2.2 mmol) and ethyl 3-
oxobutanoate (0.29 g, 2.2 mmol) was added p-toluenesulfonic acid
monohydrate (20.3 mg, 0.11 mmol). The mixture was stirred at
60 ꢁC for 24 h under nitrogen. After cooling to room temperature,
AcOEt (25 mL) and hexane (10 mL) were added to the mixture to
precipitate of the crude product, and the supernatant was removed.
The residue was purified by recrystallization from mixed solvent
of ethanol and hexane (5:1) to give 1 (0.25 g) in 39% yield.
1H NMR (CD3OD) ꢀ 7.64 (d, J ¼ 4:2 Hz, 1H), 6.73–6.70 (m, 1H),
6.53 (d, J ¼ 2:4 Hz, 1H), 6.07 (s, 1H), 4.31 (s, 4H), 2.44 (s, 3H);
13C NMR (CD3OD) ꢀ 175.0, 165.1, 157.4, 156.9, 153.7, 128.1,
113.0, 111.5, 111.1, 100.4, 55.1, 19.4. Anal. Calcd for C14H13-
NO6: C, 57.73; H, 4.50; N, 4.81%. Found: C, 57.86; H, 4.77; N,
4.61%.
Measurements. The 1H and 13C NMR spectra were measured
with a Bruker ARX-400 (400 MHz for 1H NMR) and Bruker
AVANCE 500 (125 MHz for 13C NMR) spectrometer in CDCl3
with tetramethylsilane as an internal standard. The UV absorption
and fluorescence spectra were recorded on a Shimadzu UV-1600
UV–visible spectrophotometer and on a Hitachi F-4500 fluores-
cence spectrometer, respectively. Fluorescence lifetimes were
determined with Horiba NAES-1100 time-resolved spectrofluo-
rometer. Laser flash photolysis was performed by using an exci-
mer laser (Lambda Physik LPX-100, 308 nm, 20 ns fwhm) as
the excitation light source and a pulsed xenon arc (Ushio UXL-
159) as a monitoring light source. A photomultiplier (Hamamatsu
R-928) and a storage oscilloscope (LeCroy LT264) were used for
the detection.
Results
Synthesis. Direct N,N-dialkylation of 7-amino-4-methyl-
coumarin was not successful probably because of the low reac-
tivity of anilino nitrogen due to the electron-withdrawing effect
of carbonyl group on coumarin ring. Instead of 7-amino-4-
methylcoumarin, 3-aminophenol was used as a starting mate-
rial. The four step synthesis, including the protection of the
hydroxy group,16 N,N-dialkylation, deprotection,17 and final
The fluorescence spectra of coumarin 1 were also pH
dependent. The emission band with the maximum at 440 nm
at pH 2 was ascribed to the protonated form (Fig. 3a). At
pH 5, the fluorescence bands from both the protonated and
non-protonated form were observed, depending on the excita-
tion wavelength (Fig. 3b). The fluorescence spectrum of cou-

N. Senda et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006) 1755
Fig. 4. Transient absorption spectra of coumarin 1 in
HEPES buffer solution under argon at pH 7.2.
irradiation at 308 nm light under argon atmosphere, a weak
band was observed in the range of 500–700 nm (Fig. 4). The
lifetimes of the transient species were 15 ms under argon and
2.1 ms under air. The quenching rate constant by oxygen was
calculated to be 1:4 ꢃ 109 Mꢂ1 sꢂ1. These results suggest that
the transient band is ascribed to the triplet excited state.
Discussion
A water-soluble coumarin 1 was synthesized in 18% overall
yield from readily available starting materials. Some of the
characteristics of coumarin 1, as a chromophore for caged
compounds, are mentioned below.
Fig. 2. (a) Absorption spectra of 1 in McIlavine buffer at
various pH (dotted line: pH 2.0, solid line: pH 6.4, and
thin lines: pH 2.4–6.0) at room temperature. (b) Plots of
the absorbance for 1 at 330 nm ( ), 350 nm ( ), and
390 nm ( ) as a function of pH.
(1) Solubility in aqueous buffer solution: In general, caged
compounds consist of a biological active molecule and a chro-
mophore, or a photolabile protecting group. Coumarin deriva-
tives have already been used as chromophores for cage nucleo-
tides, such as ATP, ADP, and amino acid, such as L-glutamic
acid, mainly because of their sufficient quantum yield for pho-
tolysis and large extinction coefficient at longer wavelength
region (300–400 nm). In many cases, however, the coumarin
moiety in a caged compound is hydrophobic or weakly hydro-
philic. For example, although DECM-caged compounds exhib-
ited rapid and efficient photolysis, DECM itself did not dis-
solved in aqueous buffer at pH 7.2. This result indicates that
the water-solubility of caged compounds is essentially deter-
mined by the hydrophilicity of the biologically active com-
pound part, solubility may be a problem when the biologically
active compounds are not strongly hydrophilic. Coumarin 1
has a solubility of more than 5 mM in aqueous buffer at
pH 7.2, which good for use with caged compounds because
(1) the caged compounds can be used at higher concentrations,
and (2) it can inhibit hydrophobic interactions with proteins,
lipids in membranes, or even self assembly, in aqueous solu-
tion. Furthermore, coumarin 1 did not precipitate even under
acidic conditions (pH 2.0), probably because of the proton-
ation of the anilino nitrogen below pH 4.5. Coumarin 1 also
is stable in the dark at room temperature in buffer at pH 7.2.
From these results, coumarin 1 is a good candidate for use
as a caging group not only for hydrophilic biological active
compounds but also hydrophobic compounds without solubil-
ity problems.
Fig. 3. Fluorescence spectra of coumarin 1 in HEPES
buffer (1:02 ꢃ 10ꢂ5 M) at pH 2.0 (a), at pH 5.0 (b), and
at pH 12.5 (c).
marin 1 at pH 12.5 was almost identical to that at pH 7.2. The
values of the fluorescence quantum yield in buffer at pH 7.2
were determined to be 0.32. The fluorescence lifetime of cou-
marin 1 was 2.0 ns.
Transient Absorption Spectra. The transient absorption
spectra of 1 were measured in HEPES buffer at pH 7.2. On
(2) Photochemical properties: Coumarin 1 was designed as a
water-soluble version of DECM. Since DECM-caged com-
pounds have large extinction coefficients around 370–380 nm
and undergo efficient photolysis, coumarin 1 must have similar
photochemical properties, i.e., the caged compounds with cou-
marin 1 must be photolizable with high efficiency. In a steady-

1756 Bull. Chem. Soc. Jpn. Vol. 79, No. 11 (2006)
Properties of a New Water-Soluble Coumarin
S
1
TICT*
Intersystem
crossing
T
1
hν
hν'
τf = 2.0 ns
H+
S
0
S
0
pKa 4.5
O
O
-O
-O
+HN
O
-O
-O
O
O
N
O
O
O
1
1 (protonated form)
Fig. 5. Energy diagram for 1.
state absorption study, coumarin 1 had an absorption maxi-
mum at 371 nm with the extinction coefficient of 17600 Mꢂ1
cmꢂ1 in HEPES buffer. In the fluorescence spectrum, coumar-
in 1 had a maximum at 472 nm in HEPES buffer. These values
are similar to those of DECM derivatives in MeOH–HEPES
1:4 mixture. MeOH was used for the measurements of the
absorption and the fluorescence spectra of DECM derivatives
due to their insolubility in water. The value of the Stokes shift
of 1 was 5800 cmꢂ1, suggesting that fluorescence emission of
1 in aqueous solution is mainly from a twisted intramolecular
charge-transfer (TICT) state.20–23 The fluorescence lifetime
was 2.0 ns, indicating that 1 may have enough lifetime to in-
duce photolysis, thus, releasing the biologically active mole-
cules.
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.
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