Coumarin-4-ylmethoxycarbonyls as Phototriggers for Alcohols and Phenols

Article abstract of DOI:10.1021/ol0359362

(Equation Presented) Caged compounds can be used to regulate the spatial and temporal dynamics of signaling molecules in live cells. Photochemical properties of coumarin-4-ylmethoxy carbonates (1a-d) are investigated to construct caged compounds of hydroxy-containing molecules. All the compounds possess desired properties as phototriggers for alcohols and phenols. The 6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl (Bhcmoc) group has the highest photochemical efficiency and is applied to make caged compounds of 1,2-dioctanoylglycerol (diC₈), Tyr-OMe, and adenosine.

Full text of DOI:10.1021/ol0359362

ORGANIC
LETTERS
2003
Vol. 5, No. 25
4867-4870
Coumarin-4-ylmethoxycarbonyls as
Phototriggers for Alcohols and Phenols
Akinobu Z. Suzuki, Takayoshi Watanabe, Mika Kawamoto, Keiko Nishiyama,
Hirotaka Yamashita, Megumi Ishii, Michiko Iwamura, and Toshiaki Furuta*
Department of Biomolecular Science, Toho UniVersity, 2-2-1 Miyama,
Funabashi, 274-8510 Japan
furuta@biomol.sci.toho-u.ac.jp
Received October 3, 2003
ABSTRACT
Caged compounds can be used to regulate the spatial and temporal dynamics of signaling molecules in live cells. Photochemical properties
of coumarin-4-ylmethoxy carbonates (1a−d) are investigated to construct caged compounds of hydroxy-containing molecules. All the compounds
possess desired properties as phototriggers for alcohols and phenols. The 6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl (Bhcmoc) group
has the highest photochemical efficiency and is applied to make caged compounds of 1,2-dioctanoylglycerol (diC ), Tyr-OMe, and adenosine.
8
Caged compounds are synthetic molecules whose biological
activities are masked by covalent attachment of a photo-
chemically removable protecting group (“caging group” or
“phototrigger”) to a functional group, which is of critical
importance to an activity. Intracellular distribution of signal-
ing molecules can be controlled using the chemistry of caged
compounds with high spacial and temporal resolution.1
Coumarin-4-ylmethyls are newly developed phototriggers.
They have higher photolysis efficiencies than others such
as 2-nitrobenzyls, so that the incident light intensity for the
uncaging reaction could be lowered, leading to minimization
of cell damage while maintaining higher spatial resolution.
From recent investigations, the coumarin-type cages, includ-
ing MCM2, HCM (and ACM)3, DMCM,4 BCMCM,5
DMACM (and DEACM),5,6 and Bhc7 have been successfully
applied to protect phosphates,2-7 amines,7a,b,d carboxylates,2b,7
diols,7e and carbonyl compounds.7f Furthermore, the Bhc
group was found to be photolyzed under two-photon excita-
tion conditions with practically useful absorption cross-
sections.7
We investigated photolabile protecting groups for hydroxy
functionality to construct caged compounds of sugars, amino
acids, lipid mediators, and other effector molecules. In a
previous report, we designed and synthesized four arylmethyl
carbonate-type protecting groups, among which the an-
thraquinone-2-ylmethoxycarbonyl (Aqmoc) was found to
offer a unique property as a potential phototrigger for
(5) (a) Hagen, V.; Bendig, J.; Fringe, S.; Eckardt, T.; Helm, S.; Reuter,
D.; Kaupp, U. B. Angew. Chem., Int. Ed. 2001, 40, 1046-1048. (b) Hagen,
V.; Frings, S.; Bendig, J.; Lorenz, D.; Wiesner, B.; Kaupp, U. B. Angew.
Chem., Int. Ed. 2002, 41, 3625-3628.
(6) (a) Geisler, D.; Kresse, W.; Wiesner, B.; Bendig, J.; Kettenmann,
H.; Hagen, V. ChemBioChem 2003, 4, 162-170. (b) Hagen, V.; Frings,
S.; Wiesner, B.; Helm, S.; Kaupp, U. B.; Bendig, J. ChemBioChem 2003,
4, 434-442.
(1) (a) Adams, S. R.; Tsien, R. Y. Annu. ReV. Physiol. 1993, 55, 755-
784. (b) Caged Compounds. In Methods in Enzymology; Marriott, G., Ed.;
Academic Press: New York, 1998; Vol. 291. (c) Curley, K.; Lawrence, D.
S. Pharmacol. Ther. 1999, 82, 347-354. (d) Dorman, G.; Prestwich, G. D.
Trends Biotechnol. 2000, 18, 64-77. (e) Shigeri, Y.; Tatsu, Y.; Yumoto,
N. Pharmacol. Ther. 2001, 91, 85-92.
(2) (a) Furuta, T.; Torigai, H.; Sugimoto, M.; Iwamura, M. J. Org. Chem.
1995, 60, 3953-3956. (b) Schade, B.; Hagen, Schmidt, V. R.; Herbrich,
R.; Krause, E.; Eckardt, T.; Bendig, J. J. Org. Chem. 1999, 64, 9109-
9117.
(3) (a) Furuta, T.; Momotake, A.; Sugimoto, M.; Hatayama, M.; Iwamura,
M. Biochem. Biophys. Res. Commun. 1996, 228, 193-198. (b) Furuta, T.;
Iwamura, M. Methods Enzymol. 1998, 291, 50-63.
(4) Eckardt, T.; Hagen, V.; Schade, B.; Schmidt, R.; Schweitzer, C.;
Bendig, J. J. Org. Chem. 2002, 67, 703-710.
(7) (a) 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, 1193-2000. (b) Tsien, R. Y.; Furuta, T. U.S. Patent
Application WO/00/31588, 2000. (c) Ando, H.; Furuta, T.; Tsien, R. Y.;
Okamoto, H. Nat. Genet. 2001, 28, 317-325. (d) Montgomery, H. J.;
Perdicakis, B.; Fishlock, D.; Lajoie, G. A.; Jervis, E.; Guillemette, J. G.
Bioorg. Med. Chem. 2002, 10, 1919-1927. (e) Lin, W.; Lawrence, D. S.
J. Org. Chem. 2002, 67, 2723-2726. (f) Lu, M.; Fedoryak, O. D.; Moister,
B. R.; Dore, T. M. Org. Lett. 2003, 5, 2119-2122.
10.1021/ol0359362 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/08/2003

Scheme 1. Photolysis of Coumarin-4-ylmethoxycarbonyl
Table 1. Selected Photophysical and Chemical Properties of
Coumarin-4-ylmethoxycarbonyl Phototriggers
Galactose Derivatives.
b
λmax (ꢀ)a
ꢀ350
Φ350
Φꢀ 350
solventc
1a
1b
322 (12 100)
323 (13 100)
374 (15 000)
376 (14 000)
396 (17 300)
344 (10 800)
344 (5400)
4100
3400
11 500
10 000
6300
10 400
5300
13 700
0.020
2.9 × 10-3
0.015
8.3 × 10-3
5.8 × 10-3
6.5 × 10-3
3.5 × 10-4
0.067
84
10
173
83
37
68
A
C
A
B
B
A
A
A
1c
1d
1e
3
2
918
372 (17 900)
a Extinction coefficient (cm-1 M-1). b Quantum yields for disappearance
of starting materials upon 350 nm irradiation. c Solvent A: KMOPS (pH
7.2) containing 0.1% DMSO. Solvent B: KMOPS (pH 7.2) containing 25%
acetonitrile. Solvent C: H2O containing 50% THF.
improve efficiency. We chose three types of coumarin
cages: 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc), 7-di-
ethylaminocoumarin-4-ylmethyl (DEACM), and 6,7-di-
methoxycoumarin-4-ylmethyl (DMCM). Their phosphate
esters provide favorable photochemical properties as caged
compounds. Using them, we prepared MCMoc-, Bhcmoc-,
DEACMoc-, and DMCMoc-protected galactose derivatives
(1a-d, respectively). Irradiation of the compounds with
Rayonet 350 nm lamps under simulated physiological
conditions (pH 7.2 KMOPS buffer) unmasked the parent
galactose derivative (Scheme 1).
alcohols.8 The present study is intended to evaluate whether
the carbonate derivative of coumarin-based photoremovable
protecting groups can serve as a potential phototrigger for
hydroxy-containing molecules. Toward that end, we com-
pared photochemical properties of the coumarin-4-ylmethyl
carbonates and found that the 6-bromo-7-hydroxycoumarin-
4-ylmethoxycarbonyl (Bhcmoc) group is the most suitable
candidate for producing caged compounds of alcohols and
phenols.9
One important criterion to be considered in seeking a new
photoremovable protecting group for biological applications
is photolysis efficiency. Relatively intense UV light is
required for caged compounds with lower photolysis ef-
ficiency to release a substantial amount of biological mes-
sengers, thereby causing not only desired biological responses
but also undesired cell damage. We have used the product
of the photolysis quantum yield (Φ) and molar absorptivity
(ꢀ) to compare the overall efficiency of an uncaging reaction
quantitatively.10 Previous investigations revealed that the Φꢀ
values of the coumarin-4-ylmethyl groups, MCM, HCM, and
Bhc, introduced into phosphates (as esters),2,3,7 carboxylates
(as esters),7 and amines (as carbamates)7 were sufficiently
high (Φꢀ 300-1200) for application to caging chemistry.
However, photolysis efficiency of MCM carbonate (MCMoc)
is much lower than we expected from results of other
coumarin-type caged compounds. For example, typical Φꢀ
values of phosphate esters of the MCM group are 600-800,
whereas that of MCM carbonate was only 10 when pho-
tolysis was conducted in 50% THF-H2O.8
Absorption properties (λ and ꢀ) and the photolysis quantum
yields (Φ) for disappearance of 1a-d were measured and
compared with NVOC-Gal (1e) having a 2-nitrobenzyl
phototrigger. Table 1 shows some notable findings from the
data. First, the photolytic efficiency of 1a depends strongly
on the nature of the solvent, which is consistent with the
reactivity of the MCM ester of diethyl phosphate reported
by Hagen and Bendig.2b The quantum yield of 1a in solvent
A (10 mM KMops, pH 7.2 containing 0.1% DMSO) was
about 7 times larger than that in solvent C (50% THF-H2O),
indicating that the photolytic efficiency of the MCM group
is less favorable when the reaction is performed in a solvent
system with a lower polarity or in the presence of an organic
solvent. This was further supported by the fact that the
quantum yield of 1b in solvent B (10 mM KMops, pH 7.2
containing 25% acetonitrile) was almost half of that in
solvent A. Second, under a simulated physiological environ-
ment, all tested coumarin-4-ylmethyl carbonates (1a-d) have
practically useful Φꢀ values that were more than an order
of magnitude larger than that of NVOC-Gal (1e). Third, the
Bhcmoc group (1b) has the largest Φꢀ value among the
coumarin-4-ylmethoxycarbonyl phototriggers tested. The
observed Φꢀ value of 173 was as large as the other Bhc-
caged compounds protected as Bhc carboxylates and car-
bamates.
First, we need to know whether the observed low pho-
tolysis efficiency of the MCMoc group is attributable to the
inherent reactivity of all coumarin-4-ylmethyl carbonates or
an introduction of appropriate substituents on coumarins can
(8) Furuta, T.; Hirayama, Y.; Iwamura, M. Org. Lett. 2001, 3, 1809-
1812.
(9) Part of this work was presented at the International Chemical Congress
of Pacific Basin Societies (Pacifichem 2000), Honolulu, Hawaii, Dec 14-
19, 2000; Paper no. 80925280.
(10) Lester, H. A.; Nerbonne, J. M. Annu. ReV. Biophys. Bioeng. 1982,
11, 151-175.
Furthermore, the Bhcmoc phototrigger can be applied to
protect the phenolic hydroxy group. Thus, Bhcmoc-phenol
(3) was synthesized from phenyl chloroformate and photo-
lyzed to unmask the parent phenol with a Φꢀ value of 918,
about 5 times better than that of aliphatic carbonates (Scheme
4868
Org. Lett., Vol. 5, No. 25, 2003

Photolysis reaction proceeds with the single-exponential
decay of the concentration of the starting material; otherwise,
photolysis byproducts or caged compounds themselves would
interfere with the reaction. The time course for the consump-
tion of 8 and 9 (shown in Figures 1 and 2 in Supporting
Information) indicates that photolysis of 8 and 9 in a
simulated physiological environment follows single expo-
nential decays and proceeds with no remarkable interference
by the released “Bhc” phototrigger or undesired side-
reactions. However, the photolysis quantum yield of 7
markedly decreased (by an order of magnitude) and the time
course for the consumption did not follow exponential decay
when the reaction progressed to over 50% conversion (Figure
3 in Supporting Information). Quantum yields (Φ) of
disappearance were determined as 0.014 for 7,12 0.022 for
8, and 0.012 for 9, respectively. The aliphatic carbonates,
1b, 7, and 9, have similar quantum yields (0.012-0.015),
which is approximately half that of aromatic carbonates,
0.064 for 3 and 0.022 for 8. The remarkable difference in Φ
values between the aliphatic and the aromatic carbonates
probably results from the difference in acidity of the carbonic
acids. Photoexcitation of the Bhc group must be followed
by cleavage between the C4 methylene carbon of the Bhc
group and the carbonate oxygen to produce the coumarin-
4-ylmethyl cation and the corresponding carbonate anions.13
Therefore, a difference in photolysis quantum yield (Φ)
should depend on the thermodynamic stability of the carbon-
ate anions, which is predictable from the strength of their
conjugate acids. The acid strength of the carbonic acid
derived from an aliphatic alcohol is weaker than that from a
phenol. Therefore, the photolysis quantum yields of the
aliphatic carbonates are smaller than those of the aromatic
ones. The observed decrease in quantum yield of 7 with the
progress of reaction also accounted for formation of the ion
pair intermediate. In an aqueous solution, the produced
diacylglycerol tends to form hydrophobic aggregates, provid-
ing the Bhcmoc group with a less polar environment in which
the formation of the ion pair must be less favorable.14
Quantitative production of the parent molecule concomi-
tant with the progress of photolysis is important for recovery
of the original activity. When the 2-nitrobenzyl phototriggers
are employed, we sometimes encounter difficulty in obtaining
100% recovery of the original molecule even after photolysis
is completed. This is probably the result of unwanted side
reactions resulting from production of highly reactive nitroso-
carbonyl byproducts. Thus, for photolysis of 8 and 9,
production of the parent molecules was quantified by HPLC
analysis of aliquots taken from the photolysis mixture
(Figures 1 and 2 in Supporting Information). Quantitative
production of Tyr-OMe and adenosine was observed con-
comitant with consumption of the starting materials. The
hydrolytic stability of caged compounds is often an issue in
biological applications. The Bhcmoc-caged compounds 7-9
Scheme 2. Synthesis and Photolysis of Bhcmoc-phenola
a Reagents and conditions: (a) 2b/DIEA/CH3CN (60%); (b) 350
nm/solvent A.
2). From the results, we inferred that the coumarin-type
photochemically removable protecting groups, especially
with 6-bromo and 7-hydroxy substituents (Bhcmoc), must
serve as a potential phototrigger of hydroxy-containing
molecules, both aliphatic and aromatic, with improved
photolytic efficiency.
Next, we chose three types of biologically relevant molec-
ules as model compounds to establish methods for introduc-
ing the Bhcmoc group to hydroxyl moieties and to investigate
their chemical and physical properties: diC8, a nonpolar
aliphatic alcohol; Tyr-OMe, a phenol derivative; and ad-
enosine, a polar alcohol with a free aromatic amino group.
The precursor molecules we designed were 6-bromo-7-
methoxymethoxycoumarin-4-ylmethyl chloroformate (5,
MOM-Bhcmoc-Cl) and 6-bromo-7-methoxymethoxy-cou-
marin-4-ylmethyl 4′-nitrophenyl carbonate (6, MOM-Bhc-
moc-ONp) (Scheme 3). Selective protection of the phenol
moiety of 2b was achieved by MOMCl and DIEA in
dichloromethane to yield 4 (85%). We synthesized MOM-
Bhcmoc-Cl (5) by reaction of 4 and phosgene (generated
from triphosgene and AliquatR 336) in 78% isolated yield.
Only a trace amount of the corresponding chloroformate was
obtained when the reaction was performed using unprotected
2b. The MOM protection of 7-OH of coumarin was also
necessary to achieve a better yield of the p-nitrophenyl
carbonate precursor (6).
In our previous report, Bhc p-nitrophenyl carbonate was
used to introduce a Bhcmoc group into amines. We first
attempted to protect the free hydroxy in diC8 with the
p-nitrophenyl carbonate 6. However, the isolated yield of
MOM-Bhcmoc-diC8 did not exceed 50%. In contrast, an
almost quantitative isolated yield was obtained with chlo-
roformate 5. Deprotection of the MOM group with TFA gave
Bhcmoc-diC8 (7) in 91% overall yield.11 As shown in
Scheme 3, Tyr(Bhcmoc)-OMe (8) was synthesized from Boc-
Tyr-OMe in two steps with quantitative yield, indicating that
chloroformate 5 also serves as a precursor to introduce a
Bhcmoc group into phenol. Synthesis of 5′-Bhcmoc-adenos-
ine (9) was achieved via MOM-Bhcmoc-ONp (6) starting
from 2′,3′-O-isopropylideneadenosine with 26% overall yield.
A chemoselective protection of primary alcohol (5′-OH of
ribose) over aromatic primary amine (N-6 of adenine) was
also accomplished with other p-nitrophenyl carbonates.8 In
contrast, the use of the chloroformate 5 resulted in the intro-
duction of a MOM-Bhcmoc group into both 5′-OH and N-6.
(12) Determined from the data within 50% conversion.
(13) Coumarin-4-ylmethyl cations are possible intermediates as suggested
by Bendig et al. in ref 6a, Lawrence et al. in ref 7e, and Dore et al. in ref
7f.
(14) This would not be a severe drawback when the compound is
subjected to live cell applications because the photolysis is performed in a
small volume and the complete conversion into the product is not necessary.
(11) J. W. Walker’s group reported a preliminary synthetic study and a
cell biological application of this compound. Robu, V. G.; Pfeiffer, E. S.;
Robia, S. L.; Balijepalli, R. C.; Pi, Y.; Kamp, T. J.; Walker, J. W. J. Biol.
Chem. 2003, in press.
Org. Lett., Vol. 5, No. 25, 2003
4869

Scheme 3. Synthesis and Photolysis of the Bhcmoc-Protected Hydroxyl-Containing Compoundsa
a Reagents and conditions: (a) MOMCl/DIEA/CH2Cl2 (85%); (b) phosgene (78%); (c) 4-nitrophenyl-chloroformate/DMAP; (d) 5/DMAP/
CH3CN; (e) TFA; (f) 6/DMAP/CH3CN.
show modest to good hydrolytic stabilities (see the half-lives
in the dark (t1/2) shown in Table 2).
has the highest Φꢀ values, into alcohols and phenols have
been developed and applied to synthesis of caged compounds
of biologically relevant molecules (7-9). Observed chemical
and physical properties of the compounds suggest that the
Bhcmoc group would serve as a “phototrigger” for hydroxy-
containing molecules, sugars, amino acids, and lipid media-
tors. Further study, including cell biological applications of
the compounds, are currently under way.
In summary, we characterized photochemical properties
of coumarin-4-ylmethyl carbonates (1a-d) as phototriggers
for alcohols. Photolysis efficiencies (Φꢀ value) of 1a-d were
sufficiently high to allow application to caging chemistry.
Practical methods of introducing the Bhcmoc group, which
Acknowledgment. This work was supported by grants
from Japan Science and Technology Corporation (PRESTO
to T.F.) and the Ministry of Education, Culture, Sports,
Science and Technology (13640545, 14011247 and 15011251
to T.F.).
Table 2. Selected Photophysical and Chemical Properties of
Bhcmoc-Protected Moleculesa
λmax (ꢀ)b
Φdis
Φꢀ dis
Φapp
Φꢀ app
t1/2
c
d
e
7
8
9
343 (11 600)
372 (13 900)
373 (13 700)
0.014
0.022
0.012
160
300
140
ndf
0.020
0.010
nd
270
120
37
38
467
Supporting Information Available: Experimental pro-
cedure and characterization data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
a All experiments were done in a simulated physiological saline solution
(KMOPS (pH 7.2) containing 0.1% DMSO). b Extinction coefficient (cm-1
M-1). c Quantum yields for disappearance of the starting materials upon
350 nm irradiation. d Quantum yields for appearance of the products upon
350 nm irradiation. e Half-life (h) in the dark. f Not determined.
OL0359362
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Org. Lett., Vol. 5, No. 25, 2003