A strategy for the construction of caged diols using a photolabile protecting group

Article abstract of DOI:10.1021/jo0163851

Caged analogues of biologically active compounds have received widespread attention as temporally and spatially controlled probes of cell-based processes. Recently, a coumarin-4-ylmethyl derivative has been used to cage carboxylates, sulfonates, carbamates, and phosphates. We describe herein a synthetic strategy that furnishes photosensitive caged diols and provides an entry into the protection/photodeprotection of functionality with modest leaving group abilities.

Full text of DOI:10.1021/jo0163851

J . Org. Chem. 2002, 67, 2723-2726
2723
A Str a tegy for th e Con str u ction of Ca ged
Diols Usin g a P h otola bile P r otectin g Gr ou p
Weiying Lin and David S. Lawrence*
Department of Biochemistry, The Albert Einstein College of
Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461
Under conditions of high photon flux, the 7-hydroxycou-
marin moiety can absorb and combine the energies of two
long wavelength photons. Two-photon excitation is equiva-
lent to the absorption of one photon at half the wave-
length and possesses the inherent advantage of signifi-
cantly deeper tissue penetration. Although the caging
agent 1 holds significant promise for in vivo studies, it
has been used to cage only a limited number of functional
groups [e.g., carboxylate (1a , X ) -O2CR), phosphate [1b,
X ) -O2P(OR)2], and carbamate (1c, X ) -O2CNHR)].
By contrast, aliphatic alcohols fail to undergo photocleav-
age when caged with the coumarin moiety (e.g., 2). One
possible explanation for this behavior is that the photo-
liberation process proceeds via a mechanism that gener-
ates charged intermediates. For example, the 7-methoxy
analogue of 1 photohydrolyzes via formation of an ion
pair.4 Such a mechanism would limit photodeprotection
to leaving groups that could stabilize an initially formed
anionic intermediate.3,5 As part of our program to define
the temporal role of signaling proteins in biochemical
pathways, we required access to a caging agent for diols
that also has the requisite physical stability to function
in a living animal.
Coumarin derivatives have recently been shown to
serve as caging agents for such biologically important
molecules as cAMP,5a,c,d,e cGMP,5c,d cytidine diphosphate,5b
and glutamic acid.3 Bendig and his colleagues have
recently reported that carboxylates, sulfonates, and
phosphates caged with the 7-methoxy derivative of 1 are
photoliberated via an SN1 mechanism that generates an
initially formed ion pair.4 One might predict that such a
mechanism would exclude functionality unable to stabi-
lize the negatively charged intermediate produced during
photoheterolysis. Indeed, we found that the coumarin
ether derivative 2 is completely resistant to photocleav-
age. This observation is consistent with a previous report
that coumarin caged alcohols are resistant to photo-
deprotection.6 We reasoned that it should be possible to
promote the photodeprotection of a coumarin-caged ali-
phatic alcohol if the latter were to lie adjacent to an
electron-rich center that could assist the cleavage process.
Consequently, we decided to investigate the possibility
dlawrenc@aecom.yu.edu
Received December 18, 2001
Abstr a ct: Caged analogues of biologically active compounds
have received widespread attention as temporally and
spatially controlled probes of cell-based processes. Recently,
a coumarin-4-ylmethyl derivative has been used to cage
carboxylates, sulfonates, carbamates, and phosphates. We
describe herein a synthetic strategy that furnishes photo-
sensitive caged diols and provides an entry into the protec-
tion/photodeprotection of functionality with modest leaving
group abilities.
The wide variety of roles played by metabolites,
proteins, and genes in biological systems has been
explored using approaches that range from simple inhibi-
tory molecules to transgenic animals. However, these
technologies are generally unable to assess the precise
temporal and/or spatial influence exerted by specific
biomolecules in the context of a particular biological
event. For example, the role of a specific enzyme during
the various stages of mitosis or at the onset of carcino-
genesis in an adult animal is difficult to address with a
simple dead-end inhibitor or a transgenic animal model.
Photoactivatable (“caged”) analogues of biomolecules
furnish a simple means to control, both spatially and
temporally, biological activity with light.1 A wide variety
of caged species have been described, including Ca2+, NO,
metal ion indicators, and peptides. More recently, caged
enzymes, such as protein kinases, protein phosphatases,
proteinases, and others, have been reported.2 Unfortu-
nately, the application of these caged species to living
animals has been thwarted by the short wavelengths
(<360 nm for the o-nitrobenzyl derivatives) required for
the uncaging process. Light of this wavelength has poor
tissue penetration due to the presence of both intra- and
extracellular chromophores that absorb in this region of
the electromagnetic spectrum. A photolabile-protecting
group containing a large two-photon absorbance cross-
section was recently introduced as a caging agent (1).3
(4) Schade, B.; Hagen, V.; Schmidt, R.; Herbrich, R.; Krause, E.;
Eckardt, T.; Bendig, J . J . Org. Chem. 1999, 64, 9109-9117.
(5) (a) Eckardt, T.; Hagen, V.; Schade, B.; Schmidt, R.; Schweitzer;
Bendig, J . J . Org. Chem., 2002, 67, 703-710. (b) Schonleber, R. O.;
Bendig, J .; Hagen, V.; Giese, B. Biorg. Med. Chem., 2002, 10, 97-101.
(c) Hagen, V.; Bendig, J .; Frings, S.; Eckardt, T.; Helm, S.; Reuter, D.;
Kaupp, U. B. Angew. Chem., Int. Ed. 2001, 40, 1046-1048. (d) Hagen,
V.; Bendig, J .; Frings, S.; Wiesner, B.; Schade, B.; Helm, S.; Lorenz,
D.; Kaupp, B. J . Photochem. Photobiol. B Biol. 1999, 53, 91-102. (e)
Furuta, T.; Momotake, A.; Sugimoto, M.; Hatayama, M.; Torigai, H.;
Iwamura, M. Biochem. Biophys. Res. Commun. 1996, 228, 193-198.
(f) Furuta, T.; Torigai, H.; Sugimoto, M.; Iwamura, M. J . Org. Chem.
1995, 60, 3953-3956.
(1) (a) Curley, K.; Lawrence, D. S. Cur. Opin. Cell. Biol. 1999, 3,
84-85. (b) Marriott, G.; Walker, J . W. Trends Plant Sci. 1999, 4, 330-
334. (c) Marriot, G. Methods Enzymol. 1998, 291, 1-529. (d) Adams,
S. R. Tsien, R. Y. Annu. Rev. Physiol. 1993, 55, 755-784.
(2) (a) Ghosh, M.; Ichetovkin, I.; Song, X.; Condeelis, J . S.; Lawrence,
D. S. J . Am. Chem. Soc. 2002, 124, in press. (b) Chang, C.-Y.;
Fernandez, T.; Panchal, R.; Bayley, H. J . Am. Chem. Soc. 1998, 120,
7661-7662. (c) Curley, K.; Lawrence, D. S. J . Am. Chem. Soc. 1998,
120, 8573-8574. (d) Arabaci, G.; Xiao-Chuan, Guo; Beebe, K. D.;
Coggeshall, K. M.; Dehua, P. J . Am. Chem. Soc. 1999, 121, 5085-
5086. (e) Turner, A. D.; Pizzo, S. V.; Rozakis, G.; Porter, N. A. J . Am.
Chem. Soc. 1988, 110, 244-250.
(6) Furuta, T.; Nishiyama, K.; Iwamura, M.; Tsien, R. Y. Presented
at the International Chemical Congress of Pacific Basin Societies
(Pacifichem2000), Dec 14-19, 2000. Paper no. 80925280, abstract
available online at http//www.chem.ukans.edu/Pacifichem2000/
35.HTM.
(3) 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, 1193-1200.
10.1021/jo0163851 CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/19/2002

2724 J . Org. Chem., Vol. 67, No. 8, 2002
Notes
that aliphatic ether, if present as a component of an
acetal, could undergo light-initiated cleavage.
The coumarin aldehyde 4 was prepared via oxidation
(MnO2/THF/90% yield) of the previously described cou-
marin alcohol 3.3 The aldehyde was subsequently used
to convert 1,2-, 1,3-, and 1,4-diols to their corresponding
acetals. All photolysis reactions were performed in a
F igu r e 1. Photolytic dissociation of 6 (b) and 7 (O) and
formation of diols from 6 (4) and 7 ()), respectively, in 1:1 CH3-
OH/buffer (0.01 M HEPES, 0.12 M NaCl, 3 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, pH 7.4).
quartz cuvette irradiated with a 200 W Hg arc lamp. UV
and IR filters were placed between the cuvette and the
lamp to reduce exposure to short wavelength light (<348
nm) and heat, respectively. The 1,3-dioxolanes 5-8
(Chart 1), prepared from the 1,2-diols, experience rapid
Ch a r t 1
F igu r e 2. Photolytic dissociation of 8 (b) and 11 (O) and
formation of diols from 8 (4) and 11 ()), respectively, in 1:1
CH3OH/buffer (0.01 M HEPES, 0.12 M NaCl, 3 mM KCl, 1
mM CaCl2, 1 mM MgCl2, pH 7.4).
Ta ble 1. P h otoch em ica l P r op er ties of Aceta ls 5-8 a n d
11
a
acetal
ꢀ348
φ
5
6
7
8
11
9700
8500
9400
8000
7100
0.0041
0.0053
0.0051
0.0278
0.0052
a
Extinction coefficient (M-1 cm-1) at 348 nm.
to quantum yields reported for coumarin caged phos-
phates.4 Interestingly, compound 8, which contains a
tertiary amine, is even more rapidly converted to the
photocleaved product (85% conversion in 5 min) (Figure
2). The φ for the 1,3-dioxolane 8 is 7-fold greater than
those obtained with the dioxolanes 5-7. The tertiary
amine in dioxolane 8 could potentially serve as a proton
donor during acetal decomposition (Scheme 1) and thereby
accelerate the overall rate of photouncaging.
In marked contrast, the 1,3-dioxanes 9 and 10, which
were prepared from 1,3-diols, are inert to photolysis.
Indeed, even after 40 min of irradiation, we were unable
to detect any observable free diol formation. This obser-
vation is consistent with the general observation that six-
membered-ring acetals are thermodynamically more
stable than their five-membered-ring counterparts.8 For
example, the acid-catalyzed rate of cyclic acetal hydroly-
sis is more rapid in less stable ring systems.8 However,
even given the unique stability commonly ascribed to the
photoliberation to the desired free diol products. Starting
material conversion and product formation were followed
as a function of time using HPLC (Figures 1 and 2). For
example, nearly 75% of compounds 6 and 7 are converted
to the uncaged diols after irradiation for 10 min (Figure
1). An analogous conversion rate is displayed by the
dioxolane 5 (data not shown). Quantum yields (φ) for the
photocleavage process were determined by ferrioxalate
actinometry.7 The φ values for compounds 5-7 are
between 0.004 and 0.005 (Table 1), which are analogous
(7) Hatchard, C. G.; Parker, C. A. Proc. R. Soc. London A 1956, A220,
518-536.
(8) Ferrier, R. J .; Collins, P. M. Monosaccharide Chemistry; Penguin
Books, Inc.: Baltimore, MD, 1972; pp 215, 220, 223.

Notes
J . Org. Chem., Vol. 67, No. 8, 2002 2725
Sch em e 1
derivatives) commonly produces byproducts structurally
analogous to 4 that can potentially interfere with biologi-
cal processes. However, the liberation of caged species
in cells appears to proceed in a fashion that inflicts little
or no damage. This may be due, in part, to the high
intracellular concentration of glutathione. Indeed, sulf-
hydryl reagents have previously been shown to protect
proteins from photoinitiated damage by scavenging reac-
tive intermediates, such as aryl aldehydes.11
The utility of caged species in living animals will be
dependent upon their hydrolytic stability in the dark. For
example, an o-nitrobenzyl-caged derivative of glutamic
acid displays a half-life of less than 20 h in the absence
of light.3 The corresponding coumarin-caged ester ana-
logue of glutamic acid suffers 50% hydrolysis within 2
days.3 These short half-lives seriously hamper the ap-
plication of these caged derivatives to living organisms.
By contrast, glutamic acid caged through a carbamate
linkage to the coumarin shows no detectable hydrolysis
after 35 h (pH 7.2 at room temperature).3 With these
features in mind, we examined the hydrolytic stability
of compounds 5-8 and 11 in two different systems: a
1:1 mixture of CH3OH/H2O and a 1:1 mixture of CH3-
OH/buffer (0.01 M HEPES, 0.12 M NaCl, 3 mM KCl, 1
mM CaCl2, 1 mM MgCl2, pH 7.4). The methanolic
component of these solvent systems was required to
solubilize the various acetal derivatives. In addition, the
HEPES buffer system has been previously employed by
others to mimic the intracellular environment.4 We were
unable to observe any hydrolysis of the 1,3-dioxolane and
1,3-dioxepane ring systems to the corresponding free diols
after 2 weeks.
In summary, we have developed a strategy to cage diols
using a photolabile protecting group. Surprisingly, al-
though coumarin-caged 1,2- and 1,4-diols are rapidly
photoliberated, the corresponding 1,3-derivatives appear
to be completely resistant to photolysis. We note that
photodeprotection of the coumain-acetals directly affords
the free diol. This is in marked contrast to o-nitroben-
zylidene acetals which, upon photodecomposition, yields
hydroxy monobenzoates.12 The strategy outlined herein
should prove applicable to a wide variety of diol-
substituted biologically active compounds, including spe-
cies containing the ribose ring system, various steroids,
and glycerol derivatives. Previous studies have demon-
strated that the 7-hydroxycoumarin moiety 1 possesses
a biologically useful cross-section for two photon pho-
tolysis.3 Consequently, it may be possible to photoliberate
the coumarin-caged diols described herein via multipho-
ton photolysis as well. These studies are currently in
progress.
six-membered acetal rings, it is surprising that the 1,3-
dioxane ring displays properties so dramatically different
from its 1,3-dioxolane counterpart.
We also examined the photouncaging properties of the
1,3-dioxepane ring contained in 11. This species, like the
1,3-dioxolanes, undergoes substantial uncaging (∼65%)
within 10 min of irradiation. The φ (0.005) associated
with the conversion of 11 to the free diol is essentially
identical to those displayed by the 1,3-dioxolanes 5-7.
We note that the 10 min irradiation time required for
the in vitro uncaging of the caged diols should be reduced
to <10 s inside a cell. For example, we have found that
a caged fluorescein dextran is uncaged >120 times faster
in vivo than in vitro (data not shown). This is due to a
dramatically enhanced laser-driven photon flux through
a small cell volume (∼2 pL).9
Although the mechanism of light-mediated coumarin
acetal uncaging remains to be determined, a possible
scenario is outlined in Scheme 1. Excitation of the
coumarin chromophore in 12 should generate the in-
tramolecular ion pair contained within the intermediate
13. By comparison, an intermolecular ion pair serves as
an early intermediate in the photodecomposition of
coumarin caged carboxylates, carbamates, and phos-
phates (1a -c). In the latter instances, the positive charge
produced following photoexcitation is dispersed into the
aromatic ring system. Apparently, the leaving group
potential of simple aliphatic alcohols (e.g., 2) is not
sufficient to generate the courmain-stabilized cationic
intermediate. By contrast, the adjacent ether moiety of
the acetal in 12 may serve as an electron-rich trigger that
can assist the departure of the alkoxide and simulta-
neously further stabilize the subsequently formed elec-
tron deficient center (13). The intermediate 13, when
trapped by solvent (H2O), should furnish the hemiacetal
14, which can then decompose to provide the liberated
diol and the aldehyde 4. However, other mechanistic
scenarios are also possible. For example, the triphen-
ylpyrylium tetrafluoroborate sensitized photolysis of ac-
etals may proceed via a radical cation intermediate.10
Consequently, further work will be required to elucidate
the mechanistic details of the photoliberation of cou-
marin-caged diols.
Exp er im en ta l Section
Gen er a l Meth od s. Analytical HPLC (monitored at 250 or
350 nm) employed Vydac C4 (250 mm × 3.0 mm) or Econosil SI
(250 mm × 4.6 mm) columns: a linear gradient from 70% A
(water) 30% B (methanol) to 0% A (water) 100% B (methanol)
over 20 min with flow rate at 1 mL/min.
Syn th esis of 6-Br om o-7-h yd r oxy-4-for m ylcou m a r in (4).
To a solution of 6-bromo-7-hydroxy-4-hydroxymethylcoumarin2
(2.5 g, 9.2 mmol) in anhydrous THF (30 mL) under an Ar
We note that the aldehyde 4 is a potent electrophile
that could react with and therefore damage a variety of
physiologically important biomolecules. Indeed, in gen-
eral, photoliberation of caged species (e.g., o-nitrobenzyl
(11) (a) Kaplan, J . H. Biochemistry 1978, 17, 1929-1935. (b) Walker,
J . W. Methods Enzymol. 1988, 172, 288-301.
(12) Collins, P. M.; Munasinghe, V. R. N. J . Chem. Soc., Perkin
Trans. 1 1983, 921-926.
(9) Mitchison, T. J . J . Cell Biol. 1989, 109, 637-652.
(10) Garcia, H.; Iborra, S.; Miranda, M.; Primo, J . New J . Chem.
1989, 13, 805-806.

2726 J . Org. Chem., Vol. 67, No. 8, 2002
Notes
atmosphere was added manganese oxide (3.5 g, 40.2 mmol). After
the suspension was stirred at room temperature overnight, the
black solid was removed by filtration. The filtrate was evapo-
rated under reduced pressure, and the resulting crude product
was purified by flash chromatography on silica gel (CH2Cl2/CH3-
OH ) 18:1) to afford a yellow solid (2.2 g, 90% yield).
Gen er a l P r oced u r e for th e Syn th esis of 6-Br om o-7-
h yd r oxycou m a r in Aceta ls 5-11. Compound 4 (30 mg, 0.115
mmol) was dissolved in anhydrous toluene (5 mL) under an Ar
atmosphere. After addition of a diol (1.3 equiv), pyridinium
p-toluenesulfonate (1 equiv), and anhydrous MgSO4 (100 mg),
the reaction mixture was stirred at 110 °C overnight. The
reaction was then diluted with EtOAc and washed with brine.
The organic layer was separated and dried over MgSO4. After
evaporation of the solvent under reduced pressure, the crude
product was purified by flash chromatography on silica gel (CH2-
Cl2/CH3OH ) 30:1).
Gen er a l P r oced u r e for P h otolysis of 6-Br om o-7-h y-
d r oxycou m a r in Aceta ls 5-11 a n d Qu a n tu m Yield Deter -
m in a tion s. A 180 µL 1 mM solution of 6-bromo-7-hydroxycou-
marin acetal in 1:1 CH3OH/HEPES (0.01 M HEPES, 0.12 M
NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4) was placed
in a quartz cuvette in ice. Photolysis was performed using an
Oriel 200 W Hg arc lamp (model 6283) with a 348 nm UV filter
(Oriel, lot no. 51260; 50% internal transmittance at 348 nm and
cutoff at <325 nm) to remove short wavelength light and an IR
filter (to remove heat). The spectral irradiance (at 0.5 m) of the
lamp in the region of the coumarin λmax ((50 nm) varies from
50 to 200 mW m2 nm-1. Aliquots of 10 µL were removed and
analyzed by analytical HPLC. The photochemical quantum
yields, φ, defined as the ratio of photolabile molecules converted
to the amount of photons absorbed, were determined using 6
mM potassium ferrioxate actionmetry according to the equation
φ ) ∆P/(It),7 where t is the irradiation time. ∆P, the amount of
photolabile converted, was determined using HPLC peak areas.
The value of I, the light intensity, was measured by using
chemical actionmetry.
Ha lf-Life Deter m in a tion for 6-Br om o-7-h yd r oxycou -
m a r in Aceta ls. One millimolar solutions of 6-bromo-7-hydroxy-
coumarin acetals in 1:1 CH3OH/HEPES (0.01 M HEPES, 0.12
M NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4)
(condition A) and in 1:1 CH3OH/H2O (deionized) (condition B)
were placed in the dark (wrapped with aluminum foil) at room
temperature. Aliquots of 10 µL were taken at various time
intervals for HPLC analysis.
Ack n ow led gm en t. Funding was provided by the
National Institutes of Health.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This material is available free of charge via the Internet
at http://pubs.acs.org.
J O0163851