8-Bromo-7-hydroxyquinoline as a photoremovable protecting group for physiological use: Mechanism and scope

Article abstract of DOI:10.1021/ja0555320

Two-photon excitation (2PE) of "caged" biomolecules represents a powerful method to investigate the temporal and spatial relevance of physiological function in real time and on living tissue, because the excitation volume can be restricted to 1 fL. Additi

Full text of DOI:10.1021/ja0555320

Published on Web 03/15/2006
8-Bromo-7-hydroxyquinoline as a Photoremovable Protecting
Group for Physiological Use: Mechanism and Scope
Yue Zhu,† Christopher M. Pavlos,‡ John P. Toscano,‡ and Timothy M. Dore*,†
Contribution from the Departments of Chemistry, UniVersity of Georgia,
Athens, Georgia 30602-2556, and Johns Hopkins UniVersity, Baltimore, Maryland 21218-2685.
Received August 12, 2005; E-mail: tdore@chem.uga.edu
Abstract: Two-photon excitation (2PE) of “caged” biomolecules represents a powerful method to investigate
the temporal and spatial relevance of physiological function in real time and on living tissue, because the
excitation volume can be restricted to 1 fL. Additionally, low-energy IR light is used, which minimizes tissue
destruction and enables deeper penetration into tissue preparations. Exploitation of this technology for
studying cell physiology requires the further development of photoremovable protecting groups with sufficient
sensitivity to 2PE for use in “caged” compounds. 8-Bromo-7-hydroxyquinoline (BHQ) is efficiently photolyzed
by classic 1PE (365 nm) and 2PE (740 nm) under simulated physiological conditions (aqueous buffer of
high ionic strength, pH 7.2) to release carboxylates, phosphates, and diolssfunctional groups commonly
found on bioactive molecules such as neurotransmitters, nucleic acids, and drugs. It is stable in the dark,
soluble in water, and exhibits low levels of fluorescence, which will enable use in conjunction with fluorescent
indicators of biological function. BHQ-protected effectors are synthetically accessible. Stern-Volmer
quenching, time-resolved infrared (TRIR), and 18O-labeling experiments suggest that the photolysis occurs
through a solvent-assisted photoheterolysis (SN1) reaction mechanism on the sub-microsecond time scale.
BHQ has the requisite photochemical and photophysical properties as a photoremovable protecting group
to regulate the action of biological effectors in cell and tissue culture with light, especially 2PE.
Introduction
conjugates used to study biological systems are often referred
to as “caged” compounds,6-11 because the activity of the effector
Understanding the temporal and spatial relevance of physi-
ological processes requires a set of probes that selectively act
at specific times and locations within a biological preparation.
The nonlinear optical process of two-photon excitation (2PE)1,2
enables the excitation volume of a chromophore to be confined
to 1 fL, about the volume of an Escherichia coli bacterium.
This volume is significantly smaller than the size of a typical
mammalian cell or neuron; therefore, excitation in subregions
of a cell in a complex tissue sample is conceivably possible.
Conventional one-photon excitation (1PE) using UV light
enables tight control over the timing of the excitation of a
chromophore, but 2PE makes three-dimensional spatial control
of the excitation also possible. In addition, 2PE is a particularly
noninvasive technique, because it uses IR wavelengths, which
minimizes tissue destruction, light absorption, and scattering,
facilitating much deeper penetration into complex tissue samples
than can be achieved with UV light. When a photoremovable
protecting group that is sensitive to 2PE is attached to a
biological effector, thereby inactivating it, one achieves control
over the timing and the three-dimensional space in which the
active form of the effector is released.3-5 Chromophore-effector
is “caged” by the connection of the chromophore with the
effector through a single covalent bond. Exposure to light causes
the covalent bond to break and “uncages” the effector in its
active form. Using 2PE to mediate the release of a biological
effector represents a powerful technique for probing the temporal
and location dependence of biological function in real time and
on living tissue.
To be useful in exploring a biological system, photoremovable
protecting groups for 2PE should meet the following design
criteria. The chromophore must possess a large absorption cross
section at wavelengths accessible with lasers employed in two-
photon microscopy, typically mode-locked and fs-pulsed Ti:
sapphire lasers. The photolysis reaction must proceed in high
quantum yield. The photoremovable protecting group must
render the biological effector inert to the biological system
used with minimal chemical or photochemical toxicity to living
cells. Any photoproducts other than the desired messenger
should not interact or interfere with the biological system. The
chromophore and the effector conjugate must be synthetically
accessible.
† University of Georgia.
(6) Adams, S. R.; Tsien, R. Y. Annu. ReV. Physiol. 1993, 55, 755-784.
(7) Marriott, G., Ed.; Caged Compounds; Academic Press: New York, 1998;
Vol. 291.
(8) Curley, K.; Lawrence, D. S. Pharmacol. Ther. 1999, 82, 347-354.
(9) Dorma´n, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64-77.
(10) Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125-142.
(11) Givens, R. S., Goeldner, M., Eds.; Dynamic Studies in Biology: Photo-
triggers, Photoswitches, and Caged Biomolecules; Wiley-VCH: Weinheim,
Germany, 2005.
‡ Johns Hopkins University.
(1) Go¨ppert, M. Naturwissenschaften 1929, 17, 932.
(2) Go¨ppert-Mayer, M. Ann. Phys. (Berlin) 1931, 9, 273-294.
(3) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73-76.
(4) Denk, W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6629-6633.
(5) Dore, T. M. In Dynamic Studies in Biology: Phototriggers, Photoswitches,
and Caged Biomolecules; Givens, R. S., Goeldner, M., Eds.; Wiley-VCH:
Weinheim, Germany, 2005; pp 435-459.
9
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J. AM. CHEM. SOC. 2006, 128, 4267-4276
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A R T I C L E S
Zhu et al.
Chart 1. Photoremovable Protecting Groups
Scheme 1. Synthesis of BHQ-Protected Carboxylatesa
Chromophores that can act as a photoremovable protecting
group (Chart 1) and have sufficient sensitivity to 2PE for
biological use are rare. The nitrobenzyl-based protecting groups
such as the dimethoxynitrobenzyl (DMNB or veratryl), carboxy-
nitrobenzyl (CNB), and the nitrophenylethyl (NPE) groups have
almost no sensitivity to 2PE at wavelength ranges typically
associated with two-photon microscopy, 730-980 nm.4,12,13 The
MNI-based protecting group has been used to release glutamate
in hippocampal brain slices,14,15 but the neuroscience community
has not used it widely probably because of its marginal
sensitivity to 2PE. Groups based on 8-bromo-7-hydroxycou-
marin (Bhc and Bhc-diol) have good sensitivity to 2PE, and
they have been used to cage neurotransmitters,5,12 DNA and
RNA,16 diols,17 alcohols,18 cyclic nucleotide monophosphates,19
ketones and aldehydes,20 an inhibitor of nitric oxide synthase,21,22
and an inhibitor of protein synthesis.23 Nevertheless, the
somewhat low solubility of Bhc in aqueous buffers of high ionic
strength and the high level of fluorescence observed upon
excitation might limit its use, especially in applications where
a fluorescent indicator is used to observe a physiological event.
We found that the 8-bromo-7-hydroxyquinolinyl group (BHQ)
can be efficiently photolyzed by 1PE and 2PE in aqueous buffer
at physiological pH and has the additional advantages of a large
quantum efficiency for the photolysis, good solubility in aqueous
buffers, and low levels of fluorescence.24 We report that BHQ
can release carboxylates, phosphates, and diols efficiently and
a Reagents and conditions: (a) BzCl, Et3N, DMAP, CH3Cl, rt, 12 h,
73%; (b) Br2, AcOH, rt, 24 h, 67%; (c) TBAF, THF, rt, 90%; (d)
piperonyloyl chloride, Et3N, DMAP, CH3Cl, rt, 12 h, 66%; (e) TBAF, THF,
rt, 87%; (f) Br2, AcOH, rt, 5 h, 64%.
rapidly using 1PE or 2PE. Through Stern-Volmer quenching,
time-resolved infrared (TRIR), and 18O-labeling experiments,
we demonstrate that the photochemical reaction proceeds
through a singlet excited state and that the release of the effector
likely proceeds through a solvent-assisted photoheterolysis (SN1)
reaction mechanism.25
Results
Synthesis. BHQ-protected benzoate and piperonylate were
prepared from tert-butyldiphenylsilyl-protected quinoline 124
(Scheme 1). Esterification of alcohol 1 with benzoyl chloride,
triethylamine, and 4-(dimethylamino)pyridine in chloroform pro-
vided benzoate 2 in 73% yield. Bromination of 2 with bromine
in acetic acid gave a single regioisomer in 67% yield, and
removal of the silyl protecting group with tetrabutylammonium
fluoride provided BHQ-protected benzoate 4 in 90% yield. The
1H NMR spectrum of 3 showed that the H-6 signal condensed
to a doublet (δ 7.12 ppm, J ) 8.8 Hz) from the doublet of
doublets (δ 7.10 ppm, J ) 8.8, 2.8 Hz) found in the spectrum
of 2. The disappearance of the smaller meta-coupling indicates
the addition of bromine at C-8, rather than at C-6, which would
generate a spectrum with two singlets for H-5 and H-8. The
regiochemistry of the bromination was further confirmed by the
1H NMR spectrum of BHQ-OBz (4) in which the quinolinyl
protons appear as four doublets, each with coupling constants
(J ) 8.8 Hz) indicative of having one neighboring proton in
the ortho position. No other substitution pattern is consistent
with this observation. BHQ-protected piperonylate was synthe-
sized similarly, except the tert-butyldiphenylsilyl protecting
(12) 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.
(13) Kiskin, N. I.; Chillingworth, R.; McCray, J. A.; Piston, D.; Ogden, D. Eur.
BioPhys. J. 2002, 30, 588-604.
(14) Matsuzaki, M.; Ellis-Davies, G. C. R.; Nemoto, T.; Miyashita, Y.; Iino,
M.; Kasai, H. Nat. Neurosci. 2001, 4, 1086-1092.
(15) Smith, M. A.; Ellis-Davies, G. C. R.; Magee, J. C. J. Physiol. (London)
2003, 548, 245-258.
(16) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet. 2001, 28,
317-325.
(17) Lin, W.; Lawrence, D. S. J. Org. Chem. 2002, 67, 2723-2726.
(18) Suzuki, A. Z.; Watanabe, T.; Kawamoto, M.; Nishiyama, K.; Yamashita,
H.; Ishii, M.; Iwamura, M.; Furuta, T. Org. Lett. 2003, 5, 4867-4870.
(19) Furuta, T.; Takeuchi, H.; Isozaki, M.; Takahashi, Y.; Kanehara, M.;
Sugimoto, M.; Watanabe, T.; Noguchi, K.; Dore, T. M.; Kurahashi, T.;
Iwamura, M.; Tsien, R. Y. ChemBioChem 2004, 5, 1119-1128.
(20) Lu, M.; Fedoryak, O. D.; Moister, B. R.; Dore, T. M. Org. Lett. 2003, 5,
2119-2122.
(21) Montgomery, H. J.; Perdicakis, B.; Fishlock, D.; Lajoie, G. A.; Jervis, E.;
Guillemette, J. G. Bioorg. Med. Chem. 2002, 10, 1919-1927.
(22) Perdicakis, B.; Montgomery, H. J.; Abbott, G. L.; Fishlock, D.; Lajoie, G.
A.; Guillemette, J. G.; Jervis, E. Bioorg. Med. Chem. 2005, 13, 47-57.
(23) Goard, M.; Aakalu, G.; Fedoryak, O. D.; Quinonez, C.; St. Julien, J.; Poteet,
S. J.; Schuman, E. M.; Dore, T. M. Chem. Biol. 2005, 12, 685-693.
(24) Fedoryak, O. D.; Dore, T. M. Org. Lett. 2002, 4, 3419-3422.
1
group was removed prior to bromination. H NMR confirmed
the regiochemistry of the bromination. The doublet of doublets
(25) Schade, B.; Hagen, V.; Schmidt, R.; Herbich, R.; Krause, E.; Eckardt, T.;
Bendig, J. J. Org. Chem. 1999, 64, 9109-9117.
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4268 J. AM. CHEM. SOC. VOL. 128, NO. 13, 2006

BHQ as a Photoremovable Protecting Group
A R T I C L E S
Scheme 2. Synthesis of BHQ-Protected Phosphatesa
Scheme 3. Synthesis of BHQ-Protected Diolsa
a Reagents and conditions: (a) PPTS, MgSO4, toluene, 110 °C, 48 h,
60% for 16, 55% for 17
Scheme 4. Single- and Two-Photon Photolyses of BHQ-Protected
Compoundsa
a Reagents and conditions: (a) Br2, AcOH, rt, 8 h, 71%; (b) Ac2O,
pyridine, DMAP, rt, 4 h, 83%; (c) SeO2, dioxane, 80 °C, 18 h, 60%; (d)
H2NNHTs, EtOH, rt, 48 h, 74%; (e) NaOMe, MeOH, rt, 10 min; (f) dimethyl
phosphate, CH3CN, rt, 48 h, 20%.
corresponding to H-6 (δ 7.19 ppm, J ) 8.8, 2.4 Hz) in the
spectrum of 6 gave way to a doublet (δ 7.36 ppm, J ) 8.4 Hz)
in the spectrum of 7, and the signal for H-8 (δ 7.29, d, J ) 2.0
Hz) in the spectrum of 6 was absent in the spectrum of 7.
BHQ-protected dimethyl phosphate 13 was synthesized in
six steps from quinaldine 824 (Scheme 2). Bromination of 8 with
bromine in acetic acid followed by acetylation of the phenol
with acetic anhydride in pyridine yielded bromoquinaldine 10.
a Reagents and conditions: (a) hν (365 nm), KMOPS, pH 7.2; (b) hν
(740 nm), KMOPS, pH 7.2
1
The regiochemistry of the bromination was determined by H
NMR. The peak corresponding to H-8 in 8 (δ 7.33 ppm, d, J )
1.9 Hz) was not present in the spectrum of 9, and the H-6 signal
in the spectrum of 8 collapsed from a double of doublets (δ
7.08 ppm, J ) 8.8, 1.9 Hz) to a doublet in 9 (δ 7.22 ppm, J )
8.0 Hz). Oxidation of the benzylic methyl group with selenium
dioxide generated aldehyde 11, which upon treatment with
tosylhydrazine in ethanol eventually provided the corresponding
tosylhydrazone 12 in moderate yields. Generation of BHQ-
protected phosphate was accomplished by treating 12 with
sodium methoxide to generate the diazo species, which was
trapped by dimethyl phosphate to produce the target compound
13, albeit in poor yield.
chloride titrated to pH 7.2 with potassium hydroxide) were
excited with light from a 365-nm mercury lamp passed through
a pair of filters that narrow the spectral output to 365 ( 15 nm.
The pKa of the phenolic moiety in BHQ-OAc is 6.8, and λmax
for the phenolate is ∼370 nm, compared to ∼320 for the
phenol;24 therefore, in KMOPS buffer, the excited state likely
arises from the deprotonated form of the hydroxyquinoline. UV
spectra indicated that the solutions contained a mixture of the
phenol and phenolate forms of the hydroxyquinoline. Neverthe-
less, excited-state hydroxyquinolines are superacids, and the
hydroxy group is expected to deprotonate rapidly upon excita-
tion.28,29
The BHQ-protected glycerol derivatives 16 and 17 were
synthesized in a single step from aldehyde 11 and 1-phenoxy-
propane-2,3-diol (14)26 and 1-phenthioxypropane-2,3-diol (15)27
by heating the aldehyde with the diol with pyridinium para-
toluenesulfonic acid and anhydrous magnesium sulfate in toluene
(Scheme 3). The yields for these reactions were modest (55-
60%).
The time courses for the one-photon photolysis reactions of
18a-c were monitored by HPLC analysis of 20-µL aliquots
taken at periodic intervals, using an external standard to relate
the detector response to the concentrations of 18a-c and 20b
(Figure 1). In the case of BHQ-OPip (18b), the product of the
reaction, piperonylate (20b), had sufficient UV signal to monitor
its formation in the reaction. The yield of 20b approaches 70-
75%. Similarly, HPLC was used to monitor the photolyses of
BHQ-caged diols 21d,e and the generation of 23e (Figure 2).
Comparison with the HPLC retention times under identical
conditions of authentic samples of 19, 22, 20b, and 23e
confirmed their identity in the reaction mixture. From these data,
the one-photon uncaging quantum efficiency, Qu, for each
Photochemistry. We evaluated the BHQ-protected com-
pounds for their ability to undergo one- and two-photon
photolysis under simulated physiological conditions to generate
the caging remnant 19 (BHQ-OH) or 21 (BHQ-CHO) and the
respective carboxylate, phosphate, or diol (Scheme 4). Solutions
of 18a-c and 21d,e (100 µM) in KMOPS buffer (10 mM
4-morpholinepropanesulfonic acid and 100 mM potassium
(26) Egri, G.; Kolbert, A.; Balint, J.; Fogassy, E.; Novak, L.; Poppe, L.
Tetrahedron: Asymmetry 1998, 9, 271-283.
(27) Yale, H. L.; Pribyl, E. J.; Braker, W.; Bernstein, J.; Lott, W. A. J. Am.
Chem. Soc. 1950, 72, 3716-3718.
(28) Bardez, E. Isr. J. Chem. 1999, 39, 319-332.
(29) Bardez, E.; Fedorov, A.; Berberan-Santos, M. N.; Martinho, J. M. G. J.
Phys. Chem. A 1999, 103, 4131-4136.
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A R T I C L E S
Zhu et al.
Table 1. Photophysical and Photochemical Properties of
BHQ-Caged Compoundsa
λmax
(nm)
ꢀmax
1
ꢀ365
1
δu
(GM)b
τdark
(h)c
1
1
compd
(M-
‚
cm-
)
(M-
‚
cm-
)
Qu
BHQ-OAcd
18a
18b
18c
21d
369
368
296
370
375
374
2600
2400
6400
3900
2300
2500
2580
2400
2900
3800
2200
2400
0.29
0.30
0.32
0.31
0.37
0.39
0.59
0.64
0.76
0.43
0.78
0.90
71
100
94
105
79
21e
69
a Measured in KMOPS, pH 7.2. b Measured at 740 nm, GM ) 10-50
(cm4‚s)/photon. c Time constant for hydrolysis in the dark. d Values taken
from ref 24.
Figure 1. Time course of one-photon photolysis of BHQ-protected
carboxylates 18a,b and phosphate 18c at 365 nm. The percent remaining
was determined by HPLC and is the average of 3 runs. Also shown is the
relative amount of piperonylate (20b) released from 18b, which was
determined by HPLC from 3 runs. Lines are least-squares fits of a simple
decaying exponential or exponential rise to max. Error bars represent the
standard deviation of the measurement.
Figure 3. Time course of two-photon photolysis of BHQ-protected
carboxylates 18a,b and phosphate 18c at 365 nm. Also shown is the relative
amount of piperonylate (20b) released from 18b. The percent remaining
was determined by HPLC and is the average of 3 runs. Lines are least-
squares fits of a simple decaying exponential or exponential rise to max.
Error bars represent the standard deviation of the measurement.
We followed a previously described method12,20,24 to deter-
mine the two-photon uncaging cross section, δu (in Goeppert-
Mayer, GM, which is defined as 10-50 cm4‚s/photon), of
compounds 18a-c. This is a measure of the sensitivity of a
photoremovable protecting group to undergo a photochemical
reaction initiated by 2PE. It is related to the two-photon
absorption cross-section, δa, by δu ) Qu‚δa. Briefly, 20-µL
samples were irradiated with a fs-pulsed, mode-locked Ti:
sapphire laser tuned to 740 nm (287 fs pulse width and 160-
200 mW average power). After a period, HPLC analysis of the
entire sample was used to determine the extent of photolysis,
as in the one-photon quantum efficiency measurements, and,
in the case of 18b, the yield of piperonylate (20b), which
approached 60%. The runs were graphed as a function of time
and fit to a least-squares decaying exponential (Figure 3). The
pulse parameters of the laser were estimated by using fluorescein
as an external standard, because it has a known fluorescence
quantum yield, QfF, and two-photon absorbance cross-section,
δaF.33,34 The time-averaged fluorescence photon flux, F(t) , from
the two-photon excitation of fluorescein in the same setup was
measured using a radiometer. The initial rate of photolysis was
used to determine the number of molecules photolyzed/s, Np,
which is related to δu by the following equation:12
Figure 2. Time course of one-photon photolysis of BHQ-protected diols
21d,e. The percent remaining was determined by HPLC and is the average
of at least 3 runs. Also shown is the relative amount of 1-phenthioxypropane-
2,3-diol (23e) released from 21e, which was determined by HPLC from at
least 3 runs. Lines are least-squares fits of a simple decaying exponential
or exponential rise to max. Error bars represent the standard deviation of
the measurement.
compound was determined from the following relationship:30,31
-1
Qu ) (Iσt90%
)
(1)
Here I is the irradiation intensity (determined by potassium
ferrioxalate actinometry32), σ is the decadic extinction coefficient
(1000ꢀ, the molar extinction coefficient) at 365 nm, and t90% is
the irradiation time for 90% conversion to product. The quantum
efficiencies for the BHQ-protected carboxylates and phosphates
were found to be 0.30-0.32, while the protected diols had
slightly higher values of 0.37 and 0.39 (Table 1).
(30) Adams, S. R.; Kao, J. P. Y.; Grynkiewicz, G.; Minta, A.; Tsien, R. Y. J.
Am. Chem. Soc. 1988, 110, 3212-3220.
(31) Livingston, R. In Photochromism; Brown, G. H., Ed.; Wiley: New York,
1971; pp 13-44.
NpφQfFδaFCF
δu )
(2)
(32) Hatchard, C. G.; Parker, C. A. Proc. R. Acad. London, Ser. A 1956, 235,
518-536.
<F(t)>CS
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BHQ as a Photoremovable Protecting Group
A R T I C L E S
Figure 5. TRIR difference spectra averaged over the time scales indicated
following laser photolysis (266 nm, 5 ns, 1 mJ) of BHQ-OAc (5 mM) in
argon-saturated acetonitrile-d3. The vertical bars represent B3LYP/6-31G*
calculated frequencies (scaled by 0.96)33 and relative intensities of the lowest
triplet excited state of BHQ-OAc.
Figure 4. Time course of two-photon photolysis of BHQ-protected diols
21d,e. Also shown is the relative amount of phenthioglycerol 23e released
from 21e. The percent remaining was determined by HPLC and is the
average of 3 runs. Lines are least-squares fits of a simple decaying
exponential or exponential rise to max. Error bars represent the standard
deviation of the measurement.
Table 2. Stern-Volmer Quenching of BHQ-OAc
[SNS]a
[BHQ-OAc]
M)
[PS]b
[BHQ-OAc]
M)
(µM)
(
µ
Qu
(µM)
(
µ
Qu
0
100
100
100
100
0.29
0.30
0.31
0.29
0
180
180
180
180
0.29
0.30
0.30
0.30
100
200
300
180
360
720
a SNS ) sodium 2-naphthalene sulfonate. b PS ) potassium sorbate.
Figure 6. Representative kinetic traces observed following laser photolysis
(266 nm, 5 ns, 1 mJ) of BHQ-OAc (5 mM) in argon-saturated acetonitrile-
d3 showing decay of the BHQ-OAc triplet excited state at 1532 cm-1 and
recovery of the ground-state BHQ-OAc band at 1756 cm-1. The dotted
curves are experimental data; the solid curves are the calculated best fit to
a single-exponential function.
Here φ is the collection efficiency of the detector, QfF is the
fluorescence quantum yield of fluorescein, δaF is the absorbance
cross section of fluorescein, CF is the concentration of fluores-
cein, and CS is the concentration of the BHQ-protected substrate.
Similarly, the time courses for the BHQ-protected diols 21d,e
were measured (Figure 4) and δu was calculated. Values of δu
for 18a-c and 21d,e ranged from 0.43 to 0.90 GM (Table 1).
Dark Hydrolysis Rates. Each of the BHQ-protected com-
pounds was tested for its stability in the dark at room
temperature in KMOPS. Compounds 18a-c and 21d,e were
placed in KMOPS and stored in the dark. Periodically, 20-µL
aliquots were removed to measure the extent of spontaneous
hydrolysis by HPLC (data not shown). The time constants
measured ranged from 69 to 105 h (Table 1).
Stern-Volmer Quenching Experiments. To determine
whether the reaction proceeds through a triplet or singlet excited
state, we conducted Stern-Volmer quenching experiments on
BHQ-OAc (23). The quantum efficiency, Qu, of the one-photon
photolysis of BHQ-OAc was determined in the presence of 1,
2, and 3 times the concentration of a triplet quencher, sodium
2-naphthalene sulfonate (SNS) or potassium sorbate (PS). No
statistically relevant change in Qu was observed with either
quencher (Table 2).
depletion bands match well with the ground-state IR spectrum
of BHQ-OAc. These depletion bands recover (albeit incom-
pletely) with an observed rate constant of 1.8 × 106 s-1 (Figure
6). The incomplete recovery of these bands indicates some
irreversible conversion (ca. 20%) of BHQ-OAc to products. Two
sets of partially overlapping positive bands are observed. One
set, with major signals at 1736, 1574, 1532, and 1412 cm-1, is
produced within the time resolution (50 ns) of the TRIR
experiment and subsequently decays at the same rate within
experimental error ((10%) that the BHQ-OAc ground-state
bands recover (Figure 6). A second set, with signals at 1764,
1644, 1584, 1540, 1484, 1412, 1360, and 1316 cm-1, is also
produced within the time resolution of the TRIR experiment
but appear to be stable on the microsecond time scale.
We have assigned the first set of bands to the triplet excited
state of BHQ-OAc since they agree well with B3LYP/6-31G*
calculated frequencies (Figure 5 and Supporting Information)
and are quenched by oxygen (Figure 7). Although oxygen
efficiently quenches the BHQ-OAc triplet excited state, TRIR
spectral data collected in oxygen-saturated acetonitrile (Figure
7) indicate that the stable product(s) (e.g., observed bands at
1764 and 1644 cm-1) is (are) still formed in the presence of
oxygen. This latter observation is consistent with our hypothesis
that productive chemistry occurs from a BHQ-OAc singlet
Time-Resolved IR Studies. We further analyzed the pho-
tolysis of BHQ-OAc by time-resolved infrared (TRIR) spec-
troscopy. TRIR spectra observed following 266 nm laser
photolysis (5 ns, 1 mJ) of BHQ-OAc in argon-saturated
acetonitrile-d3 are shown in Figure 5. Depletion of reactant gives
rise to negative signals, and the formation of transient inter-
mediates or products leads to positive bands. The observed
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A R T I C L E S
Zhu et al.
Figure 7. TRIR difference spectra averaged over the time scales indicated
following laser photolysis (266 nm, 5 ns, 1 mJ) of BHQ-OAc (5 mM) in
(a) argon- and (b) oxygen-saturated acetonitrile.
excited state; the BHQ-OAc triplet excited state merely decays
back to ground state. The set of stable product bands are
assigned to BHQ-OH (presumably formed via trapping by
residual water) and acetic acid on the basis of a comparison
with the known IR spectra of these products (see Supporting
Information).
The kinetics of BHQ-OAc ground-state recovery at 1756
cm-1 were further examined following triplet sensitized pho-
tolysis with xanthone. The triplet excited state of xanthone has
a broad, weak absorbance between 1800 and 1700 cm-1. Thus,
in the absence of BHQ-OAc, a positive transient signal,
attributed to the xanthone triplet excited state, is observed at
1756 cm-1 following 355 nm laser photolysis (90 ns, 1.5 mJ)
of an acetonitrile solution of xanthone (Figure 8a). However,
in the presence of 1 mM BHQ-OAc, the xanthone triplet excited
state is efficiently quenched and the decay and recovery of the
BHQ-OAc 1756 cm-1 is now observed (Figure 8b). Note that,
in contrast to direct photolysis (Figure 8c), triplet-sensitized
photolysis of BHQ-OAc results in complete recovery of the
ground-state depletion, further verifying that products are not
formed via the BHQ-OAc triplet excited state.
BHQ-phosphate (18c) was also examined by TRIR spectros-
copy. TRIR spectra observed following 266 nm laser photolysis
(5 ns, 1 mJ) of BHQ-phosphate in acetonitrile-d3 are shown in
Figure 9. Analogous to BHQ-OAc, we observe the decay and
recovery of ground-state BHQ-phosphate, the corresponding
formation and decay of BHQ-phosphate triplet excited state,
and the formation of stable product bands. Experimentally
observed IR bands of the BHQ-phosphate triplet excited state
are well reproduced by B3LYP/6-31G* calculations (Figure 9)
and are also quenched by oxygen (Supporting Information).
Similar to the case with BHQ-OAc, ground-state bleaching does
not fully recover to baseline, indicating some finite depletion
of reactant. Although kinetic data indicate that the triplet excited-
Figure 8. Kinetic traces observed following laser photolysis (355 nm, 90
ns, 1.5 mJ) of an argon-saturated acetonitrile solution of (a) xanthone (A355
) 0.25) and of (b) xanthone (A355 ) 0.25) plus 1 mM BHQ-OAc. (Under
these conditions >96% of the incident light is absorbed by xanthone.) Also
shown for comparison is (c) the kinetic trace observed following laser
photolysis (266 nm, 5 ns, 1 mJ) of an argon-saturated acetonitrile solution
of BHQ-OAc (5 mM). The dotted curves are experimental data; the solid
curves are the calculated best fit of a single-exponential function.
state lifetimes of BHQ-OAc and BHQ-phosphate are compa-
rable, in the case of BHQ-phosphate no signals were observed
(or calculated) in the region between 1800 and 1600 cm-1. The
signals observed in this region for BHQ-OAc (Figure 5) have
been assigned to the acetate portion of the molecule.
Oxygen-18 Labeling Experiment. In the photolysis of BHQ-
caged carboxylates, the oxygen on the primary alcohol of BHQ-
OH can come from the aqueous solvent or from the ester
(Scheme 5). To determine the origin of this oxygen, the
photolysis was conducted in 18O-labeled water. If the oxygen
on the remnant of the BHQ-chromophore comes from the
9
4272 J. AM. CHEM. SOC. VOL. 128, NO. 13, 2006

BHQ as a Photoremovable Protecting Group
A R T I C L E S
Scheme 6. Proposed Mechanism of the BHQ Photolysis Reaction
Figure 9. TRIR difference spectra averaged over the time scales indicated
following laser photolysis (266 nm, 5 ns, 1 mJ) of BHQ-phosphate (5 mM)
in argon-saturated acetonitrile. The vertical bars represent B3LYP/6-31G*
calculated frequencies (scaled by 0.96) and relative intensities for the lowest
triplet excited state of BHQ-phosphate.
Scheme 5. Oxygen-18 Labeling Experiment
respectively, but at wavelengths below 270 nm, which are
deleterious to biological preparations. The two-photon uncaging
cross section is greater than the 0.1 GM threshold for physi-
ological use,12 and further, it is an order of magnitude larger
than the measured values for DMNB, MNI, CNB, and NPE,
which have been used in a physiological context. The photolysis
kinetics is faster than physiological signal transduction events,
which occur on millisecond and slower time scales. Because
the kinetics were measured in acetonitrile and not aqueous buffer
of high ionic strength at neutral pH, the estimated rate constant
for product formation (g2 × 107 s-1 in acetonitrile) is likely
larger in a physiological environment. In conducting flash
photolysis experiments on Bhc-OAc, which photochemically
decomposes by a mechanism25 similar to BHQ, Magde found
that the photolysis rate constant is proportional to pH.5 BHQ-
caged carboxylates and phosphates will enable tight three-
dimensional localization of effector release with 2PE, because
the photolysis time scale is much shorter (τ ) 0.6 µs) than those
typical for diffusion through aqueous media (τ ) 113-900 µs).39
The photochemical reaction is complete and the cargo released
before any intermediates can diffuse away from the focus of
the laser. Photolysis rates of BHQ-caged diols are likely to be
slower than those for the carboxylates and phosphates.
The Stern-Volmer quenching experiments indicated that a
triplet excited state is not involved in product formation, because
the two triplet quenchers used were unable to diminish the
quantum efficiency of the photolysis of BHQ-OAc. TRIR
spectroscopy shows that recovery of the ground-state starting
material is incomplete, as would be expected for a photochemi-
cal reaction where a portion of the excited state provides product
rather than decaying back to the ground state. In the presence
of oxygen as a triplet quencher, stable products, BHQ-OH and
acetate, are still observed, also indicating that the reaction
proceeds through a singlet excited state. If the mechanistic path
to products involved a triplet state, no product bands would be
observed in the TRIR spectra in the presence of a triplet
quencher. Triplet-sensitized excitation of BHQ with xanthone
results in complete recovery of the original ground state, and
solvent, then the product observed will be the labeled BHQ-
18OH (24). Alternatively, if unlabeled BHQ-OH (25) is observed,
then the source of the oxygen would be from the ester. A
solution of BHQ-OAc (18) in either H2O or H218O (97% label)
was exposed to 365-nm light for several seconds. The reactions
were then analyzed by LC-MS (Supporting Information). For
the reaction in H2O, mass spectral analysis of the peak on the
chromatogram corresponding to BHQ-OH gave m/z 254/256,
while the reaction in H218O gave almost exclusively m/z 256/
258. A comparison of the MS data of the labeled and unlabeled
reaction revealed that 96% of the label was incorporated into
the chromophore remnant.
Discussion
The BHQ chromophore can be covalently linked to carbox-
ylates, phosphates, and diols, three common functional groups
found on bioactive molecules such as neurotransmitters, nucleic
acids, and drugs. It is efficiently removed, releasing the active
functional group, through 1PE or 2PE at wavelengths that are
not deleterious to biological preparations, and all of the
compounds tested are sufficiently stable in the dark at pH 7.2.
Compared to other chromophores for protecting carboxylates,
the sensitivity, as measured by the product of the quantum
efficiency and the molar absorptivity (Quꢀ),19 of BHQ (780) to
1PE is higher than DMNB (31),24 Bhc (555),12 and MNI (408).36
CNB37 and NPE38 have high sensitivities, 714 and 2184,
(33) Xu, C.; Guild, J.; Webb, W. W.; Denk, W. Opt. Lett. 1995, 20, 2372-
2374.
(34) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481-491.
(35) Scott, A. P.; Radom, L. J. Phys. Chem. 1996, 100, 16502-16513.
(36) Papageorgiou, G.; Corrie, J. E. T. Tetrahedron 2000, 56, 8197-8205.
(37) Wieboldt, R.; Gee, K. R.; Niu, L.; Ramesh, D.; Carpenter, B. K.; Hess, G.
P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8752-8756.
(38) Kaplan, J. H.; Forbush, B., III; Hoffman, J. F. Biochemistry 1978, 17, 1929-
1935.
(39) Kiskin, N. I.; Ogden, D. Eur. BioPhys. J. 2002, 30, 571-587.
9
J. AM. CHEM. SOC. VOL. 128, NO. 13, 2006 4273

A R T I C L E S
Zhu et al.
system with an autosampler and diode array detector using Microsorb
C-18 reverse phase columns. Mass spectrometry was performed on a
Sciex API-1 Plus quadrupole mass spectrometer with an electrospray
ionization (ESI) source or a Bruker Autoflex MALDI-TOF. HRMS
was performed on a Micromass QTOF-Ultima with ESI. KMOPS buffer
consisted of 100 mM KCl and 10 mM MOPS titrated to pH 7.2 with
KOH. Thin layer and column chromatography were performed on
precoated silica gel 60 F254 plates (Sorbent Technologies) and 230-
400 mesh silica gel 60 (Sorbent Technologies), respectively. Melting
points were determined on a Mel-Temp (Laboratory Devices, Inc.) and
are uncorrected.
no product bands are observed, consistent with our hypothesis
that the triplet state is not involved in product formation.
Intersystem crossing to form the triplet state is probably
facilitated by the presence of the bromine in BHQ, but its
presence is required to lower the pKa of the phenolic proton
and induce a shift of the absorbance maximum from ap-
proximately 320 to 370 nm. Improvements to the quantum
efficiency could potentially be made by replacing the bromine
with an electron-withdrawing group that does not facilitate
intersystem crossing but would still lower the pKa of the phenol.
Conducting the photoreaction in 18O-labeled water reveals that
the reaction is not a simple hydrolysis of an ester; the label is
incorporated into the BHQ-OH product and not the acetate. This
supports a mechanism involving a substitution reaction at the
benzylic carbon center.
Taken together, the physical data on the photolysis of BHQ-
OAc and BHQ-phosphate suggest a solvent-assisted photo-
heterolysis (SN1) reaction mechanism25 (Scheme 6). Irradiation
of BHQ-OR (24) in aqueous media at pH 7.2 leads to a singlet
excited state 25, which can undergo intersystem crossing to the
triplet state 26 followed by decay back to 24. Cleavage of the
carbon-oxygen bond in 25 proceeds from the singlet state,
either by heterolytic cleavage or homolytic cleavage followed
by single electron transfer, to generate a zwitterion-like inter-
mediate resembling 27. Trapping the cation or electropositive
carbon by water yields 28 and the carboxylate or phosphate. In
the excited state, hydroxyquinolines are protonated on the
picosecond time scale even in 9-10 M NaOH;29 therefore, a
transient intermediate such as 29 is probably formed during the
photoreaction, but its role on the path to product is unclear.
TBDPS-HQ-Benzoate (2). Under a nitrogen atmosphere, alcohol
1 (100 mg, 0.242 mmol) was dissolved in CHCl3 (10 mL) and Et3N
(45 µL, 0.36 mmol) and DMAP (10 mg, 0.08 mmol) were added.
Benzoyl chloride (30 µL, 0.26 mmol) was added dropwise. The reaction
mixture was stirred overnight. The mixture was washed with 15% citric
acid, water, and brine. The organic layer was dried over anhydrous
Na2SO4. The solvent was evaporated and the residue was purified by
flash chromatography with EtOAc/hexane (1:9) to afford 2 as a white
solid (92 mg, 0.178 mmol, 73% yield), mp ) 85-89 °C: 1H NMR
(CDCl3) δ 8.11 (2H, dm, J ) 8.4 Hz), 8.04 (1H, d, J ) 8.4 Hz), 7.76
(4H, m), 7.57 (2H, d, J ) 8.8 Hz), 7.41 (10H, m), 7.10 (1H, dd, J )
8.8-2.4 Hz), 5.55 (2H, s), 1.13 (9H, s); 13C NMR (CDCl3) δ 166.27,
156.97, 156.52, 149.02, 136.59, 135.53, 134.83, 133.22, 132.36, 130.07,
129.85, 128.46, 127.91, 127.74, 123.06, 122.59, 117.28, 116.25, 67.91,
22.68, 14.16; FTIR (neat) 3070, 2932, 2857, 2360, 1685, 1583, 1425,
1289, 1113, 933, 809, 702 cm-1; HR-MS (ESI) m/z calcd for
(C33H31NO3Si + H)+ 518.2146, found 518.2130.
TBDPS-BHQ-Benzoate (3). Ester 2 (80.0 mg, 0.155 mmol) was
dissolved in acetic acid (5 mL). Bromine (140 mg, 20% in acetic acid
solution, 0.175 mmol) was added dropwise. The reaction mixture was
stirred for 24 h. The reaction was quenched with saturated NaHCO3,
extracted with EtOAc, washed with water and brine, and dried over
anhydrous Na2SO4. The organic layer was evaporated, and the residue
was purified by flash chromatography with EtOAc/hexane (1:9) to
afford 3 as a white solid (62 mg, 0.104 mmol, 67% yield), mp ) 80-
84 °C: 1H NMR (CDCl3) δ 8.64 (1H, d, J ) 8.8 Hz), 8.06 (2H, dm,
J ) 8.4 Hz), 7.56 (6H, m), 7.41 (9H, m), 7.11 (1H, d, J ) 8.8 Hz),
6.18 (2H, s), 1.14 (9H, s); 13C NMR (CDCl3) δ 166.15, 158.91, 157.64,
147.13, 136.99, 135.43, 134.88, 132.32, 131.63, 130.17, 129.58, 128.91,
127.85, 127.06, 123.94, 122.08, 118.18, 114.05, 69.17, 22.54, 14.16;
FTIR (neat) 3071, 2955, 2858, 1723, 1618, 1506, 1427, 1257, 1109,
862, 700 cm-1; MS (MALDI-TOF) m/z calcd for (C33H30BrNO3Si +
H)+ 596.1 (79Br) and 598.1 (81Br), found 596.2 (79Br) and 598.2 (81Br).
BHQ-Benzoate (4). Ester 3 (50 mg, 0.084 mmol) was dissolved in
THF (5 mL). TBAF in THF (1 M, 90 µL, 0.09 mmol) was added
dropwise. The reaction was monitored by TLC. When complete, it was
diluted with EtOAc and washed with water and brine. The organic layer
was dried over anhydrous Na2SO4. The solvent was evaporated, and
the residue was purified by flash chromatography with EtOAc/hexane
(3:7) to afford 4 as a pale yellow solid (27 mg, 0.075 mmol, 90% yield),
Conclusions
We have demonstrated that BHQ can mediate the efficient
photorelease of carboxylates, phosphates, and diols with 1PE
or 2PE under simulated physiological conditions. The carbox-
ylates and diols are produced in good yield (60-70%) via both
1PE and 2PE. BHQ has a high sensitivity to light in 1PE and
a large 2PE uncaging cross section at convenient wavelengths,
365 and 740 nm, respectively. The carboxylate and phosphate
functional groups are discharged on the sub-microsecond time
scale, sufficient for the study of fast kinetics in physiological
processes. The physical data support a solvent-assisted photo-
heterolysis (SN1) reaction mechanism25 that proceeds from the
singlet excited state. BHQ has the requisite photochemical and
photophysical properties as a photoremovable protecting group
to regulate the action of biological effectors in cell and tissue
culture and should prove useful to the neuroscience community.
1
mp ) 180-202 °C (dec): H NMR (CDCl3) δ 8.17 (2H, dm, J ) 8.8
Hz), 8.13 (1H, d, J ) 8.8 Hz), 7.72 (1H, d, J ) 9.2 Hz), 7.61 (1H, tm,
Experimental Methods
J ) 7.6 Hz), 7.49 (3H, m), 7.33 (1H, d, J ) 8.8 Hz), 5.72 (2H, s); 13
C
General Methods. Compounds 1,24 8,24 14,26 15,27 and 2324 were
prepared by their respective literature procedure. All other reagents and
solvents were purchased from commercial sources and used without
further purification with the following exceptions. Toluene and THF
were dried by passing through activated alumina under nitrogen pressure
(Solv-Tek, Berryville, VA). Pyridine and acetonitrile were refluxed with
NMR (CDCl3) δ 166.25, 158.94, 155.80, 148.13, 137.43, 133.12,
130.19, 129.65, 128.86, 128.30, 122.19, 119.72, 116.68, 108.81, 66.96;
FTIR (neat) 2958, 1723, 1612, 1506, 1471, 1269, 1113, 842, 710 cm-1
;
HR-MS (ESI) m/z calcd for (C17H12BrNO3 + H)+ 358.0073 (79Br) and
360.0055 (81Br), found 358.0074 (79Br) and 360.0057 (81Br).
TBDPS-HQ-Piperonylate (5). Under a nitrogen atmosphere,
alcohol 1 (100 mg, 0.242 mmol) was dissolved in CHCl3 (10 mL).
Et3N (45 µL, 0.36 mmol) and DMAP (10 mg, 0.08 mmol) were added,
followed by piperonylic chloride (50 mg, 0.27 mmol). The reaction
was stirred overnight. The mixture was washed with 15% citric acid,
water, and brine. The organic layer was dried over anhydrous Na2SO4.
The solvent was evaporated, and the residue was purified by flash
1
calcium hydride under nitrogen and then distilled. H NMR and 13C
NMR spectra were recorded on a Varian MercuryPlus 400 MHz
spectrometer. FTIR spectra were recorded on an Avatar 360 spectro-
photometer (Thermo Nicolet). UV spectra were recorded on a Cary
300 Bio UV-visible spectrophotometer (Varian). HPLC analysis
(analytical and preparative) was performed on a Varian ProStar HPLC
9
4274 J. AM. CHEM. SOC. VOL. 128, NO. 13, 2006

BHQ as a Photoremovable Protecting Group
A R T I C L E S
chromatography with EtOAc/hexane (1:9) to afford 5 as a white solid
(90 mg, 0.16 mmol, 66% yield), mp ) 90-95 °C: 1H NMR (CD3OD)
δ 8.18 (1H, d, J ) 8.0 Hz), 7.76 (4H, m), 7.71 (1H, d, J ) 8.0 Hz),
7.66 (1H, dd, J ) 8.4, 2.0 Hz), 7.40 (8H, m), 7.22 (1H, s), 7.21 (1H,
d, J ) 7.2 Hz), 6.87 (1H, d, J ) 8.4 Hz), 6.03 (2H, s), 5.39 (2H, s),
1.12 (9H, s); 13C NMR (CD3OD) δ 165.48, 157.18, 156.92, 148.06,
137.39, 135.24, 131.84, 130.02, 128.88, 127.71, 125.33, 123.31, 123.23,
122.49, 117.24, 114.64, 108.85, 107.67, 102.10, 66.57, 25.50, 18.81;
FTIR (neat) 3072, 2932, 2858, 1719, 1619, 1506, 1442, 1257, 1113,
1076, 876, 842, 702 cm-1; HR-MS (ESI) m/z calcd for (C34H31NO5Si
+ H)+ 562.2044, found 562.2020.
Na2SO4. Evaporation of the solvent followed by purification through
silica gel with EtOAc/hexane (2:8) provided 10 as a white solid (980
mg, 3.5 mmol, 83% yield), mp ) 144-146 °C: 1H NMR (CDCl3) δ
8.05 (1H, d, J ) 8.4 Hz), 7.76 (1H, d, J ) 8.8 Hz), 7.34 (1H, d, J )
8.0 Hz), 7.29 (1H, d, J ) 8.8 Hz), 2.83 (3H, s), 2.45 (3H, s); 13C NMR
(CDCl3) δ 168.56, 161.06, 149.49, 145.78, 136.41, 127.73, 125.63,
122.61, 121.85, 117.12, 25.77, 20.97; FTIR (neat) 2932, 1772, 1701,
1612, 1369, 1184, 1009, 841, 722 cm-1; HR-MS (ESI) m/z calcd for
(C12H10BrNO2 + H)+ 279.9968 (79Br) and 281.9948 (81Br), found
279.9975 (79Br) and 281.9953 (81Br).
(7-Acetyl-8-bromoquinolin-2-yl)formaldehyde (11). A mixture of
SeO2 (375 mg, 3.38 mmol) and 1,4-dioxane (10 mL) was heated to
over 80 °C. 7-Acetyl-8-bromoquinaldine (10, 900 mg, 3.21 mmol) in
1,4-dioxane (5 mL) was added. After stirring at 80 °C for 18 h, the
reaction was cooled and vacuum filtered. The filtrate was collected
and concentrated, leaving a yellow solid. Purification by silica gel with
EtOAc/hexane (3:7) gave 11 as a white solid (561 mg, 1.9 mmol, 60%
HQ-Piperonylate (6). Ester 5 (80.0 mg, 0.142 mmol) was dissolved
in THF (5 mL). TBAF in THF (1 M, 150 µL, 0.15 mmol) was added
dropwise. The reaction was monitored by TLC. When complete, it was
diluted with EtOAc and washed with water and brine. The organic layer
was dried over anhydrous Na2SO4. The solvent was evaporated, and
the residue was purified by flash chromatography with EtOAc/hexane
(4:6) to afford 6 as a white solid (41.0 mg, 0.124 mmol, 87% yield),
1
yield), mp )190-196 °C (dec): H NMR (CDCl3) δ 10.31 (1H, s),
1
mp ) 130-185 °C (dec): H NMR (CD3OD) δ 8.25 (1H, d, J ) 8.4
8.36 (1H, d, J ) 8.0 Hz), 8.09 (1H, d, J ) 8.0 Hz), 7.91 (1H, d, J )
8.4 Hz), 7.50 (1H, d, J ) 9.2 Hz), 2.48 (3H, s); 13C NMR (CDCl3) δ
193.34, 168.33, 153.39, 150.50, 146.01, 138.03, 129.28, 127.93, 125.51,
118.87, 117.85, 20.98; FTIR (neat) 3054, 2848, 2360, 1751, 1708, 1369,
1194, 1157, 909, 851, 752 cm-1; HR-MS (ESI) m/z calcd for
(C12H8BrNO3 + Na)+ 315.9580 (79Br) and 317.9561 (81Br), found
315.9587 (79Br) and 317.9559 (81Br).
Hz), 7.79 (1H, d, J ) 8.8 Hz), 7.73 (1H, dd, J ) 8.0, 1.6 Hz), 7.51
(1H, d, J ) 1.6 Hz), 7.43 (1H, d, J ) 8.0 Hz), 7.29 (1H, d, J ) 2.0
Hz), 7.18 (1H, dd, J ) 8.8, 2.4 Hz), 6.92 (1H, d, J ) 8.4 Hz), 6.06
(2H, s), 5.51 (2H, s); 13C NMR (CD3OD) 164.58, 157.67, 155.03,
154.49, 148.29, 143.52, 137.59, 128.75, 125.67, 123.77, 121.83, 120.49,
118.07, 117.68, 110.05, 109.40, 101.87, 66.41; FTIR (neat) 2905, 2361,
1716, 1620, 1442, 1257, 1156, 1104, 1036, 842, 759 cm-1; HR-MS
(ESI) m/z calcd for (C18H13NO5 + H)+ 324.0866, found 324.0868.
BHQ-Piperonylate (7). Ester 6 (30 mg, 0.093 mmol) was dissolved
in acetic acid (2 mL). Bromine (100 mg, 20% in acetic acid, 0.125
mmol) was added dropwise. The mixture was stirred for 5 h. The
reaction was quenched with saturated NaHCO3, extracted with EtOAc,
and washed with water and brine. The organic layer was dried over
anhydrous Na2SO4, and the solvent was evaporated. The residue was
purified by flash chromatography with EtOAc/hexane (4:6) to afford
7 as a white solid (24 mg, 0.06 mmol, 64% yield), mp ) 186-194 °C
7-Acetyl-8-bromo-2-(formylquinolinyltosyl)hydrazone (12). p-
Toluenesulfonhydrazide (507 mg, 2.72 mmol) was added to a stirred
solution of aldehyde 11 (400 mg, 1.36 mmol) in ethanol (5 mL). After
2 d, the resulting precipitate was collected by vacuum filtration, washed
with minimum amount of ethanol, and dried under vacuum to yield 12
as a white solid (466 mg, 1.01 mmol, 74% yield), mp ) 180-190 °C
1
(dec): H NMR (DMSO-d6) δ 8.76 (1H, d, J ) 8.8 Hz), 8.19 (1H, d,
J ) 8.8 Hz), 7.89 (1H, d, J ) 8.4 Hz), 7.83 (1H, s), 7.81 (2H, d, J )
8.4 Hz), 7.74 (1H, d, J ) 8.4 Hz), 7.42 (2H, d, J ) 8.4 Hz), 2.47 (3H,
s), 2.36 (3H, s); 13C NMR (DMSO-d6) δ 168.74, 152.80, 150.92, 144.52,
143.55, 140.08, 137.23, 136.51, 130.53, 129.22, 127.63, 127.09, 125.51,
123.74, 115.88, 21.50, 21.16; FTIR (neat) 3178, 2360, 2339, 1764,
1599, 1502, 1434, 1356, 1162, 1079, 938, 895, 845 cm-1; HR-MS (ESI)
m/z calcd for (C19H16BrN3O4S + Na)+ 483.9937 (79Br) and 485.9918
(81Br), found 483.9936 (79Br) and 485.9886 (81Br).
1
(dec): H NMR (CDCl3) δ 8.15 (1H, d, J ) 8.4 Hz), 7.77 (1H, dd, J
) 8.4, 1.6 Hz), 7.72 (1H, d, J ) 8.8 Hz), 7.59 (1H, d, J ) 2.0 Hz),
7.46 (1H, d, J ) 8.4 Hz), 7.35 (1H, d, J ) 8.4 Hz), 6.87 (1H, d, J )
8.4 Hz), 6.07 (2H, s), 5.69 (2H, s); 13C NMR (CDCl3) δ 165.67, 158.58,
158.03, 157.29, 148.49, 141.59, 137.52, 128.25, 125.77, 123.67, 122.43,
120.89, 117.87, 117.40, 109.68, 108.19, 101.90, 67.52; FTIR (neat)
2909, 2359, 1716, 1622, 1504, 1444, 1259, 1229, 1157, 1037, 843,
760 cm-1; HR-MS (ESI) m/z calcd for (C18H12BrNO5 + H)+ 401.9972
(79Br) and 403.9954 (81Br), found 401.9979 (79Br) and 403.9960 (81Br).
8-Bromo-7-hydroxyquinaldine (9). 7-Hydroxyquinaldine (8, 1.0 g,
6.32 mmol) was dissolved in glacial acetic acid (15 mL). Bromine (5.5
g, 20% acetic acid solution, 6.88 mmol) was added dropwise with
vigorous stirring. A precipitate was observed. The reaction was stirred
for 8 h and then diluted with CHCl3 and washed with saturated
NaHCO3, water, and brine. The organic layer was dried over anhydrous
Na2SO4 and the solvent evaporated, leaving a brown oil, which was
purified by silica gel with EtOAc/hexane (3:7). The product 9 (1.07 g,
4.49 mmol, 71% yield) was acquired as a pale yellow solid, mp )
175-180 °C (dec): 1H NMR (CDCl3) δ 7.97 (1H, d, J ) 8.4 Hz), 7.66
(1H, d, J ) 8.4 Hz), 7.27 (1H, d, J ) 9.2 Hz), 7.21 (1H, d, J ) 8.0
Hz), 2.79 (3H, s); 13C NMR (CDCl3) δ 160.77, 153.89, 145.52, 136.39,
128.17, 122.57, 120.44, 116.79, 107.65, 25.69; FTIR (neat) 3059, 2360,
1618, 1561, 1503, 1429, 1334, 1262, 984, 836 cm-1; HR-MS (ESI)
m/z calcd for (C10H8BrNO + H)+ 237.9862 (79Br) and 239.9842 (81Br),
found 237.9856 (79Br) and 239.9833 (81Br).
8-Bromo-7-hydroxyquinolin-2-ylmethyl Dimethyl Phosphate (13).
NaOMe (80 mg, 1.48 mmol) was added to a mixture of compound 12
(300 mg, 0.65 mmol) in MeOH (5 mL). The MeOH was removed by
rotary evaporation. The resulting residue was taken up in dry CH3CN
(15 mL), and dimethyl phosphate (492 mg, 3.9 mmol) was added
dropwise. The reaction was stirred for 2 d. The solvent was evaporated,
and the residue was purified by flash chromatography using EtOAc/
hexane (4:6) to afford 13 (47 mg, 0.13 mmol, 20% yield) as a pale
yellow oil: 1H NMR (CDCl3) δ 8.02 (1H, d, J ) 8.4 Hz), 7.75 (1H,
d, J ) 8.8 Hz), 7.34 (1H, d, J ) 8.4 Hz), 7.24 (1H, d, J ) 8.4 Hz),
5.37 (2H, d, J ) 8.0 Hz), 3.80 (6H, d, J ) 10.8 Hz); 13C NMR (CDCl3)
160.87, 154.59, 144.32, 134.51, 128.47, 121.97, 120.84, 116.74, 104.76,
71.77, 49.56; FTIR (neat) 2929, 2832, 2359, 1614, 1505, 1438, 1332,
1217, 1189, 1108, 1070, 986, 909, 844 cm-1; HR-MS (ESI) m/z calcd
for (C12H13BrNO5P + H)+ 361.9788 (79Br) and 363.9768 (81Br), found
361.9808 (79Br) and 363.9782 (81Br).
BHQ-Glycerol (OPh) (16). (7-Acetyl-8-bromoquinolin-2-yl)form-
aldehyde (11, 50 mg, 0.17 mmol), 1-phenoxypropane-2,3-diol (14, 86
mg, 0.51 mmol), and pyridinium p-toluenesulfonate (85 mg, 0.34 mmol)
were dissolved in toluene (25 mL); MgSO4 (50 mg) was added as a
drying agent. The reaction mixture was heated at reflux for 48 h. After
vacuum filtration, the filtrate was concentrated, and the resulting residue
was purified by flash chromatography using EtOAc/hexane (3:7) to
yield 16 as a white solid (41 mg, 0.1 mmol, 60% yield), mp ) 210 °C
(dec). The product exists as a pair of diastereomers (1:1), according to
1H NMR: 1H NMR (CDCl3) δ [8.16 (d, J ) 8.4 Hz), 8.15 (d, J ) 8.4
7-Acetyl-8-bromoquinaldine (10). Under a nitrogen atmosphere,
8-bromo-7-hydroxyquinaldine (9, 1.0 g, 4.2 mmol) was dissolved in
pyridine (20 mL). DMAP (50 mg, 0.41 mmol) was added to the
reaction, followed by slow addition of acetic anhydride (0.600 mL,
6.23 mmol). The reaction was stirred for 4 h. It was diluted with CHCl3,
washed by 15% citric acid, water, and brine, and dried over anhydrous
9
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A R T I C L E S
Zhu et al.
Hz) (1H)], [7.72 (d, J ) 9.2 Hz), 7.72 (d, J ) 9.2 Hz) (1H)], [7.64
(dd, J ) 8.0, 1.2 Hz), 7.59 (dd, J ) 8.4, 1.2 Hz) (1H)], 7.31 (3H, m),
6.97 (3H, m), [6.24 (s), 6.12 (s) (1H)], [4.86 (m), 4.72 (m) (1H)], 4.55
(0.5H, m), [4.32 (d, J ) 6.0 Hz), 4.31 (d, J ) 6.0 Hz) (1H)], 4.26 (1H,
m), 4.15 (1.5H, m); 13C NMR (CDCl3) δ (158.48, 158.37), 157.98,
154.18, 145.24, 137.39, 129.58, 128.22, (124.64, 124.52), (121.31,
121.25), (116.86, 116.74), 114.56, 108.29, (104.95, 104.89), (104.52,
104.46), 75.09, (68.41, 68.32), 68.07; FTIR (neat) 2924, 1599, 1496,
1442, 1374, 1241, 1102, 907, 842, 728 cm-1; HR-MS (ESI) m/z calcd
for (C19H16BrNO4 + H)+ 402.0335 (79Br) and 404.0317 (81Br), found
402.0350 (79Br) and 404.0330 (81Br).
Measurement of the Two-Photon Uncaging Cross Section.
Measurements were carried out in microcuvettes (10 × 1 × 1 mm
illuminated dimensions) with an effective filling volume of 20 µL
(26.10F-Q-10, Starna, Atascadero, CA) using light from a fs-pulsed
and mode-locked Ti:sapphire laser (Mira 900 pumped by a Verdi,
Coherent, Santa Clara, CA) focused on the center of the cuvette chamber
with a 25-mm focal length lens optimized for IR lasers (06LXP003/
076, Melles-Griot, Irvine, CA). The pulse width of the laser was 144
fs, and the average powers exiting the apparatus were 330, 310, 285,
285, and 295 mW for 18a-c and 21d,e, respectively. The two-photon
uncaging cross-section (δu) was estimated by referencing to fluorescein,
a compound with a known fluorescence quantum yield (QfF ) 0.9 mol/
ein) and absorbance cross section (δaF ) 30 GM at 740 nm),33,34 using
eq 2, where Np is the number of product molecules formed/unit time
(molecules/s, determined by HPLC analysis as in the one-photon
photolysis), φ is the collection efficiency of the detector (SED033 on
an IL-1700, International Light, Newburyport, MA) used to measure
the fluorescence of fluorescein emitted at a right angle to the beam
and passed though a 535/45 nm band-pass filter (Chroma Technologies,
Brattleboro, VT), CF is the concentration of the fluorescein standard
(mol/L), F(t) is the time-averaged fluorescent photon flux (photons/
s) of the fluorescein standard collected by the detector, and CS is the
initial concentration of the BHQ-protected substrate (mol/L).
Time-Resolved IR Experiments. TRIR experiments were conducted
following the method of Hamaguchi and co-workers40,41 as described
previously.42 Briefly, the broad-band output of a MoSi2 IR source
(JASCO) is crossed with excitation pulses from a Nd:YAG laser.
Changes in IR intensity are monitored by an MCT photovoltaic IR
detector (Kolmar Technologies, KMPV11-1-J1), amplified, and digi-
tized with a Tektronix TDS520A oscilloscope. The experiment is
conducted in the dispersive mode with a JASCO TRIR-1000 spec-
trometer at 16 cm-1 resolution.
Oxygen-18 Labeling Experiment. As a control, a 100-µM solution
of BHQ-OAc in water was irradiated at 365 nm from a mercury lamp
(Spectroline SB-100P; Spectronics, Westbury, NY) for 2.5 min as
described for one-photon photolysis. The reaction was analyzed by LC-
MS (Biobasic 4 column, 150 × 1 mm, 50 µm, 300 Å; gradient elution
of acetonitrile and water/TFA, 0 min 0% acetonitrile, 8 min 40%
acetonitrile, 40 min 70% acetonitrile, 45 min 100% acetonitrile; analysis
at 214 nm), selecting the peak corresponding to BHQ-OH for mass
analysis. A 100-µM solution of BHQ-OAc in 18O-labeled water was
irradiated for 2.5 min, and the reaction was analyzed by LC-MS as in
the unlabeled control experiment.
BHQ-Glycerol (SPh) (17). (7-Acetyl-8-bromoquinolin-2-yl)form-
aldehyde (11, 50 mg, 0.17 mmol), 1-phenthioxypropane-2,3-diol (15,
94 mg, 0.51 mmol), pyridinium p-toluenesulfonate (85 mg, 0.34 mmol),
and MgSO4 (50 mg) were mixed in toluene (25 mL). After the same
reaction and workup procedure as the synthesis of compound 16,
compound 17 was acquired as a pale yellow solid (39 mg, 0.093 mmol,
55% yield), mp ) 205 °C (dec). The product exists as a pair of
diastereomers (3:2), according to 1H NMR: 1H NMR (CDCl3) δ [8.15
(d, J ) 8.4 Hz), 8.13 (d, J ) 8.0 Hz) (1H)], [7.72 (d, J ) 9.2 Hz), 7.71
(d, J ) 9.2 Hz) (1H)], [7.59 (d, J ) 8.8 Hz), 7.51 (d, J ) 8.4 Hz)
(1H)], 7.42 (1.5H, m), 7.33 (4H, m), 7.23 (0.5H, m), [6.19 (s), 6.05 (s)
(1H)], [4.62 (m), 4.50 (m) (1H)], [4.45 (m), 4.24 (m) (1H)], [4.13 (m),
3.94 (m) (1H)], [3.45 (m), 3.42 (m) (1H)], [3.20 (m), 3.10 (m) (1H)];
13C NMR (CDCl3) δ 158.05, 154.19, 145.26, 137.41, (130.08, 129.91),
129.16, 128.23, (126.81, 126.71), 124.61, 118.38, (116.82, 116.65),
105.29, 104.91, 104.26, (75.95, 75.70), 70.57, 70.08; FTIR (neat) 2925,
2359, 1613, 1508, 1439, 1374, 1337, 1304, 1187, 1092, 908, 842, 734
cm-1; HR-MS (ESI) m/z calcd for (C19H16BrNO3S + H)+ 418.0107
(79Br) and 420.0088 (81Br), found 418.0121 (79Br) and 420.0103 (81Br).
Determination of the Quantum Efficiency for One-Photon
Photolysis. KMOPS-buffered solutions (3 mL) of the substrates (100
µM) in quartz cuvettes (21-Q-10, Starna, Atascadero, CA) were
irradiated with 365-nm UV light from a mercury lamp (Spectroline
SB-100P; Spectronics, Westbury, NY) with a light filter. The spectral
output of the lamp after the glass filters (CS0-52 and CS7-60, Ace
Glass, Vineland, NJ) has a band between 350 and 380 nm. The duration
of each irradiation period ranged from 5 to 60 s. After each period of
irradiation, a 20-µL aliquot of the solution was removed for analysis
by HPLC, using an external standard method to determine concentra-
tions. The analytes were eluted isocratically (flow rate of 1 mL/min)
with acetonitrile and water containing 0.1% TFA. The solvent composi-
tion was 50% acetonitrile with 50% water/TFA for compounds 4, 7,
16, and 17 and 40% acetonitrile and 60% water/TFA for compound
13. Compounds 4, 13, 16, and 17 were monitored at 330 nm, and
compound 7 was monitored at 299 nm. The progress curves were plotted
as simple decaying exponentials. Quantum efficiencies were calculated
using eq 1,30,31 where I is the irradiation intensity in ein‚cm-2‚s-1, σ is
the decadic extinction coefficient (103ꢀ, the molar extinction coefficient)
in cm2‚mol-1, and t90% is the irradiation time in s for 90% conversion
to product. The UV intensity of the lamp I was measured by potassium
ferrioxalate actinometry32 in the same setup. Piperonylic acid and
1-phenthioxypropane-2,3-diol were monitored for their formation during
the process and plotted as an exponential rise to max curve.
Acknowledgment. We thank Dennis R. Phillips for assistance
with the ESI and LC-MS experiments. HRMS was provided
by the Washington University Mass Spectrometry Resource with
support from the NIH National Center for Research Resources
(Grant No. P41RR0954). An NSF CAREER Award (CHE-
0349059) to T.M.D. supported the work. J.P.T. also thanks the
NSF (Grant CHE-0518406) for support of this research.
Supporting Information Available: MS data for the 18O-
labeling experiment, tables of optimized geometry and calculated
and observed IR frequencies and intensities for BHQ-OAc and
Stern-Volmer Triplet Quenching Experiment. Sodium 2-naph-
thalensulfonate or potassium sorbate was dissolved in methanol and
diluted to generate a 10 mM solution in KMOPS buffer. A 30-, 60-,
and 90-µL aliquot was added to each of 3 different KMOPS-buffered
solutions of BHQ-OAc (3 mL, 100 µM). The quantum efficiencies were
determined as described for one-photon photolysis.
1
BHQ-phosphate, and H NMR spectra of synthesized com-
pounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA0555320
Determination of the Dark Hydrolysis Rate. Substrates were
dissolved in KMOPS and stored in the dark at room temperature. HPLC
analysis was carried out periodically as described for one-photon
photolysis. Data were fit to simple decaying exponentials from which
the time constant, τ, was calculated.
(40) Iwata, K.; Hamaguchi, H. Appl. Spectrosc. 1990, 44, 1431-1437.
(41) Yuzawa, T.; Kato, C.; George, M. W.; Hamaguchi, H. Appl. Spectrosc.
1994, 48, 684-690.
(42) Wang, Y.; Yuzawa, T.; Hamaguchi, H.-o.; Toscano, J. P. J. Am. Chem.
Soc. 1999, 121, 2875-2882.
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