Toward the Development of New Photolabile Protecting Groups That Can Rapidly Release Bioactive Compounds upon Photolysis with Visible Light

Article abstract of DOI:10.1021/jo0300643

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a

Full text of DOI:10.1021/jo0300643

Tow a r d th e Develop m en t of New P h otola bile P r otectin g Gr ou p s
Th a t Ca n Ra p id ly Relea se Bioa ctive Com p ou n d s u p on P h otolysis
w ith Visible Ligh t
Anamitro Banerjee,†,‡ Christof Grewer,§ Latha Ramakrishnan,† J u¨rgen J a¨ger,†,|
Armanda Gameiro,† Hans-Georg A. Breitinger,†, Kyle R. Gee,3 Barry K. Carpenter,O and
George P. Hess*,†
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853,
Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402, Max-Planck-Institute for
Biophysics, Kennedyallee 70, D60596 Frankfurt, Germany, Department of Chemistry and Chemical
Biology, Cornell University, Ithaca, New York 14853, and Institute of Biochemistry, University of
Erlangen-Nuremberg, D-91054 Erlangen, Germany
gph2@cornell.edu
Received February 19, 2003
The synthesis and characterization of a new photolabile protecting group (caging group) for
carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound
has a major absorption band in the visible wavelength region with a maximum near 400 nm
(ꢀ400 ) 9077 M-1 cm-1 at pH 7.4 and 21 °C). The caging group is attached through an ester linkage
to the carboxyl functionality of â-alanine, which activates the inhibitory glycine receptor in the
mammalian central nervous system. Such caged compounds play an important role in transient
kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged â-alanine, the caging
group is released within 5 µs. Quantum yields of 0.03 and 0.002 were obtained in the UV region
(308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments,
using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293
cells transiently transfected with cDNA encoding the R1 homomeric, wild-type glycine receptor.
The experiments demonstrated that neither DANP-caged â-alanine nor its byproducts inhibit or
activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged
â-alanine is water-soluble and stable and can be used for transient kinetic measurements.
In tr od u ction
render a bioactive compound inert until they are removed
by photolysis, thus releasing the compound rapidly
(within microseconds to milliseconds). These properties
of caged compounds, the inertness of a precursor, and
the rapid release of a bioactive compound after an
equilibrium with another reactant has been achieved
make the photolabile precursors particularly useful for
transient kinetic investigations (reviewed in ref 8). High
time resolution becomes important in the study of fast
processes (e.g., the opening of ion channels).9,10 In con-
ventional kinetic investigations of cell surface proteins,
ligand solutions flow over the cell. Equilibration of the
ligands with the cell-surface proteins is determined by
the velocity with which layers of solution covering the
cell are displaced.11,12 In kinetic investigations of neu-
rotransmitter receptors on cell surfaces, the time resolu-
tion of these flow techniques is not adequate to resolve
the elementary reaction steps in the response of the
First reported by Barltrop and Schofield in 1962,1
photolabile protecting groups2-5 have found numerous
applications in biology in the past decade.6,7 The protect-
ing groups (also known as “caging” groups in biology) can
† Department of Molecular Biology and Genetics, Cornell University.
‡ Current address: Department of Chemistry, University of North
Dakota, Grand Forks, North Dakota 58202-9024.
§ Max-Planck-Institute for Biophysics. Current address: Depart-
ment of Physiology and Biophysics, University of Miami School of
Medicine, Miami, FL 33136.
| Current address: ARPIDA AG, Dammstr. 36, CH-4142 Mu¨nchen-
stein, Switzerland.
University of Erlangen-Nuremberg.
∇ Molecular Probes, Inc.
O Department of Chemistry and Chemical Biology, Cornell Univer-
sity.
(1) Barltrop, J . A.; Schofield, P. Tetrahedron Lett. 1962, 697-699.
(2) Patchornik, A.; Amit, B.; Woodward, R. B. J . Am. Chem. Soc.
1970, 92, 6333-6335.
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(9) Eigen, M. Nobel Lectures in Chemistry 1969-1970; Elsevier
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10.1021/jo0300643 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/07/2003
J . Org. Chem. 2003, 68, 8361-8367
8361

Banerjee et al.
receptors to neurotransmitter.5,13,14 The main advantage
of photorelease of a bioactive compound from its caged
form over conventional mixing of reactants is that caged
compounds allow time resolutions of microseconds rather
than milliseconds to be achieved. In addition, photolytic
release of biologically active compounds can be performed
with high spatial resolution, and this has been exploited
for identifying specific receptors in neuronal circuits,15-18
and in the synthesis of DNA chips.19
A caged compound suitable for the investigation of
biological systems must satisfy the following criteria:
(1) The compound must be soluble and stable in water
at neutral pH.
(2) The compound should be photolyzable at long
wavelengths to avoid cell damage (visible region pre-
ferred, vide infra).
(3) The quantum yield of the photorelease should be
sufficiently high that the bioactive compound is released
in quantities adequate for the proposed studies.
(4) The release of the bioactive molecule should occur
in microseconds so the photolysis is not rate limiting for
the biological reaction to be investigated.
However, they suffer from low solubility in an aqueous
medium or, in the case of the 2-nitroveratrole group and
coumarin derivatives, from a slow rate of release of the
bioactive compound. Thus, a photolabile caging group
that has all the desired characteristics of a compound
suitable for investigating biological reaction mechanisms
(vide supra) is still to be made.
The o-nitrobenzyl group is one of the most widely
applied photolabile caging groups in use today. In 1966
Barltrop et al.26 reported the release of benzoic acid from
its nitrobenzyl ester upon photolysis. Since then, this
caging group has found a variety of applications in
biology, but it has a few shortcomings that limit its
applicability. First, short-wavelength UV light is required
for deprotection (“uncaging”).27 Second, after the initial
photochemical excitation, the molecule goes through a
series of “dark” steps before the bioactive molecule is
released.28 It is estimated that there is a lag of a few
milliseconds after photolysis before the bioactive molecule
is released.28,29 This makes the caging group unsuitable
for studying fast reactions, which frequently occur on the
sub-millisecond time scale. Third, a reactive o-nitroso-
benzaldehyde is formed as a byproduct of the photolysis
reaction, and this can damage cells.27 Introduction of the
carboxyl group in the R-position of this group overcomes
most of these problems.5 Transient kinetic investigations
with a microsecond time resolution of neurotransmitter-
receptor-mediated reactions, which regulate signal trans-
mission between ∼1012 nerve cells in the brain,30 first
became possible with the use of this caging group.5,31
However, UV light is still required for uncaging.5
The desyl32,33 and 2-methoxy-5-nitrophenyl (MNP)34,35
groups are the two other photolabile caging groups that
are in common use. The desyl group suffers from low
solubility in aqueous medium, and both the desyl and
MNP groups absorb in the UV region, thus requiring
high-energy UV radiation for the uncaging step. Substi-
tuted coumarins were used by Furuta et al.24,25,36 to cage
phosphates and carboxylic acids. The quantum yield
when the coumarin protecting group is used (photolysis
wavelength 340 nm) was found to be dependent on the
substitution at the C-7 position.24 Although the quantum
yield of the 7-methoxy-substituted coumarins is 0.12 at
340 nm, that of the 7-acetoxy, 7-propionyloxy, and
7-hydroxy derivatives is about 0.06 at the same wave-
length. These caged compounds also have a low solubility
(5) The caged compound and the photolysis byproducts
must be biologically inert.
The stability of the caged compound in water is
important as any reaction or hydrolysis in the dark would
reduce the effective concentration of the bioactive com-
pound released by photolysis, and the released compound
would activate the biological system before measure-
ments begin. A second problem is that neurotransmitter
receptors are transiently inactivated (desensitized) dur-
ing prolonged (milliseconds) exposure to neurotransmit-
ter. Dark hydrolysis would lead to liberation of the
neurotransmitter and, therefore, to transient inactivation
of the receptor before the reaction can be investigated.
The wavelength of the radiation used to photolyze the
caged compound is also an important parameter. Most
of the photolabile caging groups developed so far are
uncaged with high-energy UV light. Radiation at short
wavelengths can damage living cells.20 Moreover, expen-
sive light sources, which are not simple to operate or
maintain, are required. Recently, the substituted 2-ni-
troveratrole group,21,22 the nitroindolines,7 the phenacyl
group,23 and substituted coumarins24,25 have been studied
as caging groups that can be removed by visible light.
(13) Udgaonkar, J . B.; Hess, G. P. Proc. Natl. Acad. Sci. U.S.A. 1987,
84, 8758-8762.
(26) Barltrop, J . A.; Plant, P. J .; Schofield, P. J . Chem. Soc., Chem.
Commun. 1966, 22, 822-823.
(14) J ayaraman, V.; Thiran, S.; Hess, G. P. Biochemistry 1999, 38,
11372-11378.
(27) Amit, B.; Zehavi, U.; Patchornik, A. Isr. J . Chem. 1974, 12, 103-
(15) Dalva, M. B.; Katz, L. C. Science (Washington, D.C.) 1994, 265,
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36, 85-97.
(29) Zhu, Q. Q.; Schnabel, W.; Schupp, H. J . Photochem. 1987, 39,
317-332.
(16) Denk, W. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6629-6633.
(17) Li, H. Y.; Avery, L.; Denk, W.; Hess, G. P. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 5912-5916.
(18) Ko¨tter, R.; Staiger, J . F.; Zilles, K.; Luhmann, H. J . Neuroscience
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(30) Kandel, E. R.; Schwartz, J . H.; J essell, T. M. Principles of
Neural Science, 4th ed.; McGraw-Hill: New York, 2000; p 1414.
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31, 5507-5514.
(19) Pirrung, M. C.; Wang, L. X.; Montague-Smith, M. P. Org. Lett.
2001, 3, 1105-1108 and references therein.
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271-278.
(32) Gee, K. R.; Kueper, L. W., III; Barnes, J .; Dudley, G.; Givens,
R. S. J . Org. Chem. 1996, 61, 1228-1233.
(21) Bochet, C. G. Tetrahedron Lett. 2000, 41, 6341-6346.
(22) Bochet, C. G. Angew. Chem., Int. Ed. 2001, 40, 2071-2073.
(23) Banerjee, A.; Lee, K.; Yu, Q.; Fang, A. G.; Falvey, D. E.
Tetrahedron Lett. 1998, 39, 4635-4638.
(33) Sheehan, J . C.; Wilson, R. M. J . Am. Chem. Soc. 1964, 86,
5277-5281.
(34) Kuzmic, P.; Pavlickova, L.; Soucek, M. Collect. Czech. Chem.
Commun. 1986, 51, 1293-1300.
(24) Furuta, T.; Iwamura, M. Methods Enzymol. 1998, 291, 50-63.
(25) Furuta, T.; Wang, 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.
(35) Ramesh, D.; Wieboldt, R.; Niu, L.; Carpenter, B. K.; Hess, G.
P. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11074-11078.
(36) Furuta, T.; Torigai, H.; Sugimoto, M.; Iwamura, M. J . Org.
Chem. 1995, 60, 3953-3956.
8362 J . Org. Chem., Vol. 68, No. 22, 2003

Development of New Photolabile Protecting Groups
SCHEME 1a
a
The overall yield of compound 4 was 23% of the starting material. The overall yield of compound 5, starting from compound 4, was
about 77%.
in aqueous medium and must be dissolved in 1% DMSO
before addition to an aqueous solution, and the rates of
release of the phosphates and esters have not been
reported. Recently, brominated 7-hydroxycoumarin-4-
methyl was developed as a promising caging group for
carboxylic acids that can be removed by longer wave-
length UV light.23 Although this caging group absorbs
in the visible range (ꢀ400 ≈ 5-8000 M-1 cm-1), the one-
photon photolysis was carried out with 365 nm radiation,
with a quantum yield of 0.019. Two-photon photolysis of
coumarin derivatives with 800 nm light has been achieved,
but the efficiency of this process is not known, and no
data on the rate of release of the free compound are
available. The brominated 7-hydroxycoumarin-4-methyl
caging group is soluble in aqueous medium and can be
used for mapping neurotransmitter-evoked currents.23
Carboxylic acids have been released from phenacyl
esters upon photolysis with visible light (400 nm).23
However, the deprotection requires the use of a sensi-
tizer, 2-aminoanthracene. Moreover, the ester and the
sensitizers used are insoluble in aqueous medium at
physiological pH.
Recently, Bochet has explored nitroveratrole deriva-
tives as photolabile protecting groups.21,22 Some of these
groups can be removed in the visible wavelength region,
but the compounds are insoluble in aqueous medium and
are photolyzed in the minute time region.21,22
Thus, none of the caging groups that photolyze in the
visible region reported so far meet all the criteria
mentioned above. Current efforts in this laboratory are
directed toward the development and study of photolabile
caging groups for amino acid neurotransmitters that can
be removed by visible light, and that have all the other
desirable qualities of the caged neurotransmitters tested
previously37,38 (reviewed in ref 8). Here we report the
synthesis of 2-(dimethylamino)-5-nitrophenol and the use
of the 2-(dimethylamino)-5-nitrophenyl (DANP) group as
a caging group for the carboxyl functionality. This caging
group is analogous to the MNP caging group,34 but it
absorbs at wavelengths above 400 nm. Previous experi-
ments in which the MNP group was used to cage
â-alanine suggested that the analogous DANP group
would also be suitable for transient kinetic investigations
of neurotransmitter receptors on cell surfaces.39
Resu lts a n d Discu ssion
Syn th esis of th e Ca ged Com p ou n d . The synthesis
of DANP-caged â-alanine is described in Scheme 1.
2-(Dimethylamino)-5-nitrophenol (4) was synthesized as
previously described.40 Methylation of 2-aminophenol
with dimethyl sulfate generated the methylated com-
pound 2. Acylation of the hydroxyl group in 2 reduces
its ring-activating effect, and the nitro group was intro-
duced in the desired 5 position. The acyl group was then
removed with NaOH to form 4 with an overall yield of
(37) Breitinger, H.-G.; Wieboldt, R.; Ramesh, D.; Carpenter, B. K.;
Hess, G. P. Biochemistry 2001, 40, 8419-8429.
(38) Grewer, C.; J a¨ger, J .; Carpenter, B. K.; Hess, G. P. Biochemistry
2000, 39, 2063-2070.
(39) Niu, L.; Gee, K. R.; Schaper, K.; Hess, G. P. Biochemistry 1996,
35, 2030-2036.
(40) Amery, G. W.; Corbett, J . F. J . Chem. Soc. C 1967, 1053-1057.
J . Org. Chem, Vol. 68, No. 22, 2003 8363

Banerjee et al.
F IGURE 1. Thermal hydrolysis of DANP-caged â-alanine:
UV-vis spectrum of DANP-caged â-alanine (100 µM in 50 mM
phosphate buffer, pH 7.0, 22 °C) at times t ) 0 min (top line
at 400 nm) and t ) 7, 27, 44, 90, and 400 min after the
compound was dissolved. The path length of the cuvette was
10 mm. From the time dependence of the absorption at 400
nm, the rate constant for thermal hydrolysis was calculated
to be k ) 0.007 ( 0.001 min-1 (t1/2 ) 99 min).
23% (from 2-aminophenol). The BOC (N-tert-butoxycar-
bonyl group) protected â-alanine was introduced in the
presence of the coupling reagent DCC (1,3-dicyclohexyl-
carbodiimide)41 and DMAP (4-(dimethylamino)pyridine).
The BOC group was then removed by treatment with
trifluoroacetic acid in dichloromethane to yield the caged
compound 5.35
Th er m a l Sta bility. The absorption spectrum and the
thermal stability of the caged compound 5 were measured
in a buffered solution at pH 7 (Figure 1). The caged
F IGURE 2. Absorbance change [∆(OD)] measured as a
function of time, produced by photolysis of DANP-caged
â-alanine with a single laser pulse (A, λ ) 308 nm, 75 mJ /
cm2; B, λ ) 450 nm, 250 mJ /cm2) and averaged over 20 laser
pulses at 22 °C. Conversion to the product is complete within
5 µs. The wavelength of the analyzing light was 495 nm (A)
and 480 nm (B). The concentration of the caged compound was
250 µM in 50 mM phosphate buffer (pH 7.0). The path length
of the excitation beam was 2 mm, while that of the analyzing
beam was 10 mm. The appearance of the phenol on photolysis
is indicated by the wavy lines. The spikes immediately after
the laser pulse are instrumental artifacts due to scattered light
that saturates the detector.
compound decomposed in the dark by hydrolysis (t1/2
)
99 min at 22 °C) to form 4. On the basis of previous
studies with 2-methoxy-5-nitrophenyl glycine (MNP-
glycine) ester, the rates of thermal hydrolysis for DANP-
caged R-amino acids are expected to be much faster.35
The thermal hydrolysis rate of MAP-glycine is 6.1 min
at pH 7.1 and room temperature. Hence, MAP-glycine
was not considered for further study.
La ser -F la sh P h otolysis. Laser-flash photolysis ex-
periments were carried out with 250 µM DANP-â-alanine
(5) in 50 mM phosphate buffer (pH 7) using a 308 nm
(Figure 2A) or 450 nm (Figure 2B) laser pulse (5-10 ns
pulse duration). The transient absorbance changes were
monitored at wavelengths where the absorption coef-
ficient of the phenol 4 (Scheme 1) was significant while
that of the caged compound was negligible (ca. 500 nm;
see Figure 1). The absorbance was averaged over 20
pulses to achieve a better signal-to-noise ratio. At 22 °C,
the phenol 4 is detected immediately after the laser pulse
at both the wavelengths, within the time resolution of
the method (see Figure 2). These experiments demon-
strate that the photolysis products are formed within a
few microseconds after the laser pulse. The experiments
did not, however, reveal any reaction intermediates that
would provide clues about the mechanism of the pho-
tolysis reaction, nor are the experiments capable of
detecting the free â-alanine as it is released from its
caged precursor. As in the case of the similar MNP-caged
compound,35 the mechanism of the photolysis probably
involves photohydrolysis of the ester linkage from the
excited triplet state, as demonstrated by Kuzmic et al.34
in the case of MNP-caged acetate. It is assumed that
â-alanine is released stoichiometrically with the phenol
(4 in Scheme 1) and that the free â-alanine is released
on the same time scale (within 5 µs) as the phenol (4 in
Scheme 1). Therefore, it appears that the caged com-
pound is suitable for kinetic measurements of fast
cellular processes in the microsecond time domain.
Qu a n tu m Yield . For a photolabile caging group to be
useful for biological applications, the quantum yield of
the photochemical step should be high (criterion 3, vide
supra) so that the neurotransmitter is released in suf-
ficient quantity at the desired site at a light intensity
that is not harmful to the cell. The quantum yields of
(41) Sheehan, J . C.; Hess, G. P. J . Am. Chem. Soc. 1955, 77, 1067-
1068.
8364 J . Org. Chem., Vol. 68, No. 22, 2003

Development of New Photolabile Protecting Groups
F IGURE 3. (A) Determination of the photolysis quantum
yield. The absorbance A of a 500 µM caged â-alanine solution
in 50 mM phosphate buffer at pH 7.0 and 22 °C was measured
as a function of the number of laser flashes. For comparison
between two experiments, on the x-axis the number of laser
flashes was converted to the number of absorbed photons
(µmol-1). The excitation wavelengths were 308 nm (b) and 450
nm (O). A 40 µL portion of a total volume of 50 µL of the
solution was irradiated in a 3 × 3 mm cuvette. The solution
was stirred after each laser flash (308 nm) or after every 10th
laser flash (450 nm). The lines represent the results of a linear
regression analysis with slopes of (3.4 ( 0.1) × 105 and (2.1 (
0.1) × 104 µmol-1, respectively. (B) Quantum yield plotted as
a function of wavelength. The experiments were performed in
50 mM phosphate buffer at pH 7.0 and 22 °C. The data were
fitted to the equation5 An ) ꢀMlC0φKE exp[-φKEF(n - 1)]. An
is the absorbance after the nth pulse, ꢀM the extinction
coefficient of the product, l the path length, C0 the initial
concentration of the caged compound, φ the quantum yield,
KE the ratio of the absorbed photons to the number of target
molecules (constant), and F the fraction of the solution
containing the caged compound through which the laser beam
passes.
F IGURE 4. (A) The 2-(dimethylamino)-5-nitrophenyl ester of
â-alanine (DANP-caged â-alanine) does not inhibit the R1-
glycine receptor. 100 µM â-alanine flowed over the surface of
an HEK 293 cell transfected with R1-glycine receptor cDNA
(V ) -60 mV, pH 7.4, T ) 22 °C) in the absence (solid line) or
presence (dotted line) of 1 mM DANP-caged â-alanine, using
the cell-flow technique. The induced current was recorded in
the whole-cell configuration.44 The experiments were carried
out three times on different cells. (B) The 2-(dimethylamino)-
5-nitrophenol does not inhibit the R1-glycine receptor. 100 µM
â-alanine flowed over the surface of an HEK 293 cell tran-
siently transfected with R1-glycine receptor cDNA (V ) -60
mV, pH 7.4, T ) 22 °C) in the presence (solid line) and absence
(dashed line) of 1 mM 2-(dimethylamino)-5-nitrophenol using
the cell-flow technique, and the â-alanine-induced current was
recorded in the whole-cell configuration. The experiments were
carried out twice with the same cell.
the DANP-caged â-alanine were determined at different
wavelengths as described previously.5 A 40 µL sample of
5 (Scheme 1) was photolyzed with repetitive laser flashes
at appropriate wavelengths, and the concentration of 4
(Scheme 1) was determined spectrophotometrically. Fig-
ure 3A shows the change in the absorbance of the caged
compound as a function of the number of photons
absorbed from the laser flashes at 308 and 450 nm. The
quantum yield at a number of different excitation wave-
lengths is shown in Figure 3B; it is approximately 0.03
at 308 and 350 nm. This value is higher than that of the
brominated coumarins,25 but it drops more than 10-fold
to 0.002 at 400 nm and above. Thus, even though the
J . Org. Chem, Vol. 68, No. 22, 2003 8365

Banerjee et al.
DANP-â-alanine (Figure 4A) or in the presence and
absence of 1 mM 2-(dimethylamino)-5-nitrophenol (Fig-
ure 4B) at pH 7.4 and 22 °C. The whole-cell currents
recorded as a response to â-alanine, in both the presence
and the absence of the caged compound (Figure 4A) or
the presence and absence of 2-(dimethylamino)-5-nitro-
phenol (Figure 4B) at concentrations as high as 1 mM,
were the same. These control experiments demonstrate
that neither the caged compound before photolysis nor
its photolysis product, 2-(dimethylamino)-5-nitrophenol,
activates or inhibits the R1-glycine receptor.
La ser -P u lse P h otolysis. Laser-pulse photolysis of the
caged compound at excitation wavelengths of 337 nm
(Figure 5A) and 360 nm (Figure 5B) was carried out with
HEK 293 cells transiently transfected with cDNA encod-
ing the R1 homomeric glycine receptor. The whole-cell
currents generated due to the opening of the ion channels
induced by the released â-alanine were measured as a
function of time (Figure 5). They demonstrated that at
these wavelengths DANP-â-alanine is useful for kinetic
investigations of the opening of the glycine receptor
channel. Before and after each laser-pulse photolysis
measurement, standard solutions of â-alanine flowed over
the same cell and the maximum current amplitudes were
determined8,13 to ascertain that neither the cell mem-
brane nor the receptors were damaged. These control
experiments, together with those illustrated in Figure 4,
demonstrated that neither the caged compound nor its
photolysis byproducts affect the cells or the glycine
receptor. (We routinely use such experiments to assess
the effects of drugs and inhibitors on the kinetics of ion-
channel openings.5,8) However, upon irradiation of the
caged compound with laser light at wavelengths higher
than 360 nm glycine-receptor-associated currents could
hardly be detected. Thus, these results confirm that
efficient photolysis takes place only at or below 360 nm
and not at higher wavelengths.
F IGURE 5. Whole-cell current recorded44 from an HEK 293
cell transfected with R1-glycine receptor at V ) -60 mV, pH
7.4, and T ) 22 °C. The current was induced by the photolytic
release of â-alanine. (The concentration of liberated â-alanine
was estimated as described in the Experimental Section.) (A)
From 0.94 mM DANP-caged â-alanine, with a 337 nm pulse
used for the photolysis (500 µJ ). The concentration of liberated
â-alanine was estimated to be 50 µM. The smooth line
represents the best fit according to the equation I(t) )
I∞[1 - exp(-kobst)] with kobs ) 368 ( 3 s-1 and I∞ ) 720 pA.
I(t) is the current at time t, I∞ is the current at t ) ∞ (in the
absence of desensitization), and kobs is the apparent pseudo-
first-order rate constant of the current rise.31 (B) From 2 mM
DANP-caged â-alanine, with a 360 nm laser pulse (500 µJ ).
The concentration of liberated â-alanine was estimated to be
Con clu sion s
Laser-pulse photolysis data for the DANP-caged â-ala-
nine indicate that the DANP caging group can be used
for biological applications requiring rapid release of
carboxylic acid-containing molecules. As judged by ex-
periments with the MNP caging group,35,39 DANP-caged
R-amino acids are likely to be less stable in aqueous
media than are DANP-caged â-amino acids and are,
therefore, likely to be more difficult to use in kinetic
measurements. While the DANP-caged compound re-
leases â-alanine a few microseconds after the laser flash,
at visible wavelengths the quantum yield of the release
step is low. Ultraviolet light of wavelengths 337 and 360
nm can be used to release the active compound with
moderate efficiency. The DANP-caged compound can
easily be synthesized from readily available starting
material.
30 µM. The smooth line represents the best fit with kobs
)
115 ( 5 s-1 and I∞ ) 180 ( 2 pA.
caged compound absorbs well into the visible range,
efficient release of the bioactive compound does not occur
at 400 nm and above. In contrast, quantum yields of
different caged neurotransmitters in the UV wavelength
region were found to be in the range of 0.8-0.14.8
Biologica l Ch a r a cter iza tion . Neither the caged
compound nor its photolysis product, 2-(dimethylamino)-
5-nitrophenol (Scheme 1), should inhibit or activate the
glycine receptors on the cell surface (criterion 5, vide
supra) to be applicable for kinetic measurements of ion-
channel opening. The HEK 293 cells transfected with
R1-glycine receptor cDNA were, therefore, exposed to
100 µM â-alanine in the presence and absence of 1 mM
Exp er im en ta l Section
Syn t h esis.
2-(Dim et h yla m in o)-5-n it r op h en ol
(4)
(Sch em e 1). The phenol 4 was synthesized by methods
previously published41 starting from commercially available
2-aminophenol: 1H NMR (CDCl3) δ 2.77 (s, 6H), 7.16 (d, J )
9 Hz, 1H), 7.77-7.81 (m, 2H).
8366 J . Org. Chem., Vol. 68, No. 22, 2003

Development of New Photolabile Protecting Groups
2-(D im e t h y la m in o )-5-n it r o p h e n y l-N -t -B O C -â-a la -
n in e. N-t-BOC-â-alanine (0.172 g, 9.1 × 10-4 mol), DMAP (10
mg), and DCC (2 mL of a 1 M solution in CH2Cl2) were stirred
at room temperature for about 10 min. 4 (0.124 g, 6.8 × 10-4
mol) was added to the reaction mixture, and the resulting
reaction mixture was stirred at room temperature for about
2 h. The mixture was diluted with diethyl ether and washed
several times with saturated NaHCO3 solution. The organic
layer was dried over anhydrous MgSO4 and filtered over silica
gel to give a yellow solid: yield 0.202 g (84%); 1H NMR (CDCl3)
δ 1.48 (s, 9H), 2.84 (t, J ) 6 Hz, 2H), 2.97 (s, 6H), 3.51 (q, J )
6 Hz, 2H), 5.08 (s (br), 1H), 6.86 (d, J ) 9 Hz, 1H), 7.87 (d,
J ) 3 Hz, 1H), 8.03 (dd, J 1 ) 9 Hz, J 2 ) 3 Hz, 1H). Anal. Calcd
for C16H23N3O6: C, 54.38; H, 6.56; N, 11.89. Found: C, 55.08;
H, 6.56; N, 11.84.
2-(Dim eth yla m in o)-5-n itr op h en yl-â-a la n in e (5). 2-(Di-
methylamino)-5-nitrophenyl-N-t-BOC-â-alanine (0.202 g,
5.7 × 10-4 mol) and 1 mL of trifluoroacetic acid were dissolved
in 5 mL of dichloromethane, and the resulting solution was
stirred for about 3 h at room temperature. The solvent was
removed under reduced pressure. The residue was put under
vacuum for about 24 h. The resulting solid was washed with
small quantities of cold acetone and dried under vacuum: yield
0.194 g (92%); 1H NMR (CDCl3) δ 3.06 (s, 6H), 3.43 (t, J )
6 Hz, 2H), 4.28 (t, J ) 6 Hz, 2H), 7.05 (d, J ) 9 Hz, 1H), 7.89
(d, J ) 3 Hz, 1H), 8.04 (dd, J 1 ) 9 Hz, J 2 ) 3 Hz, 1H), 8.75
(s (br), 3H). Anal. Calcd for C13H16F3N3O6: C, 42.51; H, 4.39;
N, 11.44. Found: C, 42.28; H, 4.42; N, 11.22.
Hyd r olysis Exp er im en ts. The caged compound (100 µM)
was rapidly dissolved in the buffer solution (50 mM phosphate,
pH 7.0), immediately transferred to a cuvette (4.5 mL volume,
10 mm path length), and placed in an absorption spectrometer
(Beckman) at 22 °C. Whole spectra were recorded at times
between 1 and 400 min after the compound was dissolved.
La ser -F la sh P h otolysis. Transient absorption spectros-
copy was performed as described.5 The caged compound (250
µM in 50 mM phosphate buffer, pH 7.0) in a 2 × 10 mm sample
cuvette was photolyzed with 308 nm light from an excimer
laser, 10 ns pulse duration and 75 mJ /cm2 energy per pulse,
or with light from a dye laser pumped by the same excimer
laser. The laser dyes used were p-terphenyl (λmax ) 343 nm),
DMQ (λmax ) 360 nm), BiBuQ (λmax ) 388 nm), and coumarin
120 (λmax ) 450 nm). The dyes were available commercially.
The absorption change after excitation of the caged compound
was probed with the light output from a Xe lamp at a 90° angle
to the laser beam and monitored with a photodiode (active area
1 mm2). The output of the photodiode was amplified and
measured with a digital oscilloscope. The wavelength of the
analyzing light was selected with band-pass filters.
Cell Exp er im en ts. HEK 293 cells were transiently trans-
fected with cDNA encoding the R1-glycine receptor as described
previously.38,42 Transient transfection of exponentially growing
HEK 293 cells for control and photolysis experiments was
performed using the Effectene reagent or calcium phosphate
transfection method.43 Cells were cotransfected with cDNA
encoding the green fluorescent protein pGreenLantern to
detect transfected cells. The cells were used for electrophysi-
ological experiments between 24 and 72 h after transfection.
Whole-cell currents evoked by â-alanine (100 µM) were
recorded under voltage-clamp conditions44 at -60 mV and
amplified with an amplifier or an integrating patch clamp
amplifier. The experiments were carried out in the absence
or presence of 1 mM DANP (Figure 4A) or 1 mM 2-(dimeth-
ylamino)-5-nitrophenol (Figure 5B). The recording pipet solu-
tion contained 120 mM CsCl, 2 mM MgCl2, 10 mM TEACl, 10
mM EGTA, and 10 mM HEPES and was adjusted to pH 7.4.
The bath buffer solution contained 140 mM NaCl, 5 mM KCl,
1 mM MgCl2, 2 mM CaCl2 and 10 mM HEPES (adjusted to
pH 7.4). Typical pipet resistances were 2.5-3.5 MΩ, and the
series resistance was 4-6 MΩ. Series resistance compensation
of 60-80% was used in the whole-cell recording experiments.
Solutions were exchanged around the cell with a cell-flow
device13,45 with an effective time resolution of 10-20 ms.13
La ser -P u lse P h otolysis (F igu r e 5). The laser light pro-
duced by a dye laser pumped by an excimer-pumped dye laser
was coupled into an optical fiber (200 µm diameter), which
delivered the laser light to the cell. The dyes used for the
different wavelengths were the same as described above. In
the case of photolysis with 337 nm light, the laser light was
coupled into an optical fiber and delivered to the cell. The
concentration of DANP-caged â-alanine used was 0.94 mM in
Figure 5A and 2 mM in Figure 5B. Typical laser energies were
50-500 µJ /cm2. The amount of â-alanine liberated was cali-
brated by cell-flow experiments before and after the laser pulse
with a standard â-alanine solution (100 µM) and the known
dose-response curve for â-alanine as described.46 The pulse/
flow system was computer-controlled with pClamp software.
Data were sampled at 5-100 kHz and low-pass filtered at
2-10 kHz. Data were analyzed with Origin software.
Ack n ow led gm en t. We are grateful to Dr. Visha-
khar Shembekar (Cornell University) for providing the
2-(dimethylamino)-5-nitrophenol, and to Drs. Heinrich
Betz and Bodo Laube (Max-Planck Institute for Brain
Research, Frankfurt, Germany) for the gift of the cDNA
for the R1-glycine receptor. We are also grateful to the
National Institutes of Health Institute for General
Medical Sciences (Grant GM 04843 awarded to G.P.H.)
for financial support.
The quantum yield was determined as described earlier.5
Briefly, a 40 µL sample of the caged compound solution (500
µM caged compound in phosphate buffer, pH 7.0) was photo-
lyzed with repetitive laser pulses (λ ) 308, 345, 390, and 450
nm), and the concentration of the liberated 2-(dimethylamino)-
5-nitrophenol was determined spectrophotometrically (at wave-
lengths 400 and 492 nm). It is assumed that â-alanine is
produced stoichiometrically with the 2-(dimethylamino)-5-
nitrophenol. From these data the conversion to product after
the first laser pulse was calculated, allowing the direct
determination of the quantum yield together with the mea-
sured number of absorbed photons. This number was mea-
sured with an energy meter placed behind the cuvette.
J O0300643
(42) Grewer, C. Biophys. J . 1999, 77, 727-738.
(43) Chen, C.; Okayama, H. Mol. Cell. Biol. 1987, 7, 2745-2752.
(44) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F.
J . Pfluegers Arch. 1981, 391, 85-100.
(45) Krishtal, O. A.; Pidoplichko, V. I. Neuroscience 1980, 5, 2325-
2327.
(46) Niu, L.; Wieboldt, R.; Ramesh, D.; Carpenter, B. K.; Hess, G.
P. Biochemistry 1996, 35, 8136-8142.
J . Org. Chem, Vol. 68, No. 22, 2003 8367