Photoactivatable, biologically-relevant phenols with sensitivity toward 2-photon excitation

Article abstract of DOI:10.1039/c5pp00334b

Spatio-temporal release of biologically relevant small molecules provides exquisite control over the activation of receptors and signaling pathways. This can be accomplished via a photochemical reaction that releases the desired small molecule in response to irradiation with light. A series of biologically-relevant signaling molecules (serotonin, octopamine, capsaicin, N-vanillyl-nonanoylamide, estradiol, and tyrosine) that contain a phenol moiety were conjugated to the 8-bromo-7-hydroxyquinolinyl (BHQ) or 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting groups (PPGs). The CyHQ caged compounds proved sensitive toward 1PE and 2PE processes with quantum efficiencies of 0.2-0.4 upon irradiation at 365 nm and two-photon action cross sections of 0.15-0.31 GM when irradiated at 740 nm. All but one BHQ caged compound, BHQ-estradiol, were found to be sensitive to photolysis through 1PE and 2PE with quantum efficiencies of 0.30-0.40 and two photon cross sections of 0.40-0.60 GM. Instead of releasing estradiol, BHQ-estradiol underwent debromination.

Full text of DOI:10.1039/c5pp00334b

Photochemical &
Photobiological Sciences
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Photoactivatable, biologically-relevant phenols
with sensitivity toward 2-photon excitation†
Cite this: DOI: 10.1039/c5pp00334b
Duncan E. McLain,a,b Adam. C. Rea,b Magnus B. Widegrena and Timothy M. Dore*a,b
Spatio-temporal release of biologically relevant small molecules provides exquisite control over the acti-
vation of receptors and signaling pathways. This can be accomplished via a photochemical reaction that
releases the desired small molecule in response to irradiation with light. A series of biologically-relevant
signaling molecules (serotonin, octopamine, capsaicin, N-vanillyl-nonanoylamide, estradiol, and tyrosine)
that contain a phenol moiety were conjugated to the 8-bromo-7-hydroxyquinolinyl (BHQ) or 8-cyano-7-
hydroxyquinolinyl (CyHQ) photoremovable protecting groups (PPGs). The CyHQ caged compounds
proved sensitive toward 1PE and 2PE processes with quantum efficiencies of 0.2–0.4 upon irradiation at
365 nm and two-photon action cross sections of 0.15–0.31 GM when irradiated at 740 nm. All but one
BHQ caged compound, BHQ-estradiol, were found to be sensitive to photolysis through 1PE and 2PE
with quantum efficiencies of 0.30–0.40 and two photon cross sections of 0.40–0.60 GM. Instead of
releasing estradiol, BHQ-estradiol underwent debromination.
Received 6th September 2015,
Accepted 1st October 2015
DOI: 10.1039/c5pp00334b
www.rsc.org/pps
of vertebrates.1–3 Octopamine is primarily an insect neuro-
transmitter involved in reinforcing reward encoding and
Introduction
Biological effectors containing a phenol functional group are certain behaviors.4 Capsaicin, the active ingredient in hot
ubiquitous in nature. Phenols are found in neurotransmitters, peppers, is a TRPV1 ion channel agonist, and heterologously
synthetic neural agonists and antagonists, steroids, and amino expressed TRPV1 channels have been activated by in vivo appli-
acids, among other classes of compounds (Fig. 1). The physio- cation of capsaicin to effect behavioral changes.5 N-Vanillyl-
logical function of these phenols is quite broad. For example, nonanoylaminde (VNA) is an equipotent TRPV1 agonist.6
serotonin (5-HT) plays a role in the modulation of mood, appe- Steroids are involved in myriad signaling pathways7 and have
tite, and sleep, and there is a growing body of evidence that it been recently implicated in neurogenesis.8 Amino acids, tyro-
influences left-right patterning in the embryonic development sine in particular, are involved in the regulation of MAPK and
MEK/ERK through phosphorylation and dephosphorylation.9
Developing ways to artificially activate phenols in model bio-
logical systems would enable the conduct of experiments
aimed at understanding the role that they play in physiology at
the molecular level.
One method of activating biological effectors is through the
use of a photoremovable protecting group (PPG). This strategy
typically employs a covalent linkage between the PPG and a
biologically relevant functional group on the desired biological
effector; it has been used successfully to “cage” carboxylates,
phosphates, alcohols, aldehydes and ketones, diols, thiols,
amines, and phenols.10 A major limitation faced when using a
PPG on a phenol is that most PPGs require a carbonate linkage
Fig. 1 Some biologically active phenols.
between the PPG and the phenol to effect release. A common
aNew York University Abu Dhabi, PO Box 129188, Abu Dhabi, United Arab Emirates.
way around this design problem has been to instead protect a
different functional group, typically a primary or secondary
amine. In either case, this limitation necessitates a decarboxyla-
tion step from either the phenol (∼4 ms)11 or the amine
(∼0.4 s)12,13 to return the biological effector to full activity.
Direct release of phenols enables the study of biological pro-
E-mail: timothy.dore@nyu.edu
bDepartment of Chemistry, University of Georgia, Athens, Georgia, 30602, USA
†Electronic supplementary information (ESI) available: Synthetic procedures for
the preparations of 1b, 2–7, 10b, and 11–16; 1H NMR and 13C NMR spectra;
experimental procedures for determining ε, Qu, and δu; and individual photolysis
reaction progress curves. See DOI: 10.1039/c5pp00334b
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cesses faster than the rate(s) of decarboxylation; an example of
this is the ortho-carboxynitrobenzyl (o-CNB) PPG, which has
demonstrated the ability to directly release phenols with a time
constant of 16 μs, albeit with a low quantum efficiency (0.03).14
The kinetics of photolysis are important in design consider-
ations from two different standpoints. The first is in regard to
spatial resolution, and the second is in regard to the kinetics
of the biological process under investigation. Spatial resolution
in 3-dimensions is primarily achieved through two-photon
excitation (2PE): the nearly simultaneous absorbance of two
photons of light to reach an excited state restricts the volume
of excitation to the focal point of the laser, which can provide
spatial resolution down to one femtoliter.10 The exquisite
spatial resolution provided by 2PE can only be achieved when
the kinetics of photolysis are faster than the rate of diffusion
(∼3.3 × 10−6 cm2 s−1),15 otherwise diffusional spread results in
a release volume larger than the excitation volume. When pho-
tolysis kinetics are slower than the rate of diffusion, the sensi-
tivity of the PPG toward 2PE must raise accordingly, and a
reported threshold of 0.10 GM16 for biological application may
be insufficient for certain PPGs and a revised threshold of >3
GM17 may be warranted. The photorelease of the effector must
be faster than the physiological process under study. This is
akin to watching a video of sprinters in a 50 m dash at a low
vs. a high frame rate. If the frame rate is 0.1 frames per s, one
would see the runners lined up for the start and the next
image would show the racers post finish. Using a frame rate of
10 frames per s would capture nearly all of the action in
between the start and finish. Likewise, to investigate a biologi-
cal process, the photolysis reaction needs to be faster than the
action of the biological effector on the system.
The kinetics of biological processes involving the phenols
shown in Fig. 1 have been studied and occur on a range of
timescales. For example, the estrogen receptor activation rate
constant is ∼1.7 × 106 M−1 s−1 18
,
tyrosine phosphorylation by
the tyrosine kinase Lck occurs with an apparent Michaelis rate
constant kcat of 2.8 × 10−3 s−1 19
and octopamine binds to its
receptor with an on-rate constant of ∼3.2 × 105 M−1 s−1 20 Cap-
,
.
saicin induced channel opening of the TRPV1 channel has
three phases with the fastest occurring in 0.46 ms.21 There are
two major types of 5-HT receptors: the 5-HT3 receptor, a ligand
gated ion channel with kinetics of channel opening equal to
Fig. 2 Photoactivatable, biologically relevant phenols from the
literature.
4 × 102 s−1 22
and six G-protein coupled receptor subtypes
,
marinyl derivatives26 and a pyrenylmethyl PPG27 have been
reported. Several strategies for protecting capsaicin and VNA
have been developed. It has been protected at the phenol
using CNB and 7-bromo-8-hydroxycoumarinyl derivatives,28
whereas VNA has been protected at the phenol with NB,29
DMNB,29,30 NVOC,11,29 and MeOPA29,30 or the amide nitrogen
with NB or DMNB.11 There are two previously reported photo-
activatable forms of estradiol, one protected by DMNB at the
phenol and one protected by NPEC at the C-17 hydroxyl
group.31 Tyrosine has been protected at the phenol with CNV32
and the amine with PMOC.27
(5-HT1,2,4–7), which display binding on timescales in the
100–500 ms range.23
Photoactivatable versions of each of the biologically-rele-
vant phenols shown in Fig. 1 have been previously reported
(Fig. 2). The neurotransmitter 5-HT has been protected at the
phenol by the α-carboxynitrobenzyl (CNB)14 and the nitrophe-
nethyl (NPE)24 PPGs and at the amine with CNB,14 through a
carbamate linkage with NPE (Tocris cat. #3991), and as a
complex with the ruthenium bipyridyl PPG.25 More recently,
we reported the synthesis and photochemistry of an 8-bromo-
7-hydroxyquinoline (BHQ)-protected 5-HT (1a Fig. 3).3
Octopamine has not been protected on its phenol. Rather, a
carbamate linkage between the amine and several 7-aminocou-
Most of the photoactivatable phenols described in the pre-
vious paragraph photolyze with low quantum efficiencies (Qu =
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Results and discussion
We protected the biologically relevant phenols using two
hydroxyquinoline-based PPGs, BHQ and CyHQ (Scheme 1).
Compound 1a was prepared as previously reported.3 The other
compounds were synthesized from MOM-protected BHQ-OH
(8a) and CyHQ-OH (8b), which were prepared by published
protocols.36,38 The hydroxyl group on 8a or 8b was activated
with methanesulfonyl chloride to produce mesylate 9a, which
has been previously reported,3 or 9b, respectively, in high
yield. Displacement of the methanesulfonate with the desired
phenol using cesium carbonate in acetone produced each of
the protected caged phenols 10–16. Deprotection conditions
depended upon the protecting groups involved, but generally,
trimethylsilyl chloride in methanol effected global de-
protection, which was followed by HPLC purification to
provide the photoactivatable phenols 1–7.
Fig. 3 Photoactivatable, biologically relevant phenols prepared.
The caged capsaicinoids 3a and 4a were too insoluble in
neutral aqueous buffer, so BHQ-VAA (5a), which is sufficiently
soluble, was used as a surrogate for assessing the photo-
physical and photochemical properties of the photoactivatable
capsaicin and VNA. The CyHQ-protected capsaicinoids were
much more water-soluble, but for direct comparison,
CyHQ-VAA (5b) was photolyzed as a surrogate for 3b and 4b.
Both BHQ- (a series) and CyHQ-protected (b series) phenols
had a maximum wavelength of absorption near 370 nm,
although comparing the average values of ε (2480 vs. 5660 M−1
cm−1 for BHQ and CyHQ, respectively) shows that the CyHQ
series had around twice the molar absorptivity than the BHQ-
protected phenols (Table 1).
Irradiation of the a and b series of caged compounds 1, 2,
5, and 7 plus 6b with 365 nm light in KMOPS buffer (pH 7.2)
released the corresponding phenols in 55–65% yield and with
average quantum efficiencies of 0.29 for the BHQ series and
0.36 for the CyHQ series (Fig. 4). The chemical yields reported
are similar to those of a variety of other caged compounds.10
Several factors can reduce the chemical yield of the photolysis:
0.003–0.05)11,14,25,27,28,32 or the quantum efficiencies are not
reported.24,29–31 The value of λmax is less than 300 nm for the
NB-,11 CNB-,14 and NPEC-conjugated31 effectors, which raises
the potential for damaging biological tissues during photoacti-
vation experiments. The coumarinyl-protected compounds
have the highest quantum efficiencies at biologically useful
wavelengths (λmax > 365 nm),26,28 but they are linked to the
respective biological effectors through carbonate or carbamate
linkages, which significantly slows the release kinetics,12,13
because the resulting carbonate or carbamate must decarboxy-
late to reveal the active biological effector. Nitrobenzyl PPGs
also show slow release kinetics, especially with poor leaving
groups.33 Only the coumarinyl derivatives,26,28 NV-VNA,11 and
the [Ru(bpy)2(5HT)2]2+ complex25 photolyze readily by 2PE.
Hydroxyquinoline derivatives have demonstrated the ability
to efficiently release a number of biologically relevant func-
tional groups, including carboxylates, phosphates, diols, and
thiols, through 1PE and 2PE.34–37 The hydroxyquinoline PPG
has also been shown to directly release phenols without the
use of a carbonate linker,3 and the timescale, measured for
BHQ-OAc, is on the order of nanoseconds,38 thus making this
PPG suitable for use in studying temporally resolved signaling
events. By directly releasing phenols, photoactivatable versions
of relevant phenol-containing biological effectors with poten-
tially rapid release kinetics are now possible. Indeed, 5-HT was
recently caged and subsequently released in Xenopus embryos
to provide temporal resolution in the examination of left-right
patterning defects caused by abnormal distribution of the
monoamine in the developing embryo.3,39
We describe the synthesis of representative biologically
active photactivatable phenols (Fig. 3), their sensitivity toward
both 1PE and 2PE with subsequent release of the phenol, and
their hydrolytic stability in the dark. The phenols are com-
prised of 5-HT, octopamine, capsaicin, VNA, estradiol, and
tyrosine.
Scheme 1 Synthesis and photolysis of hydroxyquinoline-protected
phenols.
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Table 1 Photophysical and photochemical data for the photoactivatable phenols
Photoactivatable
phenol
Molar
Quantum
efficiency (Qu)
Sensitivity
(Qu × ε)
2-Photon uncaging
λmax (nm)
absorptivity (ε)a
action cross-section (δu)b
BHQ-O-5HT 1a3
CyHQ-O-5HT 1b
BHQ-octopamine 2a
CyHQ-octopamine 2b
BHQ-VAA 5a
368
372
368
372
370
368
369
373
366
371
2000
5700
2700
5400
2700
6100
2400
6200
2600
4900
0.30
0.39
0.36
0.34
0.18
0.41
—
0.29
0.32
0.38
600
2223
972
1944
486
2501
—
1798
832
0.50
0.23
0.49
0.18
0.61
0.24
—
0.24
0.61
0.36
CyHQ-VAA 5b
BHQ-estradiol 6a
CyHQ-estradiol 6b
BHQ-tyrosine 7a
CyHQ-tyrosine 7b
1862
a M−1 cm−1, λ = 370 nm. b GM = 10−50 cm4 s per photon.
absorptivity (Qu × ε) was much greater for the CyHQ-protected
compounds, owing to their much larger extinction coefficients.
BHQ-protected estradiol 6a did not photolyze to the
expected products of 17a and estradiol. Instead, debromina-
tion of 6a was the primary reaction pathway, a phenomenon
that has been observed previously.41 To further investigate the
debromination reaction, we irradiated at 365 nm a model com-
pound, BHQ-OH (17a), which did not undergo photoinduced
cleavage of the C–O bond, but lost bromine instead with a
quantum efficiency of 0.12 in pH 7.2 KMOPS buffer. The
extent of debromination in the BHQ series of photoactivatable
phenols can be significant. A factor that contributes to the
extent of debromination is the leaving group ability, and
effectors that are poor leaving groups (high pKa) conjugated to
PPGs tend to be inert.10 The calculated pKa of estradiol was
found to be 10.27
0.60 (ACD/Labs version 11.02 accessed
through SciFinder). All of the other phenols have calculated
pKa values lower than this value (10.09 0.40 for 5-HT, 9.97
0.26 for octopamine, 10.02 0.31 for VAA, and 9.09 0.10 for
tyrosine), which might be the threshold for leaving group
ability for BHQ-protected effectors. The ACD/Labs-calculated
values were used to provide an estimated pKa of the phenols
under identical conditions and avoid issues arising from the
variations in conditions found in the literature reporting pKa
values from experimental titrations.
Sufficient hydrolytic stability of the PPG-effector conjugate
is required, so that there is no problem with leakage of the
activated effector prior to photoactivation. Experiments where
the caged compound is subjected to aqueous environments
for hours or days require robust resistance to hydrolysis in the
dark. Such experiments, might include those that use CyHQ-
estradiol for neurogenesis or gene regulation studies or BHQ/
CyHQ-tyrosine when controlling kinase cascades via phos-
phorylation of tyrosine residues. All of the compounds were
examined for their ability to resist hydrolysis under simulated
physiological conditions. Solutions of the relevant compound
(100 μM) in KMOPS pH 7.2 were prepared, stored in the dark
at room temperature, analyzed periodically over a period of
1 week, and in all cases showed minimal (<3%) hydrolysis over
the measurement time frame.
Fig. 4 Time courses for the photolysis of the photoactivatable phenols
at 365 nm (1PE) and the rise of the biologically active phenols. Top: BHQ
series, Bottom: CyHQ series. The percentage remaining is the average of
at least 3 runs, and the error bars represent the standard deviation of the
measurement. Lines are least-squares fits of a single exponential decay
or a single exponential rise to max.
the caged compounds might undergo undesired photo-
reactions,38 the effectors might possess some photosensitivity
and react further to form secondary photoproducts,40 or un-
desired secondary photoproducts might be generated through the
interaction of the spent PPG and the biological effector, or a com-
bination of all of these. The BHQ- and CyHQ-protected phenols
have similar quantum efficiencies, but the sensitivity, which is
quantified by multiplying the quantum efficiency by the molar
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VNA, estradiol, and tyrosine). The CyHQ-protected phenols
were more sensitive to 1PE owing to their larger molar absorp-
tivities, whereas the BHQ-derivatives were more sensitive to
2PE. Instead of releasing estradiol, BHQ-estradiol was efficien-
tly debrominated upon exposure to 365 nm light. This photo-
chemical reaction pathway dominates when the attached
effector is a poor leaving group. Alternative reaction pathways
were not observed with the CyHQ-protected phenols. The syn-
thesis of the XHQ-phenol conjugates is modular and can be
applied to any biologically relevant phenol, not just the ones
described here. The photoactivatable phenols reported will
enable the examination of their associated biological processes
at the cellular and molecular level. BHQ-O-5HT (1a) has
already been used to study the role of 5-HT in left/right asym-
metry in the developing vertebrate embryo.3,39 Biological appli-
cations of the other photoactivatable phenols will be reported
in due course.
Experimental
Synthesis
General procedures. MOM-protected BHQ-OH (8a)38 and
CyHQ-OH (8b)36 were prepared as previously described, as
were 1a, 9a, and 10a.3 A representative experimental procedure
for preparing a photoactivatable phenol is given below. Experi-
mental procedures for preparing each caged phenol can be
found in the ESI.† uHPLC and HPLC were carried out on
Agilent 1290 and 1260 systems, respectively, with Zorbax
Eclipse C-18 reverse phase columns.
Fig. 5 Reaction progress curves for the photolysis of the photoactivata-
ble phenols at 740 nm (2PE). Top: BHQ series, Bottom: CyHQ series.
The percentage remaining is the average of at least 3 runs, and the error
bars represent the standard deviation of the measurement. Lines are
least-squares fits of a single exponential decay or a single exponential
rise to max.
(8-Cyano-7-(methoxymethoxy)quinolin-2-yl)methyl methane-
sulfonate (9b). Alcohol 8b (0.31 g, 1.2 mmol) was stirred in
THF (5 mL). Methanesulfonyl chloride (0.189 ml, 2.44 mmol)
and triethylamine (0.51 ml, 3.7 mmol) were added. The reac-
tion progress was monitored by uHPLC, and upon completion,
celite was added to the reaction mixture, which was sub-
sequently dried in vacuo, loaded onto a column and purified
via flash chromatography using EtOAc/hexane (1 : 2) to provide
9b as a white powder (0.132 g, 0.408 mmol, 34%): 1H NMR
(400 MHz, CDCl3) δ 8.23 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 9.2 Hz,
1H), 7.58 (dd, J = 12.6, 8.8 Hz, 2H), 5.55 (s, 2H), 5.47 (s, 2H),
3.59 (s, 3H), 3.29 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 162.5,
157.0, 148.2, 137.5, 133.7, 122.9, 119.1, 116.3, 114.5, 99.5, 95.1,
To assess the sensitivity of the photoactivatable phenols to
2PE, 1, 2, 5, 6b, and 7 were photolyzed at 740 nm using a
femtosecond-pulsed, mode-locked Ti:sapphire laser. HPLC or
uHPLC was used to monitor the reaction progress and from
the decay curves (Fig. 5) and the 2PE-generated fluorescence
output of fluorescein as an external standard, the 2-photon
uncaging action cross-sections (δu) were calculated as pre-
viously described (Table 1).3,16 The BHQ derivatives were more
sensitive to 2PE than the CyHQ derivatives, having average
values of δu of 0.55 GM compared to 0.25 GM for the CyHQ
derivatives. The cross-sections reported are similar to that of
previously reported quinoline- and coumarin-based PPGs and
significantly higher than most nitrobenzyl and nitroindoline
derivatives.10,42–44 BHQ and CyHQ are sufficiently sensitive to
be applied to the photoactivation of biologically relevant
phenols in vivo through 2PE.
71.6, 56.9, 38.5; HRMS-ESI (m/z) [M
C14H14N2O5S 323.06962; found, 323.06966.
tert-Butyl(2-(4-((8-cyano-7-(methoxymethoxy)quinolin-2-yl)-
methoxy)phenyl)-2-hydroxyethyl)carbamate (11b). tert-Butyl
+
H]+ calcd for
(2-hydroxy-2-(4-hydroxyphenyl)ethyl)carbamate
(0.043
g,
0.17 mmol) and 9b (0.050 g, 0.16 mmol) were stirred in
acetone (1 mL). Cs2CO3 (0.10 g, 0.33 mmol) was added, and
the reaction progress was monitored by uHPLC. Upon com-
pletion, brine was added and the resulting mixture was
Conclusion
The sensitivity of the hydroxyquinoline PPGs BHQ and CyHQ extracted with EtOAc. The extract was dried over sodium
toward 2PE, combined with sub-microsecond release kinetics sulfate, concentrated in vacuo and purified via flash chromato-
provided an opportunity to create a variety of photoactivatable graphy eluting with EtOAc/hexane (1 : 2), which yielded 11b
phenolic biological effectors (5-HT, octopamine, capsaicin, as a yellow residue upon drying (0.025 g, 0.052 mmol, 31%):
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1H NMR (600 MHz, CD3OD) δ 9.05 (d, J = 8.3 Hz, 1H), 8.26 (d, methods using fluorescein as an external standard.34 See ESI†
J = 9.1 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), for detailed experimental procedure.
7.27 (d, J = 7.9 Hz, 2H), 6.82 (d, J = 7.9 Hz, 2H), 5.27 (s, 2H),
4.82 (dd, J = 11.9, 3.7 Hz, 1H), 3.35 (s, 3H), 3.59–3.20 (m, 2H),
Hydrolysis in the dark
1.53 (s, 9H); 13C NMR (151 MHz, CD3OD) δ 167.6, 162.0, 157.3,
Substrates (100 μM) were dissolved in KMOPS and stored in
the dark at room temperature. Aliquots (20 μL or 5 μL) were
removed and analyzed periodically by HPLC or uHPLC,
147.0, 145.5, 140.8, 135.5, 131.6, 126.9, 122.6, 120.0, 117.6,
115.0, 111.9, 97.1, 88.9, 85.0, 69.3, 60.7, 53.9, 46.0, 26.2;
HRMS-ESI (m/z) [M + H]+ calcd for C26H29N3O6 480.21291;
respectively, as described for one-photon photolysis. None of
found, 480.21438.
the compounds synthesized displayed a measurable rate of
2-((4-(2-Amino-1-hydroxyethyl)phenoxy)methyl)-7-hydroxy-
dark hydrolysis over an approximately 100 h time period.
quinoline-8-carbonitrile (2b). Compound 11b (0.025 g,
0.052 mmol) was dissolved in methanol (1 mL). TMSCl
(0.115 mL, 0.918 mmol) was added. The resulting solution was
stirred in the dark and monitored by uHPLC. Upon com-
pletion, the reaction was concentrated in vacuo and purified by
HPLC using a 10 minute gradient from 5% CH3CN/95% H2O
Foundation (CHE-1012412, CHE-1317760) and the National
(0.1% TFA) to 100% CH3CN. Fractions containing only one
Acknowledgements
This work was supported by grants from the National Science
Institutes of Health (R01NS070159). The research was carried
out using Core Technology Platform resources at New York
University Abu Dhabi and mass spectrometry resources at the
Proteomics and Mass Spectrometry Core Facility at the Univer-
peak were combined and concentrated in vacuo to provide 2b
as a pale yellow solid (0.014 g, 0.042 mmol, 80% yield): 1H
NMR (600 MHz, CD3OD) δ 8.27 (d, J = 8.4 Hz, 1H), 8.01 (d, J =
9.1 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H),
sity of Georgia.
6.82 (d, J = 8.4 Hz, 2H), 5.03 (s, 2H), 4.81 (d, J = 6.5 Hz, 1H),
4.81 (d, J = 12.7 Hz, 1H), 3.12–3.02 (m, 2H); 13CNMR
(600 MHz, CD3OD) δ 163.6, 157.3, 148.4, 137.0, 133.7, 131.6,
126.8, 121.3, 117.7, 116.8, 115.1, 115.0, 94.0, 69.3, 64.7, 47.1,
45.9; HRMS-ESI (m/z) [M
Notes and references
+
H]+ calcd for C19H17N3O3
336.13427; found, 336.13471.
1 M. Levin, G. A. Buznikov and J. M. Lauder, Of Minds and
Embryos: Left-Right Asymmetry and the Serotonergic Con-
trols of Pre-Neural Morphogenesis, Dev. Neurosci., 2006, 28,
171–185.
Photochemistry
Determination of the quantum efficiency for one-photon
photolysis (Qu). Briefly, solutions (100 μM) of the relevant sub-
strate in KMOPS pH 7.2 were irradiated in quartz cuvettes (21-
Q-10, Starna, Atascadero, CA) with a 365 10 nm LED (Cairn
OptoLED Lite). Aliquots (25 μL) were removed at various time
points and analyzed by uHPLC (MeCN/H2O 0.1% TFA: 6 min
gradient 5% CH3CN to 100% CH3CN, 2 min gradient 100%
CH3CN to 5% CH3CN), using an external standard to deter-
mine concentrations. The reaction progress was plotted and
the data fit to a single exponential decay function and Qu
values were calculated according to previously reported proto-
cols.34 See ESI† for detailed experimental procedure.
Determination of the two-photon uncaging action cross-
section (δu). Solutions (100 μM) of the substrates in KMOPS
were prepared and stored in the dark. Aliquots (25 μL) of this
solution were placed in a microcuvette (10 × 1 × 1 mm illumi-
nated dimensions, 25 μL effective filling volume) and irra-
diated with a fs-pulsed and mode-locked Ti:Sapphire laser
(Chameleon Ultra II, Coherent or a Mai Tai HP DeepSee,
Spectra-Physics) with 740 nm light at an average power of
220–300 mW measured after passing through the cuvette.
Three samples of each substrate were irradiated for various
time periods and analyzed by HPLC or uHPLC as described for
one-photon experiments. The reaction progress was plotted
and the data fit to a single exponential decay function and δu
values were calculated according to previously reported
2 L. N. Vandenberg and M. Levin, Far from solved: a pers-
pective on what we know about early mechanisms of left-
right asymmetry, Dev. Dyn., 2010, 239, 3131–3146.
3 A. C. Rea, L. N. Vandenberg, R. E. Ball, A. A. Snouffer,
A. G. Hudson, Y. Zhu, D. E. McLain, L. L. Johnston,
J. D. Lauderdale, M. Levin and T. M. Dore, Light Activated
Serotonin for Exploring Its Action in Biological Systems,
Chem. Biol., 2013, 20, 1536–1546.
4 T. Roeder, Tyramine and octopamine: ruling behavior and
metabolism, Annu. Rev. Entomol., 2005, 50, 447–477.
5 A. D. Guler, A. Rainwater, J. G. Parker, G. L. Jones,
E. Argilli, B. R. Arenkiel, M. D. Ehlers, A. Bonci,
L. S. Zweifel and R. D. Palmiter, Transient activation of
specific neurons in mice by selective expression of the cap-
saicin receptor, Nat. Commun., 2012, 3, 1749.
6 C. S. J. Walpole and R. Wrigglesworth, in Capsaicin in the
Study of Pain, ed. J. N. Wood, Academic Press, London,
1993, pp. 63–81.
7 E. R. Prossnitz, J. B. Arterburn, H. O. Smith, T. I. Oprea,
L. A. Sklar and H. J. Hathaway, Estrogen signaling through
the transmembrane G protein-coupled receptor GPR30,
Annu. Rev. Physiol., 2008, 70, 165–190.
8 S. Suzuki, L. M. Gerhold, M. Bottner, S. W. Rau, C. Dela
Cruz, E. Yang, H. Zhu, J. Yu, A. B. Cashion, M. S. Kindy,
I. Merchenthaler, F. H. Gage and P. M. Wise, Estradiol
enhances neurogenesis following ischemic stroke through
Photochem. Photobiol. Sci.
This journal is © The Royal Society of Chemistry and Owner Societies 2015

View Article Online
Photochemical & Photobiological Sciences
Paper
estrogen receptors α and β, J. Comp. Neurol., 2007, 500, 21 M. Raisinghani, R. M. Pabbidi and L. S. Premkumar, Acti-
1064–1075.
vation of transient receptor potential vanilloid 1 (TRPV1)
9 A. De Luca, M. R. Maiello, A. D’Alessio, M. Pergameno and
by resiniferatoxin, J. Physiol., 2005, 567, 771–786.
N. Normanno, The RAS/RAF/MEK/ERK and the PI3 K/AKT 22 K. Solt, D. Ruesch, S. A. Forman, P. A. Davies and
signalling pathways: role in cancer pathogenesis and impli-
cations for therapeutic approaches, Expert Opin. Ther.
Targets, 2012, 16, S17–S27.
D. E. Raines, Differential effects of serotonin and dopa-
mine on human 5-HT3A receptor kinetics: interpretation
within an allosteric kinetic model, J. Neurosci., 2007, 27,
13151–13160.
10 P. Klan, T. Solomek, C. G. Bochet, A. Blanc, R. Givens,
M. Rubina, V. Popik, A. Kostikov and J. Wirz, Photoremo- 23 J.-P. Vilardaga, G. Romero, T. N. Feinstein and V. L. Wehbi,
vable Protecting Groups in Chemistry and Biology: Reac-
tion Mechanisms and Efficacy, Chem. Rev., 2013, 113,
119–191.
Kinetics and dynamics in the G protein-coupled receptor
signaling cascade, Methods Enzymol., 2013, 522, 337–363.
24 Y. O. Boahen and G. M. MacDonald, A concise approach to
caged serotonin for Fourier transform infrared (FT-IR)
difference photolysis studies, J. Ghana Sci. Assoc., 2005, 7,
54–59.
25 L. Zayat, M. Salierno and R. Etchenique, Ruthenium(II)
Bipyridyl Complexes as Photolabile Caging Groups for
Amines, Inorg. Chem., 2006, 45, 1728–1731.
26 J. Schaal, B. Dekowski, B. Wiesner, J. Eichhorst, K. Marter,
C. Vargas, S. Keller, N. Eremina, A. Barth, A. Baumann,
D. Eisenhardt and V. Hagen, Coumarin-Based Octopamine
Phototriggers and their Effects on an Insect Octopamine
Receptor, ChemBioChem, 2012, 13, 1458–1464.
11 J. Zhao, T. D. Gover, S. Muralidharan, D. A. Auston,
D. Weinreich and J. P. Y. Kao, Caged Vanilloid Ligands for
Activation of TRPV1 Receptors by 1- and 2-Photon Exci-
tation, Biochemistry, 2006, 45, 4915–4926.
12 G. Papageorgiou and J. E. T. Corrie, Synthesis and pro-
perties of carbamoyl derivatives of photolabile benzoins,
Tetrahedron, 1997, 53, 3917–3932.
13 G. Papageorgiou, A. Barth and J. E. T. Corrie, Flash photo-
lytic release of alcohols from photolabile carbamates or
carbonates is rate-limited by decarboxylation of the photo-
product, Photochem. Photobiol. Sci., 2005, 4, 216–220.
14 H.-G. A. Breitinger, R. Wieboldt, D. Ramesh, 27 M. J. G. Fernandes, S. P. G. Costa and M. S. T. Goncalves,
B. K. Carpenter and G. P. Hess, Synthesis and Characteriz-
ation of Photolabile Derivatives of Serotonin for Chemical
Kinetic Investigations of the Serotonin 5-HT3 Receptor, Bio-
chemistry, 2000, 39, 5500–5508.
Synthesis and light triggered release of catecholamines
from pyrenylmethyl carbamate cages, New J. Chem., 2013,
37, 2369–2376.
28 D. Gilbert, K. Funk, B. Dekowski, R. Lechler, S. Keller,
F. Moehrlen, S. Frings and V. Hagen, Caged capsaicins:
new tools for the examination of TRPV1 channels in soma-
tosensory neurons, ChemBioChem, 2007, 8, 89–97.
15 P. Xia, P. M. Bungay, C. C. Gibson, O. N. Kovbasnjuk and
K. R. Spring, Diffusion coefficients in the lateral intercellu-
lar spaces of Madin-Darby canine kidney cell epithelium
determined with caged compounds, Biophys. J., 1998, 74, 29 M. P. Van Ryssen, N. Avlonitis, R. Giniatullin,
3302–3312.
C. McDougall, J. L. Carr, M. N. Stanton-Humphreys,
E. L. A. Borgstroem, C. T. A. Brown, D. Fayuk, A. Surin,
M. Niittykoski, L. Khiroug and S. J. Conway, Synthesis, pho-
tolysis studies and in vitro photorelease of caged TRPV1
agonists and antagonists, Org. Biomol. Chem., 2009, 7,
4695–4707.
16 T. Furuta, S. S. H. Wang, J. L. Dantzker, T. M. Dore,
W. J. Bybee, E. M. Callaway, W. Denk and R. Y. Tsien, Bro-
minated 7-hydroxycoumarin-4-ylmethyls: photolabile pro-
tecting groups with biologically useful cross-sections for
two photon photolysis, Proc. Natl. Acad. Sci. U. S. A., 1999,
96, 1193–1200.
17 N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston and
D. Ogden, The efficiency of two-photon photolysis of a
“caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in
30 J. L. Carr, K. N. Wease, M. P. Van Ryssen, S. Paterson,
B. Agate, K. A. Gallagher, C. T. A. Brown, R. H. Scott and
S. J. Conway, In vitro photo-release of a TRPV1 agonist,
Bioorg. Med. Chem. Lett., 2006, 16, 208–212.
relation to photodamage of synaptic terminals, Eur. 31 K.-i. Hayashi, K. Hashimoto, N. Kusaka, A. Yamazoe,
Biophys. J., 2002, 30, 588–604.
H. Fukaki, M. Tasaka and H. Nozaki, Caged gene-inducer
spatially and temporally controls gene expression and plant
development in transgenic Arabidopsis plant, Bioorg. Med.
Chem. Lett., 2006, 16, 2470–2474.
18 B. M. Weichman and A. C. Notides, Analysis of estrogen
receptor activation by its [3H]estradiol dissociation kine-
tics, Biochemistry, 1979, 18, 220–225.
19 H. R. Housden, P. J. S. Skipp, M. P. Crump, 32 A. G. Russell, M. J. Sadler, H. J. Laidlaw, A. Gutierrez-Lor-
R. J. Broadbridge, T. Crabbe, M. J. Perry and M. G. Gore,
Investigation of the kinetics and order of tyrosine phos-
phorylation in the T-cell receptor ζ chain by the protein
tyrosine kinase Lck, Eur. J. Biochem., 2003, 270, 2369–
2376.
iente, C. W. Wharton, D. Carteau, D. M. Bassani and
J. S. Snaith, Photorelease of tyrosine from α-carboxy-6-nitro-
veratryl (αCNV) derivatives, Photochem. Photobiol. Sci., 2012,
11, 556–563.
33 Y. V. Il’ichev, M. A. Schwoerer and J. Wirz, Photochemical
Reaction Mechanisms of 2-Nitrobenzyl Compounds:
Methyl Ethers and Caged ATP, J. Am. Chem. Soc., 2004, 126,
4581–4595.
20 Y. Dudai, High-affinity octopamine receptors revealed in
Drosophila by binding of [3H]octopamine, Neurosci. Lett.,
1982, 28, 163–167.
This journal is © The Royal Society of Chemistry and Owner Societies 2015
Photochem. Photobiol. Sci.

View Article Online
Paper
Photochemical & Photobiological Sciences
34 O. D. Fedoryak and T. M. Dore, Brominated hydroxyquino- 39 L. N. Vandenberg, D. J. Blackiston, A. C. Rea, T. M. Dore
line as a photolabile protecting group with sensitivity to
multiphoton excitation, Org. Lett., 2002, 4, 3419–3422.
35 Y. Zhu, C. M. Pavlos, J. P. Toscano and T. M. Dore,
and M. Levin, Left-right patterning in Xenopus conjoined
twin embryos requires serotonin signaling and gap junc-
tions, Int. J. Dev. Biol., 2014, 58, 799–809.
8-Bromo-7-hydroxyquinoline as a Photoremovable Protect- 40 M. L. Gostkowski, R. Allen, M. L. Plenert, E. Okerberg,
ing Group for Physiological Use: Mechanism and Scope,
M. J. Gordon and J. B. Shear, Multiphoton-excited seroto-
J. Am. Chem. Soc., 2006, 128, 4267–4276.
nin photochemistry, Biophys. J., 2004, 86, 3223–3229.
36 M. J. Davis, C. H. Kragor, K. G. Reddie, H. C. Wilson, 41 J. Ma, S. C. Cheng, H. An, M.-D. Li, C. Ma, A. C. Rea,
Y. Zhu and T. M. Dore, Substituent Effects on the Sensi-
tivity of a Quinoline Photoremovable Protecting Group to
One- and Two-Photon Excitation, J. Org. Chem., 2009, 74,
1721–1729.
Y. Zhu, J. L. Nganga, T. M. Dore and D. L. Phillips, Com-
parison of the Absorption, Emission, and Resonance
Raman Spectra of 7-Hydroxyquinoline and 8-Bromo-7-
Hydroxyquinoline Caged Acetate, J. Phys. Chem. A, 2011,
115, 11632–11640.
37 D. Abate-Pella, N. A. Zeliadt, J. D. Ochocki, J. K. Warmka,
T. M. Dore, D. A. Blank, E. V. Wattenberg and 42 D. Warther, S. Gug, A. Specht, F. Bolze, J. F. Nicoud,
M. D. Distefano, Photochemical Modulation of Ras-
Mediated Signal Transduction Using Caged Farnesyltrans-
ferase Inhibitors: Activation via One- and Two-Photon Exci-
tation, ChemBioChem, 2012, 13, 1009–1016.
A. Mourot and M. Goeldner, Two-photon uncaging: New
prospects in neuroscience and cellular biology, Bioorg.
Med. Chem., 2010, 18, 7753–7758.
43 T. M. Dore and H. C. Wilson, in Photosensitive Molecules for
Controlling Biological Function, ed. J. J. Chambers and
R. H. Kramer, Humana Press, New York, 2011, pp. 57–92.
38 J. Ma, A. C. Rea, H. An, C. Ma, X. Guan, M.-D. Li, T. Su,
C. S. Yeung, K. T. Harris, Y. Zhu, J. L. Nganga,
O. D. Fedoryak, T. M. Dore and D. L. Phillips, Unraveling 44 G. Bort, T. Gallavardin, D. Ogden and P. I. Dalko, From
the Mechanism of the Photodeprotection Reaction of
8-Bromo- and 8-Chloro-7-hydroxyquinoline Caged Acetates,
Chem. – Eur. J., 2012, 18, 6854–6865.
One-Photon to Two-Photon Probes: “Caged” Compounds,
Actuators, and Photoswitches, Angew. Chem., Int. Ed., 2013,
52, 4526–4537.
Photochem. Photobiol. Sci.
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