Two-photon sensitive protecting groups operating via intramolecular electron transfer: Uncaging of GABA and tryptophan

Article abstract of DOI:10.1039/c4sc03775h

Improved photo-labile protecting groups, with high sensitivity to two-photon excitation, are needed for the controlled release of drugs, as tools in neuroscience and physiology. Here we present a new modular approach to the design of caging groups based on photoinduced electron transfer from an electron-rich two-photon dye to an electron acceptor, followed by scission of an ester to release a carboxylic acid. Three different electron acceptors were tested: nitrobenzyl, phenacyl and pyridinium. The nitrobenzyl system was ineffective, giving only photochemical decomposition and no release of the carboxylic acid. The phenacyl system also performed poorly, liberating the carboxylic acid in 20% chemical yield and 0.2% photochemical yield. The pyridinium system was most successful, and was tested for the release of two carboxylic acids: γ-amino butyric acid (GABA) and tryptophan. The caged GABA undergoes photochemical cleavage with a chemical yield of >95% and a photochemical yield of 1%; it exhibits a two-photon absorption cross section of 1100 GM at 700 nm, corresponding to a two-photon uncaging cross section of 10 ± 3 GM. This journal is

Full text of DOI:10.1039/c4sc03775h

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Two-photon sensitive protecting groups operating
via intramolecular electron transfer: uncaging of
GABA and tryptophan†
Cite this: Chem. Sci., 2015, 6, 2419
Karolina A. Korzycka,‡a Philip M. Bennett,‡a Eduardo Jose Cueto-Diaz,b
Geoffrey Wicks,c Mikhail Drobizhev,c Mireille Blanchard-Desce,b Aleksander Rebanecd
a
*
and Harry L. Anderson
Improved photo-labile protecting groups, with high sensitivity to two-photon excitation, are needed for the
controlled release of drugs, as tools in neuroscience and physiology. Here we present a new modular
approach to the design of caging groups based on photoinduced electron transfer from an electron-rich
two-photon dye to an electron acceptor, followed by scission of an ester to release a carboxylic acid.
Three different electron acceptors were tested: nitrobenzyl, phenacyl and pyridinium. The nitrobenzyl
system was ineffective, giving only photochemical decomposition and no release of the carboxylic acid.
The phenacyl system also performed poorly, liberating the carboxylic acid in 20% chemical yield and
0.2% photochemical yield. The pyridinium system was most successful, and was tested for the release of
two carboxylic acids: g-amino butyric acid (GABA) and tryptophan. The caged GABA undergoes
photochemical cleavage with a chemical yield of >95% and a photochemical yield of 1%; it exhibits a
two-photon absorption cross section of 1100 GM at 700 nm, corresponding to a two-photon uncaging
cross section of 10 ꢀ 3 GM.
Received 6th December 2014
Accepted 2nd February 2015
DOI: 10.1039/c4sc03775h
www.rsc.org/chemicalscience
allows the use of near-IR wavelengths which diminishes photo-
damage and improves tissue penetration by reducing scattering
Introduction
and avoiding absorption by natural pigments. Unfortunately,
most protecting groups optimized for one-photon uncaging
display low efficiency under two-photon excitation, because
their chromophores lack the specic features required for effi-
cient TPA. The process of uncaging can be divided into two
steps: light absorption followed by bond scission. The efficiency
of TPA is quantied by the TPA cross-section (da), while the
quantum yield of uncaging (fu) measures the efficiency of
photo-induced bond scission. The product of these two
parameters, the two-photon uncaging cross-section (du ¼ dafu)
is a gure of merit reecting the overall sensitivity of a pro-
tecting group to two-photon uncaging. Typically, du values
reported to date lie between 0.05 and 2.5 GM (ref. 2c, 6, 7) (1 GM
¼ 1050 cm4 s per photon) whereas du > 3 GM is desired for
efficient uncaging in living cells.8 A common strategy for
enhancing the TPA cross-section of a pre-existing protecting
platform is to extend the p-system and modulate the strength of
electron donating/accepting substituents. Dipolar, quadrupolar
and octupolar architectures have been explored for functional-
izing established caging groups such as coumarine,9 o-nitro-
benzyl,10,11c 2-(o-nitrophenyl)propyl,11 phenacyl11c and quinoline12
with vinyl, phenyl, styryl, dihydronaphthalenyl, thienyl, uorenyl
and triphenylamine groups. The strategy of incorporating
an existing protecting group into a conjugated donor–p–
acceptor system led to a dipolar protecting group within the
Light-sensitive protecting groups have continued to gain
importance as tools for investigating the role of physiologically
active compounds, ever since they were rst applied in biolog-
ical system in 1978.1 A number of UV- and visible-light cleavable
cages have been developed2 to allow rapid spatially and
temporally controlled photo-release of various biomolecules
within living cells.3 In the 1990s, the concept of uncaging was
extended to take advantage of two-photon absorption (TPA),4,5
a
nonlinear optical phenomenon in which excitation occurs by
the simultaneous absorption of two photons, each having half
the energy of the corresponding one-photon process. The main
advantage of TPA is that excitation is effectively restricted to the
focal volume, giving tight spatial control. Furthermore, TPA
aOxford University, Department of Chemistry, Chemistry Research Laboratory, 12
Manseld Road, Oxford, OX1 3TA, UK. E-mail: harry.anderson@chem.ox.ac.uk; Fax:
+44 (0)1865 285002; Tel: +44 (0)1865 275704
b
´
´
Universite de Bordeaux, Institut des Sciences Moleculaires, CNRS UMR 5255, 33400
Bordeaux, France
cDepartment of Physics, Montana State University, Bozeman, MT 59717, USA
dNational Institute of Chemical Physics and Biophysics, Tallinn 12618, Estonia
† Electronic supplementary information (ESI) available: One- and two-photon
absorption
spectra,
emission
spectra,
synthetic
procedures
and
characterization, square-wave and cyclic voltammetry, photolysis protocols. See
DOI: 10.1039/c4sc03775h
‡ K.A.K. and P.M.B. made equal contribution to this work.
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2-(o-nitrophenyl)propyl series with a record du of 11 GM at 800 was chosen as a potential electron-acceptor, since it has been
nm.11a However, as absorption and bond scission are inherently demonstrated to release carboxylic acids upon PeT.13b,c,19 Fig. 1b
related, any alteration in the caging group inuences both da shows the proposed mechanism of photo-deprotection. Light
and fu. Therefore some structurally modied cages, such as absorption generates a photosensitizer-based singlet state,
BNSF (2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-diox- which is quenched by electron transfer to the release group.
aheptyl)]-uorene, du of 5 GM at 800 nm, 65% yield of unca- The resulting charge-shied state decays by s-bond cleavage
ging),11b suffer from light-induced side reactions or decreased to liberate the physiologically active carboxylic acid. The
yield of release, compared to their parent protecting units. Since symmetrical design was chosen to simplify the synthesis, while
it proves extremely difficult to enhance da while preserving a the aniline unit was substituted with heptaethyleneglycol
high value of du, several attempts have been made to explore an chains to promote solubility in aqueous media. A model dye-
alternative approach, in which the absorption and release steps unit BEF-OH was synthesized for the purpose of photophysical
are decoupled and occur in different parts of the caging plat- and electrochemical studies (Fig. 2). We used caged tryptophan,
form. A modular design allows each process to be optimized BEF-Pyr-Trp, for testing the intramolecular PeT mediated
independently. This concept was rst demonstrated for one- uncaging mechanism. L-Tryptophan (Trp) was selected as a
photon photolysis, where two spatially separated steps of model amino acid due to the presence of an indole chromo-
uncaging were linked by intramolecular photoinduced electron phore that allows release to be quantied by HPLC, with UV
transfer (PeT)13 or triplet sensitization.14 Recently this strategy detection. We also prepared caged g-amino butyric acid (GABA),
has been implemented in two-photon uncaging systems: the BEF-Pyr-GABA, to explore the utility of our protecting group for
uorenyl–nitroindolinyl derived protecting group (du of 0.5 GM release of an inhibitory neurotransmitter.
at 730 nm), where the absorption step is followed by intra-
molecular energy transfer-mediated release15 and 2-(o-nitro-
Results and discussion
Synthesis
phenyl)propyl–thioxanthone with intermolecular FRET (du of
0.86 GM at 766 nm).16 Here, we report a study of PeT-mediated
uncaging in a two-photon excitable system.17 Drawing upon
The reference dye BEF-OH was prepared as shown in Scheme 1.
previously reported designs,13 we devised a protecting group,
the removal of which operates via intramolecular PeT between a
photoexcited electron-donor (a TPA dye with high da) and an
electron-acceptor (pre-existing release unit) to achieve efficient
release of physiologically active compounds (Fig. 1a).
The synthesis started from N-(4-iodophenyly)diethanolamine20
(1) by coupling with TIPS-acetylene to give intermediate 2,
which was subsequently alkylated with hexaethyleneglycol
monomethyl ether tosylate. Removal of TIPS group from 3
resulted in the key aniline intermediate 4, which was coupled
with the central uorene diiodide 5 (see ESI†) under Sonoga-
shira conditions to give BEF-OH. In a convergent synthesis
towards GABA and tryptophan derivatives, the uorene core was
appended with the pyridinium esters before conjugation with
the substituted aniline units 4 using Sonogashira cross-
coupling (Scheme 2). The nal step involved removal of the Boc
protecting groups to yield the triuoroacetate salts BEF-Pyr-
GABA and BEF-Pyr-Trp.
Our studies began with choosing a suitable electron-donor
with high da. We selected a symmetric banana-shaped bise-
thynyl uorene (BEF) dye, in which the core is extended with
substituted anilines via acetylene bridges.18 A pyridinium salt
One- and two-photon absorption and uorescence spectra
The one-photon absorption and emission spectra of BEF-OH in
water and polar organic solvents (EtOH and THF) are shown in
Fig. 3. The uorene-based dye exhibits a strong absorption at
Fig.
photoinduced electron transfer. Upon two-photon excitation (i) the
dye unit donates an electron to the release unit (ii), which undergoes Fig. 2 Structure of model electron-donor BEF-OH, caged tryptophan,
1 Concept of a photolabile protecting group operating via
photochemical reaction and liberates the drug (iii).
BEF-Pyr-Trp, and caged GABA, BEF-Pyr-GABA; R ¼ (CH2CH2O)7CH3.
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300–400 nm (maximum: 380 nm; 3380 ¼ 9.0 ꢁ 104 Mꢂ1 cmꢂ1).
Comparison of the absorption spectra in water and organic
solvents reveals that the one-photon absorption properties of
BEF-OH are independent of the solvent polarity. However, the
uorescence spectrum is sensitive to the environment, being
red-shied and broadened in polar solvents. The Stokes shis
are 78, 83 and 128 nm in THF, EtOH and water, respectively.
Bathochromic shis in the luminescence spectra of banana-
shaped uorene-dyes have previously been attributed to
symmetry breaking and the formation of a polar excited state,
which is stabilized in polar solvents.21 The uorescence
Scheme 1 Reagents: (i) TIPS-acetylene, Pd(OAc)2, PPh3, CuI, DIPA, 50
ꢃC, 16 h, (ii) CH3(OCH2CH2)6OTs, NaH, THF, reflux, 48 h, (iii) TBAF, THF,
20 ꢃC, 12 h, (iv) 4, Pd(OAc)2, PPh3, CuI, DIPA, MeCN, 20 ꢃC, 3 h; Ts ¼ p-
toluenesulfonate, TIPS ¼ triisopropylsilyl, THF ¼ tetrahydrofuran, DIPA quantum yield (ff) of BEF-OH is about 0.42 in organic solvents
¼ diisopropylamine, TBAF ¼ tetra-n-butylammonium fluoride, R ¼
(0.43 in THF, 0.41 in EtOH, referenced to quinine in 0.5 M
H2SO4), but it falls to 0.1 in water. The TPA spectra of BEF-OH
and its t-butyldimethylsilyl ether derivative, BEF-OTBDMS
(Fig. S28†), in water and EtOH, respectively, are compared in
Fig. 4. The TPA maxima are 1150 GM at 700 nm (for BEF-
OTBDMS in EtOH) and 1100 GM at 715 nm (for BEF-OH in
water). These cross-sections are similar to those reported
previously for closely related dyes.18,22 The spectrum is slightly
broader and red-shied in water, but the spectra are similar,
revealing that the TPA is insensitive to the solvent environment.
In both solvents, there is a shoulder in the TPA spectrum at
twice the wavelength of the one-photon allowed S0 / S1 tran-
sition. However, the TPA spectra are dominated by peaks cor-
responding to the one-photon forbidden, two-photon allowed
higher-energy electronic or vibronic transitions. This behavior
is similar to that reported for slightly non-centrosymmetric
D–p–D quadrupolar chromophores.18
(CH2CH2O)7CH3.
The model electron acceptor, N-methyl pyridinium hexa-
uorophosphate (Pyr) (Fig. 5), displays weak absorption in the
UV region (lmax ¼ 270 nm, 3270 ¼ 0.5 ꢁ 104 Mꢂ1 cmꢂ1 in THF,
Fig. S26†), with no signicant absorption at wavelengths greater
than 300 nm. The difference between the absorption spectra of
the dye unit BEF-OH and release platform Pyr, mean that the
uorene dye is the only absorbing species at wavelengths longer
than 300 nm.
Scheme 2 Reagents: (i) MeCN, reflux, 36 h, (ii) 4, Pd(OAc)2, PPh3, CuI,
DIPA, 20 ꢃC, 1 h, brine wash, (iii) BF3$OEt2, DCM, 0 ꢃC / 20 ꢃC, 5 h, (iv)
MeCN, 100 ꢃC, 24 h, (v) 4, Pd(OAc)2, PPh3, CuI, DIPA, DCM, MeCN, 20
ꢃC, 2 h, then purification in TFA-buffered solvent, (vi) HCO2H, TIPS-H,
20 ꢃC, 1 h.
Fig. 3 Absorption (solid) and emission (dashed) spectra of BEF-OH in
THF (black), EtOH (red) and water (blue). Excitation wavelength: 366
nm.
Fig. 4 TPA spectra (circles) overlaid with double-wavelength one-
photon absorption spectra (lines); blue – BEF-OH recorded in water,
black – BEF-OTBDMS (t-butyldimethylsilyl ether derivative) recorded
in EtOH.
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Fluorescence quenching
The efficiency of electron transfer in the systems reported here
can be evaluated from their uorescence quantum yields,
because PeT competes directly with uorescence. Comparison
of the uorescence quantum yields of the free donor unit (BEF-
OH; f ¼ 0.10 in water) and the donor incorporated into the dyad
(BEF-Pyr-GABA; f ¼ 0.001 in water) shows that the uorescence
of the uorene dye is severely quenched by the presence of
acceptor, implying that intramolecular PeT is fast and efficient.
Using this information and eqn (3), the quantum yield of charge
transfer is estimated to be near unity (fCT ¼ 0.98).
Fig. 5 Diagram representing the energy levels of the species involved
in electron transfer. Right: structure of N-methyl pyridinium hexa-
fluorophosphate (Pyr), the model compound used in electrochemical
measurements.
ff ðlinked systemÞ
fCT ¼ 1 ꢂ
(3)
ff ðparent dyeÞ
Hydrolytic stability
Thermodynamics of electron transfer
A prerequisite for applications of a caged drug in physiological
experiments is that it must be stable in aqueous media, in the
absence of light, at least for a few hours. The stability of BEF-
Pyr-GABA was assessed in aqueous buffeꢃrs at pH 7.4 by HPLC
and in non-buffered D2O by NMR at 20 C. We found that its
stability is sensitive to pH and to the composition of the buffer.
The half-life of BEF-Pyr-GABA in phosphate-buffered saline
(PBS) was found to be ca. 15 h (Fig. 6; [BEF-Pyr-GABA]: 20 mM). In
contrast, in NaHCO3-based articial cerebrospinal uid (aCSF
buffer, pH 7.4, which is routinely used as a medium in experi-
ments with neurons) the half-life was reduced to 1.5 h. An
extensive set of troubleshooting experiments, in which eight
versions of aCSF-buffer were prepared (each missing one
The fundamental requirement for efficient PeT is that the Gibbs
free energy (DGET) for the process must be negative. The energy
of the singlet excited state of the dye (E00) must be greater than
the energy cost of transferring an electron from the donor to the
acceptor, i.e. greater than the difference between the oxidation
potential of the donor (EOX) and the reduction potential of the
acceptor (ERED), corrected by the Coulombic stabilization of the
charges, as summarized by Fig. 5 and eqn (1),23
DGET ¼ NA{e[EOX ꢂ ERED] + w(D+cAꢂc) ꢂ w(DA)} ꢂ E00 (1)
where w(D+cAꢂc) and w(DA) are terms that factor in electrostatic
interaction in the products and reactants:
ꢂ
different component) allowed us to identify HCO3 as the
zðDþ$ÞzðAꢂ$Þe2
4p303ra
zðDÞzðAÞe2
4p303ra
detrimental component. When NaHCO3 was replaced with
HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, an
alternative component of standard buffers used in neurosci-
ence] the half-life of BEF-Pyr-GABA rose to ca. 50 h. No hydro-
lysis was detected in non-buffered D2O (Fig. S31†). These
observations highlight the necessity of evaluating the stability
À
Á
w Dþ$Aꢂ$
¼
;
wðDAÞ ¼
(2)
and NA is the Avogadro constant, e is the elementary charge, 30 is
the vacuum permittivity, 3r is the dielectric constant of the
solvent, a is the distance of charge separation and z(D/A)
is charge of the species (D: donor; A: acceptor). The excited state
energy (E00) is dened as the energy of transition between the
lowest vibrational level of the ground and excited states, and
can be estimated from the point of overlap between the
absorption and emission spectra. The oxidation potential of the
donor (EOX) and reduction potential of the acceptor (ERED) can
be measured electrochemically. The excited state energy of BEF-
OH in THF was estimated at 2.96 eV (418 nm) and the rst
oxidation potential was determined to be 0.36 V (relative to
ferrocene in THF with 0.1 M Bu4PF6). The reduction potential of
Pyr relative to ferrocene is ꢂ1.76 V under the same conditions.
The free Gibbs energy (DGET) for PeT calculated according to
eqn (1) for the pair BET-OH and Pyr is ꢂ0.84 eV indicating that
PeT is strongly favorable. The Coulombic term is zero in the
case of the pyridinium-based systems because electron transfer
does not result information of a charge-separated state but only
in the migration of a pre-existing charge. An extended study of
photoinduced electron transfer in model dyads, in which the
BEF electron donor is covalently linked to a variety of electron
acceptors, is reported separately.24
Fig. 6 Stability of BEF-Pyr-GABA (20 mM) in aqueous buffers at pH 7.4
at 20 ꢃC: HEPES-based aCSF – black, NaHCO3 based aCSF – red, PBS
– blue; HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid,
aCSF: artificial cerebrospinal fluid, PBS: phosphate-buffered saline.
Changes in concentration of BEF-Pyr-GABA were determined by
HPLC. Mono-exponential fitting curves were applied to the data.
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of caged compounds under conditions identical to those of the
nal target application. We have not investigated how bicar-
bonate catalyzes this hydrolysis reaction, but the hydrolysis of
a-amino acid esters under similar conditions has been attrib-
uted to CO2-mediated carbamate formation and intramolecular
cyclization.25 The low hydrolytic stability of GABA-pyridinium
esters in standard aCSF buffer poses a limitation for the use of
these compounds under strictly physiological conditions that
will need to be addressed in future molecular designs.
The hydrolytic stability of BEF-Pyr-Trp was monitored only
by HPLC and is different from that of BEF-Pyr-GABA. BEF-Pyr-
Trp has a half-life of only about 2 h at pH 7.4 (in both PBS and
NaHCO3 based aCSF buffers). The hydrolysis proceeds more
slowly at pH 3.0 (citric acid/citrate buffer), with a half-life of 3 h
(Fig. S36†). The close proximity of the protonated amino group
to the ester functionality probably increases the electrophilicity
of the carbonyl center, enhancing the hydrolytic instability of
the tryptophan derivative. BEF-Pyr-Trp also undergoes decom-
position at low pH, in a reaction that appears to be associated
with the uorene dye rather than ester hydrolysis (Fig. S37†).
Fig. 7 1H-NMR spectra from a representative uncaging experiment,
which shows the disappearance of caged-GABA signals with a
concurrent increase of a new set of multiplets later found to be free
GABA (signals “a-c”). Decrease in intensity of signal “d” (5.29 ppm) is not
shown.
Uncaging studies
simultaneously (300–400 nm, peak 350 nm), and their respec-
tive rates of uncaging (ku) were determined by 1H-NMR (see
ESI†). Under these concentrated conditions, the rate of uncag-
ing depends only on the light intensity and the quantum yield,
but not on the molar absorption coefficient. The uncaging
quantum yield of BEF-Pyr-GABA was calculated using eqn (4) to
give fu ¼ 0.009 ꢀ 0.003.
We evaluated the uncaging properties of BEF-Pyr-Trp and BEF-
Pyr-GABA in a series of one-photon irradiation experiments. The
method used to monitor release of the amino acid was dictated
by properties of caged species. The high aqueous solubility of
BEF-Pyr-GABA facilitated photolysis at mM concentration and we
followed the reaction by 1H NMR spectroscopy. It was not
possible to monitor the release of GABA by HPLC because this
amino acid lacks a UV chromophore. In contrast, BEF-Pyr-Trp
ꢀ
ꢁ
ku ðBEF-Pyr-GABAÞ
ku ðDPNI-GABAÞ
fu ðBEF-Pyr-GABAÞ ¼ 0:085
(4)
1
gave broad H NMR spectra at mM concentrations, presumably
due to aggregation. Photolysis studies were conducted at mM
concentration and the indole motif in the side chain of trypto-
phan enabled quantication of the released amino acid by HPLC.
One-photon uncaging of the pyridinium-based protecting
group was initially investigated by irradiating a 1 mM D2O solu-
tion of BEF-Pyr-GABA with a broad UV-A source (300–400 nm,
peak 350 nm) in an NMR tube. At this concentration, the trans-
mittance of the solution is negligible across the entire wavelength
range of the light source. 1H NMR spectroscopy (with t-butanol as
an internal reference) demonstrated the release of GABA with a
chemical yield of >95% (Fig. 7 and 8). Two GABA molecules are
released from each molecule of BEF-Pyr-GABA, which demon-
strates that the BEF chromophore is able to undergo two cycles of
photoreduction. We were unable to identify the chemical prod-
ucts generated by photolysis of the caging group.
This determination of the uncaging quantum yield of BEF-
Pyr-GABA was veried using ferric oxalate actinometry,27 which
The photolysis of BEF-Pyr-Trp (20 mM in water) with 300–
400 nm light (350 nm peak) was monitored by HPLC. Complete
consumption of the starting material resulted in release of
tryptophan with 83% chemical yield. No photochemically
generated byproducts were detected but decomposition of the Fig. 8 The change in concentration of BEF-Pyr-GABA (black squares)
and free GABA (red triangles) over time. Concentration of free GABA
and BEF-Pyr-GABA were determined by integration of signal “c” and
“d” respectively relative to t-butanol. The presence of free GABA was
confirmed by doping an irradiated solution with an authentic sample,
chromophore unit was observed aer the uncaging events.
The quantum yield of uncaging of BEF-Pyr-GABA was deter-
mined by comparison with the commercially available DPNI-
GABA, which has a known fu of 0.085.26 Solutions of BEF-Pyr-
which resulted in no new signals and an increase in the intensity of the
GABA and DPNI-GABA (1 mM, D2O) were irradiated suspected GABA signals (Fig. S30†).
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gave an uncaging quantum yield of fu ¼ 0.088 ꢀ 0.004 for DPNI-
Preliminary experiments were carried out to test the two-
GABA (in close accord with the published value) and an photon excited release of GABA in the proximity of cultured
uncaging quantum yield of fu ¼ 0.009 ꢀ 0.004 for BEF-Pyr- neurons using BEF-Pyr-GABA. Upon two-photon excitation at
GABA.
720 nm (300 fs, Ti:sapphire laser; 2–5 ms duration), we observed
The uorescence quenching experiments showed that 98% of changes in membrane potential with kinetics consistent with
absorption events lead to charge transfer, while the overall activation of GABA-A receptors, indicating GABA release.
uncaging quantum yield is only about 1%, which suggests that Further experiments are needed to test the effectiveness of this
decay of the excited state is dominated by back electron transfer caged compound, and to quantify side-effects, such as the
to the ground state. Nevertheless, due to the high value of the TPA biological activity of the caged drug.
cross section (1100 GM at 700 nm), the calculated two-photon
uncaging cross section for BEF-Pyr-GABA is du ¼ dafu ¼ 10 ꢀ 3
Non-pyridinium designs
GM at 700 nm, which is comparable to the highest reported value
(11 GM for the 2-(o-nitrophenyl)propyl caged GABA).11a
The uncaging quantum yield for BEF-Pyr-Trp was also
determined by ferric oxalate actinometry. A solution of BEF-Pyr-
Trp (ꢄ1.5 mM, pH 3.0) was irradiated at 360 nm with a uo-
rimeter and photorelease of tryptophan was monitored by
HPLC (Fig. 9), using a protocol designed to take account of
competing background hydrolysis (see ESI†). The quantum
yield of uncaging was measured for BEF-Pyr-Trp as fu ¼ 0.0025
(at 360 nm), so the calculated two-photon uncaging cross-
section for BEF-Pyr-Trp at 720 nm is only du ¼ 2.5 GM. The
reasons for the difference in uncaging quantum yield between
BEF-Pyr-Trp and BEF-Pyr-GABA are unclear and will require
further investigation.
To prove that photo-cleavage of our new protecting group is
sensitized by the absorption of the uorene-based dye, rather
than by direct excitation of pyridinium unit, we investigated the
efficiency of uncaging as a function of irradiation wavelength.
For this purpose, solutions of BEF-Pyr-Trp (1.5 mM, pH 3.0) were
irradiated at 340, 360, 380, 400 and 420 nm for 3800 s, and dark
control experiments were carried out, to account for back-
ground hydrolysis (Fig. 9). The extent of uncaging correlates
closely with the absorption spectrum of the BEF chromophore
(Fig. 10), conrming the active role of the uorene-dye in
uncaging and PeT mediated release of tryptophan from BEF-
Pyr-Trp.
The pyridinium group is an excellent electron-acceptor, and the
results presented above show that it gives very efficient PeT,
however the efficiency of cleavage of the charge-separated
state is disappointing (fu z 1%). The pyridinium group may
also exhibit undesirable reactivity towards nucleophiles, so we
decided to explore other electron acceptors. We chose to
investigate phenacyl, for which uncaging via PeT has been
previously reported13b and nitrobenzyl esters (Fig. 11). The
reduction potentials of both methyl 4-acetylbenzoate (Phen)
and methyl 4-nitrobenzoate (NB) were measured by cyclic and
square wave voltammetry, giving ERED of Phen: ꢂ2.18 V; ERED of
NB: ꢂ1.47 V (vs. Fc/Fc+ in THF with 0.1 M Bu4PF6). We estimated
the values of the Coulombic term, eqn (2), in BEF-Phen-Ind and
BEF-NB-Trp from the distance of photoinduced charge separa-
tion (a) using molecular mechanics calculations (see ESI†). This
gave w(D+cAꢂc) ¼ ꢂ0.21 eV for both compounds. Calculation of
Gibbs energy of the photoinduced electron transfer (DGET),
according to eqn (1), revealed that it is energetically favorable in
both systems, giving DGET of ꢂ0.54 eV and ꢂ1.25 eV for Phen
and NB respectively. To evaluate the photo-release properties of
phenacyl and nitrobenzyl derived groups, we synthesized their
tryptophan analogues for HPLC-monitored uncaging experi-
ments. In the case of the phenacyl group, attack of the free
amino group of tryptophan on the ketone group to form a
6-membered ring posed a limitation for protection of a-amino
acids. This issue was overcome by use of the alternative
Fig. 9 The change in concentration of tryptophan upon photolysis of
BEF-Pyr-Trp at pH 3.0 and 360 nm; red circles – photolyzed sample, Fig. 10 Normalized absorption spectrum of BEF-Pyr-Trp in citric acid/
blue squares – control samples stored in the dark, black triangles – net sodium citrate buffer (pH 3.0) (black line) plotted in the scale with
concentration of tryptophan released upon uncaging. The concen- extent of photolysis, expressed as yield of released tryptophan upon
tration of tryptophan was determined by HPLC.
3800 s of irradiation of BEF-Pyr-Trp at 340–420 nm (blue circles).
2424 | Chem. Sci., 2015, 6, 2419–2426
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electron transfer to the pyridinium; charge-transfer leads to
bond scission, to liberate a carboxylic acid. Our protecting
group has been demonstrated to release the neurotransmitter
GABA and amino acid L-tryptophan upon irradiation with light
of wavelength 340–420 nm, in aqueous solution in nearly
quantitative chemical yields. This group exhibits a high TPA
cross-section (1100 GM at 700 nm) and highly efficient charge-
transfer between the electron donor and acceptor was observed
(98%). The fast back electron transfer from the charge-shied
state reduces the overall quantum efficiency of uncaging to
around 1% which, when combined with the TPA cross-section,
results in a two-photon uncaging cross-section of approximately
du ¼ 10 GM (700 nm) for BEF-Pyr-GABA. Wavelength-dependent
uncaging experiments conrmed electron-transfer mediated
release of caged tryptophan with efficiency of release propor-
tional to the extinction coefficient of the uorene dye within
340–420 nm.
Fig. 11 The structures of alternative electron acceptor units: methyl
4-acetylbenzoate (Phen) and methyl 4-nitrobenzoate (NB) and cor-
responding protecting groups based on the banana-shaped fluorene
dye BEF-Phen-Ind and BEF-NB-Trp; R ¼ (CH2CH2O)7CH3.
A key objective for future research will be to apply the
modular design strategy demonstrated in this study to create an
electron donor–acceptor pair for which the back electron
transfer is suppressed, so that bond-scission becomes the main
decay pathway. The susceptibility of pyridinium esters towards
hydrolysis can lead to practical difficulties for uncaging studies
in aqueous media, and it would be useful to extend these
systems to non-ester linking unit that is are more stable to
aqueous hydrolysis, such as carbamates.
structure: 3-indolepropionic acid (Ind), which possesses an
indole chromophore but lacks the amino group, allowing us to
preserve the absorption properties of tryptophan while avoiding
cyclization. Structures of nitrobenzyl tryptophan (BEF-NB-Trp)
and phenacyl protected 3-indolepropionic acid (BEF-Phen-Ind)
are shown in Fig. 11 (for synthesis see ESI†).
Photolysis of BEF-Phen-Ind was investigated by irradiating
a solution in water (17 mM) with a broad UV-A light source (300–
400 nm; peak: 350 nm), monitoring the progress of uncaging by
HPLC. 3-Indolepropionic acid was liberated with a modest 20%
chemical yield, despite full consumption of the starting mate-
rial. HPLC analysis did not reveal formation of any other
products of photolysis and we were unable to dene the fate of
the remaining 80% of starting material. The quantum yield of
uncaging was determined by use of ferric oxalate actinometry,
giving fu ¼ 0.0022, corresponding to a two-photon uncaging
cross section of du ¼ 2.4 GM at 700 nm. The limited scope of
substrates that can be caged (due to the reactivity of the ketone
group), the low chemical yield of photorelease and the suscep-
tibility to hydrolysis under physiological conditions make ester-
linked phenacyl platforms unattractive release units.
Acknowledgements
We thank Prof. Seth R. Marder for valuable discussion, Prof. Ole
Paulsen, Dr Yu Zhang and Dr Golnaz Borghei for carrying out
preliminary electrophysiology experiments with BEF-Pyr-GABA.
This work was funded by the European Commission Seventh
Framework Programme (TOPBIO ITN, project-grant agreement
no. 264362). We thank the EPSRC Mass Spectrometry Service
(Swansea) for mass spectra.
Notes and references
Photolysis of BEF-NB-Trp was tested in a range of solvents
(5 mM concentration in water, NaHCO3-aCSF, acetonitrile,
methanol and THF) using a broad UV-A light source (300–
400 nm; peak: 350 nm), but no release of tryptophan was
observed. Photochemical decomposition of BEF-NB-Trp
occurred, but it did not result information of free tryptophan.
The same result was observed when irradiation was carried out
at 280 nm. Tryptophan was liberated cleanly in 75% yield by
hydrolysis of BEF-NB-Trp in the dark over 40 h in NaHCO3-
based aCSF buffer.
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