Water-soluble, donor-acceptor biphenyl derivatives in the 2-(o-nitrophenyl)propyl series: Highly efficient two-photon uncaging of the neurotransmitter γ-aminobutyric acid at λ=800 nm

Article abstract of DOI:10.1002/anie.201106559

Rattling the cage: The two γ-aminobutyric acid (GABA) derivatives 1 and 2 exhibit efficient and rapid (<5a- 10^-6 s) GABA photorelease upon one-photon excitation combined with two-photon uncaging cross-section at λ=800 nm. Compounds 1 and 2 were

Full text of DOI:10.1002/anie.201106559

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DOI: 10.1002/anie.201106559
Caged Compounds
Water-Soluble, Donor–Acceptor Biphenyl Derivatives in the 2-(o-
Nitrophenyl)propyl Series: Highly Efficient Two-Photon Uncaging of
the Neurotransmitter g-Aminobutyric Acid at l = 800 nm**
Loꢀc Donato, Alexandre Mourot, Christopher M. Davenport, Cyril Herbivo, David Warther,
Jꢁrꢁmie Lꢁonard, Frꢁdꢁric Bolze, Jean-FranÅois Nicoud, Richard H. Kramer,
Maurice Goeldner,* and Alexandre Specht*
By using two-photon (TP)[1] photoreleasable neurotransmit-
ters like glutamate (Glu) or g-aminobutyric acid (GABA),
neuronal processes can be activated or inhibited with high
temporal and spatial control (with around one micron three-
dimensional precision) with reduced photodamage to cells or
organs, and with deeper penetration of the light beam into
living tissue compared to that of UV photoactivation.[2]
Whereas TP excitation of fluorophores can be achieved
with high efficiency to allow accurate imaging in living
systems,[3] TP uncaging is still in its early stages. This challenge
requires the design of TP-sensitive caged neurotransmitters
having a high TP uncaging action cross-section (expressed in
Goeppert-Mayer GM, 1 GM = 1050 cm4 s per photon), ideally
with excitation wavelengths around l = 800 nm.[2] To be
useful, caged neurotransmitters must also be stable and
biologically inert in the dark, and have good aqueous
solubility (usually in the mM range in electrophysiological
buffers). Additionally, the photolytic reaction should be fast
(ꢀ 0.1 ms) and clean, thus leading to a stoichiometric release
of the neurotransmitter.
The most commonly used caged Glu, 4-methoxy-7-nitro-
indolinyl-amino (MNI-Glu; see Scheme 1 for MNI struc-
ture),[4b–c] has enabled mapping of synaptic receptors,[4c]
activation of individual spines[5] and neurons,[6] and has very
recently been used in the cortex in vivo to look at the
functional distribution of AMPA-type glutamate receptors
(AMPA = a-amino-3-hydroxy-5-methyl-4-isoxazole propi-
onic acid)[7] and to induce continuous growth of a functional
spine.[8] However, because none of the caged Glu systems
described so far combine high TP uncaging action cross-
sections with good quantum efficiency and sufficient buffer
solubility, elevated concentrations of caged compounds and
relatively high laser powers are needed, both of which can
have deleterious effects. Accordingly, we have developed the
(2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxa-
Because of its primary role in neuronal excitation, the
neurotransmitter Glu has been extensively targeted for one-
photon and more recently for TP uncaging investigations.[4a–g]
[*] L. Donato, Dr. C. Herbivo, D. Warther, Prof. Dr. M. Goeldner,
Dr. A. Specht
Laboratoire de Conception et Application de Molꢀcules Bioactives
UMR 7199, CNRS/UDS, Facultꢀ de Pharmacie
74 Route du Rhin, 67400 Illkirch (France)
E-mail: specht@bioorga.u-strasbg.fr
goeldner@bioorga.u-strasbg.fr
Dr. A. Mourot,[+] Dr. C. M. Davenport,[+] Prof. Dr. R. H. Kramer
University of California Berkeley, Department of Molecular and Cell
Biology, Berkeley, CA 94720-3200 (USA)
Dr. J. Lꢀonard
Institut de Physique et Chimie des Matꢀriaux de Strasbourg
UMR 7504, CNRS—Universitꢀ de Strasbourg (France)
Dr. F. Bolze, Prof. Dr. J.-F. Nicoud
Laboratoire de Biophotonique et Pharmacologie, UMR 7213, CNRS/
UdS, Facultꢀ de Pharmacie, 67400 Illkirch (France)
[+] A.M. and C.M.D. contributed equally to the work.
[**] We thank Dr. M. Banghart for helpful discussions and critical
comments on the manuscript. This work was supported by the
Universitꢀ de Strasbourg, the CNRS, the France-Berkeley Fund, and
ANR (Contract No. 09-BLAN-0425-01 and PCV 07 1-0035).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106559.
Scheme 1. Two-photon-sensitive photoremovable groups. LG=leaving
group.
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heptyl)]fluorene (BNSF) photoremovable group[9] which
displays to date the highest (5 GM) TP uncaging action
cross-section at l = 800 nm. Nevertheless, BNSF-Glu does not
allow for a quantitative release of the neurotransmitter
(60%) because of a competing photodegradation pathway,
and it is sparingly soluble in aqueous media (0.1 mm).
Despite the primary role of GABA in neuronal inhibition,
only a small number of caged GABA systems have been
engineered so far, all with unfavorable photochemical (low
TP uncaging action cross-section) and pharmacological
properties.[10] CDNI is one cage used for TP uncaging of
GABA in intact brain tissue, but it has a modest TP uncaging
cross-section even at a low excitation wavelength (around
0.3 GM at l = 720 nm[4d]). To our knowledge only N-DCAC-
GABA (N-DCAC = 7-(dicarboxymethyl)-aminocoumarin)
has been used for the TP release of GABA at l = 800 nm
on hippocampal neurons in brain slices,[11] but it suffers from
slow GABA release with a long half time of about 4 ms and a
moderate efficiency upon TP excitation (0.37 GM at l =
800 nm[4a]). Therefore the development of efficient TP-
sensitive caged GABA is one of the current challenges in
the field of chemical neuroscience.[2e]
tion of CANBP-GABA (1) and EANBP-GABA (2) enabled
rapid and spatially controlled GABA release in intact brain
slices.
The syntheses of the probes 1 and 2 are summarized in
Scheme 2. The key intermediate 2-(5-bromo-2-nitrophenyl)-
propan-1-ol (3) was prepared in good yield by a modification
of our previously described procedure,[4f] using a vicarious
nucleophilic substitution[12] as the key reaction. Starting from
4-bromonitrobenzene, this latter reaction afforded the tert-
butyl 2-(5-bromo-2-nitrophenyl)acetate intermediate quanti-
tatively, which was then selectively methylated on the
benzylic carbon atom and reduced with DIBAL-H, to
afford 3 in three steps with an overall yield of 64%. The
intermediate 3 was coupled to 4-aminophenylboronic acid
hydrochloride by a Suzuki reaction using microwave activa-
tion,[13] thus leading to the 4-amino-4’-nitrobiphenyl 5, which
could be conveniently bis(alkylated) with a-bromo tert-
butylacetate to lead to the new protected cage 6.
A similar bis(alkylation) of the cage 10 occurred with very
low yields ( ꢀ 5%; data not shown). Accordingly, we
elaborated an alternative synthesis starting from 3. The
nitro-substituted pinacolato borate 8 was synthesized by
We have been developing
new
TP
photoremovable
groups in the 2-(o-nitrophenyl)-
propyl series by applying the
molecular engineering of one-
dimensional
donor–acceptor
nonlinear optical chromophores
for the optimization of the TP
absorption cross-section. Such a
strategy has already led to the
PMNB cage[4f] as an efficient TP
photolabile protecting group for
glutamate, with a TP uncaging
efficiency (du=da.fu) of 3.1 GM
at l = 740 nm.[4f] We report
herein an important develop-
ment related to this biphenyl
donor–acceptor
platform:
replacing the alkoxy donor
group with a functionalized di-
alkylamino moiety leads to new
photoremovable groups with
improved aqueous solubility
(up to 10 mm) and unprece-
dented (up to 11 GM) TP
uncaging cross-sections at l =
800 nm. We present here the
synthesis and photophysical
properties of two new photo-
removable groups for carboxylic
acids: 2-(4’-(bis(carboxymeth-
yl)amino)-4-nitro-[1,1’-
Scheme 2. Synthesis of 1 and 2. a) 1. tert-butylchloroacetate, tBuOK, DMF, 2 h, RT; 2. HCl, 96%.
b) 1. NaH, CH3I, THF, 24 h RT, 83%; 2. DIBAL-H, THF, 3 h, 08C, 80%. c) 4-aminophenylboronic acid
hydrochloride, K2CO3, Bu4NBr, Pd(OAc)2, EtOH/H2O (2:1), microwave 1508C, 10 min, 81%. d) DIEA,
tert-butyl bromoacetate, DMF, 908C, 5 h, 40%. e) 1. N-Boc GABA, DCC, DMAP, CH2Cl2, 19 h, RT; 2. TFA/
CH2Cl2 (4:6), 5 h or 1 min, 94–96%. f) [PdCl2(dppf)], KOAc, bis(pinacolato)diboron, DMSO, 808C, 15 h,
49%. g) I2, 1,4-dioxane/pyridine (1:1), 08C–RT, 2 h, 84%. h) K2CO3, Bu4NBr, Pd(OAc)2, EtOH/H2O (2:1),
microwave 1508C, 10 min, 70%. DCC=N,N’-dicyclohexylcarbodiimide, DIBAL-H=diisobutylaluminum
hydride, DIEA=diisopropylethylamine, DMAP=4-dimethylaminopyridine, DMF= N,N’-dimethylform-
amide, DMSO=dimethylsulfoxide, dppf=1,1’-bis(diphenylphosphanyl)ferrocene, TFA=trifluoroacetic
biphenyl]-3-yl)propan-1-ol
(CANBP) and 2-(4’-(bis((2-
methoxyethoxy)ethyl)amino)-4-
nitro-[1,1’-biphenyl]-3-yl)pro-
pan-1-ol (EANBP).TP excita- acid, THF=tetrahydrofuran.
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borylation of 3 using pinacolatodiboron in the presence of a
palladium catalyst at 808C.[14] The alkylated 4-iodoaniline 9
was also synthesized by a selective para iodination[15] of the
N,N-bis((2-methoxyethoxy)ethyl)aniline.[16] Then a Suzuki
coupling reaction, using microwave activation,[13] between 9
and 8 led to the cage 10 with improved yields (30% overall).
Finally, N-Boc-protected GABA (N-a-tert-butoxycar-
bonyl GABA) was grafted onto the cages 6 and 10 to provide
the new caged GABA analogues 1 and 2, respectively, after
deprotection in acidic media.
The solubility of 1 and 2 at room temperature in
phosphate buffer pH 7.4 (without any cosolvent) was 10 and
1.3 mm, respectively. The hydrolytic stability was explored by
HPLC in phosphate buffer (pH 7.4) at room temperature; less
than 1% hydrolysis was measured after 24 h for 1 and 2. The
absorption maximum and molecular coefficient for both 1 and
2 are l = 397 nm and 7500mÀ1 cmÀ1, respectively.
The TP uncaging action cross-section (daFu) of 1 and 2
were determined at l = 800 nm by HPLC analysis of the
photolysis products and compared to the 3-(2-propyl)-4’-tris-
ethoxy(methoxy)-4-nitrobiphenyl caged 1,3-dichloro-9,9-
dimethyl-9H-acridin-2(7)-one (PENB-DDAO) as a reference
(0.45 GM).[19] The TP photolysis curves of 1, 2, and PENB-
DDAO are presented in Figure 2, and show TP uncaging
action cross-section values of 7.4 GM and 11 GM for 1 and 2,
respectively.
The uncaging kinetics for ANBP photoremovable groups
were estimated using a caged fluorescent derivative[19] ANBP-
DDAO (see the Supporting Information). A time scale
shorter than 5 ms was obtained for the release of the
fluorescent DDAO, a value in accord with the reported
kinetics for other 2-(o-nitrophenyl)propyl photoremovable
groups,[4f,19] and in agreement with temporal control of
neuronal processes.
The one-photon photochemical properties of 1 and 2 were
investigated by UV/visible spectroscopy. Photolysis was
carried out by irradiation of samples (around 40 mm) at l =
405 nm in phosphate buffer 50 mm pH 7.4. Both 1 and 2
showed a new and broad absorbance at l = 450 nm, and the
initial absorbance at l = 397 nm gradually diminished and the
isobestic points at l = 350 and 450 nm appeared, thus
indicating a homogenous photolysis reaction (Figure 1). The
photolytic release of GABA was analyzed by UV and HPLC.
To do so, 1 and 2 were coupled to a 4-methoxy benzoic acid
chromophoric derivative by using an activated ester (see the
Supporting Information). An almost quantitative (ꢁ 95%)
formation of the 4-methoxybenzoic amide GABA derivatives
was measured by HPLC after complete photoconversion of
both 4-methoxybenzoic amide caged GABA derivatives.
Altogether, this is in agreement with an almost quantitative
photolytical reaction following b-elimination pathways with
formation of biphenyl 2-(o-nitrophenyl)propene deriva-
tives[17]. The quantum yield for GABA release, as determined
by competition with the 2-(nitrophenyl)ethyl-ATP[18] refer-
ence molecule, was 15% for both 1 and 2, thus indicating a
good photochemical efficiency (f.e ꢂ 1100mÀ1 cmÀ1) at l =
400 nm. Altogether, the good water solubility, the high
photolytical efficiency, and quantitative release of GABA
make the molecules remarkable caged GABA systems for
visible light photolysis.
Finally, we used 1 and 2 for TP uncaging of GABA at l =
800 nm in intact brain tissue. We performed whole-cell
voltage clamp recordings from Layer 2/3 pyramidal cells in
rat cortical brain slices (Figure 3A). Cells were held at 0 mV
to record outward GABAergic currents. A 1 mm amount of 1
was applied focally to the proximal apical dendrite using a
glass pipette. The l = 800 nm laser light was scanned over an
apical dendrite and resulted in an outward current (Fig-
ure 3B; mean current peak = (6.4 Æ 0.3) pA, n = 3 cells). The
GABAA receptor antagonist picrotoxin (100 mm) completely
abolished the current, thus confirming that it was mediated
entirely by GABAA receptors (Figure 3B; n = 3 cells).
Experiments with 2 gave currents with similar amplitude
(data not shown). Both caged GABA compounds were found
to decrease the amplitude of miniature inhibitory post-
synaptic currents before photolysis, thereby supporting the
idea that like most other caged GABA systems described so
far[9] these new cages still display antagonist properties at
GABAA receptors (see the Supporting Information).
In conclusion, the replacement of the alkoxy donor group
by a dialkyl amino group in the donor–acceptor biphenyl 2-(o-
nitrophenyl)propyl series led to a 90 nm bathochromic shift
(lmax = 397 nm), with no significant modification on the
efficiency and kinetics of the photolytic reaction (95%
Figure 1. Changes in the UV/visible spectrum of 2 during photolysis
(l=405 nm) in phosphate buffer (50 mm pH 7.4) using a LUMOS 43
(Atlas Photonics Inc.).
Figure 2. Two-photon photolysis of 1 and 2 at l=800 nm followed by
HPLC analysis and compared to the reference molecule PENB-DDAO
(green triangles).
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Figure 3. Uncaging of 1 activates GABA receptors in brain slices.
A) Alexa 488-filled L2/3 pyramidal cell. Uncaging location is within the
white box. The puffer pipette containing 1 is visible as a shadow
(arrow). B) Outward current evoked by l=800 nm uncaging flash
(blue line) in the presence of 1 (black), which is blocked by picrotoxin
(red). Scale bar=2 pA/200 ms.
release with a 15% quantum yield for one-photon photo-
conversion, and 5 ꢀ 10À6 s time range kinetic for uncaging).
Most importantly, the substitution of the methoxy group by a
better electron-donating group (NR2) significantly increased
the TP sensitivity of this photoremovable group, thus leading
to an unprecedented TP uncaging action cross-section at l =
800 nm (up to 11 GM). In addition, the presence of the
dialkylamino group opens the possibility for improving
significantly the aqueous solubility of these probes by grafting
either oligoethyleneglycol chains or 2-carbonylmethyl groups
on the amino functional group. The two new caged GABA
compounds we have developed, CANBP-GABA (1) and
EANBP-GABA (2), indeed have millimolar range buffer
solubility, and were successfully used for TP GABA release in
intact brain tissue at l = 800 nm.
Altogether, the remarkable chemical and photophysical
properties of the ANBP chromophore open the way for
future developments using TP uncaging in neuroscience,
cellular, and molecular biology. While the extension to the
synthesis to caged glutamate or glycine is straight forward, the
chemical strategy developed herein can be transposed to the
modification of other functional groups such as phosphates
(not shown), thereby leading to the synthesis of original caged
nucleotides (ATP, cAMP) or inositolphosphates. Final efforts
in the design of ANBP-GABA derivatives, fully devoid of
antagonist properties, are currently under investigation for
further study of the synaptic signaling both in slices and
in vivo.
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Received: September 15, 2011
Published online: January 11, 2012
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Keywords: biaryls · caged compounds · neurotransmitters ·
photolysis · synthetic methods
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