The donor-acceptor biphenyl platform: A versatile chromophore for the engineering of highly efficient two-photon sensitive photoremovable protecting groups

Article abstract of DOI:10.1039/c2pp05360h

Different photoremovable protecting groups in the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl series with a donor-acceptor biphenyl backbone, known to display excellent two-photon absorption cross-sections, were investigated in order to develop efficient two-photon sensitive photoremovable protecting groups. The 2-(o-nitrophenyl)propyl series was a more versatile platform to increase the two-photon sensitivity of photoremovable protecting groups, leading to the p-alkoxy and p-bisalkylamino-4-nitro-[1,1′- biphenyl]-3-yl)propyl derivatives: PENB and EANBP respectively. Those two photoremovable protecting groups are to date the best caging groups for two-photon excitation at 800 and 740 nm respectively, offering attracting perspectives in chemical biology.

Full text of DOI:10.1039/c2pp05360h

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PAPER
The donor–acceptor biphenyl platform: Aversatile chromophore for the
engineering of highly efficient two-photon sensitive photoremovable
protecting groups†
Alexandre Specht,*a Frédéric Bolze,b Loïc Donato,a Cyril Herbivo,a Sébastien Charon,a David Warther,a
Sylvestre Gug,a,b Jean-François Nicoudb and Maurice Goeldner*a
Received 27th October 2011, Accepted 20th December 2011
DOI: 10.1039/c2pp05360h
Different photoremovable protecting groups in the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl
series with a donor–acceptor biphenyl backbone, known to display excellent two-photon absorption
cross-sections, were investigated in order to develop efficient two-photon sensitive photoremovable
protecting groups. The 2-(o-nitrophenyl)propyl series was a more versatile platform to increase the two-
photon sensitivity of photoremovable protecting groups, leading to the p-alkoxy and p-bisalkylamino-4-
nitro-[1,1′-biphenyl]-3-yl)propyl derivatives: PENB and EANBP respectively. Those two photoremovable
protecting groups are to date the best caging groups for two-photon excitation at 800 and 740 nm
respectively, offering attracting perspectives in chemical biology.
only by the absorption of a single photon of energy E = hν, but
also by the simultaneous absorption of two photons of half
Introduction
The search for probes possessing suitable properties for an
efficient and controlled photolytic release of biomolecules (un-
caging) has been thoroughly developed in the last few decades
leading to an impressive panel of different photoremovable pro-
tecting groups.1 The uncaging of biomolecules requires several
well-defined properties which include a rapid and efficient
photolytic release of the biomolecule, an irradiation wavelength
which should be compatible with the biological environment
(preferably UV-Visible or visible excitation wavelengths), the
formed side product should not interfere with the ongoing photo-
lytic reaction and should be non-toxic for the biological system.
Lastly, the biomolecule modified by the photoremovable group
should be inert to the targeted biological system and remain
freely soluble in aqueous media. None of the published probes
satisfy fully all these properties at once, but one has to keep in
mind that the requirements for biological applications are fairly
different according to their issues. A more recent outcome for
the uncaging of biomolecule is to use two-photon excitation
(TPE).2 Indeed, TPE can intrinsically provide fine spatio-tem-
poral control during a photochemical cleavage. In such a photo-
physical process a chromophore can reach an excited state not
energy E′ = hν/2. The interaction between the electric field
linked to the electromagnetic wave of the excitation light (E) and
the polarisable electronic cloud of a conjugated molecule
induces a charge redistribution that induces a modification of the
molecular dipole moment (μ).3 The latter is usually described as
a development of increasing powers of the electric field E, as
described in eqn (1):
μ ¼ μ0 þ αE þ βE2 þ γE3 þ …
ð1Þ
The static dipole moment of the molecule is μ0, when α·E rep-
resents the effects related to the linear polarisability. βE2 and γE3
are related to the first and second order molecular non-linear
optical properties, respectively. Noticeably the theory shows that
the two-photon absorption phenomenon is related to the imagin-
ary part of the cubic hyperpolarisability γ. The excitation prob-
ability of a molecule P is in this case proportional to the square
of the light intensity, δa being the two-photon absorption cross-
section as in eqn (2) (The two-photon absorption cross-section
δa is often mentioned as σ2 in most chemical papers):
P ¼ 1=2 ꢀ δaI 2
ð2Þ
This quadratic dependence of P versus I is crucial for the
spatial localisation of this non-linear optical phenomenon. The
excitation will occur only where the light intensity is maximum,
typically at the focal point of an optical system and only using a
pulsed laser as excitation source to take benefit of the high peak
power of the pulses. δa is generally expressed in Göppert-Mayer
unit (GM) in honour of Maria Göppert-Mayer who first
predicted theoretically this phenomenon in 1931 (1 GM =
aLaboratoire de Conception et Application de Molécules Bioactives,
UMR 7199, CNRS/UDS, Faculté de Pharmacie, 74 Route du Rhin,
67401 ILLKIRCH Cedex, France. E-mail: specht@bioorga.u-strasbg.fr,
goeldner@bioorga.u-strasbg.fr; Fax: (+33) 368854306
bLaboratoire de Biophotonique et Pharmacologie, UMR 7213, CNRS/
UdS, Faculté de Pharmacie, 74 Route du Rhin, 67401 ILLKIRCH
Cedex, France
†This paper is part of a themed issue on photoremovable protecting
groups: development and applications.
578 | Photochem. Photobiol. Sci., 2012, 11, 578–586
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10−50 cm4 s photon−1 molecule−1).4 In addition to an intrinsic
fine 3D localisation of the excitation, this nonlinear process is
obtained with low energy wavelength (typically with IR light for
molecules which absorb classically in the UV). This induces
limited phototoxicity for cells, tissues or organs, and an
increased penetration depth (tissues transparency windows from
700 to 1000 nm),5 for example, in highly scattering tissues such
as brain slices. Most of the photoremovable groups described for
one photon uncaging have been tested for their two-photon exci-
tation properties, leading, overall, to rather disappointing results
in terms of two-photon uncaging efficiencies defined by the
uncaging action cross-section (δaΦu) also expressed in GM. In a
seminal article,6 the group of Tsien and colleagues has initiated
this theme by describing brominated 7-hydroxycoumarin-4-
ylmethyl caged carboxylic acid derivatives possessing improved
two-photon uncaging action cross-sections over previously
described chromophores. Two-photon uncaging of neurotrans-
mitters is of particular interest in neurobiology for a better
control of in vivo synaptic signalling and this has prompted the
synthesis of different two-photon sensitive caged glutamate
(MNI-Glu,7 DMNPB-Glu,8 PMNB-Glu9) and caged γ-amino-
butyric acid derivatives (CDNI-GABA10 and N-DCAC-
GABA11). Nevertheless, none of the described chromophores
displayed satisfactory efficiencies at 800 nm, excitation wave-
length which corresponds to standard Ti:sapphire lasers justify-
ing continuous efforts to improve the two-photon uncaging
cross-sections at this wavelength.12 In this article we investigate
the connection of different photoremovable protecting groups
with a donor–acceptor biphenyl backbone, known to display
excellent two-photon absorption cross-sections, to form potential
probes for two-photon uncaging of biomolecules.
Purification by flash chromatography using heptane/EtOAc 8/2
in vol. gave 170 mg of the title compound in 54% yield.
1H NMR (400 MHz, MeOD): δ (ppm) = 2.64 (s, 3H), 6.90
(d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.4
Hz, 2H), 8.04 (d, J = 8.8 Hz, 2H).
2-Bromo-1-(4′-hydroxy-[1,1′-biphenyl]-4-yl)ethanone (2). A
mixture
of
1-(4′-hydroxy-[1,1′-biphenyl]-4-yl)ethanone
(160 mg, 0.755 mmol) and CuBr2 (286 mg, 1.28 mmol) in anhy-
drous ethyl acetate (15 mL) was stirred at reflux for 19 h. Water
(100 mL) was added. The aqueous phase was extracted by
EtOAc (2 × 200 mL). Purification by flash chromatography
using heptane/EtOAc 8/2 in vol. gave 124 mg of the title com-
pound in 56% yield.
1H NMR (400 MHz, MeOD): δ (ppm) = 4.67 (s, 3H), 6.91
(d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.4
Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H).
1-(4′-(Bis(2-(2-methoxyethoxy)ethyl)amino)-[1,1′-biphenyl]-4-
yl)ethanone (3). A mixture of 4-iodo-N,N-bis(2-(2-methoxy-
ethoxy)ethyl)aniline (827 mg, 1.95 mmol), 4-acetylphenylboro-
nic acid (213 mg, 1.30 mmol), K2CO3 (254 mg, 1.95 mmol),
Bu4NBr (1.42 g, 2.92 mmol), and Pd(OAc)2 (catalytic) in EtOH
(10 mL) and water (5 mL) was heated under microwave con-
ditions at 150 °C for 10 min. Water (100 mL) was added and the
aqueous phase was extracted by EtOAc (200 mL). Purification
by flash chromatography using gradient elution of heptane/
EtOAc 1/1 in vol. gave 674 mg of the title compound in 80%
yield.
1H NMR (400 MHz, CDCl3): δ (ppm) = 2.54 (s, 3H), 3.30 (s,
6H), 3.44–3.80 (m, 16H), 6.73 (d, J = 8.8 Hz, 2H), 7.45 (d, J =
8.8 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H).
1-(4′-(Bis(2-(2-methoxyethoxy)ethyl)amino)-[1,1′-biphenyl]-4-
yl)-2-bromoethanone (4). A mixture of 1-(4′-(bis(2-(2-methoxy-
ethoxy)ethyl)amino)-[1,1′-biphenyl]-4-yl)ethanone (233 mg,
0.56 mmol), diisopropylethylamine (166 μL, 0.95 mmol) and
tert-butyldimethylsilyl trifluoromethanesulfonate (257 μL,
1.12 mmol) in anhydrous CH2Cl2 (15 mL), were stirred at room
temperature for 4 h. A saturated NaHCO3 solution (100 mL) was
added. The aqueous phase was extracted by CH2Cl2 (2 ×
200 mL). The organic phases were combined and evaporated.
The crude product was dissolved in anhydrous CH2Cl2 (15 mL)
and bromine (29 μL, 0.56 mmol) was added at 0 °C. After 1 h at
room temperature, a saturated NaHCO3 solution (100 mL) was
added. The aqueous phase was extracted by CH2Cl2 (2 ×
200 mL). Purification by flash chromatography using gradient
elution of heptane/EtOAc: 1/1 to pure EtOAc gave 190 mg of
the title compound in 68% yield.
Experimental
General procedures
All chemicals were purchased from Sigma–Aldrich, Alfa Aesar
or Fluka in analytical grade. The NPE-ATP was purchased from
Jena Bioscience. An Agilent MM-ESI-ACI-SQ MSD 1200 SL
spectrometer or an Agilent LC-MS Agilent RRLC 1200SL/ESI
QTof 6520 were used for ESI analysis. H NMR and 13C NMR
1
were run at 300 or 400 and 75 or 100 MHz, respectively. Coup-
ling constants (J) are quoted in Hz and chemical shifts (δ) are
given in parts per million (ppm) using the residue solvent peaks
as reference relative to TMS. A Zorbax C18 column (4.6,
250 mm) or an Acclaim C18 column (4.6, 250 mm) were used
for HPLC analysis. Absorption spectra were recorded on a
UVIKON XS spectrometer. Microwave reactions were performed
in a Biotage Initiator EXP EU at 2.45 GHz.
1H NMR (400 MHz, CDCl3): 3.41 (s, 6H), 3.51–3.70 (m,
16H), 4.48 (s, 2H), 6.92 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz,
2H), 7.67 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H).
Synthesis
1-(4′-Hydroxy-[1,1′-biphenyl]-4-yl)ethanone (1). A mixture of
1-(4-bromophenyl)ethanone (298 mg, 1.5 mmol), 4-hydroxy-
phenylboronic acid (310 mg, 2.25 mmol), K2CO3 (518 mg,
3.75 mmol), Bu4NBr (489 mg, 1.5 mmol), and Pd(OAc)2 (cata-
lytic) in EtOH (10 mL) and water (5 mL) was heated under
microwave conditions at 150 °C for 10 min. Water (100 mL) was
added and the aqueous phase was extracted by EtOAc (200 mL).
General procedure for coupling and deprotection of N-Boc-
GABAs in the phenacyl series. The bromide caged precursors 2
or 4 (0.35 mmol) were dissolved in dry benzene (15 mL), then
N-Boc-GABA (146 mg, 0.7 mmol) and 1,8-diazabicycloundec-
7-ene (DBU) (63 μL, 0.42 mmol). The solution was further
stirred for 19 h at room temperature. A saturated solution of
NaHCO3 was then added. The mixture was extracted with
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dichloromethane (200 mL), and the protected caged-GABAs
were purified by silica gel chromatography using a mixture of
heptane/EtOAc: 6/4 or 1/1 to pure EtOAc as eluent for the syn-
thesis of 2 or 4 respectively. Finally the protected caged GABAs
were dissolved in anhydrous CH2Cl2 (8 mL) and TFA (6 mL).
After 1 min at room temperature under argon the solution was
evaporated to yield 90 mg (60%) and 190 mg (86%) of the TFA
salts of 5 and 6 respectively.
13C NMR (400 MHz, CDCl3): δ (ppm) = 160.0, 149.5, 145.6,
140.6, 138.8, 130.1, 126.9, 125.8, 124.8, 119.8, 113.6, 113.4,
67.9, 55.4, 36.4, 17.5.
oMNB-OH (8c). 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.84
(d, J = 8.4 Hz, 1H), 7.65 (d, J = 1.2 Hz, 1H), 7.53 (d, J = 8.4 Hz
and 1.8 Hz, 1H), 7.38–7.44 (m, 1H), 7.34 (dd, J = 7.8 Hz and
1.2 Hz, 1H), 7.02–7.10 (m, 2H), 3.83–3.85 (m, 5H), 3.64–3.68
(m, 1H), 3.50 (m, 1H), 1.37 (d, J = 6.9 Hz, 3H).
13C NMR (400 MHz, CDCl3): δ (ppm) = 156.8, 149.4, 143.8,
138.3, 131.0, 130.4, 129.9, 128.9, 128.7, 124.5, 121.5, 111.9,
68.3, 56.0, 36.9, 18.0.
pHBP-GABA (5). 1H NMR (400 MHz, Acetone-d6): δ (ppm)
= 2.17 (t, J = 7.2 Hz, 2H), 2.67 (t, J = 7.2 Hz, 2H), 3.93 (t, J =
7.2 Hz, 2H), 5.50 (s, 2H), 6.99 (d, J = 8.4 Hz, 2H), 7.61 (d, J =
8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H).
13C NMR (100 MHz, Acetone-d6): δ (ppm) = 28.1, 35.4,
51.4, 71.5, 121.2, 131.5, 133.5, 133.6, 135.6, 137.4, 151.4,
163.6, 176.9, 197.2.
General procedure for coupling and deprotection of N-Boc-
Glu-OtBu in the 2-(o-nitrophenyl)-propyl series. The caged
alcohols 8a–c (0.5 mmol) were dissolved in dry CH2Cl2
(15 mL), then N-Boc-Glu-OtBu (150 mg, 0.5 mmol), DCC
(110 mg, 0.55 mmol) and DMAP (6 mg) was added at 0 °C.
The solution was further stirred for 19 h at room temperature. A
saturated solution of NaHCO3 was then added. The mixture was
extracted with dichloromethane (200 mL), and the protected
caged-Glutamates were purified by silica gel chromatography
using a mixture of heptane/EtOAc 8 : 2 in vol. as eluent. Finally
the protected caged glutamates were dissolved in anhydrous
CH2Cl2 (8 mL) and TFA (6 mL). After 5 h at room temperature
under argon the solution was evaporated to yield the caged gluta-
mates 9a–c.
MS (ESI-ACI): m/z = 314.2 [M + H].
pABP-GABA (6). 1H NMR (300 MHz, CDCl3): δ (ppm) =
2.17 (m, 2H), 2.61 (m, 2H), 3.12 (m, 2H), 3.36 (s, 6H), 3.50-
3.63 (m, 16H), 5.31 (s, 2H), 6.75 (d, J = 8.8 Hz, 2H), 7.47 (d, J
= 8.4 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H)
13C NMR (100 MHz, CDCl3): δ (ppm) = 30.8, 39.2, 51.0,
59.1, 66.3, 68.4, 70.6, 72.0, 112.1, 125.9, 126.6, 128.2, 128.5,
131.1, 148.1, 149.3, 172.2, 192.0.
MS (ESI-ACI): m/z = 517.2 [M + H].
PENB-Glu (9a). 1H NMR (400 MHz, DMSO-d6): δ (ppm) =
7.79 (d, 1H), 7.64 (m, 4H), 7.00 (m, 2H), 4.25 (m, 4H), 3.85 (m,
1H), 3.70 (m, 8H), 3.50 (m, 3H), 2.55 (br, 2H), 2.07 (m, 2H),
1.30 (m, 3H).
Synthesis of alkoxy-4-nitro-[1,1′-biphenyl]-3-yl)propan-1-ol
(8a–c). 2-(5-Iodo-2-nitrophenyl)propan-1-ol13
(507
mg,
1.66 mmol) was suspended in dry toluene (30 mL) under argon.
Tetrakistriphenylphosphine palladium (200 mg, 0.19 mmol) was
then slowly added to the mixture and stirred until it was fully
dissolved. Na2CO3 in water (20 mL) was then added. The
mixture was then heated at 110 °C during 30 min. 4-(2-(2-Meth-
oxyethoxy)ethoxy)phenylboronic acid,14 3-methoxyphenylboro-
nic acid or 2-methoxyphenylboronic acid (3.29 mmol) was
dissolved in ethanol (3 mL) and dropwise added to the mixture.
The reaction was stirred at 110 °C and followed by TLC. After
reaction, the mixture was cooled to room temperature, diluted
with a saturated NaCl solution, and extracted with ethyl acetate.
The organic layer was then washed with water and dried on
MgSO4 and the solvent was removed under vacuum. The crude
product was purified on column chromatography (SiO2; cyclo-
hexane/AcOEt 2 : 8 v/v). PENB-OH (8a) (320 mg, 46%),
mMNB-OH (8b) (434 mg, 90%) and oMNB-OH (8c) (448 mg,
91%) were obtained as slight yellow viscous oils.
13C NMR (400 MHz, DMSO-d6): δ (ppm) = 173.5, 160.3,
149.7, 145.9, 138.8, 132.1, 129.5, 127.0, 126.3, 125.8, 116.0,
99.2, 72.3, 71.2, 70.8, 70.5, 70.4, 69.5, 68.6, 59.2, 55.2, 53.8,
34., 30.4, 25.8, 17.9.
mMNB-Glu (9b). 1H NMR (400 MHz, CDCl3): δ (ppm) =
7.73 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.42 (d; J = 8.1 Hz, 1H),
7.28–7.34 (m, 1H), 7.4–7.11 (m, 1H), 6.88 (d; J = 8.4 Hz, 1H),
4.11–4.19 (m; 2H), 3.69–3.81 (m; 5H), 2.52 (br, 2H), 2.05–2.14
(m, 2H), 1.24–1.30 (m, 3H), 0.85–0.92 (m, 1H).
13C NMR (400 MHz, CDCl3): δ (ppm) = 173.7, 173.2, 160.0,
149.0, 145.5, 140.3, 137.9, 130.0, 126.7, 125.9, 124.8, 119.7,
113.7, 113.1, 68.8, 55.2, 53.7, 32.9, 30.0, 25.6, 17.3.
oMNB-Glu (8c). 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.72
(d, J = 8.2 Hz, 1H), 7.58 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H),
7.26–7.37 (m; 2H), 6.95–7.04 (m, 2H), 4.05–4.25 (m, 2H),
3.68–3.88 (m; 5H), 2.40–2.62 (m, 2H), 2.09–2.15 (m, 2H),
1.28–1.31 (m; 3H), 0.82–0.92 (m, 1H).
PENB-OH (8a). 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.79
(d, 1H), 7.58 (d, 1H), 7.45 (m, 3H), 6.98 (d, 3H), 4.14 (m, 2H),
3.85 (m, 4H), 3.65 (m, 6H), 3.50 (m, 4H), 3.31 (s, 3H), 1.30
(t, 3H).
13C NMR (400 MHz, CDCl3): δ (ppm) = 174.0, 173.9, 156.8,
148.9, 144.0, 137.3, 131.0, 130.5, 129.8, 128.9, 128.6, 124.5,
121.5, 111.9, 69.7, 55.9, 33.3, 30.4, 25.7, 17.8, 17.7.
13C NMR (400 MHz, CDCl3): δ (ppm) = 159.0, 148.4, 144.9,
138.8, 131.1, 128.1, 125.9, 124.6, 124.6, 114.8, 99.2, 71.5, 70.4,
70.2, 70.1, 69.3, 67.2, 58.3, 36.1, 17.3.
Quantification of GABA release
A chromophoric derivative was coupled to the GABA moiety in
pHBP-GABA and pABP-GABA to quantify GABA release by
HPLC.15
mMNB-OH (8b). 1H NMR (400 MHz, CDCl3): δ (ppm) =
7.86 (d, J = 8.4 Hz, 1H), 7.66 (dd, J = 8.4 Hz and 1.8 Hz, 1H),
7.42 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H), 7.17 (d, J =
7.2 Hz, 1H), 7.11 (s, 1H), 6.97 (dd, J = 8.1 Hz and 1.8 Hz, 1H),
3.84–3.89 (m, 5H), 3.66 (m, 1H), 1.39 (d, J = 6.9 Hz, 3H).
Synthesis. 1H-Benzo[1,2,3]triazol-1-yl-4-methoxybenzoate
(4-methoxybenzoic [MBA] activated ester:
580 | Photochem. Photobiol. Sci., 2012, 11, 578–586
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Quantification of glutamate release
A mixture of 4-methoxybenzoic acid (200 mg, 1.31 mmol),
PyBop (685 mg, 1.31 mmol) and diisopropylamine (230 μL,
1.9 mmol) in 10 mL of anhydrous DMF was stirred under argon
at room temperature over night. Water (50 mL) was added and
the organic compounds were extracted into EtOAc (50 mL).
Purification by flash chromatography using heptane/EtOAc 1 : 1
in vol. gave 335 mg of a white powder in 95% yield.
The glutamic acid formation was quantified by HPLC using a
chromophoric derivative formed quantitatively after conden-
sation with o-phthaldialdehyde and mercaptoethanol in a 50 mM
Borate buffer pH 9.16 The chromophoric adduct was detected by
absorbance at 340 nm. HPLC retention time on a analytical
SB-C18 Zorbax (4.6 × 250 mm) column using a 30 min linear
gradient from 0 to 100% Acetonitrile in a 0.1% TFA water sol-
ution at 1 mL min−1 of the glutamate o-phthaldialdehyde
adduct: 16.7 min.
1H NMR (400 MHz, CDCl3): δ (ppm) = 3.95 (s, 3H), 7.07 (d,
J = 8.9 Hz, 2H), 7.42–7.57 (m, 3H), 8.10 (d, J = 8.2 Hz, 1H),
8.24 (d, J = 8.9 Hz, 2H).
General procedure for coupling GABA or caged GABA’s to
the 4-methoxybenzoate activated ester. A mixture of 1H-benzo
[1,2,3]triazol-1-yl-4-methoxybenzoate (4 mg, 0.015 mmol) and
GABA or caged GABAs (pHBP-GABA and pABP-GABA)
(0.01 mmol) with DIEA (0.01 mmol) in anhydrous DMF, were
stirred over night at room temperature. Purification by semi-
preparative C-18 HPLC with a 250 × 10 BetaBasic-18 column,
from Thermo Scientific, using elution at a flow rate of 4 mL
min−1 with a linear gradient of acetonitrile in 0.1% TFA in water
from 0 to 100% (v/v) over 30 min gave respectively pHBP-GA-
BA-MBA, pABP-GABA-MBA and GABA-MBA after evapor-
ation. Retention time: 19.3 min for the pHBP-GABA-MBA,
19.7 min for the pABP-GABA-MBA and 13.7 min for
GABA-MBA.
GABA-MBA. 1H NMR (400 MHz, CDCl3): δ (ppm) = 1.96
(m, 2H), 2.48 (t, J = 6.1 Hz, 2H), 3.54 (m, 2H), 3.85 (s, 3H),
6.92 (d, J = 8.7 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H).
pHBP-GABA-MBA. 1H NMR (400 MHz, CDCl3): δ (ppm) =
2.10 (t, J = 6.4 Hz, 2H), 2.66 (t, J = 6.8 Hz, 2H), 3.61 (t, J = 7.2
Hz, 2H), 3.85 (s, 3H), 5.40 (s, 2H), 6.92 (d, J = 8.8 Hz, 2H),
6.97 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.67 (d, J =
8.4 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H).
MS (ESI-ACI): m/z = 314.2 [M + H].
One photon quantum yield determination
The quantum yields for the photoconversion of pHBP-GABA,
pABP-GABA, PENB-Glu, mMNB-Glu and oMNB-Glu were
determined by comparison with the photolysis of 1-(2-nitrophe-
nyl)ethyl-ATP (NPE-ATP) (Φu = 0.63)17 which was taken as
reference in a phosphate buffer (0.1 mM, pH 7.4) at 25 °C.
These compounds were tested at identical optical densities at the
used irradiation wavelengths. Accordingly, mixtures of caged
neurotransmitters and 0.2 mM of NPE-ATP reference were used.
Those mixtures were photolyzed by continuous irradiation at
315 nm using a 1000 W Hg Lamp from Hanovia focused on the
entrance slit of a monochromator, and aliquots were subjected to
reversed-phase HPLC to determine the extent of the photolytic
conversions. HPLC analysis was carried out on an Acclaim
Analytical SB-C18 (4.6 × 250 mm) column; elution was per-
formed at a flow rate of 1 mL min−1 with a linear gradient of
acetonitrile in 0.1% TFA in water from 0 to 100% (v/v) over
30 min. Disappearances quantum yields Φd were calculated by
considering the conversions up to 20%, in order to limit, as
much as possible, errors due to undesired light absorption during
photolysis.
Uncaging quantum yields are given by the following eqn (3):
pABP-GABA-MBA. 1H NMR (400 MHz, CDCl3): δ (ppm) =
2.06 (t, J = 6.4 Hz, 2H), 2.62 (t, J = 6.8 Hz, 2H), 3.15 (t, J = 7.2
Hz, 2H), 3.37 (s, 6H), 3.51–3.64 (m, 16H), 3.80 (s, 3H), 5.36 (s,
2H), 6.88 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 7.50 (d,
J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.8 Hz,
2H), 7.90 (d, J = 8.4 Hz, 2H).
Φu ¼ Φd ꢀ photocleavage yield
ð3Þ
Two photon uncaging action cross-section determination
The two-photon uncaging action cross-section δaΦu was deter-
mined by comparison with that of CouOAc (1.07 GM at
740 nm)6 as a known reference, in the same illumination con-
ditions. Solutions of caged neurotransmitters and CouOAc were
irradiated during 10–40 min by a mode-locked titanium:sapphire
laser (Tsunami, Spectra Physics, 100 fs, 80 MHz) at 740 nm.
The measurements were performed at P = 250 mW, in the quad-
ratic dependence range for such chromophore.9 After irradiation,
80 μL of each solution is analyzed by HPLC to determine the
extent of the photolytic conversions. HPLC analysis was carried
out on an Acclaim Analytical SB-C18 Zorbax (4.6 × 250 mm)
column; elution was performed at a flow rate of 1 mL min−1
with a linear gradient of acetonitrile in 0.1% TFA in water from
0 to 100% (v/v) over 30 min. The ratio between the slopes deter-
mined from the graphical representation of the photolytic conver-
sion allows measurement of two-photon uncaging action cross-
section (using CouOAc6 as primary standard, for which the two-
photon uncaging action cross-section of 1.07 at 740 nm is accu-
rate within a factor of 2).
MS (ESI-ACI): m/z = 651.2 [M + H].
Released GABA quantification. The yield of released
GABA-MBA from irradiated pHBP-GABA-MBA and pABP-
GABA-MBA is determined by HPLC analysis. A 1.0 mL
portion of a 50 μM solution of pHBP-GABA-MBA or pABP-
GABA-MBA in phosphate buffer (50 mM) was irradiated at 315
or 365 nm respectively for pHBP-GABA-MBA or pABP-GA-
BA-MBA, to ensure a complete conversion of the starting com-
pound using a LUMOS 43 (Atlas Photonics Inc.). 200 μL of
each irradiated solutions were analyzed by HPLC and compared
to the HPLC calibration curve obtained by injection of 200 μL
of 20–150 μM solutions of GABA-MBA in phosphate buffer.
HPLC analysis was carried out on Acclaim Analytical
SB-C18 (4.6 × 250 mm) column; elution was performed at a
flow rate of 1 mL min−1 with a linear gradient of acetonitrile in
0.1% TFA in water from 0 to 100% (v/v) over 30 min.
Retention time: 22.9 min for pHBP-GABA-MBA, 23.0 min
for pABP-GABA-MBA and 15.8 min for GABA-MBA.
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Results and discussion
To optimize the two-photon uncaging action cross-section δaΦu
we have to improve the two-photon absorption process, linked to
δa, as well as the efficiency of the photochemical reaction,
related to Φu. As δa is related to the cubic hyperpolarisability γ,
all the molecular engineering strategies classically used to opti-
mize γ can be applied to the design of our new cages. Because
of potential limitations for biological applications (i.e. the size
and the aqueous solubility of the probe), we limited our investi-
gations to non-centrosymmetric one-dimensional (1D) donor–
acceptor (D–A) systems. (i.e. other geometries are possible, such
as centrosymmetrical 1D A–A or A–D–D–A systems, see refer-
ence 12) For such systems it is well known that the optimization
of γ requires an elongation of the conjugated π system and/or a
modulation of the end groups D,A properties.6,18 Previous works
on the o-nitrobenzyl and o-nitrophenylbutyl series have pointed
out the importance of these two parameters. Indeed Goeldner’s
group has described a caged glutamate based on the [3-(4,5-
dimethoxy-2-nitrophenyl)-2-butyl] (DMNPB) cage, showing
low two-photon uncaging action cross-section (0.17 GM at
720 nm) but high photocleavage efficiency (Φu = 0.26).8 On the
contrary Jullien’s group has described a caged coumarin based
on 5-(4-methoxyphenylethynyl)-2-nitrobenzyl cage which pre-
sents a low two-photon uncaging action cross-section (0.045
GM at 730 nm), the later being probably due to a too low
efficiency for the photochemical reaction (Φu = 0.001), when δa
was certainly improved by the introduction of a long conjugated
donor–acceptor system.19 We thus decided to design new cages
based on the biphenyl central core ended by oxygen or nitrogen
atoms as electron donors, and nitro or carbonyl groups as elec-
tron acceptors. We will describe here three caging platforms
based on D–A system centred on a biphenyl core, successively
the phenacyl series, the o-nitrobenzyl series and at last the
o-nitrophenyl propyl series, deciphering unprecedented two-
photon uncaging properties within the latter series.
Scheme 1 Structure of the donor–acceptor biphenyl derivatives in the
phenacyl series: pHBP and pABP.
Scheme 2 Synthesis of pHBP-GABA (5) and pABP-GABA (6). i)
K2CO3, Bu4NBr, Pd(OAc)2, EtOH/H2O, microwave 150 °C, 10 min, ρ
= 46–91%. ii) CuBr2, AcOEt, Δ, 19h, ρ = 56%. iii) a) TBDMS-OTf, di-
isopropyl ethyl amine (DIEA), CH2Cl2, 4 h, RT. b) Br2, CH2Cl2, 1 h,
RT, ρ = 68%. iv) a) N-Boc GABA, DBU, benzene, 19 h, RT. b) TFA/
CH2Cl2, 1 min, ρ = 60–86%.
Donor–acceptor biphenyl derivatives in the phenacyl series
In this series the caging platform is based on a biphenyl central
core surrounded by a hydroxy or bis((bis-ethoxy)methoxy)amino
electron donating group and the carbonyl function of the phena-
cyl group as electron acceptor (Scheme 1).
grafted to the biphenyl α-bromoketones (2, 4) to provide the new
caged GABA analogues, pHBP-GABA (5) and pABP-GABA
(6) respectively, after deprotection in acidic media.
Synthetic pathways. The syntheses of two donor–acceptor
biphenyl phenacyl cages: p-hydroxybiphenylacyl (pHBP) and
p-bisalkyl-aminobiphenylacyl (pABP) are summarized in
Scheme 2. The biphenyl moieties were obtained using Suzuki
cross-coupling reactions with microwave activation20 between 1-
(4-bromophenyl)ethanone and 4-hydroxyphenylboronic acid or
4-iodo-N,N-bis(methoxyethyl)aniline and 4-acetylphenylboronic
acid for the synthesis of pHBP or pABP cages respectively. The
1-(4′-hydroxy-[1,1′-biphenyl]-4-yl)ethanone (1) was converted
to the corresponding α-bromoketone (2) by bromination with
cupric bromide.21
Photochemical and photophysical properties. The solubilities
of pHBP-GABA (5) and pABP-GABA (6) at room temperature
in phosphate buffer pH 7.4 (without any co-solvent) were 40 μM
and 13 mM, respectively. The hydrolytic stability was explored
by HPLC in phosphate buffer (pH 7.4) at room temperature; a
half-time of 10 h was measured for the hydrolysis of 5 and 6.
The absorption maxima of pHBP-GABA and pABP-GABA are
313 and 369 nm with molecular extinction coefficients of 14800
and 18000 M−1 cm−1 respectively (Fig. 1). The photolytic
release of GABA was analyzed by UV-visible spectroscopy and
HPLC. Photolysis was carried out by irradiation of samples
(around 50 μM), in phosphate buffer 50 mM pH 7.4, at 315 nm
or 365 nm for pHBP-GAGA and pABP-GABA respectively.
However, 1-(4′-(bis(2-(2-methoxyethoxy)ethyl)amino)-[1,1′-
biphenyl]-4-yl)-2-bromoethanone (4) was synthesized by treat-
ment with bromine of the TBDMS silyl enol ether derivative
of 3. Protected GABA (N-α-tert-butoxycarbonyl-GABA) was
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action cross-section value of 0.24 GM at 740 nm was obtained,
limiting further developments. (Table 1).
Donor–acceptor biphenyl derivatives in the o-nitrobenzyl series
One example has been selected in this series, that is based on a
methoxy group as donor, and a nitro group as acceptor
(Scheme 3).
The two-photon optimization of the donor–acceptor o-nitro-
benzyl platform was investigated by the group of Jullien.19
Using molecular engineering on this platform several strategies
were investigated in order to induce a red-shifted absorbance that
could allow an efficient photolytic reaction upon two-photon
excitation. The donor–acceptor biphenyl platform was therefore
investigated. The p-methoxynitrobiphenyl (pMNB) photoremo-
vable group showed an interesting shift in absorbance compared
to the usual o-nitrobenzyl (oNB) group (317 nm versus 262 nm)
but with a very low quantum yield for the photolytic reaction.
On the other hand, this series showed a poor hydrolytic stability
on caged carboxylic acids like glutamate and GABA. Clearly, it
Fig. 1 UV spectra of donor–acceptor biphenyl chromophores in the
phenacyl series at 50 μM in PBS pH = 7.4, 50 mM.
pHBP-GABA showed a new absorbance at 399 nm, whilst the
initial absorbance at 313 nm gradually diminished, with the
appearance of isobestic points (data not shown). Alternatively,
pABP-GABA only showed a gradual diminution of the initial
absorbance at 369 nm during irradiation.
To be able to quantify the released GABA by HPLC, our
caged GABA derivatives 5 and 6 were coupled to an activated
ester of 4-methoxybenzoic acid used as a chromophoric deriva-
tive (data not shown).15 An almost quantitative (≥93%) for-
mation of the GABA 4-methoxybenzamide derivative was
measured by HPLC after complete photo-conversion of
pHBP-GABA-MBA. However, only a 55% yield formation of
the GABA 4-methoxybenzamide derivative was measured after
complete photo-conversion of pABP-GABA-MBA, indicating
that these functional groups induce some competitive photoche-
mical pathways, which do not contribute to the photorelease of a
carboxylic acid function. Quantum yields of the photolytic reac-
tions for pHBP-GABA (5) and pABP-GABA (6) were deter-
mined by competition with the 2-(nitrophenyl)ethyl-ATP taken
as reference molecule.17 A good uncaging quantum yields
(Table 1) was determined in phosphate buffer (50 mM, pH 7.4)
only for the for pHBP. Altogether, the high photolysis quantum
yield and the significant molar absorption coefficient (εΦu
=
3100 M−1cm−1 at 315 nm) together with the efficient release of
GABA (≥ 93%) make this photoremovable group an interesting
cage for near UV photolysis. Therefore, we decided to fully
characterize the two-photon sensitivity of pHBP-GABA. The TP
uncaging action cross-section (δaΦu) of caged pHBP-GABA (5)
was determined at 740 nm by HPLC analysis of the disappear-
ance of the starting material during TP photolysis and compared
to the disappearance of CouOAc as a reference (δaΦu = 1.07 GM
at 740 nm).6 Unfortunately a modest two-photon uncaging
Scheme 3 Structure of the pMNB donor–acceptor biphenyl derivative
in the o-nitrobenzyl series.
Table 1 Photochemical and photophysical properties of donor–acceptor biphenyl caged GABAs in the phenacyl series
δaΦu (GM)
740 nm
Solubility
(PBS pH 7.4)
Hydrolysis
(t1/2 in PBS pH 7.4 at RT)
Compound
λmax (nm)
ε (M−1 cm−1
)
% photo-release
Φu
pHBP-GABA
pABP-GABA
313
369
14800
18000
93
55
0.21
0.015
0.24
n.d.
40 μM
13 mM
10 h
10 h
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was very difficult to optimize both the absorbance and the un-
caging properties in this series. Therefore, we decided to investi-
gate the 2-(o-nitrophenyl)propyl series which, besides a good
hydrolytic stability offered improved photolytic properties in the
UV-visible region.8
syntheses of three donor–acceptor biphenyl 2-(o-nitrophenyl)
propyl cages: 3-(2-propyl-1-ol)-4′-tris-ethoxy(methoxy)-4-nitro-
biphenyl (PENB), 3-(2-propyl-1-ol)-m-methoxy-4-nitrobiphenyl
(mMNB) and 3-(2-propyl-1-ol)-o-methoxy-4-nitrobiphenyl
(oMNB) are summarized in Scheme 5. The 4-iodo-2-(2-propyl-
1-ol)nitrobenzene13 was coupled to the corresponding phenyl-
boronic acids to give the biphenyl derivatives 8. Protected gluta-
mate (N-α-t-Boc-L-glutamic acid γ-t-butyl ester) was then grafted
to this cage to give the caged glutamates 9 after deprotection in
acidic media.
Donor–acceptor biphenyl derivatives in the 2-(o-nitrophenyl)
propyl series
In this series the caging platform is based on a biphenyl central
core surrounded by a hydroxy, methoxy, tris-ethoxy(methoxy) or
bis((bis-ethoxy)methyl)amino electron donating groups and nitro
as electron attracting group (Scheme 4).
Photochemical and photophysical properties. The one-photon
properties of caged glutamate 8a–c have been investigated by
UV-visible spectroscopy and HPLC. The absorption maxima and
the molar extinction coefficients of PENB-Glu, mMNB-Glu
and oMNB-Glu are summarized in Table 2. The photolytic
release of glutamate was analyzed quantitatively by HPLC using
derivatization with o-phthaldialdehyde and mercaptoethanol16
after irradiation in neutral buffered medium. All caged gluta-
mates 9a–c afforded a 90% yield of glutamate release. The
photochemical and photophysical properties of EANBP-GABA,
PMNB-Glu and PHNB-Glu, are summarized in Table 2 as pre-
viously reported by Donato et al.15 and Gug et al.,9 respectively.
The 2-(o-nitrophenyl)propyl series was an ideal photoremova-
ble protecting group to be connected to the donor–acceptor
biphenyl plateform to generate powerful TPE-sensitive caging
groups. First, we were able to improve the two-photon sensitivity
by elongating the conjugated system on methoxynitrobiphenyl
platforms, without substantially affecting the yield of photo-
release and the quantum yield of the 2-(o-nitrophenyl)propyl
photolytical reaction.9 The p-methoxynitrobiphenyl platforms
showed the best two-photon properties at 740 nm compared to
the other methoxy regioisomers. However, the substitution of the
methoxy group by a better electron donating hydroxy group
which should also increase the δa, did not increase the two-
Synthetic pathways. The synthesis of 2-(4′-((di(tris-ethoxy
(methyl))amino)-4-nitro-[1,1′-biphenyl]-3-yl)propyl (EANBP),
3-(2-Propyl-1-ol)-4′-methoxy-4-nitrobiphenyl (PMNB) and 3-
(2-propyl-1-ol)-4′-hydroxy-4-nitrobiphenyl (PHNB) cages and
their corresponding caged neurotransmitters were previously
reported by Donato et al.15 and Gug et al.9 respectively. The
Scheme 5 Synthesis of donor–acceptor biphenyl caged glutamates in
the 2-(o-nitrophenyl)propyl series. i) Pd(PPh3)3, NaHCO3, toluene,
110 °C, 2–10 h, ρ = 54–80%. ii) a) N-Boc-Glu-OtBu, DCC, DMAP,
CH2Cl2, 19 h, RT, b) TFA, CH2Cl2, RT, 5 h, ρ = 80–90%.
Scheme 4 Structure of the donor–acceptor biphenyl derivatives in the
2-(o-nitrophenyl)propyl series: pHBP, PENB, EANBP, mMNB and
oMNB.
Table 2 Photochemical and photophysical properties of donor–acceptor biphenyl caged neurotransmitters in the 2-(o-nitrophenyl)propyl series
δΦu
(GM) (λ)
Solubility
(in PBS pH 7.4)
Hydrolysis
(24 h in PBS pH 7.4)
Compound
λmax (nm)
ε (M−1 cm−1
)
% photo-release
Φu
PHNB-Glu
mMNB-Glu
oMNB-Glu
PMNB-Glu and
PENB-Glu
330
296
302
317
n.d.
<10%
90
90
n.d.
n.d.
n.d.
0.09
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3%
7150
6300
9900
1.8 (740 nm)
2.2 (740 nm)
3.2 (740 nm)
90
> 5 mM for PENB-Glu
EANBP-GABA
397
7500
95
0.15
11 (800 nm)
10 mM
≤ 1%
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photon uncaging action cross-section, mainly due to undesirable
photochemical reactions leading to an inefficient photolytic reac-
tion. The p-dialkylaminonitrobiphenyl platforms, on the other
hand, led to a 90 nm bathochromic shift compared to the p-meth-
oxynitrobiphenyl platforms (Fig. 2), with no significant
efficiency modification of the photolytic reaction (95% release
with a 15% quantum yield for one photon photoconversion).
Most importantly, the substitution of the methoxy group by a
better electron-donating group (NR2) significantly increased the
TP sensitivity of this photoremovable group, leading to an
unprecedented TP uncaging action cross-section at 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 cages by grafting oligoethyleneglycol chains
on the amino function. Finally, the photolytic kinetics for the
more interesting photoremovable EANBP and PENB protecting
groups were investigated using a fluorescent reporter: 1,3-
dichloro-9,9-dimethyl-9H-acridin-2(7)-one (DDAO). This red
emitting fluorophore showed a large and interesting spectral
difference between the caged DDAOs and the free DDAO,
allowing an easy estimation of the photolytic reaction kinetics by
following the appearance of a red fluorescent signal after laser
flash photolysis.14–15 A time scale shorter than 5 μs and 15 μs
for EANB-DDAO and PENB-DDAO respectively, were
obtained for the release of the fluorescent DDAO, value in con-
cordance with the reported kinetics for other o-(nitrophenyl)
propyl photoremovable groups,8–9 in agreement with temporal
control of most neuronal processes.
In summary, the best photophysical properties in the 2-(o-
nitrophenyl)propyl series, were observed for the p-alkoxy and
p-bisalkylamino-4-nitro-[1,1′-biphenyl]-3-yl)propyl derivatives.
Therefore oligoethylene glycol chains were used to alkylate
those two scaffolds, leading the more soluble PENB and
EANBP cages. The EANBP and PENB photoremovable pro-
tecting group are to our knowledge the best caging group for
two-photon excitation at 800 and 740 nm respectively, offering
attracting perspectives in chemical biology. They have already
been used for the two-photon GABA release in intact brain
tissue at 800 nm,15 and for live-cell 740 nm two-photon un-
caging of a far-red emitting acridinone fluorophore,14 respectively.
In addition PENB-Glu has been used for live-cell two-color
photoactivation with 740 nm two-photon glutamate uncaging in
combination with optogenetic methods (800 nm two-photon
channel Rhodopsin activation) on brain slices.22,23
Conclusion
Molecular engineering of photoremovable protecting groups
using the donor–acceptor biphenyl backbone was evaluated for
the development of probes displaying highly efficient two-
photon uncaging properties. In an early article19 by the group of
Jullien, the o-nitrobenzyl protecting group was investigated.
Besides an interesting red-shift of the absorption maximum
wavelength observed for the donor–acceptor biphenyl backbone,
the quantum yield of uncaging dramatically dropped, leading to
an inefficient two-photon uncaging process. Therefore the o-
nitrobenzyl series seems not to be an adapted protecting group to
enlarge the two-photon cross-section. The phenacyl protecting
group was subsequently investigated. The p-hydroxybiphenyl
backbone (pHBP) showed an interesting uncaging quantum
yield but without an significant increase of the absorption
maximum wavelength compared to the p-hydroxyphenacyl
(pHP)24 protecting group, also leading to an inefficient two-
photon uncaging process at 740 nm. On the other hand, the p-
dialkylaminobiphenyl backbone (pABP), a better electron
donating group displaying
a very interesting absorption
maximum wavelength at 370 nm, showed a low uncaging
quantum yield and only 55% yield of photocleavage, indicating
that these functional groups induce some undesired competitive
photochemical pathways. The 2-(o-nitrophenyl)propyl series was
a more versatile platform to increase the two-photon sensitivity
of photoremovable protecting groups, leading to the p-alkoxy
and p-bisalkylamino-4-nitro-[1,1′-biphenyl]-3-yl)propyl deriva-
tives: PENB and EANBP respectively. Those two photoremova-
ble protecting groups are up to date the best caging groups for
two-photon excitation at 800 and 740 nm respectively, offering
attracting perspectives in chemical biology. In addition, they also
could provide interesting applications using their two-photon
orthogonality in order to trigger independently another effector
with spatio-temporal control. The two-photon excitation around
800 nm of the EANBP cages could be selectively triggered
without inducing a photocleavage of the PENB cage. But it will
be unlikely that excitation around 740 nm will selectively cleave
the PENB or any other cage. Therefore, a dual wavelength TP
photoactivation can only be achieved through a spatial orthogon-
ality of the biological process under investigation.
Acknowledgements
This work was supported by the Université de Strasbourg, the
CNRS, the France-Berkeley Fund and ANR (Contracts No.
09-BLAN-0425-01 and PCV 07 1-0035).
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