Molecular engineering of photoremovable protecting groups for two-photon uncaging

Article abstract of DOI:10.1002/anie.200803964

(Chemical Equation Presented) Open the cage: Symmetrical photoremovable bis-glutamate cages have been designed, synthesized, and characterized that can release a neurotransmitter with unprecedented two-photon efficiencies (see picture), up to 5 GM for BNSF-Glu (BNSF=2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl] }-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene) at 800 nm, the optimal window both for tissue transparency and classically available laser sources.

Full text of DOI:10.1002/anie.200803964

Angewandte
Chemie
DOI: 10.1002/anie.200803964
Two-Photon Photolysis
Molecular Engineering of Photoremovable Protecting Groups for
Two-Photon Uncaging**
Sylvestre Gug, Frꢀdꢀric Bolze,* Alexandre Specht, Cyril Bourgogne, Maurice Goeldner, and
Jean-Franꢁois Nicoud
Photoremovable protecting groups have become a mainstay
for dynamic studies in various biological systems, from
neuroscience to genetics,[1] mainly because photoinduced
activation is orthogonal to other techniques used to detect
biological responses.[2] The photochemical release of the
active molecule is usually induced by an initial one-photon
absorption process, leading to a limited spatial localization of
the released substance. To overcome this obstacle, two-
photon (TP) excitation has recently emerged as a very
promising technique to obtain spatial control.[3,4] Indeed this
nonlinear optical (NLO) process takes place only where the
light intensity is at a maximum, typically by focusing an
infrared pulsed laser beam. In this case, the excited state
yields to the photolytic reaction by the simultaneous absorp-
tion of two low-energy photons (infrared instead of ultra-
violet, in classical absorption), which also limits the photo-
toxicity of the excitation beam. Unfortunately, the various
photoremovable groups (“cages”) that have been developed
for one-photon photoactivation exhibit very low efficiency in
two-photon excitation.[5] Some chemical modifications have
been performed on these chromophores to improve their TP
sensitivity, and new platforms have also been described.[6–10]
These approaches have led to moderately efficient TP cages
with uncaging cross-sections (dufu) of about 1 Goeppert-
Mayer (1 GM = 10À50 cm4 sphotonÀ1) at best. However, this
value remains insufficient for use in biological studies, for
which a 3 GM minimum value has been suggested.[11]
applications in material[14,15] and biological[16,17] sciences.
These efforts have led to various possible approaches for
increasing the TPA properties of chromophores or fluoro-
phores. Different chromophore geometries have been inves-
tigated, with linear (1D),[18,19] planar (2D),[20,21] and tetrahe-
dral (3D)[22] structures. The typical dipolar architecture of a
1D TPA chromophore, the smallest system to be useful in
biology, is generally composed of two electron-donor or
electron-acceptor groups (D or A) linked to a central core by
conjugated systems. Donor or acceptor groups can be added
on the central core to give quadrupolar architectures
(Figure 1).
Figure 1. Typical structure of a 1D two-photon absorption chromo-
phore (black=dipolar, gray=quadrupolar).
The TPA properties of such systems can be improved by
lengthening the conjugated system and/or increasing the
electron-donating or -withdrawing effect of the side groups.
We recently described the 3-(2-propyl)-4’-methoxy-4-nitro-
biphenyl (PMNB) cage as an efficient TP photolabile
protecting group for glutamate (duFu = 0.45 GM at
800 nm).[23] Its uncaging cross-section has been increased in
comparison with the well-known methoxynitrobenzyl plat-
form by extending the p system. Another approach was
proposed by Andraud, Baldeck, and co-workers, who pointed
out that TPA cross-sections of oligomers can be enhanced by
biexcitonic coupling between two weakly conjugated mono-
mers.[24–26] We applied this concept to the molecular engineer-
ing of new linear caging platforms. The first designed
We report herein the design, synthesis, and character-
ization of highly efficient TP cages, and their application to
glutamate photorelease. During the last decade, the optimi-
zation of chromophores for TP absorption (TPA) became an
important goal for organic chemists,[12,13] and give rise to many
[*] S. Gug, Dr. F. Bolze, Prof. Dr. J.-F. Nicoud
Institut Gilbert Laustriat, Pharmacologie et Physicochimie
Facultꢀ de Pharmacie, 74 route du Rhin, 67401 Illkirch (France)
Fax: (+33)3-90-24-43-13
E-mail: Frederic.bolze@pharma.u-strasbg.fr
molecule
(4,4’-bis-{8-[4-nitro-3-(2-propyl)-styryl]}-3,3’-di-
S. Gug, Dr. A. Specht, Prof. Dr. M. Goeldner
Institut Gilbert Laustriat, Chimie Bio-organique
Facultꢀ de Pharmacie, 74 route du Rhin, 67401 Illkirch (France)
methoxybiphenyl or BNSMB, Figure 2) was composed of
two vinylogues of PMNB linked together to take advantage of
a possible interaction between the two monomers. A double
bond was introduced in the system to improve its solubility in
organic solvents. Clearly, this increase in the length of
conjugation is beneficial to the TPA properties, but can be
detrimental to the uncaging quantum yield, as it might induce
photochemical side reactions. To obtain a linear bis(donor–
acceptor) system (according to Figure 1), the methoxy group
was moved from the para to the ortho position, allowing the
Dr. C. Bourgogne
Institut de Physique et Chimie des Matꢀriaux de Strasbourg
23 rue du Loess, 67034 Strasbourg cedex 2 (France)
[**] We wish to acknowledge Prof. Dr. Y. Mꢀly, Dr. Y. Arntz, and Dr. P.
Didier for two-photon irradiations, and Dr. C. Andraud for
calculations. This work was financially supported by the ANR and
MNERT.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200803964.
À
two monomers to be linked by a single C C bond. This
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Figure 3. Molecular structures of model compounds used for semi-
Figure 2. Structure of new TP photolabile cages: quadrupolar bis-
empirical AM1 calculations
(donor–acceptor) BNMSB (top), dipolar acceptor–acceptor BNSF
(middle), and the parent molecule PMNB (bottom).
structure can be considered as a biphenyl core with two
electron-donating methoxy groups, surrounded by two styr-
enic p systems bearing electron-withdrawing nitro groups.
The nitro groups also play a fundamental role in the uncaging
process, as they are involved in the mechanism of the
photochemical reaction.[8] The presence of a noncentrosym-
metric push–pull system, which is very important in quadratic
NLO properties such as second harmonic generation, is not
necessary for cubic NLO properties, such as TPA. Thus, we
designed another original cage based on a dipolar architec-
ture, in which the methoxy groups were removed and the
interaction between the two acceptor groups was enforced by
retaining the planarity of the central core. For this purpose,
we designed a caging platform with a fluorenyl central core,
substituted in the 9-position by 1-(3,6-dioxaheptyl) chains, to
increase aqueous solubility (2,7-bis-{4-nitro-8-[3-(2-propyl)-
Figure 4. Calculated two-photon absorption spectra of model chromo-
phores 1 (green), 2 (blue), 3 (black), and 4 (red).
styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene
or
BNSF,
Figure 2). A parent chromophore has been reported with a
TPA cross-section in excess of 5000 GM at 520–570 nm.[27] In
addition, such dipolar or quadrupolar structures seem prom-
ising for the uncaging process, as they carry two photo-
removable moieties that can photorelease two biomolecules
per cage.
To validate these strategies, TPA properties were tested
on model chromophores 1–4 (Figure 3) by using a semi-
empirical calculation method AM1 (Austin Method 1) for
geometry optimization and a CNDO/S method (CNDO =
complete neglect of differential overlap) modified by
Andraud et al, for the calculation of TPA properties.[28]
Chromophore 1 represents a model for the recently devel-
oped cage PMNB, which we used for TP uncaging of
glutamate.[23] Chromophore 2 acts as a model compound of
a vinylogue of 1. The two other systems, 3 and 4, represent
models for the new cages reported in this paper. Compound 3
is a quasi-dimeric form of 2, and 4 represents the symmetrical
linear system designed in this work.
increased TPA, in comparison with 1. For the dimeric system
3, there is a red shift, mainly resulting from the increased
length of conjugation, and a threefold increase of the TPA
cross-section compared to 2. These effects are even more
pronounced in the case of 4, even in the absence of electron-
donating methoxy groups. A threefold increase of the TPA
cross-section is evident for 4, in comparison with the
quadrupolar chromophore 3.
In this theoretical study, we compared only the TPA
efficiencies of the model chromophores. However, in dealing
with TP uncaging of biomolecules, the quantum yield of the
photolytic reaction must also be taken into account.
Caged glutamates
8 (4,4’-bis-{8-[4-nitro-3-(2-propyl)-
styryl]}-3,3’-di-methoxybiphenyl, BNSMB) and 14 (2,7-bis-
{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-
fluorene, BNSF) were synthesized according to the methods
outlined in Scheme 1 and Scheme 2, respectively. The bis-
(stilbene) chromophore 3 was synthesized by the Heck
coupling of 3-ethyl-4-nitrostyrene[6] and 4,4’-diiodo-3,3’-dime-
thoxybiphenyl 5, itself readily obtained from the commer-
cially available diazonium salt fast blue B. To synthesize the
As expected, the calculated two-photon absorption spec-
trum for 2 (Figure 4) showed a red-shifted absorption and an
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Scheme 1. Synthesis of caged glutamate 8 (BNSMB–Glu). a) Xylenes, Pd(OAc)2, EtN3, tri-o-tolylphosphine, 3-ethyl-4-nitrostyrene; 5 days, 1408C,
68%; b) tBuOK/tBuOH, paraformaldehyde, dimethylsulfoxide (DMSO), 808C, 3 h; c) Boc-l-Glu-O-tBu (boc=tert-butoxycarbonyl), 4-dimethyl-
aminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), CH2Cl2, room temperature, 2 h; d) 20% trifluoroacetic acid (TFA), CH2Cl2, room
temperature, 6 h.
Scheme 2. Synthesis of caged glutamate 14 (BNSF–Glu). a) NaH, N,N-dimethylformamide, 1-bromo-2-(2-methoxyethoxy)ethane; b) xylenes,
Pd(OAc)2, Et3N, tri-o-tolylphosphine, 3-ethyl-4-nitrostyrene, 5 days, 1408C, 68%; c) tBuOK/tBuOH, paraformaldehyde, DMSO, 808C 3 h, 20%;
d) Boc-l-Glu-O-tBu, DMAP, DCC, CH2Cl2, room temperature, 2 h; e) 20% TFA, CH2Cl2, room temperature, 6 h.
fluorenyl chromophore 11, the first step was the functional-
ization of 2,7-dibromofluorene 9 at the 9-position with two 1-
(3,6-dioxaheptyl) chains, to improve the water solubility of
the molecule. The Heck coupling was then carried out in the
same conditions as for the biphenyl compound, to give 11.
Chromophores 3 and 11 were then treated with a tBuOK/
tBuOH mixture in the presence of paraformaldehyde, to give
cages 6 and 12, respectively. Protected glutamate (N-a-tert-
butyloxycarbonyl-l-glutamic acid-a-tert-butyl ester) was cou-
pled to these cages, prior to deprotection in acidic media
(20% trifluoroacetic acid), to give caged glutamates 8 and 14.
All compounds were fully characterized by UV/Vis spectros-
copy, HPLC, 1H and 13C NMR spectroscopy, and high-
resolution mass spectrometry (see the Supporting informa-
tion).
The one-photon photophysical properties of caged gluta-
mates 8 and 14 were measured (Table 1) and compared to
those of MNI–Glu (MNI = 4-methoxy-7-nitroindolinyl),[29]
DMNPB–Glu
(DMNPB =
3-(4,5)-dimethoxy-2-nitro-
phenyl)-2-butyl),[8] PMNB–Glu,[23] and Bhc–Glu (N-(6-
Bromo-7-hydroxycoumarin-4-yl)methoxycarbonyl-l-gluta-
mic acid). A significant red-shift is evident for the two new
cages, as well as a strong increase in molar extinction
coefficient. The photolytic release of glutamate was analyzed
quantitatively by HPLC.[30] Caged glutamates 8 and 14
afforded 60% and 65% yields of glutamate release per
glutamate unit, respectively. As each caging system incorpo-
rates two caged glutamates, the overall yield of glutamate
release reaches 120% per molecule. The disappearance
quantum yields (F) of 0.3 for 8 and 0.25 for 14, respectively,
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Table 1: One- and two-photon photophysical properties of caged glutamates 8 (BNSMB–Glu), 14
(BNSF–Glu), MNI–Glu, DMNPB–Glu, PMNB–Glu, and Bhc–Glu.
and two-photon excitation: eF >
10000mÀ1 cmÀ1 and duFu up to
5.0 GM. Furthermore, these two
new cages present a two-photon
uncaging cross-section greater than
2 GM in the range 740–850 nm, the
optimal window both for tissue
transparency and classically avail-
able laser sources. The successful
use of these new cages is promising,
owing to adequate solubility in
water, especially for BNSF–Glu, as
a result of the presence of 1-(3,6-
dioxaheptyl) chains (ꢀ 0.1 mm in
0.1m phosphate buffer). Further
Caged
glutamate
lmax
[nm]
e (lmax
)
Yield
F
eF(lmax
)
duFu
[GM][c]
[mÀ1 cmÀ1
]
[% Glu][a,b]
[mÀ1 cmÀ1
]
BNSMB–Glu
(8)
BNSF–Glu (14)
400
415
39340
60
65
0.30 11800
(6600)[b]
0.9
5.0
63960
0.25 16000
(7500)[b]
MNI–Glu[30]
DMNPB–Glu[8]
PMNB–Glu[23]
Bhc–Glu[7]
350
350
317
368
4300
4500
9900
–
95
90
–
0.08
0.26
0.1
366
1170
990
0.06[d]
0.17[d]
0.45
17470
0.02
350
0.37
[a] Yield of glutamate release per glutamate unit. [b] At 354 nm. [c] At 800 nm. [d] At 740 nm.
were determined by comparison with the reference molecule,
2-(nitrophenyl)ethyl–ATP [31] at 315 nm, monitored by HPLC
analysis. Clearly, the one-photon photolytic efficiencies of
glutamate release at 354 nm is at least one order of magnitude
higher for the new probes compared to the previously
described cages (Table 1). This increase is even more
accentuated at higher wavelengths (l ꢀ 400 nm).
investigations on the effect of 8 and 14 on neurostimulation
are underway.
Received: August 11, 2008
Published online: October 29, 2008
Keywords: glutamate · neurotransmitters · photolysis ·
.
synthetic methods · two-photon absorption
The two-photon uncaging cross-sections duFu of 8 and 14
have been determined in the range 740–900 nm (Figure 5). At
800 nm, the two-photon uncaging cross-sections of 8 and 14
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*
Figure 5. Two-photon photolysis cross-sections (duFu) of Bhc–Glu ( ),
~
&
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