Cooperative Dyads for Two-Photon Uncaging

Article abstract of DOI:10.1021/ol5033046

A series of dyads that combine a photolabile protecting group (PPG) 4,5-dimethoxy-2-nitrobenzyl and different bis-donor or bis-acceptor dissymmetric chromophores acting as two-photon (2P) absorbers were synthesized. Even for low energy transfer efficiency

Full text of DOI:10.1021/ol5033046

Letter
pubs.acs.org/OrgLett
Cooperative Dyads for Two-Photon Uncaging
Eduardo Jose Cueto Díaz,† Sebastien Picard,† Vincent Chevasson,† Jonathan Daniel,† Vincent Hugues,†
́
́
Olivier Mongin,‡ Emilie Genin,† and Mireille Blanchard-Desce*,†
†Univ. Bordeaux, Institut des Sciences Molec
́
ulaires, CNRS UMR 5255, 351 Cours de la Liber
́
ation, F-33450 Talence Cedex, France
‡Institut des Sciences Chimiques de Rennes, UMR CNRS 6226, Universite
́
de Rennes 1, 35042 Rennes Cedex, France
S
* Supporting Information
ABSTRACT: A series of dyads that combine a photolabile
protecting group (PPG) 4,5-dimethoxy-2-nitrobenzyl and different
bis-donor or bis-acceptor dissymmetric chromophores acting as two-
photon (2P) absorbers were synthesized. Even for low energy
transfer efficiency from the 2PA subunit to the uncaging one,
improvement of the 2P uncaging sensitivity in the NIR is achieved as
compared to isolated PPG. Moreover enhancement of the 2PA
response is achieved by tuning the electronic dissymmetry of the 2PA
subunit and the arrangement of the complementary subunits in the
dyads.
ight induced deprotection reactions have attracted signifi-
cant attention, as they offer applications in various fields
photorelease kinetics, etc. A proof of concept has been realized
recently with symmetrical tandem systems having two uncaging
7-nitroindolinyl (NI) moieties grafted onto a quadrupolar 2PA
module, enabling a 10-fold enhancement of 2P uncaging of
glutamate derivatives as compared to MNI, leading to a δu of 0.7
GM at 730 nm.3 These systems however still show a modest 2PA
cross section in the biological spectral window (with a maximum
of ∼40−70 GM at 710−720 nm and vanishing 2PA response at
800 nm). Based on this observation, our goal was thus the design
of synergic modular systems whose 2PA responses would be
significantly improved with 2PA activity maintained at a longer
wavelength (typically 800 nm) to allow improved penetration
depth in thick biological samples. The 4,5-dimethoxy-2-nitro-
benzyl (NV) photolabile protecting group (PPG) was selected as
a model PPG, as it has been used in a wide range of
photoactivated systems for drug or bioactive molecules delivery4
and has relatively good dark stability. This PPG however shows a
modest 2P uncaging action cross section (δu = 0.03 GM at 730
nm).2a To confer improved 2PA responses, instead of using
symmetrical quadrupolar 2PA structures built from a fluorene
core5 as done earlier,3,6 we chose to impart electronical
dissymmetry and designed A1−π−A2 or D1−π−D2 two-photon
absorbing modules (Figure 1). The symmetry breaking was
meant to relieve the symmetry interdiction of the 2P transition to
the lowest excited state and thus to promote enhanced 2PA
characteristics in the 700−800 nm region.7 Based on earlier work
on organic nanodots, we chose a phosphorus-based clip to
control the relative positioning of 2PA and uncaging units within
the dyads. This clipping module has been shown to allow
cooperative increase of two-photon absorption in dipolar
chromophore dimers.8
L
such as multistep organic synthesis, polymerization, volatiles
release, and biology. They offer significant advantages in terms of
possible external triggering with spatial and temporal control,
absence of chemical additives, and compatibility with a wide
range of common protecting groups. Various families of
protecting groups that can be photoactivated under near-UV
irradiation have been described in the literature including
arylcarbonylmethyl, nitroaryl, coumarin-4-ylmethyl, arylmethyl,
and metal complexes.1 They show diverse properties in terms of
photorelease kinetics, uncaging sensitivity, and dark stability
which are key criteria for applications.
In recent years, two-photon excitation (2PE) has emerged as a
popular tool for biological applications, as it permits performing
photoactivation in the near-IR region (700−1000 nm). 2PE
offers several advantages including intrinsic 3D resolution,
reduced out-of focus photodamage, and improved penetration
depth in tissues. However, one of the main current limitations for
its widespread application in biology is the poor two-photon
absorption (2PA) ability (quantified by the 2PA cross section σ2)
of most classical caging groups resulting in low 2P uncaging
sensitivity (quantified by the 2P uncaging action cross section δu
= σ2Qu, where Qu is the uncaging quantum yield). The
development of improved 2P cages displaying large δu values
that enabled efficient 2P photorelease to be performed at
nontoxic excitation intensities is thus required. Various attempts
toward new uncaging moieties specifically engineered for 2PE
have been conducted recently, but only very few have δu values
over 1 GM.2 Rather than designing new uncaging moieties, we
have been interested in modular approaches where classical
uncaging groups are combined with a better 2P absorber liable to
transfer its excitation energy to the adjacent uncaging module.
This alternative strategy was aimed at retaining critical
characteristics of the uncaging unit such as dark stability,
Received: November 13, 2014
© XXXX American Chemical Society
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Letter
Scheme 2. Synthesis of Dyads 1−4
Figure 1. Design of dyads for two-photon induced acetic acid uncaging
by combination of a fluorene-cored two-photon absorber (in blue) and
4,5-dimethoxy-2-nitrobenzyl (NV) uncaging moiety (in red).
The synthesis of two-photon absorbing modules 10a−d was
achieved in two steps by means of an in situ deprotection/double
Sonogashira coupling of the known 2,7-diiodofluorene deriva-
tive5 6 with the corresponding silylated alkynes 5a−c and 7a−d
with moderate to good yields for each step (Scheme 1). The
Scheme 1. Synthesis of Two-Photon Absorbing Modules
10a−d
carbonate in THF in the dark. The reaction was monitored by
1
31P and H NMR. The first substitution was observed in 31P
NMR to be completed by the presence of a singlet at 69.0 ppm
compared to the 31P NMR signal of 12 (63.4 ppm). The dyads
1−4 were easily obtained by purification on silica gel with
moderate yields. All compounds were characterized by 1H, 13C,
31P NMR and mass spectroscopy.
The photophysical properties of dyads 1−4 and their
corresponding fluorene-cored chromophoric subunits 10a−d
have been investigated, and experimental data are gathered in
Table 1. Both chromophoric subunits and dyads show an intense
absorption band in the near-UV, with a maximum molar
extinction coefficient more than 1 order of magnitude larger than
that of the isolated NV uncaging subunit. All 2P absorbing
subunits (10a−d) display strong fluorescence emission in the
violet-blue visible region (Figure 2). Increasing strength of the
end groups induces a red shift of both the absorption and
emission bands (Table 1). Interestingly a slight overlap between
NVOAc absorption and 2PA subunits emission is observed
(Figure 2), paving the way for energy transfer from the excited
2PA subunit to the close uncaging subunit within dyads. The
overlap integral values however decrease drastically as the
emission band of the 2PA subunit becomes more red-shifted (see
SI). The energy transfer is thus theoretically possible in dyads 1−
4, but with markedly decreasing efficacy on going from 1 to 4. In
agreement with these expectations, the fluorescence quantum
yields (except dyad 2) and lifetimes are found to decrease in
dyads 1−4 as compared to their 2PA subunits 10a−d. The
relative decrease of fluorescence is ∼40% for dyad 1 and falls to
4% for dyad 4, following the decrease of the overlap J between
acceptor (NVOAc) absorption and donor (2PA module)
emission spectra. Similarly, the fluorescence lifetimes decrease
by ∼40% for dyad 1 but only by 4% for dyad 4. From these
acetyl group was removed by saponification to yield the phenol
moiety. The NV derivatives 11 and 13 were prepared in six steps
from the same starting material, vanillin, with respectively a 29%
and 33% overall yield (Scheme 2). The key steps are the
introduction of the nitro group by nitration, the incorporation of
the acetyl group by simple esterification, and the grafting of
phenol moieties by click chemistry or by nucleophilic
substitution. The experimental protocols are reported in detail
in the Supporting Information (SI).
The synthesis of the targets dyads is based on highly efficient
nucleophilic substitution of phenolic derivatives 10a−d, 11, and
13 on the known scaffold 129 (Scheme 2). To obtain dyads 1−4,
we developed a one-pot procedure involving first the substitution
of one Cl by a stoichiometric amount of the phenolic anion of 11
or 13, followed by the substitution of the second Cl in the
presence of the phenolic anion of 10a−d (Scheme 2). These
phenolic anions were generated by the action of cesium
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Table 1. Photophysical Data of Dyads 1−4 and Their Corresponding Two-Photon Absorbing Subunits 10a−d in CHCl3
a
b
c
d
e
compd
λabsmax [nm]
ε
max [M−1·cm−1
]
λemmax [nm]
Φf
τf [ns] ΦET
ε
maxQu [M−1·cm−1
]
λ2PAmax [nm] σ2max [GM] σ2maxQu [M−1·cm−1
]
f
NVOAc
341
358
358
368
369
368
367
375
373
5.9 × 103
7.2 × 104
9.0 × 104
7.7 × 104
7.8 × 104
7.8 × 104
7.6 × 104
6.5 × 104
6.8 × 104
−
−
−
−
35
730
720
730
740
730
750
750
800
800
−
0.03
−
10a
1
384
383
428
426
435
433
446
446
0.70
0.45
0.60
0.68
0.69
0.61
0.64
0.60
0.66
0.40
0.89
0.74
0.97
0.86
1.04
1.00
30
0.38
0.17
0.11
0.05
195
85
50
0.11
10b
2
132
110
195
160
240
310
−
0.12
−
10c
3
50
0.10
10d
4
−
0.25
55
a
b
c
Fluorescence quantum yield, standard: quinine in 0.5 M H2SO4 (Φ = 0.546). Fluorescence lifetime (excitation at 370 nm). Fluorescence energy
d
transfer efficiency estimated from fluorescence quantum yields and/or lifetimes decrease in dyads. One-photon uncaging values derived from
comparative one-photon photolysis experiments at 365 nm, using Qu = 0.006 for NVOAc. 2PA cross section at λ2PA derived from TPEF
experiments. From ref 2a.
e
max
f
sensitivity. Yet, we note that dyad 4 retains similar uncaging
sensitivity (εmaxQu) as dyad 3, suggesting that additional
mechanisms such as photoinduced electron transfer might also
be involved in the uncaging mechanism of dyad 4.
The 2PA responses of the dyads and corresponding modules
10a−d were investigated by performing two-photon excited
fluorescence (TPEF) experiments in solution in the 700−1000
nm range using a Ti-sapphire laser delivering 140 fs pulses at a 80
MHz repetition rate. The 2PA spectra are shown in Figure 4, and
2PA maxima characteristics in the NIR region are collected in
Figure 2. Absorption spectrum of veratryl uncaging module and
Table 1.
emission spectra of 2PA modules (in CHCl3).
variations, fluorescence energy transfer efficiency values could be
estimated (Table 1). Whereas the energy transfer efficiency
remains acceptable for dyad 1 (40%), it decreases steeply on
going to dyads 3 (11%) and 4 (∼5%). Since all dyads have close
maximum extinction coefficients, we were thus expecting dyad 1
to have the largest one-photon uncaging sensitivity (εmaxQu) due
to the largest FRET efficiency.
Based on these observations, the uncaging ability of dyads 1−4
was investigated by monitoring the acetic acid photorelease upon
excitation at 365 nm and comparing with the uncaging behavior
Figure 4. Two-photon absorption spectra of dyads 1−4 (in CHCl3).
of NVOAc (see SI for experimental details). First-order kinetics
was evidenced in all cases (Figure 3). As slope values are
All chromophoric 2PA modules as well as dyads show a 2PA
band located at about twice the wavelength corresponding to
maximum one-photon absorption indicating that the symmetry
interdiction is lowered due to the inherent dissymmetry7,10 of
both the 2PA module and dyads. As anticipated, this effect is
more pronounced when the electronic dissymmetry of the 2PA
chromophoric subunits is more pronounced. The 2PA band is
red-shifted and the maximum 2PA cross-section (σ2max) increases
significantly following the 1 → 2 → 3 → 4 and 10a → 10d
sequences. Consequently, although modules 10a−d and dyads
max
1−4 have similar 1PA cross sections, module 10d has a σ2
value almost 1 order of magnitude larger than that of module 10a
while dyad 4 has a σ2max value which is 6 times larger than that of
dyad 1 and over 300 GM. Moreover, we observe that the 2PA
responses of the 2PA chromophoric subunits are significantly
affected by the proximity of the uncaging unit within the dyads.
This leads to a 2PA enhancement of ∼65% in the case of dyad 1
and 30% in the case of dyad 4 as compared to isolated modules
10a and 10c whereas a relative decrease of 17% is observed in the
case of dyads 2 and 3. This phenomenon can be ascribed to
directional through-space electrostatic interactions within dyads.
Such interactions which are known to influence the 2PA
response of both dipolar8 and quadrupolar10 type two-photon
Figure 3. Kinetics of acetic acid photorelease upon excitation at 365 nm
(X: conversion rate).
proportional to ε365Qu values, this allowed deriving the relative
Qu values with respect to that of NVOAc. Using the Qu reported
for NVOAc in the literature,2a one-photon uncaging sensitivities
could then be derived (Table 1). We observe that all dyads show
improved one-photon uncaging sensitivity as compared to
isolated NVOAc, and their ordering shows a similar trend to that
for overlap J (i.e., 1 ≫ 2 ≫ 3) indicating that FRET efficacy is
indeed a critical parameter that determines dyads 1−3 uncaging
C
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Organic Letters
Letter
absorbers are favored by the close proximity of the (dipolar)
uncaging and (dissymmetrical quadrupolar) 2PA subunits within
dyads. Hence, when the topology is favorable (as in dyads 1 and
4), a positive cooperative 2PA enhancement is achieved in dyads.
Using 2PA data, we derived estimated values of the 2P
uncaging action cross section (δu) of dyads approximating that
one- and two-photon uncaging quantum yields (Qu) are the same
and using δu = Quσ2max. As observed from Table 1, and contrary
to one-photon excitation at 365 nm, all dyads show much larger
two-photon uncaging sensitivity than isolated NVOAc, typically
enhanced by a factor of 5 or 6. Quite interestingly, dyad 4 though
having the lowest energy transfer efficacy and thus being the less
efficient dyad for 1PP appears as a promising dyad for 2P
photolysis both having the largest 2PA cross-section and being
the most red-shifted chromophore allowing 2PE at 800 nm. We
thus selected dyad 4 for conducting 2PP experiments at 800 nm.
NMR monitoring provides evidence that 2P uncaging of acetic
acid occurs upon excitation at 800 nm (Figure 5).
ACKNOWLEDGMENTS
■
This work was supported by funding from the European
Community’s Seventh Framework Program under TOPBIO
Project-Grant Agreement No. 264362. We also acknowledge
financial support from Agence Nationale pour la Recherche
(Grant 2010 ANR-10-BLAN-1436). M.B.D. thanks the Conseil
́
Regional d’Aquitaine for generous funding (Chaire d’Accueil
grant and fellowship to V.H.). The authors are grateful to Jean-
Pierre Majoral and Anne-Marie Caminade (LCC UPR 8241,
Toulouse, France) for the generous gift of MMH-PSCl2.
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In conclusion, we have shown that the engineering of dyads
that combine a dissymmetrical bis-donor chromophore that acts
as the 2P absorber and a dipolar PPG can lead to synergic systems
with improved 2P uncaging sensitivity at 800 nm and enhanced
2PA response. Although the reported uncaging 2P sensitivities
are still moderate due to the poor uncaging quantum yield of NV
and limited FRET efficacy, this opens an interesting route for the
design of more efficient dyads by replacing the PPG by more
efficient ones such as for instance MNI,11 DMNPB,12 and
CDNI,13 which have much larger uncaging quantum yields. In
addition the energy transfer efficiency could be enhanced by
using recently developed red-shifted PPGs that absorb in the
blue visible region14 and show large uncaging quantum yields
(such as DEAC450).14a This would open the way to dyads with
2P uncaging sensitivity over 10 GM. In addition with the purpose
of using such systems in cellular environments, water-soluble
versions of these dyads will be developed.
ASSOCIATED CONTENT
* Supporting Information
■
S
Methods, synthetic details, and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: mireille.blanchard-desce@u-bordeaux.fr.
Notes
The authors declare no competing financial interest.
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