DNA switches on the two-photon efficiency of an ultrabright triphenylamine fluorescent probe specific of at regions

Article abstract of DOI:10.1021/ja404422z

We report on the design and synthesis of two-photon fluorescent triphenylamines bearing two or three vinyl branches terminated by a N-methyl benzimidazolium moiety. The new compounds (TP-2Bzim, TP-3Bzim) are light-up fluorescent DNA probes with a long wavelength emission (>580 nm). Compared to their pyridinium models, the TP-Bzim dyes exhibit a remarkable improvement of both their DNA affinity and fluorescence quantum yield, especially for the two-branch derivative (TP-2Bzim: Φ_F = 0.54, K_a = 10⁷ M^-1), resulting in a large fluorescence emission turn-on ratio of up to 140. Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ = 1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time. This is attributed to a tight fit of the molecule inside the minor groove of AT-rich DNA which induces geometrical rearrangements in the dye ground state as supported by circular dichroism and molecular modeling data. Consequently, TP-2bzim displays an exceptional two-photon molecular brightness (δ×Φ_F = 583 GM), a value unrivalled for a small biofluorophore. These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant, and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.

Full text of DOI:10.1021/ja404422z

Article
pubs.acs.org/JACS
DNA Switches on the Two-Photon Efficiency of an Ultrabright
Triphenylamine Fluorescent Probe Specific of AT Regions
Blaise Dumat,†,∥ Guillaume Bordeau,†,§,∥ Elodie Faurel-Paul,† Florence Mahuteau-Betzer,†
Nicolas Saettel,† Germain Metge,‡ Celine Fiorini-Debuisschert,‡ Fabrice Charra,‡
́
and Marie-Paule Teulade-Fichou*,†
†Institut Curie, CNRS UMR-176, Centre Universitaire d’Orsay, Paris-Sud 91405 Orsay Cedex France
‡CEA- Saclay, DSM-IRAMIS/SPCSI/Laboratoire NanoPhotonique, 91191 Gif-sur-Yvette, France
S
* Supporting Information
ABSTRACT: We report on the design and synthesis of two-
photon fluorescent triphenylamines bearing two or three vinyl
branches terminated by a N-methyl benzimidazolium moiety. The
new compounds (TP-2Bzim, TP-3Bzim) are light-up fluorescent
DNA probes with a long wavelength emission (>580 nm).
Compared to their pyridinium models, the TP-Bzim dyes exhibit a
remarkable improvement of both their DNA affinity and
fluorescence quantum yield, especially for the two-branch
derivative (TP-2Bzim: ΦF = 0.54, Ka = 107 M−1), resulting in a
large fluorescence emission turn-on ratio of up to 140.
Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ =
1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time.
This is attributed to a tight fit of the molecule inside the minor groove of AT-rich DNA which induces geometrical
rearrangements in the dye ground state as supported by circular dichroism and molecular modeling data. Consequently, TP-
2bzim displays an exceptional two-photon molecular brightness (δ×ΦF = 583 GM), a value unrivalled for a small biofluorophore.
These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to
visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant,
and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a
bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.
imaging (up to 1 mm). Moreover, the two-photon excitation is
limited to a small femtoliter volume at the focal point of the
laser resulting in an intrinsic 3D image resolution and a limited
irradiation of the sample. A high density of photons is required
to enable simultaneous absorption of two photons, and
excitation is usually performed using femtosecond lasers.
Despite the lower energy of the IR light compared to the
visible light, this excitation mode has been proven to induce
damages to the samples and bleaching of the dyes, especially
when using classical fluorophores that display generally low
two-photon absorption (2PA) capacities.17 Thus the laser
power has to be kept as low as possible to avoid too high peak
powers in the excitation volume, which may result in modest
imaging performances. Hence, there is a strong need of
dedicated fluorophores with a high two-photon absorption
cross-section to exploit the full potential of two-photon laser
scanning microscopy, in particular for fluorescence lifetime
imaging (FLIM) and intravital imaging. Over the past decades,
considerable efforts have been carried out to provide new dyes
INTRODUCTION
■
Fluorescent probes have become invaluable tools in cell biology
and medical imaging, and new fluorescent reporters, spanning
from small organic dyes1,2 to inorganic nanocrystals (quantum
dots)3 and fluorescent proteins (FPs),4 are continuously
proposed by industrial and academic research, while consid-
erable efforts are concomitantly made to develop new
ultrasensitive instrumentation.5 These two research domains
are tightly intertwined as the rapid evolution of optical
microscopy techniques is calling constantly for new dyes with
optimized optical properties to fulfill the promise of increased
temporal and spatial resolutions.6,7
The emergence in the 1990s of two-photon laser scanning
microscopy8−10 has been one of the most prominent
amelioration in fluorescence imaging, allowing the development
of in-depth11−13 and/or intravital14−16 tissue imaging. In this
technique, excitation is induced by the simultaneous absorption
of two photons with half the energy required for the
corresponding one photon absorption process. Therefore, the
excitation wavelength is in the near-infrared (NIR) range
(700−1000 nm) where the absorption and diffusion of light by
biological samples is minimized thereby allowing deep tissue
Received: May 14, 2013
© XXXX American Chemical Society
A
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with optimized two-photon absorption cross-sections
(δ),13,18−22 but due to the difficulty to find the right balance
between the optical properties and the biological constraints,
the challenge remains quite topical.
Indeed, when selecting the appropriate 2PA dye for
fluorescence imaging, many factors should be considered
depending on the application. In particular, certain photo-
physical and chemical characteristics will be of prime
importance: these include (i) a high 2PA molecular brightness
(i.e., product of 2PA absorption cross-section and quantum
yield = δ·ΦF also called action cross-section) to improve the
signal-to-noise (S/N) ratio thus enabling low dye loading and
reduced laser power; (ii) a high photochemical stability, which
is crucial to follow processes on a long time scale; and (iii) a
NIR/long wavelength emission to avoid overlap with
fluorescence of endogeneous cell components and to improve
collection of the emitted light in thick sample. These basic
characteristics, although prerequisite, are hardly simultaneously
fulfilled by the same molecular agent. Additionally, biological
criteria, such as water solubility cell permeation, low toxicity,
specific affinity for a given cell type or cellular compartment,
come into play thus adding to the difficulty and making the
design of “the ideal 2PA dye” highly demanding. More
specifically, in the class of DNA imaging probes, most
commercial dyes have low 2PA absorption (δ values from 5
GM to <50 GM).23,24 In addition, the red emitting dyes like
TO-PRO cyanines or propidium iodide (PI) have inherent
drawbacks, such as very fast bleaching for the former and poor
cell permeation and poor DNA vs RNA specificity for the latter.
Imaging of DNA is thus essentially performed with blue-
emitting dyes (DAPI and Hoechst compounds) which are more
resistant but require either harmful short-wavelength excitation
that is cytotoxic per se or huge two-photon excitation
intensities, also leading to cell damages. Overall, there is a
lack of a ubiquitous 2PA DNA probe with a long wavelength/
NIR emission displaying a satisfying combination of the above-
mentioned criteria and therefore usable for cellular imaging in
various circumstances.
Figure 1. General structure of the TP-Py and TP-Bzim dyes.
We show herein that the introduction of a more extended π-
deficient heterocyclic system, i.e., N-methyl benzimidazolium
moiety, in place of the pyridinium unit results in strong and
concomitant optimization of both optical performances and
DNA binding parameters. Consequently, this led to the
identification of a new 2PA probe called TP-2Bzim (Figure
1) that possesses an unprecedented two-photon molecular
brightness inside DNA combined with high affinity and
exquisite selectivity for AT-rich DNA regions. Importantly we
show that the DNA matrix has a dramatic influence not only on
fluorescence emission but also on the two photon absorption.
Moreover the new TP-2Bzim dye is easily accessible on a large
preparative scale, possesses exceptional photostability under
one- and two-photon excitation, is live cell permeant, and
consequently fulfills the above-mentioned characteristics
making it an outstanding fluorescent DNA 2PA probe.
RESULTS AND DISCUSION
■
Design and Synthesis. Inspired by the well-established
molecular guidelines in the design of 2PA molecular systems,
we turned our attention toward the use of π-deficient
heterocyclic systems, such as benzothiazole and benzimida-
zole.37,38 These motifs can be, in principle, easily methylated
which will afford the cationic charge inducing DNA binding and
should reinforce the internal charge transfer. However, in our
hands the benzothiazolium motif once branched on the TP
core appeared rapidly unstable in water where spontaneous
demethylation occurred.39 Therefore we focused our attention
on the N-methyl benzimidazolium series, which resulted in the
preparation of the two compounds TP-2Bzim and TP-3Bzim
achieved through a simple Wittig reaction between the bis- and
tris-formyl triphenylamine (3a,b) and the (N-methyl benzimi-
dazole)-methyltriphenylphosphonium chloride 2 (Scheme 1).
This method is more economical and easier to achieve than
the Heck reaction previously used for the pyridinium
derivatives.28 The phosphonium chloride 2 was readily
prepared in two steps by condensation of N-methyl-1,2-
benzenediamine and chloroacetic acid, followed by nucleophilic
substitution of the chlorine by triphenylphosphine (64% overall
yield). Reaction of 2 with 3a,b in methanol in the presence of
sodium methanolate led to the neutral intermediates with good
yields (65−85%), and subsequent methylation afforded TP-
2Bzim and TP-3Bzim in 55% and 86% yields, respectively. As
expected from the Wittig reaction only the E conformation was
obtained.
In our ongoing research on nucleic acid probes,25−27 we have
shown recently that π-conjugated triphenylamines (TP), typical
2PA materials, may behave as DNA-specific long wavelength/
NIR fluorescent emitters provided that a cationic acceptor is
linked at the TP branch termini.28 This study has led to the
emergence of vinyl TPs as biocompatible labels,29 while this
chemical class was confined so far to model systems30,31 and
material applications.32−34This first generation of vinyl
triphenylamines was based on the introduction of pyridinium
acceptors tethered to the central triphenylamine donor via vinyl
arms which resulted in the classical V-shaped and octupolar
(TP-2Py, TP-3Py) arrangements (Figure 1).30,31,35,36 These
compounds were shown to display high two-photon absorption
cross-sections (from 200 to 700 GM at 800 nm) and in
addition are nonfluorescent in water whereas featuring a long
wavelength red emission (650 nm < λmax < 680 nm) upon
binding to DNA.28 However, this first series exhibits modest
fluorescence quantum yields in DNA (0.07 and 0.01 for TP-
2Py and TP-3Py, respectively), leading to low brightness which
could severely limit the scope of their application for staining
nuclear DNA or oligonucleotides. We thus speculated that
exploring the chemical space around the triphenylamine core
should enable optimization of the optical properties of this
chemical class.
The two TP-Bzim compounds are dark-red powders,
displaying high water solubility. Of note the compounds obey
Beer’s law over a wide concentration range (up to 100 μM,
B
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Titration of TP-Bzim compounds by DrewAT induces
significant red-shift of the absorption (Δλmax = 20−45 nm)
with a neat isosbestic point in the case of TP-2Bzim indicating
the formation of at least one ligand/DNA complex species
(Figures 2A and S2). In the meantime, the fluorescence signal
Scheme 1. Synthesis of the TP-Bzim Derivatives
Figure S1) indicating that they do not aggregate in aqueous
media.
Light-Up Fluorescence in DNA. The one-photon
absorption and emission properties of the new TP-Bzim
compounds were characterized in glycerol and in a buffered
aqueous solution (pH 7.2) with and without DNA. In our
previous studies,28,40 we observed that both absorption and
fluorescence can be somewhat influenced by the nature and the
origin of the double stranded DNA (native polymers, synthetic
oligomers). To simplify the measurements of affinity constants
and understand more thoroughly the sequence selectivity of the
TP dyes, we studied them in presence of two custom-made self-
complementary duplex oligonucleotides of variable sequence
and length: DrewAT, a 14bp duplex with a 6AT central core,
previously used as a model of duplex AT-rich DNA41 and ds26,
a 26bp duplex with mixed AT/GC content42 (see base
sequences in the Material and Methods section).
Figure 2. (A) Absorption and fluorescence spectra of TP-2Bzim (at 10
and 0.25 μM, respectively) upon addition of DrewAT in sodium
cacodylate buffer 10 mM pH 7.2, NaCl 400 mM. (B) Titration curves
obtained by plotting the fluorescence enhancement F/F0 as function of
Drew AT concentration (F = integrated fluorescence intensity of the
complex, F0 = fluorescence of the free dye), [drewAT] is expressed in
molar concentration (i.e., per duplex).
As expected, the TP-Bzim dyes exhibit high molecular
absorption coefficients (Table 1) with absorption maxima
strongly blue-shifted (∼52−58 nm) as compared to the TP-Py
series, which may reveal a lower degree of conjugation of the
Bzim branches originating in their lower planarity (vide infra).
is restored with a pronounced fluorescence enhancement and a
significant blue-shift of the emission wavelength in DNA with
regard to buffer (35−50 nm, Figures 2A and S2). The same
trend is observed in glycerol, meaning that the Bzim dyes are
rather responsive to the constraints of the environment
(viscosity/DNA interaction). Most remarkably, the amplitude
of the fluorescence increase is dramatically larger for the new
dyes in particular for TP-2Bzim (Fmax/F0 of 140 vs 24 for TP-
2Py). Calculation of quantum yields indicates very high values
(ΦF/TP-2Bzim = 0.54; ΦF/TP-3 Bzim = 0. 34) thereby
representing a 6−34-fold increase as compared to the
pyridinium counterparts (Table 1). This remarkable improve-
ment of ΦF is also apparent in glycerol albeit more modest (3-
fold increase), suggesting that this effect lies not only in the
chemical characteristics of the benzimidazolium group (in-
creased rigidity, increased number of π-electrons) but also in a
more favorable interaction with the DNA matrix. The influence
of DNA is particularly striking when considering the number of
branches. In glycerol, the quantum yields are quite similar for
the two- and three-branch analogs in both series, respectively.
This is consistent with the observation that for V-shaped and
octupolar molecules the emitting state seems to be localized on
a single branch, yielding similar quantum yields irrespective of
the number of branches.30 In presence of DNA however, the
photophysics of the excited state is clearly modified, since the
two-branch compounds exhibit significantly higher quantum
efficiency than their three-branch counterparts. Moreover, the
Table 1. Linear Optical Properties of the New Series TP-
Bzim and of the Reference Compounds TP-Py in pH 7.2
a
Cacodylate Buffer, Glycerol, and DrewAT
λabs/λem
ε
Δλ
ΦF
buffer
glycerol
DrewAT
buffer
474/650
496/649
509/656
474/684
492/666
499/684
432/620
448/602
476/585
430/640
440/604
468/591
39 900
37 400
31 400
59 000
66 000
51 000
42 500
40 400
44 400
68 900
57 200
62 400
176
153
147
210
174
185
188
154
109
210
164
123
<0.005
0.11
TP-2Py
TP-3Py
0.07
<0.005
0.13
glycerol
DrewAT
buffer
0.01
<0.005
0.35
TP-2Bzim
TP-3Bzim
glycerol
DrewAT
buffer
0.54
<0.05
0.42
glycerol
DrewAT
0.34
a
λabs: maximum absorption wavelength (nm); λem: maximum emission
wavelength (nm); ε: molar absorption coefficient (mol−1·L·cm−1); Δλ:
Stokes’s shift expressed in nm; ΦF: fluorescence quantum yields were
measured using rhodamine 101 in ethanol as reference. Cacodylate
buffer: 10 mM sodium cacodylate buffer (pH 7.2, 100 mM NaCl).
DrewAT: 10 mol equiv of DrewAT.
C
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quantum yield of TP-2Bzim is further increased when shifting
from glycerol to DNA (0.34 vs 0.54), whereas it is decreased for
the three other compounds pointing to the influence of specific
dye/DNA interactions.
constants (Ka) were determined from the fluorimetric titrations
using nonlinear curve fitting analysis (Figures 2B and S3−S4).
In most cases, with the exception of TP-3Bzim, the 1:1
stoichiometry model fitted the experimental data with excellent
correlation coefficients (Table S2). Remarkably, the highest Ka
value (1.2 × 107 M−1) was obtained for TP-2Bzim in Drew AT
and is 1 order of magnitude larger than that obtained for TP-
2Py (Ka = 1.2 × 106 M−1) in the same duplex. Significantly
lower Ka were determined in ds26, and this trend is particularly
striking for TP-2Bzim [Ka(DrewAT)/Ka(ds26) = 12], while a
factor of 3.5 is observed for TP-2Py (Table S1). These data are
fully consistent with the fluorescence response confirming the
pronounced preference of TP-2Bzim for AT-rich DNA.
Although drewAT has been previously used as a model for
AT-rich DNA, it also contains two GC-tracts, so to
unambiguously confirm the AT preference of the dye, we
performed experiments in presence of the polynucleotides
poly(dA-dT)2 and poly(dG-dC)2 (Figure S5). The increase in
fluorescence of TP-2Bzim in the former is remarkably high and
similar to that observed in DrewAT (F/F0 = 140), while it is
very low in presence of the latter (F/F0 < 15−10). In addition
in this case the binding is very sluggish as judged by the shape
of the curve that does not display saturation.
The light-up effect of DNA on the fluorescence of TP-dyes is
attributable to several processes: the emission of the free dyes is
likely quenched by vibrations and rotations around the double
bond and around the central nitrogen atom as the scaffold is
rather flexible. On the other hand the interaction with water
molecules of highly polar excited states is known to deactivate
the radiative pathway. Consequently, the strong emission
enhancement of TP-dyes upon DNA binding is likely the result
of two contributions: restriction of intramolecular rotations of
the excited state through immobilization inside the DNA matrix
and water desolvation. In addition, conformational changes
induced by DNA binding can also be involved.
DNA Structure Selectivity. To gain insight into the
structure preference of the TP compounds, we evaluated the
ability of various nucleic acid matrices to restore the dye
fluorescence. Thus a 17-mer DNA single strand (ssDNA) and
total calf liver RNA (cl-RNA) that contains a wide variety of
secondary structures were added to the duplex oligonucleotide
set.
Remarkably, TP-2Bzim shows a strong and selective
fluorescence enhancement in the presence of duplex DNA
(Fmax/F0 = 140, Figure 3 and Table S1), whereas RNA and
Globally the three-branch derivatives exhibit DNA affinities
increased by a factor of 2−3 as compared to their two-branch
counterparts (Table S1). However, the curves could not be
fitted properly with the 1:1 model especially in the case of TP-
3Bzim and DrewAT (Figure S3, Table S2). As the sharp profile
of the curve is indicative of a strong binding, the Ka was
assumed to be >107 M−1 but not measurable in our conditions.
Obviously TP-3Py and TP-3Bzim feature a three-bladed
propeller topology a priori less prone to interact in a well-
defined manner with the double B-helix structure as compared
to their two-branch analogues, and contribution of secondary
sites and/or nonspecific electrostatic interactions cannot be
excluded albeit these are minimized by the high-salt conditions
(400 mM NaCl, Figures S3−4) used on this purpose.
Nonetheless the AT preference of TP-3Bzim, although less
clear from titrations with oligonucleotides, was also evidenced
by the experiments with homopolynucleotides (Figure S5).
TP-2Bzim, a perfect match in the groove. Based on the
nonplanar twisted V-shape of the two-branch molecules and
their apparent preference for AT-sequences, the TP dyes were
assumed to interact with duplex DNA via insertion in the minor
groove.43−45 To support this hypothesis, circular dichroism
(CD) experiments were conducted (Figure 4). Upon addition
of increasing amounts of TP-2Bzim to a solution of Drew AT, a
strong induced CD (ICD) signal in the dye absorption band
was observed. ICD signal was substantially the same with TP-
3Bzim but with a slightly lower intensity, and the same behavior
is observed with the TP-Py series (Figure S6). Strong and
positive ICD in the ligand absorption band is a characteristic
feature of DNA minor groove binders. On the other hand,
intercalation between base pairs is usually accompanied by
weak negative ICD signals.46,47 Moreover the CD signal in the
DNA absorption region (around 260 nm) remains unchanged
upon addition of the TP derivatives, which is again consistent
with minor-groove binding as this mode is known to affect
poorly the DNA structure contrary to intercalation between
bases.
Figure 3. Maximum enhancement of the fluorescence signal of TP-
dyes in presence of various forms of nucleic acids in saturation
conditions, [TP dye] = 1 μM, cacodylate buffer pH 7.2, 400 mM
NaCl, DNA 10−20 molar equiv, see sequences in the Material and
Methods section.
ssDNA induce a dramatically lower response (Fmax/F0 = 2 and
25, respectively, Table S1). In addition the dye exhibits a clear
preference for the AT-rich duplex as compared to ds26 (Fmax
/
F0 = 86). The same trend is observed with the original TP-2Py
but with a considerably lower intensity (Fmax /F0 = 24 for Drew
AT and see Table S1). Finally, the three-branch analogues of
both series (TP-3Bzim, TP-3Py) display a much less
discriminating behavior that might be attributable to their
triscationic charge favoring nonspecific electrostatic-driven
association with any polyanionic matrix. The fluorescence
enhancement factor Fmax/F0 is a semiquantitative indicator of
selectivity and thus represents a key parameter for high contrast
imaging in view of fluorescence microscopy applications. In this
regard TP-2Bzim appears the best candidate for staining duplex
DNA, as its strong and preferential fluorescence response
should result in high brightness and excellent imaging contrast.
However, one should not expect a systematic correlation
between the fluorescence enhancement and the DNA affinity,
thus to further rationalize the TP-dye/DNA interaction binding
More intriguingly, the interaction is featuring a bisignate
(negative/positive) ICD signal. This is frequently associated to
interchromophoric interactions reflecting excitonic coupling in
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6AT tract, whereas ds26 has a mixed sequence with only a 4AT
site. This model emphasizes the flexibility of the triphenylamine
core that adopts a slightly twisted V-shaped conformation,
enabling perfect geometric match with the groove curvature
and thereby optimization of the interactions. Interestingly in
the case of TP-3Bzim, the position inside the DNA is very
similar, while the third branch is sticking out of the helix
without direct contact with the DNA matrix (Figure 5B). This
demonstrates that the third branch is not immobilized and
furthermore is in contact with the aqueous solvent, which is
consistent with the lower quantum yield observed for TP-
3Bzim. Furthermore this indicates that the DNA recognition
motif is constituted by only two branches of the TP scaffold.
The same holds for the TP-Py compounds (Figure S7). Finally,
this observation provides a clue to understand the higher
binding affinity measured for the three-branch scaffolds as the
latter offer three binding motifs as compared to one for the
two-branched scaffold, which may confer a gain in the binding
entropy (Table S1).
Several contributions are involved in the binding of small
molecules in the AT-rich minor groove, the complex formation
being driven by hydrophobic, van der Waals, electrostatic, and
H-bonding components.49 The preference of ligands for AT-
rich groove is commonly attributed to the smaller groove width
of AT tracts and to steric repulsion induced by the guanine
NH2 in GC tracts. In addition the AT minor grooves display a
more negative electrostatic potential as compared to other
sequences, which is favorable to cation trapping. More recently
the role of the spine of hydration in AT grooves has been
documented.50 The highly ordered water molecules present in
the grooves could either mediate the interaction via H-bonding
between the two partners51,52 and/or create an important
entropic gain when they are displaced out in the bulk solvent.
In that sense enhanced hydrophobicity of the ligand and H-
bonding capacity are recognized to be advantageous. In the
present case both the Py and Bzim series do not present H-
bonding donor sites, and thus the other contributions should be
prominent. The larger aromatic surface of the Bzim motif as
compared to the Py motif may result in enhanced hydrophobic
effect and optimized van der Waals contacts with the walls of
the grooves. This is furthermore supported by the significantly
larger size of TP-2Bzim as compared to TP-2Py (Figures 5 and
S7).53 Moreover, due to the symmetry of the Bzim motif, the
cationic charge is distributed on the two N-methyl ring
nitrogens, while it is localized on one nitrogen atom in the Py
motif (Figure S8). This difference in the spatial localization of
the charge may significantly influence the interaction with the
anionic phosphate backbone.
Figure 4. CD spectra of drewAT (10 μM) upon addition of TP-2Bzim
and TP-3Bzim (0−10 mol equiv) in 10 mM sodium cacodylate buffer
pH 7.2, 100 mM NaCl.
dimers formed by nearby chromophores in the groove or at the
surface of the DNA.48 However, the triphenylamine scaffold
owing to its propeller shape is not prone to form dimers; as
well this situation is not consistent with the 1:1 stoichoimetry
inferred from the fluorimetric titrations. Therefore we
hypothesize that the bisignate ICD signal is reflecting the
positioning of two branches lying in the groove in a twisted
geometry thereby mimicking a dimeric arrangement between
adjacent chromophores. Each branch may thus behave like a
single dipole resulting in excitonic coupling between the 2
branches.
On the basis of the data obtained by CD, we conducted
molecular modeling calculations.
The docking results obtained in DrewAT (Figure 5) indicate
that the complex formed either by the two- or three-armed TP
is based on the insertion of two branches into the minor
groove. The two-branch moiety adopts a volume and shape
which fit snugly into the minor groove, occupying a 5−6 AT bp
site (Figure 5A). This binding site size is consistent with the
stronger binding affinity for DrewAT that features a central
In conclusion, the modeling studies indicate a perfect
geometric match between the TP-2Bzim compound and the
AT minor groove which is made possible by the adaptability/
flexibility of the two-branch scaffold. This tight fit inside DNA
and the associated conformational changes are likely to impact
the nonlinear optic (NLO) response in DNA.
NLO Properties: Influence of the DNA Matrix. The two-
photon absorption spectra of the TP derivatives were measured
in presence of DrewAT by two-photon excited fluorescence
(Figure 6).
The shape of the 2PA spectra are almost identical for the four
compounds with maximum intensity peaking at 840 nm in
contrast to their one photon absorption spectra that are
significantly blue-shifted for TP-Bzim compounds compared to
TP-Py compounds (Figure S9). Remarkably all four com-
Figure 5. Models obtained after molecular modeling for the
interaction of TP-2Bzim (A) and TP-3Bzim (B) with duplex DrewAT
(AT bases in pale blue). Side view and models obtained for TP-Py
compounds are shown in Figure S7.
E
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and (ii) the two-photon absorption is switched-on by DNA,
and this effect is especially spectacular for TP-2Bzim.
Altogether these data are in line with conformational changes
occurring upon DNA binding as suggested by the modeling
studies.
It is known that the amplitude of the 2PA response in
multibranched systems is influenced by the coupling between
the dipolar arms and by the ground-state geometry of the
individual arms.36 Presumably, the tight immobilization of the
dye inside the DNA matrix induces geometrical changes in both
the ground and excited states compared to the structure of the
free dye in solvent. Indeed the superposition of the structure of
the free and bound dye shown in Figure 7 fully confirms this
Figure 6. Nonlinear absorption spectra of the TP-dyes in presence of
10 mol equiv of DrewAT, 10 mM sodium cacodylate sodium buffer
pH 7.2, 100 mM NaCl.
pounds show a high nonlinear response with two-photon
absorption cross-section values (δ) ranging from 250 to 1080
GM (Table 2). The particularly high 2PA cross-section value of
the TP-2Bzim is worth noting, in particular as compared to that
of the three other compounds.
Table 2. NLO Properties of the TP Derivatives and Hoechst
a
33258 in Presence of DrewAT
max
λ2PA (nm)
δ (GM)
δ×ΦF (GM)
Figure 7. Modeling of TP-2Bzim: superposition of the docking pose in
DrewAT (gold) and minimized structure without DNA (green).
TP-2Py
TP-3Py
840
840
840
840
760
260
460
1080
250
19
18
5
hypothesis: It appears clearly that for the free dye, the Bzim
motif is twisted with regard to the plane of the double bound
obviously due to the steric hindrance of the two N-methyl
groups, while the branch planarity is re-established for the
bound dye. Thus TP-2Bzim is adopting a more flattened
conformation upon “induced fit” in the minor-groove pocket,
which results in an increased electronic conjugation in the
branches. Altogether these conformational changes should
enhance both the internal charge transfer in each branch and
also the interarm coupling between the transition moments of
the individual branches, thus causing a strong increase of the
2PA. This also explains why the fluorescence quantum yield of
TP-2Bzim is further increased in DNA. All these effects are
minimized or counterbalanced for TP-3Bzim due the third
branch sticking out of the helix, thus retaining a lower
conjugation and inducing higher molecular vibrations and
stronger interactions with solvent.
This unprecedented and striking effect of the DNA matrix on
the two photon absorption could not be evidenced with the
TP-Py compounds due to the higher conjugation of the
branches in the free state and a lower conformational
modification upon binding to DNA. As well related 2PA
probes possessing more rigid structures such as carbazoles,
which have been explored by us and several other groups, show
no strong 2PA sensitivity with respect to the DNA matrix.40
The measure of 2PA cross-section is subject to a strong
dispersion depending mainly on the experimental conditions.55
Consequently, we have also measured for comparison purposes
the 2PA cross-section of Hoechst 33258 with the same
experimental setup. Hoechst 33258 is a blue-emitting dye
classically used for labeling nuclear DNA with excitation in UV
or at 760 nm.56 Remarkably, the two-photon cross-section of
this dye is considerably lower (δ= 19GM) than that of TP-
2Bzim (Table 2). If we compare now the dye performances in
term of action cross-section (δ×ΦF), which is the relevant
TP-2Bzim
TP-3Bzim
Hoechst 33258
583
85
7
a
δ: two-photon absorption cross-section (GM), λ2PAmax: maximum
two-photon absorption wavelength (nm), δ×ΦF: two-photon action
cross-section. The values of the fluorescence quantum yields are those
of Table 1.Conditions: 10 mM sodium cacodylate buffer pH 7.2, 100
mM NaCl, 10 mol.equivalents of DrewAT.
Pioneering studies on triphenylamine derivatives demon-
strated the role of dimensionality and hence of the number of
branches on the 2PA performances. In this line octupolar
arrangements are expected to have a much higher two-photon
absorption cross-section than their V-shaped or quadrupolar
analogs.31,54 This structure−property relationship is verified in
the TP-Py series and is consistent with our previous results
obtained in calf thymus DNA.28 Unexpectedly in the TP-Bzim
series the opposite situation is observed, as the 2PA cross-
section of TP-2Bzim is four times higher than that of TP-
3Bzim. This striking difference points to the influence of the
DNA environment on the nonlinear absorption. To support
this conclusion, the 2PA cross-section of TP-2Bzim was
measured in glycerol. Remarkably, a particularly low value
was determined (δ = 110 GM, λexc 840 nm) thereby confirming
the crucial role of the DrewAT matrix that indeed induces a 10-
fold enhancement of the 2PA ability of the dye. Notably, this
enhancement was not observed previously in the TP-Py series
for which the 2PA cross-sections were not significantly affected
when passing from glycerol to DNA.28 Likewise a low δ value
(96 GM, λexc 840 nm) was found for TP-3bzim in glycerol
confirming the influence of DNA but to a minor extent. Two
important conclusions can be inferred from these observations:
(i) In absence of DNA the TP-Bzim compounds show lower
2PA performances than the Py compounds (400−900 GM),28
and the general quadrupole < octupole trend is not verified;
F
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figure of merit for imaging (also called molecular brightness),
we find a spectacular enhancement of 83-fold for TP-2Bzim
(δ×ΦF = 583 vs 7 GM for Hoechst). This indicates that a
considerably lower laser intensity can be applied when using
TP-2Bzim, theoretically divided by a factor of 9 taken into
account the quadratic dependendance of the 2PA absorption.
Low to fair action cross-section values were obtained for the
three other TP dyes, which fall in the range (10−50 GM) of
most classical fluorophores used in biological imaging
(fluorescein, DAPI, Cy3).57
These results emphasize the remarkable advantage brought
by the TP-2Bzim scaffold to improve 2PA properties. Resulting
from the concomitant optimization in quantum yield and in
2PA cross-section, the new dye TP-2bzim exhibits a remarkably
elevated value of 2PA molecular brightness inside DNA yet
unrivalled for a small-size molecule.58
Cellular Imaging of DNA with TP-2Bzim Dye. The TP-
2Bzim dye was used to image DNA in a cellular context.
Observations in confocal microscopy showed a bright and
specific staining of nuclear DNA with excellent signal-to-noise
ratio (Figure 8). Remarkably, the exceptional 2PA brightness of
Figure 9. Labeling of mice fibroblasts (3T3) by TP-2Bzim. Foci
represent AT-rich centromeres. Live cells were incubated with dye at a
1 μM concentration for 4 h, then washed several times with PBS buffer
(1×), and fixed with paraformaldehyde (PFA); λexc: 488 nm.
2Bzim in the centromeric AT regions of 3T3 cells. Compounds
that exploit the nature of the AT-rich DNA in nucleus are of
particular interest for mapping AT stretches which are found in
critical regions, such as replication origins, minisatellite
centromeric regions, and provide binding sites for nuclear
proteins.59,60 Therefore detection of AT-rich regions is of
particular importance to study phenomena involved in
chromatin remodeling associated to senescence and tumoro-
genesis,61 which opens perspectives toward further applications
of TP-2Bzim.
Finally, like their parent TP-Py compounds, the TP-Bzim
dyes show a remarkable photostability in cells under one- and
two-photon excitation as shown in Figures 10 and S10. Indeed,
Figure 8. Confocal microscopy imaging of fixed HT29 cells incubated
with TP-2Bzim at two different concentrations (A) 1 μM and (B) 100
nM under two-photon excitation (λexc: 800 nm). Light collected from
500 to 720 nm. Incubation made before PFA treatment of the cells.
TP-2Bzim allowed to image nuclear DNA in cells at very low
dye loading (100 nM, Figure 8B) while keeping excitation light
intensity at the level used for micromolar concentration (i.e.,
10−30 MW/cm2) (Figure 8A). Notably, although the TP-
2Bzim has a somewhat blue-shifted fluorescence as compared
to TP-Py, the emission can still be recorded in the red channel
(600− 700 nm) while retaining a high sensitivity.
The absence of cytoplasmic and nucleoli labeling is very clear
in consistency with the low binding to RNA shown in vitro
(Figure 3). Such pattern showing dark nucleoli is a typical
feature of DNA minor groove binders resulting of their strong
specificity for B-helix duplexes over ssDNA and ssRNA.50
Finally it is worth noting that two staining protocols were
assayed: incubation on cells already fixed or incubation with live
cells for 2 h before fixation. The same results were obtained
thereby indicating that the dyes are cell permeant.
Figure 10. MRC-5 cells stained by TO-PRO-3 (2 μM, λexc =633 nm),
PI (2 μM, λexc =561 nm), and TP-3Bzim (2 μM, λexc =488 nm)
observed by confocal microscopy over a time lapse of 25 min.
Because TP-2Bzim shows a pronounced AT sequence
specificity, we were curious to verify if this property observed
on a short oligonucleotide can be transposed for imaging at a
macroscopic level in the context of chromatin. With this in
mind we performed imaging of a cell line (3T3) known to
contain AT-rich centromeric domains (Figure 9).
for all TP-dyes, the fluorescence signal remains stable over a
period of 30 min (Figure S10). For purpose of comparison the
experiment was reproduced with the two commercially
available red DNA stainers the cyanine TO-PRO3, and the
PI derivative. The former faded in only 3 min (Figure 10),
confirming its improper use for imaging. Only slight bleaching
is observed with PI, but it accumulates strongly in the nucleoli
As seen in Figure 9 we observed bright spots that are typical
of high dye density foci resulting from accumulation of TP-
G
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linear gradient of acetonitrile versus water containing 0.1% of FA
ranging from 10 to 55% over 5 min then to 100% over 6 min at a flow
rate of 1 mL/min. Melting points were determined on a Kofler
apparatus. Mass spectrometry was performed at the Institut Curie for
Electrospray Ionization (ESI). High-resolution mass spectrometry was
performed at the Institut de Chimie des Substances Naturelles (ICSN)
in Gif-sur-Yvette. DAPI and PI were generous gifts from Marie Dutreix
and also provides strong background in the cytoplasm (Figure
10). Thus, although widely used, PI requires ribonuclease
(RNase) digestion before imaging and moreover is known to
be cell impermeant. Consequently, the TP-2Bzim dye offers
significant advantages over the commercial red DNA stainers: a
RNase free staining protocol, an increased cell permeation, and
a higher photostability under one- and two-photon excitation.
́
(Equipe recombinaison, reparation et cancer, Institut Curie). TO-
PRO-3 was purchased from Molecular Probes, and Hoechst 33258 was
purchased from Sigma-Aldrich. For all the tested compounds, stock
solutions (from 1 to 10 mM depending on the compounds) were
prepared in H2O or in DMSO depending on the product solubility
and kept at −18 °C.
CONCLUSION
■
We obtained a new generation of vinyl triphenylamine-based
dyes shaped for two-photon absorption by introducing two and
three N-methyl benzimidazolium groups on the TP core which
afforded the two derivatives TP-2Bzim and TP-3Bzim. These
exhibit a bright orange-red light-up fluorescence in DNA and
present huge fluorescence quantum yields (ΦF = 0.34−0.54)
which compare favorably with those of the best DNA stainers
currently in use (SYBR green, Hoechst).62 Molecular modeling
indicates that the size and shape of the two-branch TP-2Bzim
dye ensure a perfect geometric match with the minor groove of
a 6AT tract resulting in a strong and selective fluorescence
enhancement (140-fold) in AT-rich DNA. Unexpectedly the
two-photon absorption of TP-2Bzim is dramatically increased
(10-fold) in presence of DNA as compared to glycerol. This
effect is attributed to a conformational switch induced by the
DNA matrix, which modifies favorably the photophysics of the
excited state and the geometry of the ground state thereby
increasing the internal charge transfer favorable to the 2PA.
This switch-on effect on two-photon absorption of dye inside a
DNA matrix is unprecedented. As a consequence of improve-
ment in quantum yield and in 2PA cross-section, TP-2Bzim
bound to DNA is characterized by an exceptionally high two-
photon brightness (δ×ΦF = 583 GM). This enabled selective
staining of nuclear DNA in fixed cells at submicromolar dye
concentration. In addition the TP-2bzim dye was also shown to
have substantial advantages as compared to the usual red DNA
markers, such as a higher photostability, a better cell
permeation, and a RNase-free staining protocol treatment.
Finally, the AT-sequence preference of TP-2bzim was shown to
afford AT-region specificity as revealed by imaging the AT-rich
centromeric chromatin in 3T3 cells.
Nucleic Acids and Oligonucleotides Sequences. MisTA (5′- GGG-
TTA-CTA-CGA-ACT-GG-3′), ds26 (5′- CAA-TCG-GAT-CG A-
ATT-CGA-TCC-GAT-TG-3′), and DrewAT (5′-CGCG-AAATTT-
CGCG-3′) were purchased from Eurogentec (Belgium). The
autocomplementary strands (DrewAT and ds26) were heated at 90
°C for 5 min in a 10 mM sodium cacodylate buffer pH 7.2, 100 mM
NaCl (approximately duplex concentration 200 μM) and then slowly
cooled to room temperature over 12 h. Once prepared, oligonucleo-
tides solutions were stocked at 4 °C. Before use, the concentrations
were evaluated by UV (after thermal denaturation 5 min at 90 °C)
using the supplier’s values for the molar extinction coefficients at 260
nm. Calf liver RNA was purchased from Sigma-Aldrich (U.S.A.). The
lyophilized RNA was dissolved in 10 mM cacodylate buffer pH 7.2,
100 mM NaCl at a concentration of 5 mg·ml−1. Concentration was
checked by UV considering a molar absorption coefficient of 8700
M−1·cm−1 at 260 nm (Mol. Cell Biol. 1977, 18, 81).
UV−vis Absorption, Fluorescence and CD Spectroscopies.
Measurements were performed in 1 or 3 mL Hellma quartz cuvette
(material code QS blue, 200−2000 nm). UV−vis experiments were
monitored on a Uvikon XL (Secomam, France) UV spectropho-
tometer. Fluorescence spectra were recorded on a FluoroMax-3 (Jobin
Yvon) at room temperature. Data were corrected with a blank and
from the variations of the detector with the emitted wavelength. The
CD spectra were recorded on a Jasco J-710 spectrometer thermo-
statted at 22 °C. Each spectrum is the accumulation of four scans
performed with a 1 nm step and a 200 nm/min scanning speed. All
experiments were performed in 10 mM sodium cacodylate buffer pH
7.2 prepared by dissolving sodium cacodylate trihydrate (Sigma-
Aldrich, U.S.A.) in Milli-Q water.
Synthesis. Synthesis of 2-(Chloromethyl)-1-methyl-1H-benzo[d]-
imidazole 1. N1-methylbenzene-1,2-diamine (1g, 0.930 mL, 8.19
mmol) and 2-chloroacetic acid (1.160 g, 12.28 mmol, 1.5 equiv) were
added in 2 N HCl. The resulting dark-purple solution is stirred at
reflux overnight. After cooling at room temperature, the solution is
brought to pH = 8 with NaOH 1 M and extracted twice with ethyl
acetate. The combined organic phases are washed with brine dried
over MgSO4 and concentrated under reduced pressure. Raw product
was purified by flash chromatography using Companion Combiflash
automated apparatus (ethyl acetate/cyclohexane 50/50 to 80/20) to
afford 1.116 g of the title compound as a white powder (75%). CAS
number: 4760-35-4. 1H NMR (300 MHz, chloroform-d) δ 7.77 (d, J =
7.2 Hz, 1H), 7.46−7.03 (m, 3H), 4.86 (s, 2H), 3.89 (s, 3H).
Synthesis of ((1-Methyl-1H-benzo[d]imidazol-2-yl)methyl)-
triphenylphosphonium Chloride 2. A solution of 2-(chloromethyl)-
1-methyl-1H-benzo[d]imidazole (883 mg, 4.89 mmol) and triphenyl-
phosphine (1.923 g, 7.33 mmol, 1.5 equiv) in 15 mL toluene was
heated at reflux. A white precipitate forms. After one night of stirring,
the precipitates are filtered under reduced pressure, washed with
pentane, and dried under reduced pressure to afford 1.206 g of the
expected product as a white solid (56%). CAS number: 68426-76-6.
1H NMR (300 MHz, chloroform-d) δ 7.96 (dd, J = 13.0, 7.8 Hz, 6H),
In summary, the new TP-2Bzim dye features a unique
combination of optical and biological properties which makes it
the molecule of choice for DNA staining as well as the best
two-photon DNA-specific probe currently available. In
particular its high photostability and fluorescence brightness
per molecule are remarkable as these parameters remain prime
figures of merit for virtually all ultrasensitive fluorescence
spectroscopy and microscopy techniques. Ultimately, this new
dye presents chemical versatility and scalability that are both
desired for industrial development.
EXPERIMENTAL SECTION
■
Materials and Methods. Chemical and Analyses. All chemicals
were purchased from Sigma-Aldrich (U.S.A.) or Acros Organics
(Belgium) and used as received. All other chemicals were used as
received from the appropriate suppliers. Preparative flash chromatog-
raphies were carried out with Merck silica gel (Si 60, 35−70 μm).
NMR spectra were recorded on a Bruker Advance 300 spectrometer.
1H NMR spectra were recorded at 300 MHz and 13C NMR spectra at
75 MHz. Chemical shifts are reported in ppm downfield to TMS (δ =
0.00) for 1H NMR and to central CDCl3 resonance (δ = 77.16) or to
[d6]DMSO resonance (δ = 39.52) for 13C NMR. High-performance
liquid chromatography was carried out on a Waters Alliance equipped
with a photodiode array detector using a XterraMS column with a
7.75 (t, J = 7.2 Hz, 3H), 7.69−7.53 (m, 6H), 7.47 (d, J = 7.8 Hz, 1H),
7.32−7.08 (m, 3H), 6.03 (d, J = 14.8 Hz, 2H), 3.93 (s, 3H).
Synthesis of 4-((E)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl)-
N-(4-((E)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl)phenyl)-N-
phenylaniline 4a. To a solution of 4,4′-bisformyltriphenylamine (50
mg, 0.166 mmol) and 2 (162 mg, 0.365 mmol, 2.2 equiv) in 5 mL
H
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methanol is added 5 mL of a freshly prepared solution of sodium
methanolate in methanol (19 mg Na, 0.830 mmol, 5 equiv). After one
night of stirring, the solution is left to cool at room temperature and
concentrated under reduced pressure. The residue is dissolved in
DCM. Pentane is added until precipitation of a yellow solid. The
precipitate is filtered under reduced pressure, washed with pentane,
and dried under vacuum to afford 79 mg of the title compound (85%).
Molecular Modeling. Ligand drawing, standardization (proto-
nation state at physiological pH, aromatization, hydrogen treatment),
and 3D generation were done with Chemaxon Marvin Beans. Docking
was performed with the Surflex-Dock engine of the Tripos SYBYL-X
2.0 suite. The modified GeomX mode was used throughout the
calculations with the option Allow DNA movement: six additional
starting conformations were generated, pre- and postdock minimiza-
tions were performed, a maximum of three final poses were saved, with
a minimum of 0.5 RMSD between poses. The spin alignment method
was used, with an exhaustive accuracy (density of search 9.0) and 12
number of spins per alignment. A DrewAT-like structure was built
using SYBYL-X 2.0 (biopolymer builder module), and the binding site
centered on the central AATT sequence was determined by protomol
probes (CH4 as a steric hydrophobic probe, NH as a H-bond donor
probe, CO as a H-bond acceptor probe). The docking results are
ranked based on an empirical scoring function including hydrophobic,
polar, repulsive, entropic, solvation, and crash terms and expressed in
−log10(Kd) units to represent binding affinities.
1
mp °C: 150−152; H NMR (300 MHz, chloroform-d) δ 7.93 (d, J =
15.8 Hz, 2H), 7.83−7.69 (m, 2H), 7.52 (d, J = 8.3 Hz, 4H), 7.43−7.23
(m, 8H), 7.21−7.15 (m, 2H), 7.12 (d, J = 8.2 Hz, 4H), 7.00 (d, J =
15.8 Hz, 2H), 3.87 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 151.56,
148.22, 146.86, 143.29, 136.90, 136.18, 130.68, 129.76, 128.53, 125.76,
124.46, 123.80, 122.77, 122.58, 119.33, 111.27, 109.23, 29.93. HRMS
(ESI+): m/z calcd for [C38H31N5]+, 558.2658; found, 558.2657
Synthesis of Tris(4-((E)-2-(1-methyl-1H-benzo[d]imidazol-2-yl)-
vinyl)phenyl)amine 4b. According to the same procedure, 200 mg
of 4,4′,4″-triformyltriphenylamine (0.607 mmol) led to 282 mg of the
1
title compound as a yellow solid (65%). mp °C: 192−194; H NMR
The minimized conformation of TP-2Bzim was found using the
genetic algorithm-based global optimizer in Sybyl-X 2.0 with the
default parameters.
(300 MHz, chloroform-d) δ 7.95 (d, J = 15.6 Hz, 3H), 7.85−7.66 (m,
3H), 7.55 (d, J = 7.7 Hz, 6H), 7.40−7.22 (m, 9H), 7.17 (d, J = 7.6 Hz,
6H), 7.03 (d, J = 15.8 Hz, 3H), 3.87 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 151.3, 147.5, 143.1, 136.5, 136.0, 131.3, 128.5, 124.4, 122.6,
122.5, 119.1, 111.6, 109.1, 29.8. HRMS (ESI+): m/z calcd for
[C48H39N7]+, 740.3345; found, 740.3373.
Cell Culture and Fluorescence Microscopy. Cells were grown
on coverslips at 37 °C in monolayer cultures in complete DMEM
(Gibco, Cergy Pontoise, France) with 10% FCS and antibiotics (100
mg/mL streptomycin and 100 mg/mL penicillin) under conditions of
100% humidity, 95% air, and 5% CO2 for 24 h. The cells were then
incubated for 2 h with the compounds (concentration indicated in the
text). After washing twice with PBS, the cells were fixed with 4%
paraformaldehyde and washed with PBS twice. The coverslips were
then mounted on microscope slides using Prolong Gold antifade
reagent (Invitrogen).
The confocal and biphotonic imaging was performed using a
confocal laser scanning microscope DMI 6000 with a SP5-AOBS unit
(both Leica) equipped with a 63× (NA = 1.4) objective (oil
immersion), an argon gas laser for one-photon excitation, and a
Chameleon Ti:sapphire laser delivering pulses in the 100−200 fs range
at an 80 MHz repetition rate with a tunability ranging from 705 to 980
nm for two-photon excitation.
Synthesis of 2,2′-((1E,1′E)-((phenylazanediyl)bis(4,1-phenylene))-
bis(ethene-2,1-diyl))bis(1,3-dimethyl-1H-benzo[d]imidazol-3-ium)
Iodide TP-2Bzim. To a solution of 4a (48 mg, 0.086 mmol) in 2 mL
dichloromethane and 2 mL methanol is added a large excess of methyl
iodide (1 mL). The solution turns red. After stirring overnight at 40
°C, pentane is added until a red precipitates forms. The solid is filtered
under reduced pressure, washed with pentane, and dried under
1
vacuum to afford 40 mg of the pure product (55%). H NMR (300
MHz, DMSO-d6) δ 8.05 (dd, J = 6.0, 2.9 Hz, 4H), 7.90 (d, J = 8.4 Hz,
4H), 7.79 (d, J = 16.8 Hz, 2H), 7.70 (dd, J = 6.2, 2.9 Hz, 4H), 7.53−
7.36 (m, 4H), 7.34−7.23 (m, 1H), 7.23−7.13 (m, 6H), 4.14 (s, 12H).
13C NMR (75 MHz, DMSO-d6) δ 149.0, 148.6, 145.8, 145.7, 132.0,
130.3, 129.0, 126.4, 126.1, 125.5, 123.0, 112.9, 105.9, 33.0. HRMS
(ESI+): m/z calcd for [C40H37N5]2+, 293.6525; found, 293.6520.
Synthesis of 2,2′,2″-((1E,1′E,1″E)-(nitrilotris(benzene-4,1-diyl))tris-
(ethene-2,1-diyl))tris(1,3-dimethyl-1H-benzo[d]imidazol-3-ium) Io-
dide TP-3Bzim. According to the same procedure, 40 mg of 4b
(0.056 mmol) led to 55 mg of the pure compound as a red solid
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Detailed methods for fluorescence quantum yield measurement
and nonlinear curve analysis and dditional figures and tables.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(86%). H NMR (300 MHz, DMSO-d6) δ 8.07 (dd, J = 6.2, 3.1 Hz,
6H), 7.99 (d, J = 8.4 Hz, 6H), 7.84 (d, J = 16.7 Hz, 3H), 7.71 (dd, J =
6.2, 3.0 Hz, 6H), 7.51 (d, J = 16.7 Hz, 3H), 7.28 (d, J = 8.0 Hz, 6H),
4.16 (s, 18H). HRMS (ESI+): m/z calcd for [C51H48N7]3+, 252.7990;
found, 253.1304.
Two-Photon Absorption Cross-Section Measurements. Two-
photon induced fluorescence measurements were performed at the
CEA (Saclay) using an inverted microscope setup coupled to a mode
locked Ti-sapphire laser (Tsunami, Spectra Physics) delivering 100 fs
pulses with a 76 MHz repetition rate over the spectral range covering
740−950 nm. The Ti-sapphire excitation beam was focused in a cell
filled with low-concentration solutions using a 40× microscope
objective, the same objective being also used for the two-photon
fluorescence signal collection. The signal is then sent either to a
channel plate multiplier (Perkin-Elmer MP-993-CL) or to a
spectrometer coupled to a CCD camera (Andor DU401-BR-DD)
for detailed study of the emission spectra. The laser beam was linearly
polarized, and a set of half-wave plate and polarizer was used to vary
the fundamental beam intensity (700 μW at the level of the sample).
Excitation spectra were determined from measurements of the whole
emitted light using a photomultiplier proceeded with filters cutting the
fundamental beam (SemRock razor edge 785, stopline 785 and 808,
FF735 and FF-01-750). The TPIF intensities of the samples were
measured relative to a solution of fluorescein in water (pH = 13), the
ratio of the fluorescent signals enabling further determination of the
2PA cross-section (δ) assuming equal one- and two-photon
fluorescence quantum yields.
AUTHOR INFORMATION
■
Corresponding Author
mp.teulade-fichou@curie.fr
Present Address
§Universite
́ ̂
P. Sabatier, Lab IMRCP, CNRS UMR 5623, Bat
2R1, 118 route de Narbonne, 31062 Toulouse cedex 09 France.
Author Contributions
∥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
ANR-07-PCVI-0005−01 (EPATAN) and NanoBioIdf (Label
DNA) are acknowledged for fundings and in particular for post
doctoral and Ph.D. fellowship to E.F.-P. and G.M. The authors
wish to thank Drs. Maria Quanz, Nathalie Gagey, and Florent
Poyer for their help in microscopy imaging and cell culture.
I
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