Design, synthesis, and time-gated cell imaging of carbon-bridged triangulenium dyes with long fluorescence lifetime and red emission

Article abstract of DOI:10.1039/c8sc00089a

Time-resolved fluorescence offers many advantages over normal steady-state detection and becomes increasingly important in bioimaging. However, only very few fluorophores with emission in the visible range and fluorescence lifetimes above 5 ns are available. In this work, we prepare a series of new aza/oxa-triangulenium dyes where one of the usual oxa or aza bridges is replaced by an isopropyl bridge. This leads to a significant redshift of fluorescence with only moderate reductions of quantum yields and a unique long fluorescence lifetime. The fluorescence of the isopropyl bridged diazatriangulenium derivative CDATA⁺ is red-shifted by 50 nm (1400 cm^-1) as compared to the oxygen-bridged DAOTA⁺ chromophore and has intense emission in the red region (600-700 nm) with a quantum yield of 61%, and a fluorescence lifetime of 15.8 ns in apolar solution. When the CDATA⁺ dye is used as cell stain, high photostability and efficient time-gated cell imaging is demonstrated.

Full text of DOI:10.1039/c8sc00089a

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Design, Synthesis, and Time Gated Cell Imaging of Carbon-Bridged
Triangulenium Dyes with Long Fluorescence Lifetime and Red
Emission
Received 00th January 20xx,
Accepted 00th January 20xx
M. Rosenberg,a* K. R. Rostgaard,a Z. Liao,a A. Ø. Madsen,b K. L. Martinez,a T. Vosch,a and
B. W. Laursena*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Time-resolved fluorescence offers many advantages over normal steady state detection and becomes increasingly
important in bioimaging. However, only very few fluorophores with emission in the visible range and fluorescence
lifetimes above 5 ns are available. In this work, we prepare a series of new aza/oxa-triangulenium dyes where one of the
usual oxa or aza bridges is replaced by an isopropyl bridge. This leads to a significant redshift of fluorescence with only
moderate reductions of quantum yields and a unique long fluorescence lifetime. The fluorescence of the isopropyl bridged
diazatriangulenium derivative CDATA+ is red-shifted by 50 nm (1400 cm–1) as compared to the oxygen-bridged DAOTA+
chromophore and has intense emission in the red region (600-700 nm) with a quantum yield of 61 %, and a fluorescence
lifetime of 15.8 ns in apolar solution. When the CDATA+ dye is used as cell stain, high photostability and efficient time-
gated cell imaging is demonstrated.
performance of rhodamine type dyes for applications as
probes in for example super-resolution microscopy.12-21 These
dyes possess high brightness and photostability, and have red
INTRODUCTION
Organic dyes emitting in the red and near-infrared part of the
visible spectrum are particularly desired as probes in
fluorescence microscopic techniques because the high
penetration depth of light in the 650-1000 nm range in tissue
allows for non-invasive monitoring of the fluorescent signal in
biological material.1-3 Moreover, a fluorescent signal in this
spectral region can be measured with reduced auto-
fluorescence from the biological media.4-6 Several types of
fluorescent dyes that effectively emit light in the red and the
near-infrared regions have been developed with rhodamine,
BODIPY, rylene, and cyanine motifs being the most popular
designs.7-10 In these cases, the fluorophores possess high molar
absorptions and fluorescence quantum yields. Several of these
fluorophores are commercially available and have found broad
application in life-sciences.11 Recently, particular interest has
been aimed at improving the optical properties and the
to near-infrared fluorescence. However, a common feature
these dyes are also short fluorescence lifetimes in the range of
1 to 5 ns. Fluorophores with longer fluorescence lifetimes have
advantages in important applications such as; fluorescence
23
lifetime bioassays and imaging (FLIM),22,
suppression of
auto-fluorescence by time-gated detection,24 in polarization
assays,25 and time-gated stimulated emission depletion
microscopy (g-STED).26-28 Despite the obvious applications for
fluorescent dyes covering a broad range of fluorescence
lifetimes and emission wavelengths only very few dyes with
lifetimes above 5 ns are available in the visible range.22, 25, 29
The lack of such fluorescent dyes may be due to the large
focus on high brightness, which eventually leads to short
lifetimes,‡ as well as the fundamental problem of increasing
rates of non-radiative deactivation associated with redshifted
fluorescence, as predicted by the energy gap law.30
We have recently shown that the aza/oxa-triangulenium dyes;
ADOTA+ and DAOTA+ (Chart 1) have remarkable low rates of
non-radiative deactivation and low susceptibility to quenching
by oxygen,31 which explains how these organic dyes with
fluorescence peaks in the 550 nm – 600 nm range, can display
unusually long fluorescence lifetimes, close to 20 ns, and high
quantum yields (Φf ≈ 0.7 – 0.8).
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Chart 1. Azadioxatriangulenium (ADOTA+), diazaoxatriangulenium (DAOTA+) and
dimethoxyquinacridinium (DMQA+). X– refers to an arbitrary anion.
DAOTA+ chromophore is recognized as belonging to the group
of odd alternant π-systems, i.e. a conjugated system that is, or
can be described as, consisting of an odd number of sp2
hybridized carbon atoms. This is illustrated for DAOTA+ and the
rhodamine analogue Pyronin
Y in Chart 2. All other
triangulenium and helicenium dyes, including ADOTA+ and
DMQA+ also belong to this group. For such chromophores, the
HOMO is distributed on the starred positions and vanishing
small at the non-starred positions. Contrary, the distribution of
the LUMO is most pronounced at the non-starred positions.§
Since the oxygen bridge in Pyronin Y is connected to non-
starred positions it is, to a first approximation, only affecting
the LUMO energy. From this, it can be predicted that
The aza/oxa-triangulenium dyes are highly stabilized
carbenium ions, with
a
rigid and planar heterocyclic
In comparison to rhodamine and cyanine
fluorophores the aza/oxa-triangulenium dyes are less bright,
as they possess moderate molecular absorption coefficients (
33
framework.32,
replacement of the oxygen bridge by less π-electron donating
groups, such as the isopropyl bridge, should lower the LUMO
energy and thus lead to a redshift in carbopyronin as
compared to Pyronin Y,53 which has indeed proven to be the
case (Chart 2).7, 16, 57-59 In similarity, PMO theory predicts that
replacement of the oxygen bridge in DAOTA+ by an isopropyl
group should lead to the desired redshift. This was further
confirmed by TDDFT calculations (see below) and by the
spectroscopic data obtained after synthesis of this new
ε)
in the range of 10,000-16,000 M–1cm–1.34 However, the
combination of long fluorescence lifetime, high quantum
yields, and chemical stability makes them unique probes in
FLIM and time-gated fluorescence imaging,23, 24, 35, 36 and for
fluorescence lifetime and polarization assays.36-40 A redshifted
congener of DAOTA+ is the dimethoxyquinacridinium (DMQA+)
system (Chart 1). This [4]-helicenium precursor of DAOTA+,32, 41
display absorption and emission in the attractive range from
600 nm – 700 nm.42, 43 Lacour and co-workers have intensively
functionalized and extended this system demonstrating
carbon-bridged triangulenium dye CDATA+
containing congeners; CDOTA+ ( ) and CAOTA+ (
(
8
) and its oxygen
6
7
) (See Chart 2
and Scheme 1). The helical DMQA chromophore is
fundamentally very similar to the planar DAOTA+, but in this
case its redshift can be assigned to the twisting of the
π-
spectral engineering, circular polarized luminescence (CPL), pH
44-51
system, which also causes lower radiative rates.54 It is noted
that PMO analysis also can rationalize the observed effects of
both electron donating and withdrawing groups in various
positions of DMQA+ and related [6]-helicenium systems
recently reported by Lacour and co-workers.45, 50
switching, and lipophilic staining of mitochondria
.
While
DMQA+ displays long fluorescence lifetime in non-polar
solvents it display faster non-radiative deactivation than the
planer aza/oxa-triangulenium dyes and is practically non-
fluorescent in aqueous solutions.31, 42
In the search for new organic fluorophores with long
fluorescence lifetimes and emission beyond 600 nm, we turn
our attention to modification of the aza/oxa triangulenium
dyes, with the expectation that the planarity and rigidity of
these systems will keep the rate of non-radiative deactivation
low and thus allow long fluorescence lifetime and high
quantum yields. In this regard, the DAOTA+ dye (Chart 1) is
particularly interesting as it display higher chemical stability32
and less susceptibility to quenching40, 52 compared to ADOTA+.
In present work, structural variations are introduced in the
DAOTA+ chromophore motif, which redshifts the absorption
and fluorescence spectra as compared to those of DAOTA+ and
with only moderate increase in the rate of non-radiative
deactivation, thus conserving the desired long fluorescence
lifetime.
RESULTS AND DISCUSSION
Chromophore analysis and design
The structural modification of the DAOTA+ chromophore is
chosen on the basis of Dewar’s perturbational molecular
orbital (PMO) approach, which provides simple, yet powerful,
predictions of the change in energy of optical transitions as
function of chromophore modifications.53-56 Firstly, the
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Chart 2. Chromophore analysis of Pyronin Y, carbopyronin, DAOTA+, and CDATA+.
The values of the lowest energy absorption maximum (Abs) and emission
maximum (Em) are from this work and ref. 7.
The
hexafluorophosphate salt (
reaction in melted pyridine hydrochloride at 190-200 oC, which
effectively converted into in five minutes reaction time.
carbon-bridged
dioxatriangulenium
(CDOTA+)
, by
6
) was obtained directly from
5
5
6
With this stabilized carbenium salt in hand the corresponding
azaoxa- (CAOTA+) and the diazatriangulenium (CDATA+)
derivatives (7 and 8, respectively) were prepared by aromatic
nucleophilic substitution with primary amines following a
procedure similar to that reported for the synthesis of
aza/oxa-triangulenium salts.32 The CAOTA+ salt
7
was
and two equivalents of
methylamine and benzoic acid in ethanol/NMP solution. The
optimal conditions for preparation of were found when the
synthesized by reaction of
6
7
reaction was carried out in a sealed tube at 70 oC for three
days. The presence of benzoic acid elevates the reflux
temperature of the reaction mixture,43 and it protonates and
eases the dissociation of the leuco adduct between
methylamine and 6 32
was found to be very challenging using column
chromatography. However, compounds and could be
separated from taking advantage of the different reactivity
of , and towards nucleophilic attacks on the center
carbon atom. Addition of triethylamine decolorizes a solution
of , indicating that this compound forms the leuco adduct
with triethylamine. This was found to be useful in effectively
separating the traces of from the crude reaction mixture.
Addition of excess triethylamine to the reaction mixture
followed by addition of diethyl ether precipitated and the
trace amounts of , while the leuco adduct between
triethylamine remained in solution. Compound
. Separation of the traces of 6 and 8 from
7
6
8
7
6
,
7
8
Synthesis
6
While the aza and oxa bridges in triangulenium dyes efficiently
are introduced by aromatic nucleophilic substitution on
methoxysubstituted triarylmethane precursors,33,
6
43, 60, 61
the
isopropyl-bridge has to be introduced by a stepwise buildup of
the triangulenium ring system. For this, we follow a synthetic
strategy similar to that reported in the synthesis of sulfur
bridged triangulenium structures.62 The syntheses of the
7
8
6
and
was
7
separated from traces of
recrystallization to give
8
7
using a similar strategy followed by
in 53-78 % yields. The dimethyl
carbon-bridged triangulenium salts
Scheme 1.
The sp3-hybridized carbon-bridge was introduced into the
6, 7, and 8 are outlined in
CDATA+ dye
8 was obtained in 24 % yield by reacting 6 with
excess methylamine and benzoic acid in refluxing
ethanol/NMP mixture for five days in a closed system.
triangulenium skeleton using tetramethylated dithranol (
4) as
building block. Dithranol was prepared in multigram scale by
To demonstrate that different amines can be introduced
sequentially to yield asymmetric substituted CDATA+ dyes
containing functional groups, N-Boc-cadaverine was reacted
reduction of 1,8-dihydroxyantraquinone
procedure reported by Prinz et al.63 The four methyl groups
(
1
)
using the
were introduced sequentially in a two-step procedure with
easy purifications. Using conditions of similar reactions,64-66
with compounds
results of this investigation showed, that 10 was most
effectively obtained via , with an overall yield up to 42 % as
compared to 6 %, when going via compound . A more
detailed investigation of the step-wise introduction of groups
to yield asymmetric versions of containing functional groups
is currently in progress. Functional derivatives and 10 can
6 and 7 to give compound 10 (Scheme 2). The
2
was dimethylated in the 10-position giving
yields using two equivalents of methyl iodide and K2CO3 in
refluxing acetone. The hydroxy groups of were easily
methylated in a reaction with slight excess of dimethyl sulfate
using NaH as base in refluxing THF solution giving in 71-81 %
yield. The third aromatic unit was introduced by nucleophilic
addition of 2,6-dimethoxyphenyllitium using procedure
similar to that reported for related reactions,62, 67 giving
in 66
% yield. Conversion of into the corresponding carbenium ion
3 in moderate
7
9
3
8
4
9
easily be converted into maleimides and thus allow for
efficient bioconjugation of the CAOTA+ and CDATA+ dyes to
a
5
cystines.40 Reacting
6
with an amino acid, instead of a diamine,
5
would allow for synthesis of activated ester (e.g. NHS)
would potentially open up a versatile synthetic route for
introduction of various substituted aza-bridges into the new
triangulenium system in analogy to that described for known
triangulenium systems.43 However, this carbenium ion proved
to be highly unstable and attempts to isolate the carbenium
derivatives of CAOTA (7) and CDATA (8) targeting free amines,
as previously reported for aza/oxa-triangulenium dyes.24, 68
salt were unsuccessful. Thus,
a different approach was
followed in order to obtain the carbon-bridged triangulenium
salts.
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Scheme 1. Synthesis of the carbon-bridged dioxa-, azaoxa-, and
diazatriangulenium salts ( , and , respectively).
6
,
7
8
Scheme 2. Synthesis of an asymmetric substituted CDATA+ dye.
Structural effects of carbon-bridge in triangulenium ions
The molecular structures of DAOTA+,
, and 8 were
6,
7
determined by single crystal X-ray diffraction and by
computational methods (see ESI for details). Selected bond
lengths and angles are given in Table S8 and show very good
agreement between the calculated structures and those found
in the crystal structures. The most interesting observation is
the effect of the introduction of the sp3 hybridized isopropyl
bridge where the C-C bond lengths are found to be significant
longer (
of similar length as the sp2 C-C bonds in the triangulenium
system 140 pm). While longer bonds easily are
∼150 pm) than both the C-N and C-O bonds, which are
(
∼
accommodated in linear ring systems like carbopyronin, 2-
dimensional fused ring systems like triangulene are much
more constrained. Thus, triangulenes with
a central
phosphorus atom adopts a non-planar cone shape,69-71 and
attempts to synthesize a triangulenium system with three
sulfur bridges failed due to ring tension.72
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shift of ∼
500 cm-1. Clear vibrational bands are seen on the blue
side of the lowest energy peaks in the absorption spectra and
are mirrored by vibrational shoulders in the fluorescence
spectra. This indicates that all of the absorption spectra above
the dip at
∼
450 nm can be assigned to the S0
→
and the weaker bands around 400 nm to the S0
S1 transitions
S2
→
transitions. This assignment is supported by the results of
TDDFT calculations of the optical transitions (see ESI for
details), and by steady-state excitation- and fluorescence
o
measured in glycerol at 0 C
Figure 1. Side view of the non-plane-r structure found for cationic part of CDATA+
(
8) in the crystal structure of the PF6 salt.
anisotropy spectra of
6,
7, and
8
(Figure S24). The results of the calculations show that the
S0
S1 transitions for DAOTA+, 6+ +, and 8+ involves spatial
The crystal structures of
with expansion of the angle between the rings linked by the
isopropyl bridge (125 ) (Figure S38 and Table S8). For , this
tension leads to a non-planar structure with the isopropyl
bridge extending 50 pm out of plane in the crystal structure
6 and 7 are found to be planar, yet
→
, 7
similar orbitals (Tables S3-S6 and Figures S29-S32) and that the
orientation of the transition dipole moments in the
triangulenium framework are dictated by the relative donor
strength of the heteroatom bridges as also found for ADOTA+
°
8
∼
and DAOTA+.34 Thus, for CDATA+
S0 S1 transition is polarized along the axis connecting the two
nitrogen bridges, while the S0 S2 transition is orthogonal to
this, in direction of the isopropyl or oxygen bridge (see Figure
S33). When comparing CDATA+ (
) to the reference compound
(8
), similar to DAOTA+, the
(Figure 1). In the calculated structure, strain is released by
bending both nitrogen bridges out of plane (Figure S39). Such
non-planar conformations could lead to enhanced non-
radiative deactivation and thus reduce the fluorescence
lifetime and quantum yield.
→
→
8
DAOTA+, it is found that replacement of the oxygen bridge
with an isopropyl bridge conserves the fundamental electronic
structure of the chromophore yet leads to a significant redshift
of approximately 50 nm in both absorption and fluorescence
spectra, confirming our design principle guided by Dewar’s
PMO approach.
Photophysical Properties
The spectral properties of 6, 7, and 8 are shown in Figure 2 and
Table 1 (where data for DAOTA+ and DMQA+ are included for
reference).
Fluorescence quantum yields (
Φf) and fluorescence lifetimes
×103
A
(τf) measured by time correlated single photon counting
(TCSPC) are listed in Table 1. Firstly, it is observed that the new
family of carbon-bridged triangulenium dyes all display good
quantum yields in the range of 0.5-0.6, which however are
lower than those of the aza/oxa-triangulenium dyes ADOTA+
20
10
0
and DAOTA+ Φf ≈ 0.7-0.8) under similar conditions.31 Most
(
importantly, the desired long fluorescence lifetimes are indeed
found for the carbon-bridged triangulenium dyes, 23 ns for 6,
18 ns for
7 and 15 ns for 8, respectively. For a more detailed
400
500
600
700
analysis and comparison to DAOTA+, the rate constants for
fluorescence (krad) and non-radiative deactivation (knr) were
calculated (Table 1). It is found that krad show moderate
variations that, as expected,73 follows the molar absorption
λ (nm)
1,0
0,5
0,0
B
coefficients (εmax) and the calculated oscillator strengths (Table
S3-S6). The shorter fluorescence lifetime of CDATA+ (
8) relative
to DAOTA+ (16 ns vs. 22 ns) is mainly a result of a twofold
increase of the rate of non-radiative deactivation (knr). Within
the series of 6, 7, and 8, knr only show a small increase with
decreasing transition energy (Table 1). This suggests that the
increased knr compared to DAOTA+, and thus reduced quantum
yield and fluorescence lifetime, is related to the isopropyl
bridge and not to the redshift (energy gap law). We tentatively
assign this increased knr to out-of-plane structural distensions
driven by the ring tension in the isopropyl bridged
triangulenes, as discussed above. In this regard it is noted that
the non-planar helical DMQA+ display significantly higher rates
of non-radiative deactivation (Table 1). Despite the increase in
500
600
700
800
λ (nm)
Figure 2. Absorption (A) and normalized fluorescence spectra (B) of
(green), and (red) recorded in CH2Cl2 solution.
6 (blue), 7
8
The absorption spectra display a continuous redshift and
increase in the molar absorption coefficient ( ) as the number
of nitrogen bridges is increased going from to and . The
ε
6
7
8
non-radiative deactivation, CDATA+ (
8) to a large extend fulfil
fluorescence spectra follow the same trend with a small Stokes
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A
B
E
C
our objectives: with a quantum yield of 0.6 and a fluorescence
lifetime of 16 ns.
D
F
Figure 3. Confocal microscope imaging of stained HEK293 cells A-C: cell stained
with and MitoTracker Green. A: Transmission image. B: Fluorescence signal
from . C: Fluorescence signal from MitoTracker Green. D-F: cell stained with
and Calcein AM. D: Transmission image. E: Fluorescence signal from
Fluorescence signal from Calcein AM.
Table 1. Photophysical properties of
solution.
6, 7, 8
, DAOTA+ and DMQA+ in CH2Cl2
7
7
8
8. F:
Compound λabs
εmax
λf
nm
Φf
τf
ns
krad
×106 s-1
23
knr
×106 s-1
20
M–1cm-1
Figure 3D-F show a cell stained with CDATA+ (
8), which appears
nm
6 CDOTA+
7 CAOTA+
8 CDATA+
DAOTA+ (a)
DMQA+ (b)
535
568
603
555
616
9470
554
584
624
575
662
0.54
0.57
0.61
0.76
0.17
23.1
17.9
15.8
22.2
11.3
to preferentially stain the nucleus of the cell (Figure 3E). The
cells appeared to be healthy based on their morphology and
the co-staining of the cells cytosol with Calcein AM (Figure 3F)
11600
19700
14700
18100
32
24
39
25
34
11
–
a
commonly fluorescence indicator of cell viability.
Moreover, was found to be very photostable (vide infra) and
for time-gated fluorescence microscopy
was thus investigated in HEK293 cells. The increased
photostability going from via to follows the electron
15
70
8
Columns list:
λ
abs: Wavelength at the maximum intensity of the S0→S1 transition. the applicability of
λf Wavelength at the maximum
fluorescence intensity. Φf: Fluorescence quantum yield. f: Fluorescence lifetime.
rad = Φf / f. knr = (1/
f) - krad. See ESI for details. (a) Data from ref. 31 In an earlier
study,34 an underestimated quantum yield of 0.44 was reported for DAOTA+. (b)
Data from ref. 42
8
εmax
:
Molar absorption coefficient at λabs
.
:
τ
6
7
8
k
τ
τ
donor strength of the heteroatoms, which from other
triangulenium dyes is known to be correlated to cation
stability and redox potentials.32, 52 TCSPC experiments on cells
.
Applications in Time-Gated Fluorescence Imaging
The long fluorescence lifetimes of , and make these dyes
stained with 8 showed that the fluorescence decay (Figure 4A),
collected from scanning over a whole cell, was best fitted with
three exponential decay components of 1.4 ns, 4.6 ns, and
11.6 ns (Table S2). The dominating and longest fluorescence
lifetime component of 11.6 ns contributes more than half of
the overall fluorescence intensity. This fluorescence lifetime
component is shorter than the fluorescence lifetime measured
in apolar CH2Cl2 solution (15.8 ns, Table 1), but much longer
than the fluorescence lifetime measured in polar DMSO and
aqueous solutions (4.9 ns and 3.4 ns, respectively, Table S1).
Thus, the longest fluorescence lifetime component does most
6,
7
8
excellent candidates for use in e.g. time-gated fluorescence
detection.74 Initially, Human Embryonic Kidney (HEK293) cells
were stained with each of the three fluorophores and imaged
by confocal microscopy. The fluorescence signal of
found to be completely quenched in the cellular environment,
while the fluorescence signal of and could be monitored
(Figure 3). Compound tends to stain specific parts of the cell
(Figure 3A-C). The fluorescence signal of is detected from
6 was
7
8
7
7
some of the same cellular compartments as the fluorescence
signal from cells co-stained with MitoTracker Green (for
likely originate from
8 located in the more lipophilic parts of
the cell, while the 4.6 ns component may originate from
more polar environments. This long decay component of
8
8
in
in
overlap see: Figure S26), and thus
located in the mitochondria. The fluorescence intensity of
was however found to decrease significantly within the
recording of few pictures (Figure S27), indicating that
7 appears to be partly
7
specific cellular environments makes this fluorophore suitable
as probe for time-gated fluorescence microscopy
applications. To demonstrate the power of the long
fluorescence lifetime of , cells were co-stained with and
a
7
undergoes relative fast photobleaching in the cellular
environment, and is therefore less suited as probe or stain in
microscopy experiments.
8
8
Calcein Red-Orange. Calcein Red-Orange is a bright BODIPY
derived cell-tracker stain,11 and was chosen as a co-stain for
several reasons. It reports on the cells viability and its
fluorescence spectrum overlaps with the fluorescence
spectrum of
8 (Figure S25). As a result of the latter, the
fluorescence signals of the two dyes are hard to resolve by
fluorescence microscopy using standard filter combinations,
which makes difficult the investigation of fluorescently labeled
molecules in the cytosol in the presence of this indicator of cell
viability. Calcein Red-Orange displayed biexponential
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fluorescence decay in the HEK293 cells, with fluorescence
lifetime components of 1.8 ns and 4.2 ns (Figure 4A and Table
S2). The dominant 4.2 ns fluorescence lifetime component,
which contributes 78 % to the overall fluorescence intensity
fits well with the fluorescence lifetime of Calcein Red-Orange
measured in DMSO solution (3.7 ns, Table S1).
Figure 5. Photobleaching of
Integrated fluorescence intensity decay upon continuous irradiation (545 nm) of
8
and Calcein Red-Orange measured in cells.
8
(blue) and Calcein Red-Orange (red).
The results of the photobleaching experiments show that
8 is
much more photostable than Calcein Red-Orange, and thus
should be applicable in many types of fluorescence imaging.
The initial increase of the fluorescence intensity within the first
30 seconds of irradiation of
8 in the bleaching experiments is a
reproducible feature. The exact reason for the increase is not
immediately clear but it is not associated with changes in the
fluorescence spectrum (Figure S28). A similar feature has been
observed in bleaching studies of modified rhodamine 101
analogues and ATTO 647N.13, 58 Overall, these results make
8
very attractive for future fluorescent probe applications, which
require red emission, high photostability, and long
fluorescence lifetime. The time-gated experiments conducted
on cells, using 8, showed that its application is currently
limited to lipophilic targets, as the fluorescence lifetime drops
significantly in polar and aqueous environments (Table S1).
However, the introduction of water solubilizing groups may
change this limitation, as has been shown e.g. for the Alexa
dyes,11 and the development of this chemistry is in progress.
Figure 4. A: Fluorescence decays for HEK293 cells stained with
8
(black), Calcein
and Calcein
Red-Orange (blue). B: Fluorescence decay for a cell co-stained with
8
Red-Orange. The colored areas under the decay curve show photon arrival times
used for the construction of the time-resolved images (C-F). C: Fluorescence
intensity image created from all detected photons (19-90 ns). D: Time-resolved
image based on photons from the green range (19-21.5 ns) in the decay shown
in B. E: Time-resolved image based on the photons from the red range (35.5-90
ns) in the decay shown in B. F: Photon arrival time imaging (PArTI) image based
on the photons from the green (19-21.5 ns) and red (35.5-90 ns) range in the
decay shown in B.
CONCLUSION AND PERSPECTIVES
Three new triangulenium chromophore systems containing a
saturated carbon bridge were prepared from a dithranol ring
system. Introduction of one or two aza-bridges was achieved
via nucleophilic replacement of xanthenium type oxygen
bridges with primary amines. The procedure allows for
formation of asymmetric derivatives with functional side
Figure 4C-E show the fluorescence signal collected in different
time intervals: 19-90 ns, 19-21.5 ns, and 35.5-90 ns (Excitation
pulse arrives at approximately 19 ns in the measurement
window). The photon arrival time imaging (PArTI) is shown in
Figure 4F.75 The time-resolved images clearly demonstrate that
chains (
bioconjugative derivatives. All three carbon bridged dye
systems ( , and ) display redshifted emission compared to
9 and 10) that may be used for preparation of
8
can be used for time-gated fluorescence detection (and FLIM
microscopy) to yield high contrast pictures of cellular
environments, even when co-stained with a bright spectrally
overlapping dye like Calcein Red-Orange. The photostability of
6,
7
8
the oxygen bridged analogues, fluorescence quantum yields
>50% and long fluorescence lifetimes (>16 ns). Crystal
structures and computational results suggest that the
saturated carbon bridge introduce ring tension and possible
out-of-plane distortions in the triangulenium system, which
may be responsible for reduced quantum yields and
fluorescence lifetimes. The optical properties demonstrate
that it is possible by strategic modifications to redshift the
triangulenium dyes without significant loss of their unique long
fluorescence lifetimes, but also that larger structural
distortions of triangulenium system are likely to deteriorate
8
was tested in comparison to that of Calcein Red-Orange.
Cells were separately stained with the two dyes and
continuously irradiated with intense 545 nm light over a period
of 10 min in a wide field microscope setup. The fluorescence
intensity photobleaching curves of
are shown in Figure 5.
8 and Calcein Red-Orange
the attractive fluorescence properties. While the dioxa (6) and
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ARTICLE
Journal Name
6.
7.
J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626-634.
K. Umezawa, D. Citterio and K. Suzuki, Anal. Sci., 2014, 30
327-349.
J. O. Escobedo, O. Rusin, S. Lim and R. M. Strongin, Curr.
Opin. Chem. Biol., 2010, 14, 64-70.
Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014,
43, 16-29.
azaoxa (
7
) derivatives are sensitive to quenching or bleaching,
) has proven to possess very
the diaza derivative CDATA+
(
8
,
promising properties as fluorescent stain for time-gated
microscopy and FLIM in the red part of the visible spectrum.
Future investigations will focus on improving the performance
of the CDATA+ system in aqueous environment, through the
introduction of water solubilizing groups, and to investigate
the applications of the dyes in time-resolved imaging and
detection.
8.
9.
10.
11.
T. Weil, T. Vosch, J. Hofkens, K. Peneva and K. Müllen,
Angew. Chem. Int. Ed., 2010, 49, 9068-9093.
I. Johnson, The Molecular Probes Handbook: A Guide to
Fluorescent Probes and Labeling Technologies, 11th
Edition, Life Technologies Corporation, 2010.
G. Lukinavičius, K. Umezawa, N. Olivier, A. Honigmann, G.
Yang, T. Plass, V. Mueller, L. Reymond, I. R. Corrêa Jr, Z.-G.
Luo, C. Schultz, E. A. Lemke, P. Heppenstall, C. Eggeling, S.
Conflicts of interest
12.
The authors declare the following competing financial
interests: Bo W. Laursen is associated with the company KU-
dyes, which produces and sells dyes (including triangulenium
dyes).
Manley and K. Johnsson, Nat. Chem., 2013, 5, 132-139.
13.
14.
G. Y. Mitronova, S. Polyakova, C. A. Wurm, K. Kolmakov, T.
Wolfram, D. N. H. Meineke, V. N. Belov, M. John and S. W.
Hell, Eur. J. Org. Chem., 2015, 2015, 337-349.
J. B. Grimm, B. P. English, J. Chen, J. P. Slaughter, Z. Zhang,
A. Revyakin, R. Patel, J. J. Macklin, D. Normanno, R. H.
Acknowledgements
Singer, T. Lionnet and L. D. Lavis, Nat. Methods, 2015, 12
,
The work was supported by the Danish Council of Independent
Research (DFF – 6111-00483, DFF – 1337-00005 and DFF –
1335-00773). T.V. gratefully acknowledges financial support
from the “Center for Synthetic Biology” at Copenhagen
University funded by the UNIK research initiative of the Danish
Ministry of Science, Technology and Innovation (Grant 09-
065274) and bio-SYNergy, University of Copenhagen’s
Excellence Programme for Interdisciplinary Research.
244-250.
15.
K. Kolmakov, E. Hebisch, T. Wolfram, L. A. Nordwig, C. A.
Wurm, H. Ta, V. Westphal, V. N. Belov and S. W. Hell,
Chem. Eur. J., 2015, 21, 13344-13356.
16.
J. B. Grimm, A. J. Sung, W. R. Legant, P. Hulamm, S. M.
Matlosz, E. Betzig and L. D. Lavis, ACS Chem. Biol., 2013,
1303-1310.
8,
17.
18.
M. Heilemann, S. van de Linde, A. Mukherjee and M.
Sauer, Angew. Chem. Int. Ed., 2009, 48, 6903-6908.
M. K. Lee, P. Rai, J. Williams, R. J. Twieg and W. E.
Moerner, J. Am. Chem. Soc., 2014, 136, 14003-14006.
Notes and references
19.
L. M. Wysocki, J. B. Grimm, A. N. Tkachuk, T. A. Brown, E.
‡
The brightness of a fluorophore is given by the product of the molar absorption
coefficient at the excitation wavelength and the quantum yield of fluorescence (ε ×
). For dyes not undergoing excited state reactions or large structural changes the
rate of fluorescence (krad) is expected to be proportional to the oscillator strength
of the S0 S1 transition and thus to . From this follows that large values for
S0 S1 will not only favour high brightness but also fast radiative deactivation and
Betzig and L. D. Lavis, Angew. Chem. Int. Ed., 2011, 50
,
11206-11209.
Φ
20.
21.
J. B. Grimm, T. A. Brown, A. N. Tkachuk and L. D. Lavis,
ACS Cent. Sci., 2017, , 975-985.
3
→
ε
ε
→
J. B. Grimm, A. K. Muthusamy, Y. Liang, T. A. Brown, W. C.
Lemon, R. Patel, R. Lu, J. J. Macklin, P. J. Keller, N. Ji and L.
D. Lavis, Nat. Methods, 2017, 14, 987-994.
thus short lifetimes see ref. 73 (S. J. Strickler, R. A. Berg, J. Chem. Phys., 1962, 37
,
814-822).
§
For a description of the PMO approach, including starred and non-starred
22.
23.
M.-A. Franz-Josef, Methods Appl. Fluoresc., 2017,
042002.
5,
positions see ref. 54 and 55 (Griffiths, J., Colour and Constitution of Organic
Molecules. Academic Press, 1976. M. J. S. Dewar and R. C. Dougherty, The PMO
Theory of Organic Chemistry Plenum Press, New York, 1st edn., 1975). For the
A. Shivalingam, M. A. Izquierdo, A. L. Marois, A.
Vysniauskas, K. Suhling, M. K. Kuimova and R. Vilar, Nat.
Commun., 2015, , 8178.
R. M. Rich, D. L. Stankowska, B. P. Maliwal, T. J. Sørensen,
B. W. Laursen, R. R. Krishnamoorthy, Z. Gryczynski, J.
Borejdo, I. Gryczynski and R. Fudala, Anal. Bioanal. Chem.,
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DAOTA+, CDOTA+ ), CAOTA+ ), and CDATA+
(6 (7 (8) chromophores this simple
6
qualitative LCAO prediction was confirmed by the Kohn-Sham orbital plots
calculated by DFT, see ESI Figures S35 – S38.
24.
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Ff
C
C
C
O
O
O
N
N
N
tf
23 ns
18 ns
16 ns