Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye

Article abstract of DOI:10.1021/acs.jpca.5b07898

Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a

Full text of DOI:10.1021/acs.jpca.5b07898

Article
pubs.acs.org/JPCA
Photophysics of a Live-Cell-Marker, Red Silicon-Substituted
Xanthene Dye
†
†
†
‡
†
†
Luis Crovetto, Angel Orte, Jose M. Paredes, Sandra Resa, Javier Valverde, Fabio Castello,
‡
‡
†
,†
Delia Miguel, Juan M. Cuerva, Eva M. Talavera, and Jose M. Alvarez-Pez*
†
Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Cartuja Campus, 18071 Granada, Spain
Department of Organic Chemistry, Faculty of Sciences, University of Granada, C. U. Fuentenueva s/n, 18071 Granada, Spain
‡
*
S Supporting Information
ABSTRACT: Dyes with near-red emission are of great interest because of their
undoubted advantages for use as probes in living cells. In-depth knowledge of their
photophysics is essential for employment of such dyes. In this article, the photophysical
behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)-
dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state,
and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant
of the dye, pKN‑A, was determined by absorbance and steady-state fluorescence at very low
buffer concentrations. Next, we determined whether the addition of phosphate buffer
promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion
form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the
steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were
employed. The TRES experiments demonstrated a remarkably favored conversion of the
neutral form to the anion form. Then, the values of the excited-state rate constants were
determined by global analysis of the fluorescence decay traces recorded as a function of
pH, and buffer concentration. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate
constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N‑A
and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a
sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.
1
. INTRODUCTION
fluorescence, have been used as fluorescent indicators of
phosphate ions in living cells in fluorescence lifetime imaging
The family of fluorescein derivatives has been widely employed
for use as fluorescent probes because of their high water
solubility and favorable spectral parameters, including the high
molar extinction coefficient of the dianion form at a wavelength
of 490 nm, which matches the 488 nm spectral line of the argon
9
,10
microscopy (FLIM).
Although the FLIM methodology is
adequate for removing the contribution of the autofluorescence
of cells and biological fluids, it is also interesting to explore dyes
that emit in other color regions for their use in bioimaging.
Since the first red-emission silaanthracene fluorophore
1
,2
ion laser, and their high fluorescence quantum yield.
1
1
(
TMDHS) was synthesized by Fu et al., fluorescent probes
Fluorescein in aqueous solution can exist in four different
prototropic forms, depending on the pH. At near-neutral pH
values, the two predominant forms are the dianion and
monoanion, each with different molar extinction coefficients,
12,13
both silicon-substituted rhodamines
fluoresceins
and silicon-substituted
have been synthesized and developed as
14−16
labeling and bioimaging reagents. The interested reader can be
3
directed to a topical minireview covering recent progress on
and fluorescence quantum yields. The spectral changes
1
7
silicon-substituted xanthene dyes. In this context, one
significant dye silicon-substituted xanthene, the 7-hydroxy-5,5-
dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me
associated with the monoanion-dianion transition in fluores-
cein, and its derivative 2′,7′-bis(2-carboxyethyl)-5-(and-6)-
carboxyfluorescein, BCECF, have been employed for measuring
pH values in biological systems.
Various fluorescein derivatives, in which methyl, methoxy, or
tert-butyl groups have been introduced into the benzene
moiety, have been synthesized in the past decade. These dyes
have the additional advantage of providing a single anion
species in aqueous solution, and some behave as on/off
fluorescent probes. Among the latter, 9-[1-(2-methyl-4-
methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe
TG) and 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-
xanthen-3-one (Granada Green, GG), which both emit green
18
4
−6
TM) was recently synthesized. This dye exhibits absorption
and emission wavelengths that are approximately 90 nm longer
than those of fluorescein derivatives.
7
,8
As is recognized, the use of fluorescent probes as these
silicon-substituted xanthene derivatives as sensors, requires a
full understanding of the complex photophysics of the dyes. It
Received: August 13, 2015
Revised: October 13, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jpca.5b07898
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Scheme 1. Synthesis of the 2-Me TM Derivative
has been well established that fluorescein derivatives have the
ability to undergo ESPT reactions, thereby rapidly interconvert-
ing between two protonated forms in the presence of an
appropriate proton donor−acceptor. In particular, the
phosphate buffer acts as the right proton donor−acceptor,
the pKN‑A was measured by means absorption and fluorometric
titrations. Next, both steady-state and TRES methodologies
were employed to reveal the buffer-mediated two-excited-state
photophysics system. Finally, the pertinent kinetic ESPT
reaction model and its dynamics were resolved in detail by
collecting the fluorescence decay traces, and determining the
fundamental kinetic rate constants that describe the dynamic
behavior of the system. As a final point, the applicability of 2-
Me TM for bioimaging was determined using HeLa 229 cells
by collecting the emitted near-red signal with a fluorescence
microscope system.
2
6
stimulating the ESPT reaction in fluorescein or BCECF,
whereas a lower-pK buffer, as is acetate buffer, stimulates ESPT
a
reactions in 2′,7′-difluorofluorescein (Oregon Green 488,
1
9,20
OG488).
Because the presence of the ESPT reactions
influences the fluorescent decay from excited prototropic forms,
exhibiting complex kinetics, and decay times that depend on
both the pH and buffer concentration, it is necessary to know in
depth the photophysics of these new silicon-substituted
xanthene derivatives in what is referred to as the excited-state
dynamics of the molecular forms present at near physiological
pH values. Nevertheless, the presence of ESPT reactions could
be advantageous because in these reactions, the decay times are
tuned by the phosphate buffer concentration at a determined
pH, thus sensing the phosphate concentration of either a
2. EXPERIMENTAL METHODS
2
.1. Materials and Preparation of Solutions. For the
preparation of phosphate buffer solutions, NaH PO ·H O and
2
4
2
Na HPO ·7H O (both Fluka, puriss p.a.) were used. To adjust
2
4
2
the pH of the unbuffered aqueous solutions, NaOH 0.01 M
platelets, Sigma-Aldrich, Spain) and HClO 0.01 M (Sigma-
(
4
Aldrich) both spectroscopic grade were employed. Milli-Q
water was used as solvent. The chemicals were used as received.
9
,10
complex solution or living cells.
21
The time-correlated single-photon counting (TCSPC)
2-Me TM was synthesized with a good yield (85%) by the
technique supplies fluorescence data from which both the
time-resolved emission spectra (TRES) and other relevant
photophysical parameters of the system can be obtained. TRES
are fluorescence spectra obtained at distinct times in the course
of the fluorescence decay. To perform TRES and determine the
appropriate relaxation model to describe the photophysical
system, a tridimensional fluorescence decay surface that
represents the fluorescence intensity at all wavelengths and
times during the fluorescence decay is measured. Thus, the
reconstructed emission is defined in terms of both the spectral
addition of commercially available o-tolylmagnesium bromide
to 3,7-bis((tert-butyldimethylsilyl)oxy)-5,5-dimethyldibenzo-
[
b,e]silin-10(5H)-one I and subsequent dehydration with
aqueous hydrochloric acid (Scheme 1). The corresponding
TBDMS-derivative I was prepared by following the protocol
25
described by Best et al. The purity of 2-Me TM was
1
13
confirmed by H NMR and C NMR (data and figures in the
Supporting Information).
A stock solution of 2-Me TM (10− M) in diluted NaOH
was prepared. From this stock solution, the required
concentrations of 2-Me TM at the suitable pH and phosphate
concentrations were prepared. For fluorescence measurements,
4
22
and time resolution. Once it is demonstrated that the spectral
family corresponds to a system in two excited states, a suitable
model of ESPT reaction in the company of a proton donor/
acceptor should be proposed to determine the significant
solutions with absorbance at λ less than 0.1were employed. All
ex
the solutions were freshly prepared.
2
photophysical parameters. To resolve the model, a multi-
For the pK calculation using the absorbance or steady-state
a
−5
dimensional fluorescence decay data surface under various
excitation (λ ) and emission (λ ) wavelengths, buffer
fluorescence experiments, 11 solutions of 2-Me TM (5 × 10
M or 5 × 10− M, respectively) in 0.05 M phosphate buffer
with pH values in the range 6.18−10.14 were prepared. For the
TRES experiments, eight solutions of 2-Me TM (5 × 10− M)
in 0.7 M phosphate buffer with pH values in the range 3.97−
8.72 were prepared, and each solution was excited at either 485
or 530 nm to preferentially excite the neutral or anion form.
The decay traces from each solution at each excitation
wavelength were registered at each 5 nm in the emission
wavelength range from 550 to 640 nm. To construct the
multidimensional fluorescence decay surface to determine the
fundamental kinetic parameters, 11 unbuffered solutions and 44
6
ex
em
B
concentration, C , and pH is measured. Finally, to achieve an
accurate estimation of the parameters, the fluorescence decay
6
traces should be analyzed in terms of their decay times (τ ) and
i
associated amplitudes (p ) by global analysis, in which the τ
i
i
values are linked parameters in all the decay traces collected at
various excitation and emission wavelengths from the same
2
3
sample. A full study of the variation of the decay times with
pH and buffer concentration enables determination of the
underlying rate constants (k ) that define the excited-state
ij
2
4
kinetics. Once the constants are known, both the steady-state
B
fluorescence intensity and fluorescence decay time values could
phosphate buffered solutions (C = 0.05, 0.15, 0.30, and 0.6
2
,20
be calculated at any pH, and buffer concentration.
M), all in the pH range 4.0−9.5, were prepared.
In this paper, aqueous solutions of 2-Me TM were
investigated via absorption, steady-state and time-resolved
emission spectroscopy. The ground-state equilibrium between
the neutral and anionic forms of 2-Me TM was examined, and
HeLa 229 (ECACC 86090201) cells were grown in
Dulbecco’s Modified Eagle’s medium (DMEM) supplemented
with 10% (v/v) FBS, 2 mM glutamine, 100 U/mL penicillin,
and 0.1 μg/mL streptomycin in a humidified 5% CO2
B
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incubator. For the fluorescence imaging microscopy experi-
ments, HeLa 229 cells were seeded onto 20 mm diameter
coverslips in 6-well plates, then transferred to the MicroTime
3. RESULTS AND DISCUSSION
.1. Absorption Measurements and Ground-State
3
Equilibria of 2-Me TM. The visible absorption spectra of
2
00 fluorescence microscope system, and washed with Krebs−
−5
aqueous solutions of 2-Me TM (5 × 10 M) in a pH range
Ringer−phosphate (KRP) buffer before addition of the working
solutions. The KRP buffer was prepared at pH 7.4 and
between −0.40 and +10.14 were recorded at very low ionic
strength (0.01 M phosphate buffer). These spectra exhibited
pH-induced transitions due to ground-state proton-transfer
contained the following: NaCl 118 mM, KCl 5 mM, CaCl 1.3
2
mM, MgCl 1.2 mM, KH PO 1.2 mM, and Hepes 30 mM (all
2
2
4
reactions dictated by the ground-state pK values. Figure 1A
a
chemicals from Sigma-Aldrich). The working solutions were
−7
prepared using KRP buffer and 2-Me TM (10 M).
.2. Instrumentation. Absorption and steady-state fluo-
rescence spectra were recorded using a PerkinElmer Lambda
50 UV/vis spectrophotometer, and a JASCO FP-6500
2
6
spectrofluorometer, respectively, both equipped with a temper-
ature controller. All measurements were recorded at 20 °C
using 10 × 10 mm cuvettes. The pH of the solutions was
measured immediately after recording each spectrum.
Fluorescence decay traces were recorded via the time-
21,26
correlated single photon counting (TCSPC)
method using
a FluoTime 200 fluorometer (PicoQuant GmbH, Germany).
The excitation source consisted of either an LDH-485 or LDH-
530 pulsed laser (PicoQuant) with 88 ps pulse width. The rate
of pulse repetition was 20 MHz. Both detection polarizer set at
the magic angle and detection monochromator are located
before the photomultiplier detector. The fluorescence decay
traces were collected in 1320 channels with a time increment of
36 ps/channel. The instrument response functions (using
LUDOX as scatter) and sample decays were recorded until they
4
reached 2 × 10 counts on the peak channel. In the TRES
experiments, when the count rate was very low in the extreme
portion of the emission wavelength range chosen (550 to 640
nm), the decay traces were collected for 5 min to achieve a
count number sufficient for analysis.
Confocal fluorescence microscopy was carried out on a
MicroTime 200 instrument (PicoQuant), using the aforemen-
tioned LDH-530 pulsed laser as the excitation source. The laser
was directed into the specimen using a dichroic mirror
(
Z532RDC, Chroma) and the oil immersion objective (1.4
Figure 1. (A) Absorption spectra from 5 × 10− M aqueous solutions
of 2-Me TM in the pH range between −0.4 and +10.14. (B)
Recovered molar absorption coefficients versus wavelength for the
neutral (black) and anionic (red) form of 2-Me TM.
6
NA, 100×) of an inverted microscope (IX-71, Olympus). The
emitted fluorescence was filtered by a cutoff filter (550LP,
AHF/Chroma) and focused onto a 75 μm pinhole. The
detection filter was a FB600-40 bandpass filter (Thorlabs), and
the fluorescence was detected using a single-photon avalanche
diode (SPCM-AQR 14, PerkinElmer). Photon counting,
imaging reconstruction, and data acquisition were performed
with a TimeHarp 200 TCSPC module (PicoQuant). Raster
scanned images were recorded with a 512 × 512 pixel
resolution. The fluorescence images were analyzed using an
shows two isosbestic points, one occurring at approximately
05 nm with moderate acid or basic pH values, and the other,
at low pH values, arises at 485 nm. The similarity of the
chemical structure of 2-Me TM to those of 2-OMe-5-Me TG
and the presence of two isosbestic
points suggest that the aqueous solutions of 2-Me TM present
three absorbing protonated species, the cation (C), neutral
N), and anion (A), and two pK describing the proton
5
28,29
and 2-Me-4-OMe TG
27
open-source platform for biological-image analysis.
.3. Fluorescence Decay Traces Analysis. The fluo-
2
(
a
rescence decay traces for TRES were individually analyzed
using exponential models by means of the FluoFit software
package (PicoQuant). The impulse response curves, collected
either with the same number of counts at the peak or in a
discrete period of time when the emission intensity was low,
were corrected and then normalized to a steady-state emission
spectrum recorded on the same instrument.
The decay traces of samples at the same pH, buffer
concentration, and excitation wavelength were globally
analyzed using the FluoFit package. The decay times were
linked parameters, whereas the pre-exponential factors were
kept as local adjustable parameters.
transfers (Scheme 2). The N and A forms are the only relevant
species at near-neutral pH values.
As shown in Figure 1A, at a basic pH, the spectrum is
basically composed of a band centered at 585 nm and a
shoulder at approximately 550 nm, which is identical to the
spectrum observed in NaOH (1 M). With decreasing pH, the
shoulder disappears and the band that peaks at 585 nm is blue-
shifted, exhibiting a little pronounced maximum and a very
broad profile. At a pH of approximately 4.00, the spectrum
shape is a wide band with a diffuse maximum at approximately
475 nm. The absorption spectra at pH values ranging between
6.50 and 10.14 exhibit only one transition at near-neutral pH,
C
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Scheme 2. Prototropic Forms of 2-Me TM: A (anion), N
a
(neutral), and C (cation)
a
The neutral form is represented by two resonance structures.
Figure 2. Normalized fluorescence spectral profiles of the three
prototropic forms of 2-Me TM: anion (blue), neutral (black), and
cation (red).
which is characterized by the ground-state constant KN‑A. The
recovered spectra in this pH range, and their appointment of
them to both anion and neutral prototropic forms, are
form shown in Figure 2 was recorded in a 2.0 M HClO4
aqueous solution.
15
concordant with those recently reported. At pH values of
less than 6.50, in the pH range −0.4 to +3.60, a wide band with
a diffuse maximum at approximately 475 nm was red-shifted as
a new band arises, peaking at 530 nm. In that pH range,
another isosbestic point at 485 nm can be obviously
distinguished, thus indicating that another species is involved
in this other acid−base equilibrium, characterized by the
The ground-state acid−base equilibrium constants can also
be obtained by fluorometric titrations whenever there is
constant intensity of excitation, lower absorbance of 0.100,
and negligible rates of ESPT reactions. Consequently, the
steady-state fluorescence spectra of 2-Me TM solutions in the
pH range from 6.18 to 9.00 were recorded at two excitation
wavelengths (485 and 580 nm). To minimize the rate of
possible ESPT reactions mediated by the presence of a proton
ground-state constant K . The absorption spectrum of the
C−N
30
cation form was confirmed using a 2.0 M HClO aqueous
donor/acceptor, we used phosphate (0.005 M) as a buffer.
4
solution, and it exhibited a peak at 530 nm. Thus, the
experimental visible absorption spectra of 2-Me TM aqueous
solutions in the pH range between −0.4 and +10.14 is
consistent with the ionic equilibria between the prototopic
forms represented in Scheme 2.
Only the A and N emission spectral shapes were noticed in that
pH range. The recovered pKN‑A value among the neutral and
anion species of 2-Me TM was 7.41 ± 0.02. Examples of the
emission spectra and fitting of the fundamental equations to the
experimental data are shown in Figures S5 and S6 in the
Supporting Information.
The absorbance of the aqueous solutions of 2-Me TM
depends on the pH according to the acid−base equilibrium
The pK values from the absorbance and emission measure-
a
theory and Beer’s law. Because the only pK of interest for
ments were consistent and slightly greater than the previously
reported value of 6.8, although this latest value was determined
in the presence of phosphate buffer (0.1 M) and 1% dimethyl
a
biological applications is that which the dye presents at near−
neutral pH, the selected absorption spectra at pH values
ranging between 6.50 and 10.14 were analyzed. The nonlinear
global fitting of the complete surface of A vs pH and λabs to
Beer’s law and the equations of the acid−base equilibria
18
sulfoxide (DMSO).
3.3. Buffer-Mediated ESPT Reactions. Xanthene deriva-
tives undergo fast ESPT reactions when an appropriate proton
donor/acceptor is present in solution at an adequate
concentration. These reactions may radically alter the result
of steady-state or time-resolved fluorescence experiments. 2-Me
TM can undergo such reactions when facilitated by the
presence of phosphate buffer, and we study these reactions
here.
(
Supporting Information eqs S1−S3) allows us to determinate
3
the molar absorption coefficients ε (λ ) and pK . In the
i
abs
N−A
fitting, the pKN−A was linked over the whole surface, whereas
ε (λ ) was a locally adjustable parameter at each wavelength
i
abs
for each species. The absorbance data surface was composed of
1 pH values and 224 λabs values between 400 and 624 nm at
1
each pH. Beer’s law for two protonated species provided the
best fit to the experimental absorption data. Plots of the some
individual absorbance values vs pH at different wavelengths
along with the curves generated from the fitting are shown in
Figure S3 (Supporting Information). In the fitting process, the
recovered parameters were independent of the values initially
assigned to these parameters. Figure 1B shows the estimated
values of the molar absorption coefficients of the neutral (εN)
The steady-state fluorescence spectra from aqueous 5 × 10−
M solutions of 2-Me TM at pH 6.10 in the presence of different
concentrations of phosphate buffer in the range 0.010−0.75 M
were recorded from 500 to 700 nm using an excitation
wavelength of 485 nm. The observed spectra are shown in
Figure 3. This figure shows that with these experimental
conditions, an increase in the buffer concentration resulted in
both a red shift and an increasing intensity of the emission
band, which peaked at 600 nm. Moreover, a concomitant
decrease in the emission band that peaks at 560 nm, which is
attributable to the neutral species, is also observed. The above
results reveal an ESPT reaction stimulated by the phosphate
buffer. Indeed, the 485 nm excitation wavelength with the pH
of 6.10 preferentially excites the much-less-fluorescent 2-Me
TM neutral form. Then, the buffer-mediated ESPT reaction
6
and anionic forms (ε ) at low phosphate buffer concentration
A
(
0.01 M). The recovered pKN−A value was 7.31 ± 0.03.
3
.2. Steady-State Emission Spectra. In Figure 2, the
three different emission spectral profiles that correspond to the
three prototropic forms are shown. In contrast with the other
xanthene dye, the cation exhibited no photoacid behavior
2
0,29
(Figure S4).
Nevertheless, the spectral profile of the cation
D
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could be adjusted using biexponential functions. It should be
mentioned that for the emission wavelengths corresponding to
the ends of the experimental range, the decay traces had low
fluorescent signals. Therefore, the decay traces were recorded
for different collection times, and hence the pre-exponential
coefficients at each wavelength were corrected by the collection
time and then normalized to the same count number in the
decay. Thus, the decay traces were proportional to the steady-
state fluorescence intensity. The pre-exponential factors were
also corrected for the different sensitivity of the detector using a
factor that related the total emission during the decay (p × τ
1
1
+
p × τ ) to the steady-state-intensity emission.
2 2
Figure 4 shows the TRES generated at pH 5.90 and both λex
Figure 3. Steady-state emission spectra from 5 × 10−6 M 2-Me TM
aqueous solutions at a pH of 6.10 and different phosphate buffer
concentrations ranging from 0.010 to 0.75 M (λex = 485 nm).
= 485 and 530 nm. The TRES are represented by a plot of the
quickly happens during the lifetime of the excited-state system,
thus forming the much more fluorescent anion form and
causing variation in the spectral profile and fluorescence
intensity.
To observe the dynamic properties of the fluorescent
emission, we also collected the fluorescence decay traces from
the same solutions employed in the previous experiment. The
excitation wavelength was 485 nm, and the emission wave-
length was 610 nm, which preferentially detects the anionic
species. The results are shown in Figure S7, in which only four
decay traces are shown for clarity. It is apparent from the figure
that increasing the phosphate concentration caused an increase
in the fluorescence decay time. This result is consistent with the
idea that the neutral species (which was excited preferentially in
this experiment) undergoes an ESPT reaction during the
lifetime of the excited state, which is influenced by the
concentration of phosphate buffer. In the ESPT reaction, a
portion of the excited neutral species becomes an excited anion,
and if the reaction occurs sufficiently quickly, both species
decay simultaneously. As previously mentioned, the reaction
rate depends on the phosphate concentration; therefore, at a
higher buffer concentration, the reaction will be faster, and the
corresponding amount of the anion, which is dictated by the
pK*N‑A and pH of the experiment, will be formed earlier. Thus,
the subsequent fluorescence decay corresponds to the two
coupled species. If the neutral/anion relationship correspond-
ing to the pH of the experiment is achieved faster, the coupled
fluorescence of the system will be detected for longer times.
Figure 4. Contours of fluorescence intensity of 2-Me TM after a δ-
pulse excitation as a function of time and λem in 0.7 M phosphate
buffer at pH 5.90 (A, λex 485 nm; C, λex 530 nm). Corresponding
normalized emission spectra at different times after the excitation pulse
(B, λex 485 nm; D, λex 530 nm).
fluorescence intensity level as a function of time and emission
wavelength (Figure 4A,C), along with the normalized emission
spectra at different times after the excitation pulse in the range
of 0−4 ns (Figure 4B,D).
The spectrum profile at a shorter time after the pulse in
Figure 4B reflects a large amount of the neutral species, which
was preferentially excited under the experimental conditions
3.4. TRES. The ESPT reaction mediated by phosphate
employed (λ = 485 mm). With increasing emission time, the
ex
should exhibit a double-exponential decay behavior, with decay
times independent of the wavelength of emission but
dependent on the forward and reverse rates of the ESPT
spectral profile was gradually red-shifted, thus reflecting the
successive greater abundance of the anionic species. Finally,
after a longer time, the profile remained virtually unchanged
2
reaction. Therefore, the fluorescence spectra observed at
because the neutral/anion relationship dictated by the pK * and
a
distinct times during the fluorescence decay (TRES) can
provide detailed information about the photophysical two-
excited-state system. To accomplish this goal, we performed
TRES with decay traces collected from aqueous solutions of 2-
Me TM in 0.7 M phosphate buffer and at eight pH values in the
range 4.00−8.70, and using the excitation wavelengths of 485
and 530 nm to preferentially excite either the neutral or the
anionic species, respectively. Emission in the wavelength range
pH of the experiment was achieved. When the anion species
was preferentially excited at a wavelength of 530 nm (and the
same pH of 5.90), the initial spectral profile exhibited a small
contribution of the neutral species. Remarkably, the spectral
profiles also reflect a successive greater abundance in the
anionic species with increasing emission time (Figure 4D),
thereby demonstrating that the pK*N‑A of the excited state is
significantly less than the pH of the experiment. Different
fluorescent spectral shapes were obtained by determining the
spectra at each excitation wavelength using several pH values.
In the Supporting Information (Figures S8 and S9), different
illustrative examples of plots of fluorescence intensity versus
time and emission wavelength, along with the normalized
5
50−640 nm was recorded in 5 nm steps. The complete data
matrix is composed of 304 decay traces and can be described by
a three-dimensional surface, I(λ ,λ ,t), that represents the
ex em
fluorescence intensity at all excitation and emission wavelengths
and times during the fluorescence decay. All of the decay traces
E
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emission spectra at different times of emission, are shown. In
brief, the spectral profiles were consistent with the phosphate-
mediated ESPT reaction between the neutral and anionic
species of 2-Me TM. Moreover, the TRES experiments
demonstrated a preferred conversion of the neutral form
Information). To achieve a unique determination of the rate
constants, fluorescence decay traces under different exper-
imental conditions (i.e., the absence or presence of diverse
phosphate buffer concentrations, different values of pH, and
λ ) were recorded, obtaining a multidimensional decay trace
em
surface.
24
(
defined by the shoulder at 560 nm) to the anion form (which
exhibited an emission maximum at 590 nm). This finding
indicates that buffer-mediated deprotonation of the neutral
form is the main process, whereas buffer-mediated protonation
of the anion form is a much slower process.
The decay traces were analyzed in terms of the decay times,
and associated pre-exponential factors, by means global analyses
B
of traces recorded at the same pH and C but at different λ
ex
and λ . The decay times were linked for decay traces collected
em
3.5. Kinetic and Spectral Parameters of the Phos-
with these characteristics, whereas the pre-exponentials were
locally adjustable parameters.
phate-Mediated ESPT Reaction. Once experimental evi-
dence of the photophysical two-excited-state system was
obtained, a multidimensional fluorescence decay surface was
used to determine the kinetic parameters of the system in the
excited state, gaining insights into the kinetic behavior. Scheme
Without phosphate buffer, the decay times were biexponen-
tial with positive pre-exponential factors, and independent of
both the emission and excitation wavelengths and the pH.
Therefore, ESPT does not happen in the absence of buffer.
3
shows the reactions involved in the ESPT model along with
B
Global analysis of 66 curves corresponding to C = 0 M in the
the emission for the two species of 2-Me TM present at near-
neutral pH.
pH range between 4.50 and 10.26 at λ = 485 and 530 nm and
ex
λem = 560, 580, and 600 nm provided consistent decay time
2
estimates: τ = 0.990 ± 0.003 ns and τ = 3.78 ± 0.03 ns (χ =
1
2
g
Scheme 3. Kinetic Model of Ground- and Excited-State
Proton-Transfer Reactions of Aqueous 2-Me TM Added of
Phosphate Buffer
1
.15). At pH values greater than 8, only τ was present. At these
2
pH values, the reprotonation reaction in the excited state is very
slow because of the low concentration of protons and does not
compete with the radiative constant. Therefore, this decay time
unambiguously defines the value for k . Nevertheless, the
02
shorter lifetime τ consists of the sum of the excited-state
1
dissociation rate constant, k , and the radiative constant of the
21
neutral form, k . From global analyses of the decay traces, we
0
1
8
−1
9
determined that k = 2.64 × 10 s and k + k = 1.08 × 10
02
01
21
−
1
s .
To obtain the ESPT rate constants kB and k , 366 decay
B
12
21
traces were collected in the pH range 4.00−10.26 with different
phosphate buffer concentrations (0, 0.15, 0.3, and 0.6 M) at
two λ (485 and 530 nm) and three λ (560, 580, and 600
ex
em
nm) values. The theoretical equations (eqs S8 and S9 in the
Supporting Information) were fit with the decay times
previously determined to obtain the rate constants. Figure 5
shows the decay times determined, along with the fitted curves
2
(
χg = 1.18). Global analysis provided all of the rate constants
of Scheme 3 (Table 1). During the model fitting, the value of
k21 was always close to 0 and exhibited a very large associated
In Scheme 3, species 1 and 2 correspond to the neutral and
anionic forms of 2-Me TM, respectively. Following excitation
by a fast δ-pulse of light, the excited-state species 1* and 2* are
generated. These excited species can decay through fluo-
rescence (F) and nonradiative (NR) processes. The composite
rate constants for these processes are denoted by k01 (=kF1
+
kNR1) and k02 (=kF2 + kNR2). k21 denotes the rate constant for
+
the dissociation of 1* into 2* and H . Because in the
+
experimental pH range, [H ] is sufficiently small, the rate of
+
association of 2* + H → 1* (k ) can be considered negligible.
1
2
The ground-state proton exchange reaction of 2-Me TM is
described by the acidity constant K . The acidity constant of
N‑A
B
2−
+
−
the buffer is denoted by Ka = [HPO4 ][H ]/[H PO ]. The
2
4
B
2−
rate constant k21 characterizes the reaction of 1* with HPO4
−
to form 2* and H PO in the excited state. The reverse
2
4
Figure 5. Global fitting (solid lines) of the theoretical equations (eqs
S8−S11 in the Supporting Information) to the decay times at different
−
2−
that yields 1* and HPO4
B
reaction of 2* and H PO
is
2
4
designated by the rate constant k .
□
△
○
◇
1
2
buffer concentrations (0 M, ; 0.15 M, ; 0.3 M, ; 0.6 M, ) and
pH values. Note the change in scale in the two sections of the ordinate
axis.
The theory and methods of solving buffer-mediated ESPT
2
,20,24,31
reactions are well-established
(see also the Supporting
F
DOI: 10.1021/acs.jpca.5b07898
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The Journal of Physical Chemistry A
Article
Table 1. Rate Constant Values for the Phosphate-Mediated
ESPT Reaction of 2-Me TM
assistance. In addition, in contrast with the pattern observed
9
,10
for previous xanthene-based dyes, characteristic fluorescence
of 2-Me TM was also observed in the interior of the nucleus.
To examine this new accumulation pattern, we measured the
intensity profile of the probe in different cellular compartments
inside HeLa 229 cells. Parts E−H of Figure 6 show the profile
of the fluorescence intensity signal, which corresponds to the
arrows in Figure 6A−D, respectively. In every cell examined,
the cytosol and nuclei exhibited a similar intensity of
fluorescence. This result suggests that good nuclear membrane
penetration of 2-Me TM allows homogeneous and analogous
accumulation of the probe in the cytosol and nuclei. Moreover,
the probe exhibited strong accumulation in different
cytoplasmic structures. This noticeable signal will permit
isolation, differential treatment, and tracking or study of these
structures independent of the cytosol/nucleus fluorescence.
Therefore, on the basis of the fluorescence intensity upon
excitation of the cells at 530 nm during dye loading, it can be
established that 2-Me TM readily accumulates inside
cytoplasmic structures and less effectively but homogeneously
accumulates in the cytosol and nucleus.
rate constant
value
−
1
1.08(±0.01) × 109
2.646(±0.009) × 10
1.02 (±0.43) × 10
7.88 (±0.66) × 109
k /s
01
−
1
8
k /s
02
B
−1 −1
7
k /M
s
12
B
−1 −1
k /M
s
21
error. Hence, we considered the spontaneous excited-state
deprotonation negligible.
The relative values of the rate constants k2 and kB indicate
1
12
that buffer-mediated deprotonation of the neutral state is 2
orders of magnitude faster that the excited-state phosphate-
assisted protonation of the anion form. This is in perfect
agreement with the TRES results, which mainly exhibited
excited-state transformation to the anion form. Finally, the rate
B
B
B
constants k21 and k , along with the known value of K , can
12
a
B
provide the excited-state pK*N‑A. Using 7.2 as the pK , the
a
previously determined rate constants, and eq S10 (Supporting
Information), we obtained a value of 4.31 for the pK*N‑A. This
value indicates that 2-Me TM is a stronger photoacid than the
green xanthene derivatives, in which the excited-state pK* did
not vary substantially with respect to the ground-state
4
. CONCLUSIONS
2
,20,29,30
equilibrium constant.
.6. Fluorescence Imaging Microscopy of 2-Me TM in
To investigate the use of the new silicon-substituted xanthene
derivative 2-Me TM as a sensor, we have performed a study of
the photophysics of the dye. Three absorbing and emitting
species have been identified, and the acidity constant of the dye
at near-neutral pH was determined using both absorbance and
steady-state fluorescence methods. Using steady-state and time-
resolved fluorescence experiments, we demonstrated that 2-Me
TM undergoes a phosphate-mediated ESPT reaction and
rapidly interconverts between the neutral and anion species.
TRES experiments demonstrated a preferred conversion of the
neutral form to the anion form, thereby indicating that the
buffer-mediated deprotonation of the neutral form is the main
process, whereas buffer-mediated protonation of the anion form
is a much slower process. The kinetic parameters determined
were consistent with the TRES results, thus indicating a higher
rate constant for deprotonation than protonation, which
3
HeLa Cells. Water solubility and membrane permeability are
crucial properties for a fluorescent sensor that functions inside
live cells. Some xanthene-based dyes have exhibited good
penetration in cells, and they could be used as intracellular
9
,10
sensors.
In fact, 2-Me TM βgal was applied to visualize β-
18
galactosidase activity in HEK293 cells. To test the penetration
and accumulation of 2-Me TM in the various locations of the
cell interior, measurements on live HeLa 229 cells were
performed. We monitored the loading (1 × 10 M) of the
probe in the intracellular compartments. Immediately after the
loading, confocal fluorescence imaging was performed. As
shown in Figure 6A−D, the intracellular accumulation of 2-Me
TM suggests spontaneous, fast, and good membrane
penetration without a need for any external molecular
−
7
Figure 6. (A)−(D) Illustrative fluorescence images of HeLa 229 cells with 2-Me TM (10−7 M). (E)−(H) Intensity profiles of the arrows from
Figures 6A−D, respectively. Scale bars represent 10 μm.
G
DOI: 10.1021/acs.jpca.5b07898
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The Journal of Physical Chemistry A
Article
implies a very low value of pK*N‑A. Finally, we used the dye
inside cells to examine its future application as a cell sensor.
Excitation of the cells at 530 nm indicated that 2-Me TM
accumulates visibly inside cytoplasmic structures and less
effectively but homogeneously accumulates in the cytosol and
nucleus.
Phosphate Detection in Live Cells. Org. Biomol. Chem. 2014, 12,
6
(
432−6439.
10) Paredes, J. M.; Giron, M. D.; Ruedas-Rama, M. J.; Orte, A.;
Crovetto, L.; Talavera, E. M.; Salto, R.; Alvarez-Pez, J. M. Real-Time
Phosphate Sensing in Living Cells using Fluorescence Lifetime
Imaging Microscopy (FLIM). J. Phys. Chem. B 2013, 117, 8143−8149.
(11) Fu, M.; Xiao, Y.; Qian, X.; Zhao, D.; Xu, Y. A Design Concept of
Long-Wavelength Fluorescent Analogs of Rhodamine Dyes: Replace-
ment of Oxygen With Silicon Atom. Chem. Commun. 2008, 1780−
1782.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jpca.5b07898.
■
*
S
(12) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T.
Evolution of Group 14 Rhodamines as Platforms for Near-Infrared
Fluorescence Probes Utilizing Photoinduced Electron Transfer. ACS
Chem. Biol. 2011, 6, 600−608.
1H and 13C NMR data together with a copy of both
spectra (Figures S1 and S2); analysis of the absorbance
vs pH; analysis of the experimental fluorescence vs pH;
Figures S3−S9 of absorbance and fluorescence vs pH
traces, absorbance and emission spectra, fluorsecence
decay traces, contours of fluorescence instensity; and
evaluation of kinetic parameters (PDF)
(13) Pastierik, T.; Sebej, P.; Medalova, J.; Stacko, P.; Klan, P. Near-
Infrared Fluorescent 9-Phenylethynylpyronin Analogues for Bioimag-
ing. J. Org. Chem. 2014, 79, 3374−3382.
(14) Egawa, T.; Hirabayashi, K.; Koide, Y.; Kobayashi, C.; Takahashi,
N.; Mineno, T.; Terai, T.; Ueno, T.; Komatsu, T.; Ikegaya, Y.; et al.
Red Fluorescent Probe for Monitoring the Dynamics of Cytoplasmic
Calcium Ions. Angew. Chem., Int. Ed. 2013, 52, 3874−3877.
(15) Hirabayashi, K.; Hanaoka, K.; Takayanagi, T.; Toki, Y.; Egawa,
AUTHOR INFORMATION
Corresponding Author
Jose M. Alvarez-Pez. E-mail: jalvarez@ugr.es. Tel. +34
58243831.
■
T.; Kamiya, M.; Komatsu, T.; Ueno, T.; Terai, T.; Yoshida, K.; et al.
Analysis of Chemical Equilibrium of Silicon-Substituted Fluorescein
and Its Application to Develop a Scaffold for Red Fluorescent Probes.
Anal. Chem. 2015, 87, 9061−9069.
*
9
(16) Koide, Y.; Urano, Y.; Hanaoka, K.; Piao, W.; Kusakabe, M.;
Author Contributions
Saito, N.; Terai, T.; Okabe, T.; Nagano, T. Development of NIR
Fluorescent Dyes Based on Si−rhodamine for in Vivo Imaging. J. Am.
Chem. Soc. 2012, 134, 5029−5031.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(
17) Kushida, Y.; Nagano, T.; Hanaoka, K. Silicon-Substituted
Xanthene Dyes and Their Applications in Bioimaging. Analyst 2015,
40, 685−695.
18) Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.;
Notes
1
(
The authors declare no competing financial interest.
Nagano, T. Development of a Fluorescein Analogue, Tokyo Magenta,
as a Novel Scaffold for Fluorescence Probes in Red Region. Chem.
Commun. 2011, 47, 4162−4164.
ACKNOWLEDGMENTS
This research was funded by MINECO (project CTQ2014-
6370-R), and MINECO (project CTQ2014-53598).
■
5
(
19) Orte, A.; Bermejo, R.; Talavera, E. M.; Crovetto, L.; Alvarez-Pez,
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Correlation between Time-Resolved Emission and Steady-State
Fluorescence Intensity. J. Phys. Chem. A 2005, 109, 2840−2846.
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