Synthesis and spectroscopic characterization of a fluorescent phenanthrene-rhodamine dyad for ratiometric measurements of acid pH values

Article abstract of DOI:10.1039/d1nj01573g

We present the rational design, synthesis and spectroscopic characterization of a novel dual excitation, three color emitting, pH-responsive fluorescent probe consisting of two phenanthrene and one rhodamine B units linked by click chemistry. The rhodamine moiety, excitable atλ_Ex= 315 nm and atλ_Ex= 560 nm in its ring-opened form, provides the pH-responsive fluorophore, while the pH-insensitive phenanthrene, excited atλ_Ex= 315 nm, serves as inert internal reference, The presence of two phenanthrene moieties enables a blue monomer and a blueish green excimer emission at 351 nm and 500 nm, respectively. Opening of the rhodamine B spirolactam ring at an acidic pH below 5.0 (pK_a= 2.59 ± 0.04) switches on its emission at 580 nm. Simultaneously, the phenanthrene excimer emission decreases caused by a change in orientation of the phenanthrene units, while the monomer emission is barely affected. This sensor design enables ratiometric measurements in the low acidic pH range utilizing the intensity ratios of the rhodamine B and phenanthrene excimer emission at 580 nm and 500 nm. Alternatively, also the intensity ratios of the rhodamine B and the phenanthrene monomer emission could be exploited or the sum of the phenanthrene monomer and excimer fluorescence. To the best of our knowledge, this is the first report of ratiometric sensing utilizing such a versatile type of tricolor emissive dyad probe bearing phenanthrene moieties and showing phenanthrene monomer and excimer emission.

Full text of DOI:10.1039/d1nj01573g

Volume 45
Number 31
21 August 2021
Pages 13651–14240
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New Journal of Chemistry
A journal for new directions in chemistry
rsc.li/njc
ISSN 1144-0546
PAPER
Priyanka Srivastava, Ute Resch-Genger et al.
Synthesis and spectroscopic characterization of a
fluorescent phenanthrene-rhodamine dyad for ratiometric
measurements of acid pH values

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PAPER
Synthesis and spectroscopic characterization of a
fluorescent phenanthrene-rhodamine dyad for
ratiometric measurements of acid pH values†
b
Cite this: New J. Chem., 2021,
45, 13755
Priyanka Srivastava, *a Paul Christian Furstenwerth,a Jan Felix Witte
and
¨
a
Ute Resch-Genger
*
We present the rational design, synthesis and spectroscopic characterization of a novel dual excitation,
three color emitting, pH-responsive fluorescent probe consisting of two phenanthrene and one
rhodamine B units linked by click chemistry. The rhodamine moiety, excitable at lEx = 315 nm and at
lEx = 560 nm in its ring-opened form, provides the pH-responsive fluorophore, while the pH-insensitive
phenanthrene, excited at lEx = 315 nm, serves as inert internal reference, The presence of two
phenanthrene moieties enables a blue monomer and a blueish green excimer emission at 351 nm and
500 nm, respectively. Opening of the rhodamine B spirolactam ring at an acidic pH below 5.0 (pKa
=
2.59 Æ 0.04) switches on its emission at 580 nm. Simultaneously, the phenanthrene excimer emission
decreases caused by a change in orientation of the phenanthrene units, while the monomer emission is
barely affected. This sensor design enables ratiometric measurements in the low acidic pH range
utilizing the intensity ratios of the rhodamine B and phenanthrene excimer emission at 580 nm and
500 nm. Alternatively, also the intensity ratios of the rhodamine B and the phenanthrene monomer
emission could be exploited or the sum of the phenanthrene monomer and excimer fluorescence. To
the best of our knowledge, this is the first report of ratiometric sensing utilizing such a versatile type of
tricolor emissive dyad probe bearing phenanthrene moieties and showing phenanthrene monomer and
excimer emission.
Received 31st March 2021,
Accepted 8th June 2021
DOI: 10.1039/d1nj01573g
rsc.li/njc
pH can be measured electrochemically or optically,15,16 in the
latter case utilizing an optical probe, i.e., a sensor molecule or a
Introduction
pH is one of the most relevant parameters in the life and material chemodosimeter, that signals changes in pH by changes in its
sciences indicating e.g. proper functioning of cells, diseases like absorption1 and/or fluorescence properties.17–20 Very sensitive
inflammation and cancer, and corrosion processes.1 Also, pH pH measurements with a high spatiotemporal resolution can
regulates several metabolic pathways that include endocytic pro- be best done with pH-responsive molecular or nanoscale
cesses, signaling, apoptosis and cell defense mechanisms.2–5 An optical probes20–26 which can be also exploited for intracellular
acidic environment is for example essential for the functioning and pH studies.1 Suitable pH-responsive probes can be rationally
activation of various enzymes and process involved in protein designed utilizing different signaling mechanisms such as
degradation, and organelles like lysosomes (pH 4.5–5.5) and endo- (intramolecular) charge transfer, photo-induced electron trans-
somes (pH 4.5–6.8) have an acidic pH for proper functioning.6–12 fer (PET), or energy transfer (ET)27–30 processes, that provide
Abnormalities in intracellular pH can affect the various cell func- the basis for reversible pH-induced changes in the spectral and/
tions, like muscle contraction and relaxation,6 apoptosis,13,14 ion or intensity of the probe’s absorption and/or fluorescence.2
transport, and cellular homeostasis and signal or accompany several Alternatively, a chemical reaction to form an optically detect-
diseases including cancer and neurodegenerative disorders.6,13,14
able species can be exploited.31 In addition, suitable probes
should be chemically stable, show high molar absorption
coefficients, and high fluorescence quantum yields as a pre-
requisite for a high sensitivity. Moreover, they should reveal no
or only very little interferences with other analytes such as metal
ions typically present in biological systems. As fluctuations of
the excitation light source and changes in probe concentration
induced by, e.g. photobleaching can directly affect measured
a Division Biophotonics, Federal Institute for Materials Research and Testing (BAM),
Richard-Willstatter-Strasse, 11, 12489 Berlin, Germany.
E-mail: priyanka.srivastava@bam.de, ute.resch@bam.de
b Institute of Chemistry and Biochemistry, Freie University Berlin, Kaiserswerther
Str. 16-18, 14195 Berlin, Germany
† Electronic supplementary information (ESI) available: Synthesis, characteriza-
tion of and photophysical data. See DOI: 10.1039/d1nj01573g
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fluorescence intensities, pH-responsive probes are favored that
enable ratiometric pH measurements. This can be realized with
molecules showing a dual emission which is, however, rare,32–35 or
which exhibit strong pH-induced spectral changes in absorption as
revealed e.g., by the near-infrared emissive pH-probe Cypher 5.36,37
Alternatively, two spectrally distinguishable fluorophores can be
combined to a ratiometric sensor. This can be achieved with
bichromophoric or dyad-type molecular probes,32–35 that are,
however, challenging to synthesize, or with the aid of nano-
particles doped and labeled with two different fluorophores.38
A dye class that is particularly well suited for the design of
fluorescent probes are the rhodamines. Also, rhodamines can
reveal an acid-induced ring opening mechanism occurring at pH
values of B5–4 that provides the basis for OFF–ON fluorescence
switching.26,31 Such an OFF–ON or ON–OFF signaling mechanism,
however, does not enable ratiometric, i.e., self-referenced fluores-
cence measurements. In order to utilize this acidic ring opening
mechanism of rhodamine dyes for the design of a ratiometric
sensor for pH measurements,25–31 we designed a novel dyad probe
by combining a pH-responsive rhodamine B derivative with two
pH-inert phenanthrene fluorophores in one molecule. Phenan-
threne was used here since it can form emissive excimers and can
provide a blue monomer and a green excimer emission at the
same time which can be both used as a reference of the
pH-responsive rhodamine emission. Thereby, the dyad probe
is always fluorometrically detectable even when the rhodamine
fluorophore is in its non-emissive spirolactam form. In the
following, we present the synthesis of this first example of a
bichromophoric, three color-emissive ratiometric pH probe
and its spectroscopic properties as well as selectivity studies.
Fig. 1 Synthetic scheme of pH-responsive dyad 8 (i) NaBH4/methanol/RT
(ii) SOCl2/DCM/RT (iii) NaN3/DMF/100 1C (iv) ethylene diamine/ethanol/
reflux (v) propargyl bromide/K2CO3/THF/reflux (vi) CuSO4Á5H2O, sodium
ascorbate/ethanol–water/70 1C.
one molecule of propargyl derivative 7 were coupled by a Click
reaction in the presence of CuSO4 and sodium ascorbate in an
ethanol water mixture [36] to form pH sensor 8 with a yield of
50%. Comp 8 was purified by column chromatography on
neutral Al2O3 using a mixture of hexane, ethyl acetate, and
methanol as an eluent. Purified 8 was characterized by 1HNMR,
13C NMR, and ESI-MS analysis (Fig. S1–S10, ESI†).
Dyad 8 is designed to exploit the pH-induced ring opening
mechanism of the rhodamine spirolactam unit,26,40–45 that turns
this initially colorless and non-emissive moiety into a strongly
absorbing pink fluorophore, and combine this ON/OFF
pH-sensing with the pH-inert fluorescence of phenanthrene.
Phenanthrene was used as this dye can form emissive excimers
spectrally distinguishable from the monomer emission, Excimer
formation requires the interaction of two neighboring phenan-
threne molecules that is favored by the chemical structure of 8.
Thereby, fluorescence intensity-based sensing is combined with
the blue monomer and green excimer emission of phenanthrene
for single wavelength excitation. Our ratiometric approach for pH
sensing utilizes the ratio of the two emission bands of the dyad
observed upon excitation at 315 nm, measured in the wavelength
range of 420–540 nm and 540–620 nm.
Results and discussion
Design and synthesis of the pH-sensitive tri-color emissive
dyad 8
The synthesis of the bichromophoric pH sensor 8, obtained
from two phenanthrene and one rhodamine B units via Click
chemistry, is summarized in Fig. 1.39 A detailed description of
the preparation procedures of the different intermediates and
the challenging purifications is given in the (ESI†). The synth-
esis of the azide derivative of phenanthrene starting from
phenanthrene carboxaldehyde 1 involves three steps. First,
the carboxaldehyde phenanthrene derivative 1, was reduced
to its hydroxy derivative 2, then a chlorine substituent was
Photophysical behavior of dyad 8
introduced by reaction with SOCl2 yielding 3, and in next step, The absorption spectra of dyad 8 in dichloromethane (DCM),
substituted by an azide group by reaction with NaN3 in tetrahydrofuran (THF), and ethanol (EtOH) reveal absorption bands
dimethylformamide providing 4. The propargyl derivative of at 298 nm and 315 nm originating from the phenanthrene and
rhodamine B, compound 7, was prepared in two steps. In the rhodamine moieties, respectively. The corresponding emission
first step, rhodamine B, i.e., 5, was reacted with ethylene spectra recorded at an excitation wavelength (lEx) of 315 nm exhibit
diamine in ethanol to form the spirolactam ring derivative 6 phenanthrene monomer and excimer emission bands (Fig. 2a and
of rhodamine B with one free amino group. In the second step, b). At neutral or basic pH to the colorless and non-emissive
the free amine group of 6 reacted with two moles of propargyl spirolactam form of rhodamine B derivative 6, absorbing below
bromide to compound 7. Compound 7 was purified by column 350 nm, is fluorometrically silent. The phenanthrene excimer
chromatography using neutral aluminium oxide to give a faintly fluorescence is most pronounced in DCM where the monomer
yellow resin. Finally, two phenanthrene azide 4 molecules and emission is absent. In THF, the excimer emission still exceeds the
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Fig. 3 Emission spectra of dyad 8 (5 mM) at different pH values varied from
7.5 to 1.0 (a) excitation at lEx = 315 nm and (c) excitation at lEx = 560 nm,
respectively. Calculation of the pKa was done by a sigmoidal fit of (b) a
ratiometric approach (readout of I582/I500 nm at lEx = 315 nm) and (d) for
the emission intensity at 580 nm recorded at lEx = 560 nm, thereby solely
exciting the rhodamine unit. All measurements were done in THF–H2O
solvent mixtures (1 : 1) in a B–R buffer (25 mM).
Fig. 2 (a) Normalized absorption and (b) normalized emission spectra of
dyad 8 (5 mM) in different solvents (lEx = 315 nm; DCM, THF, EtOH). (c)
Absorption (8.0 mM) and (d) normalized emission spectra of compounds 2,
3, 4, 6, and dyad 8 in THF–H2O mixtures (1 : 1) in a Britton–Robinson (B–R)
buffer (25 mM).
weak monomer emission, and in ethanol, excimer and monomer
fluorescence are equally intense (Fig. 2a, b and Fig. S11, ESI†). For
some phenanthrene derivatives, a modest solvent dependence of
the optical properties was reported in low or medium polar solvents
similar to some reports on the spectroscopic features of pyrene and
naphthalene.46,47 Our dyad molecule reveals shifts in emission in
DCM and THF similar to reports from other research groups
whereas the red shift observed in the highly polar solvent ethanol is
unusual.48 The spectral position of the monomer emission of the
dyad is independent of solvent polarity while the spectral position of
the excimer fluorescence undergoes modest changes for low and
medium polar solvents and a strong red shift in highly polar protic
solvents like ethanol as shown in Table 1. The absorption spectrum
of compound 8 is barely affected by solvent polarity.
The absorption and emission spectra of all compounds
synthesized for the preparation of dyad 8 are provided in
Fig. 2c and d and their quantum yields are given in Table 1.
Further studies of this dyad were performed in a mixture of
50% THF and H2O in a Britton Robinson (B–R) buffer for pH
control (ESI†). The absorption spectra of dyad 8 reveal absorp-
tion bands at 298 nm and 315 nm (e = 11 430 MÀ1 cmÀ1
;
Fig. S13, ESI†) and the emission spectra (lEx = 315 nm) exhibit
two bands at 351 nm and 500 nm (overall quantum yield of
both phenanthrene emission bands F = 0.026; 65% and
35% contribution from phenanthrene excimer and monomer
emission) as shown in Fig. 3. Please not here that the fluores-
cence quantum yields of the phenanthrene unit in dyad 8 are
considerably underestimated upon excitation at 300 nm as
the absorbance at this wavelength contains also considerable
contributions from the non-emissive ring-closed rhodamine
unit as shown in Fig. 2c.
Table 1 Absorption maxima (lmax,Abs) and emission maxima (lmax,Em) of
pyrene (used as quantum yield standard; quantum yield value taken from
ref. 49 and 50) and compounds 2, 3, and 4 as well as dyad 8 in different
solvents. all phenanthrene derivatives are excited at 300 nm. The absor-
bances at the excitation wavelengths were typically between 0.1 and 0.2.
The fluorescence quantum yields of the phenanthrene unit in dyad 8 are
considerably underestimated in all cases upon excited at 315 nm as the
absorbance at this wavelength contains also considerable contributions
from the non-emissive ring-closed rhodamine unit as shown in Fig. 2c
pH studies of dyad 8
The absorption and emission properties of dyad, 8 (5 mM) were
studied in the pH range of 7.5 to 1.0 in THF–H2O mixtures (1 : 1)
in a Britton Robinson buffer51 using excitation wavelengths of
315 nm (phenanthrene and rhodamine absorption) and 560 nm
(rhodamine absorption), thereby examining also the pH-dependence
of the emission behavior of the different fluorophores constituting
the dyad sensor. Excitation at 315 nm results in two emission bands
at lEm = 351 nm and lEm = 500 nm at pH 7.5 originating solely from
the phenanthrene moieties as the rhodamine exists in its ring-closed
form under these conditions that is non-emissive. A change in pH
from 7.5 to 4.5 barely affects the intensity of both the phenanthrene
monomer and excimer emission indicating the suitability of
phenanthrene as pH-inert reference dye. A further decrease of
Refractive lmax,Abs/ lmax,Em/ Quantum
Sample
Solvent
index
nm
nm
yield F
Pyrene49,50
Compound 2 THF–water (1 : 1) 1.3801
Compound 3 THF–water (1 : 1) 1.3801
Compound 4 THF–water (1 : 1) 1.3801
Dyad 8
Dyad 8
Dyad 8
Dyad 8
Dyad 8
Dyad 8
Cyclohexane (CH) 1.42662 333
384
367
370
434
0.320
0.214
0.034
0.067
298
301
296
315
315
315
315
315
315
THF–water (1 : 1) 1.3801
THF–water (2 : 1) 1.3801
THF–water (1 : 2) 1.3801
351, 500 0.026
350, 499 0.039
353, 503 0.024
466
501
458
DCM
Ethanol
THF
1.4241
1.36
0.141
0.025
0.086
1.4072
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Table 2 Fluorescence quantum yields of dyad 8 and compound 6 at pH 7.0 and pH 2.0 with pyrene (pyrene in cyclohexane: F = 0.32; ref. 49 and 50)
and rhodamine B (rhodamine in ethanol: F = 0.7; ref. 53) as quantum yield standards determined at different excitation wavelengths. The absorbances of
the samples at the excitation wavelength were between 0.1 and 0.2. The emission range given equals the emission range used for integration of the
fluorescence bands
Sample
Solvent
Refractive index
Emission range/nm
lmax,Abs/nm
lmax,Em/nm
Quantum yield F
Pyrene49,50
Dyad 8 pH 7.0
CH
1.42662
1.3801
1.3801
1.3801
1.3801
1.3801
1.3801
1.3801
1.360
350–600 nm
330–620 nm
330–540 nm
540–620 nm
330–620 nm
330–540 nm
540–620 nm
540–620 nm
565–750 nm
570–750 nm
570–750 nm
335
315
0.32
THF–water (1 : 1)
THF–water (1 : 1)
THF–water (1 : 1)
THF–water (1 : 1)
THF–water (1 : 1)
THF–water (1 : 1)
THF–water (1 : 1)
Ethanol
351, 500
351, 500
580
351, 580
351
580
580
590
582
582
0.026a
0.022
0.004
0.045
0.017
0.028
0.061
0.7
Dyad 8, pH 2.0
315
Compound 6 pH 2.0
Rhodamine B46
Dyad 8 pH 2.0
315
543
560
560
THF–water (1 : 1)
THF–water (1 : 1)
1.3801
1.3801
0.451
0.507
Compound 6 pH 2.0
a
35% contribution monomer; 65% excimer.
the pH from 4.5 to 2.0, however, leads to a diminution o350 nm (see Fig. 2, compound 6) and is not emissive. As the
particularly of the phenanthrene excimer emission at 500 nm pH decreases further to pH 6.0, a new absorption band at about
and the appearance of a new fluorescence band at 582 nm 560 nm appears and a new emission band at 582 nm. The
corresponding to the rhodamine B derivative with the open intensity of both bands is gradually enhanced at more acidic
spirolactam ring (Table 2 and Fig. 4, Table S1, ESI†). The pH values (Fig. 3c). A further decrease of pH leads to a large
intensity of this band further increases with decreasing pH fluorescence enhancement. The fluorescence quantum yield of
(Fig. 3a and Fig. S14, ESI†) and saturates at pH 1.78. the resulting ring opened rhodamine B amounts to F = 0.45
The ratiometric response (I582/I500 nm) of the fluorescence (pH 2.0). The pKa value of the rhodamine system is calculated to
intensities of the rhodamine B and phenanthrene excimer of pKa = 2.59Æ 0.04 utilizing a dose response equation (Fig. 3d;
the dyad sensor at different pH yields a pKa as 2.09 Æ 0.05 rhodamine excitation at 560 nm).
(Fig. 3b; lEx = 315 nm) by applying a dose response equation for
The pH-dependence of the three different emission bands of
dyad 8 observed upon 315 nm excitation was subsequently
the sigmoidal fit of the pH curve.52
The influence of pH on the spectroscopic properties of 8 was studied in more detail. The pH-dependence of the emission
also assessed for an excitation wavelength of 560 nm, where bands at 351 nm, 500 nm, and 582 nm is shown in Fig. 4 for the
solely the rhodamine B moiety absorbs. At pH 7.5 the spiro- pH range of 7.5 to 1.5. The monomer emission undergoes only
lactam rhodamine B derivative absorbs only at wavelengths minimum intensity changes up to a pH of 2.0 (Fig. 3 and 4a)
and can serve as a constant pH-inert reference to monitor the
changes exhibited either by the excimer fluorescence of phe-
nanthrene at 500 nm (see Fig. 3 and 4b) or the rhodamine
emission intensity at 582 nm (Fig. 3 and 4d). The intensity ratio
of the phenanthrene monomer and excimer fluorescence
(Fig. 4c) showed a pKa = 1.74 Æ 0.41 (351/500 nm) while the
intensity ratio (Fig. 4e) of the rhodamine emission at 582 nm
and the phenanthrene monomer fluorescence at 351 nm
revealed a pKa = 2.38 Æ 0.05 (582/351 nm). A plot of the intensity
of the 582 nm rhodamine emission excited at 315 nm as a
function of pH gives a pKa = 2.33 Æ 0.04 (Fig. 4d).
The response time of dyad 8 to pH was determined by
addition of dyad 8 to a THF–water solution of pH 2.0 (Fig. 5).
The emission spectra of the dye measured immediately after
addition (0 min) showed a complete disappearance of the
excimer emission. Subsequently, the rhodamine emission band
at 582 nm started to appear and increased steadily in intensity
Fig. 4 Emission intensity changes of dyad 8 (lEx = 315 nm) as a function of
pH shown for (a) the 351 nm emission of the phenanthrene monomer, with time. After 16 min, only marginal changes were observed,
(b) the 500 nm fluorescence of the phenanthrene excimer, (c) the intensity
and the emission spectra reached constant values about at
20 min. Hence, although the overall response time of dyad 8 for
a complete ring opening is 20 min. at pH 2.0 as reflected by
ratio of the phenanthrene monomer and excimer emission at 351 nm and
500 nm, (d) the 582 nm rhodamine emission, and (e) the intensity ratio of
the 582 nm rhodamine and the 351 nm phenanthrene monomer fluores-
constant emission spectra resulting at this time, the appear-
ance and growing in of the rhodamine emission started
cence. All measurements were done with dyad 8 dissolved in THF–H2O
mixtures (1 : 1) in a B–R buffer (25 mM).
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Fig. 5 (a) Emission spectra and (b) bar graph of the response time of the
fluorescence of dyad 8 observed upon addition of dyad 8 to a THF–H2O
mixture (1 : 1) in B–R buffer (25 mM) of pH 2.0; excitation lEx was at 315 nm
and the fluorescence intensity was recorded at an emission wavelength
Fig. 6 Emission spectra recorded at excitation wavelength of 315 nm and
560 nm at pH 7.0 and 2.0 (3.3 mM) of (a) dyad 8 and (b) compound 6 in a
THF–H2O mixture (1 : 1) in B–R buffer (25 mM).
l
em of 582 nm. The emission spectra of dyad 8 were recorded every 4 min.
The data obtained at pH 7.2, where no ring opening should occur, are
included as control.
immediately upon dye–proton interaction. This principally
provides the basis for shorter measurement times.
Mechanistic studies and considerations
As previously discussed, pH sensing with the rhodamine moiety
utilized in dyad 8 is based upon the pH-induced opening of the
spirolactam ring of the rhodamine. This ring opening reaction
at pH o 5 involves the protonation of the amino group of the
xanthene core, which is signaled by the appearance of an
absorption band at 560 nm responsible for the pink color of
the dyad solution and an increasingly enhanced emission
located at 582 nm (Fig. 7). The opening of the spiro-lactam
ring simultaneously changes the distance and possibly also the
orientation of the two phenanthrene units, which is reflected by
a decrease of the phenanthrene excimer emission by about
66%. Although phenanthrene itself is not sensitive to pH as
demonstrated by us (Fig. S14, ESI†), this also renders the
phenanthrene excimer emission pH-responsive at acidic
pH values r4.5 (Fig. 4b). The decrease in excimer emission
automatically leads to an increase in the monomer emission of
phenanthrene as shown in Fig. 3 (panel a) by about 23%
(Fig. S15, ESI†). These changes barely affect the ratiometric
readout of the sensor dyad. Moreover, by utilizing the sum of
the monomer and excimer emission of phenanthrene as a
reference for the pH-dependent rhodamine emission, e.g.
by using integral fluorescence intensities (integration of the
rhodamine emission from 540 nm to 620 nm; integration of the
phenanthrene emission from 330 nm to 540 nm) instead of
intensity ratios measured at single wavelengths of 580 nm and
500 nm, these effects can be considered.
Fig. 7 The spirolactam and open ring derivative of the dyad 8.
ratio amounts to only 1.28 (Fig. S17, ESI†). The emission
intensity at 582 nm resulting upon excitation at 560 nm exceeds
that observed upon excitation at 315 nm by a factor of 6.95 in
the case of dyad 8 as shown in Fig. 6 while for compound 6 a factor
of 5.86 was obtained under similar experimental conditions. This
difference between dyad 8 and compound 6 is ascribed to the
presence of the two phenanthrene moieties in the dyad. When
considering the fluorescence quantum yields of the 580 nm
rhodamine emission band (wavelength range of 540–620 nm)
excited at 315 nm and 560 nm, the difference between the two
emission ratios obtained at 580 nm of 1.3 correlates with the
contribution of the phenanthrene moieties to the absorption of
the dyad at 315 nm. Similarly, we observed a 1.33-fold decrease in
fluorescence quantum yield of the 580 nm band of the rhodamine
(540–620 nm range) excited at 330 nm for dyad 8 in comparison to
compound 6 lacking the phenanthrene units (Table 2).
In addition, we performed time-resolved fluorescence studies
with dyad 8 and compound 6 at pH 2.0. The resulting fluorescence
decay curves are shown in the ESI,† in Fig. S18 and S19. Fitting
these decay curves with biexponential decay kinetics provided
fluorescence lifetimes t of 1.71 ns (84%) and 1.66 ns (76%) for
dyad 8 of the main decay components using a biexponential fit of
the fluorescence decays obtained at excitation wavelengths of
330 nm and 510 nm (detection at 580 nm) and fluorescence
lifetimes t of 1.63 ns (monoexponential fit) and 1.73 ns (84%;
main component) for compound 6 (biexponential) for excitation at
330 nm and 510 nm and detection at 580 nm (Fig. S18 and S19,
ESI†). The lifetimes are almost similar in the case of dyad 8 and
compound 6 indicating the absence of electronic interaction
between the rhodamine and phenanthrene units.
Subsequently, we tried to quantify the contribution of the
phenanthrene and rhodamine absorption to the dyad’s absorption
at 315 nm chosen as excitation wavelengths for ratiometric pH
measurements. For this purpose, we compared the absorption
and fluorescence excitation spectra of dyad 8 and compound 6
(Fig. S16, ESI†). This comparison reveals a ratio of the absorp-
tion values at 315 nm and 560 nm at pH 2.0 of 2.9 for dyad 8
while for compound 6 which has only a rhodamine unit, this
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the opening of spirolactam ring of rhodamine B as expected
(Fig. 8, panels a and d). The DFT optimized structures
also showed an increased in the distance between the two
phenanthrene units from 8.92 Å for dyad 8, and 11.22 Å for
the protonated dyad, respectively. As the observed change in
distance is not large, we can conclude that the orientational
changes of phenanthrene units are responsible for the observed
excimer emission decrease at acidic pH values.
Analyte selectivity and photostability
To assess the analyte selectivity of the dyad sensor, we per-
formed selectivity studies in THF–H2O mixture in B–R buffer
with metal ions typically present in biological systems or in other
application-relevant environments. Thereby, always 10 equiv
(10 fold excess; 50 mM) of different metal ions (K+, Na+, Ca2+,
Ba2+, Mg2+, Cd2+, Zn2+, Co2+, Ni2+, Cu2+, Hg2+, Fe2+, and Fe3+,
1.5 mL of 10À1 M) were separately added to dyad solutions (5 mM)
and the resulting emission spectra obtained at lEx of 315 nm
(phenanthrene excitation) and 560 nm (rhodamine excitation)
were measured for each [Dyad + Metal+] solution. These screen-
ing studies revealed no significant changes for any of these metal
ions as highlighted in Fig. 9 (Fig. S20 ESI†). We also performed
selectivity and interaction studies of dyad 8 with different anions
and selected amino acids (10 equiv; 1.5 mL of 10À1 M). None of
these anions or amino acids led to changes in the emission
intensity of our dyad as shown in Fig S21 in the ESI.† These
results underline the high selectivity of dyad 8 for protons/pH.
The advantage of using phenanthrene as a reference fluor-
ophore follows from the photographs of the sensor solutions.
Fig. 8 DFT optimized structures of dyad 8 (a) and its 8 + H+; protonated
form (d); HOMO (b) and LUMO (c) isosurfaces of dyad 8 and protonated
form HOMO (e) and LUMO (f) obtained at the oB97X-DÀ33/d5e4–f257-TZVP level.
Plots were generated using ORBKIT, isovalue = 0.02a0
.
Quantum chemical calculations
Dyad 8 and its protonated form have been optimized at the The violet color of phenanthrene is visible at pH 7.5 to 5.5 while
PBEh-3c54 level of density functional theory (DFT) using the the rhodamine signal is absent at pH 7.5 (Fig. 10; left). Thereby,
Turbomole programme package (version 7.3)55 and COSMO56 dyad 8 can b always detected fluorometrically which would
to implicitly include solvation effects (Fig. 8). Fig. 8 shows the otherwise not feasible. An acidic pH of 5 or less is signaled by
HOMO and LUMO isosurfaces of dyad 8 and protonated 8 + H+ the appearance of the orange-red color of the rhodamine
obtained at the oB97X-D3 level. For 8, the HOMO is localized absorption and emission.
on the rhodamine structural motif (Fig. 8b), while the LUMO is
Other factors that govern the suitability of a sensor are its
delocalized over the two phenanthrene moieties (Fig. 8b).57 The response time, reversibility and chemical and photochemical
degree of localization in the LUMO is governed by the amount stability. To assess the reversibility and hence reusability of
of ‘‘exact’’ exchange included in the density functional approxi- dyad 8, we ran pH cycles with 8, varying the pH of dyad
mation that is employed. In the case of PBE0 (20% exact
exchange), for example, the LUMO is fairly localized on the
phenanthrene units. For protonated dyad [8 + H+], both HOMO
and LUMO are localized on the rhodamine motif (Fig. 8, panels
e and f). A closer inspection reveals that the major difference
between the two orbitals is given by the distribution of the
nodes at the central pyran unit. Not very surprisingly, while the
HOMO contains contributions from a conventional p-orbital
located at the pyran moiety, the LUMO encompasses its
p*-orbital analogue.
The most useful values are likely acquired with the PBE0
Fig. 9 (a) Emission spectra and (b) bar diagram of the selectivity studies of
functional which results in a HOMO–LUMO gap of 4.2 eV for 8
dyad 8 with the application-relevant metal ions K+, Na+, Ca2+, Ba2+, Cd2+
,
and 3.10 eV for 8 + H+. In general, the HOMO–LUMO gap
decreases by roughly 1–1.5 eV when protonating the dyad. The
front view of dyad 8 and its protonated form revealed a clear
change in the orientation of the phenanthrene units caused by
Mg2+, Zn2+, Co2+, Ni2+, Cu2+, Hg2+, Fe2+, and Fe3+ always separately
adding 10 equiv. (10 fold excess) of the respective metal ion to dyad 8
(5 mM). Excitation was at lEx of 315 nm (phenanthrene). in THF–H2O
mixture (1 : 1) in B–R buffer (pH 7.0; 25 mM).
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In the future, we plan to improve the water solubility of our
dyad sensor by introducing hydrophilic substituents such as a
2-chloro methane sulfonate substituent. Moreover, to bind the
dyad to different types of nanoparticles acting as carriers, this
substituent can be used in conjunction with an additional
reactive carboxylic group introduced into the rhodamine part of
the dyad. For the simple application in aqueous environments
dyad 8 without the need for chemical modification, compound 8
can be encapsulated in mesoporous silica nanoparticles58 or
polymer-based nanoparticles like PEBBLE59 type nanoparticles.
This provides also a simple signal enhancement strategy and
enables, e.g. a straightforward approach to combine dyad 8 with
targeting bioligand like biomarker-specific antibodies.60 Further-
more, the dye can also be integrated into a D4 hydrogel yielding a
pH-responsive sensor film as previously done for water-insoluble
BODIPY dyes.52
Fig. 10 (a) Color changes of the pH sensor 8 under UV light detected by
naked eye at pH 7.0 and pH 2.0; (b) reversibility of the fluorescence
response of dyad 8. All measurements were done with 5 mM of dyad 8
dissolved in a THF–H2O mixture (1 : 1) in B–R buffer (pH 7.0; 25 mM).
solutions from 7.0 to pH 4.0 back and forth up to three times
and measured the fluorescence intensity at 582 nm corres-
ponding to the open ring form of rhodamine B (lEx = 560 nm).
These measurements did not reveal a loss in fluorescence after
these pH cycles (Fig. 10; right). Moreover, the response times
are very short, in the order of a few seconds. To determine the
photostability of dyad 8, dyad solutions (5 mM; THF–water
solution) of pH 7.2 and pH 2 were illuminated with a 450 W
xenon lamp of a fluorometer for up to 420 mins and the
emission spectra of 8 were recorded every 30 minutes, using
an excitation wavelength of 315 nm. The emission intensities at
350 nm and 500 nm at pH 7.2 and 350 nm and 582 nm at pH
2.0 revealed only marginal changes, underlining the high
photostability of our dyad (see ESI,† Fig. S22).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
P. S. acknowledges the Adolf Marten fellowship of the Federal
Institute for Material Research and Testing (BAM), Berlin,
Germany. F. W. gratefully acknowledges the Deutsche For-
schungsgemeinschaft for financial support through research
grant PA 1360/16-1. The North-German Supercomputing Alliance
¨
(Norddeutscher Verbund fu¨r Hoch-und Hochstleistungsrechnen)
is acknowledged for providing computational resources. We are
also grateful for support by the NMR and mass spectrometry
facilities of Freie University Berlin measuring the NMR and MS
spectra of the compounds prepared.
Conclusion
We designed and synthesized a new sensor dyad combining two
pH-insensitive phenanthrene fluorophores and one pH-sensitive
rhodamine B derivative in one molecule and spectroscopically
assessed its pH responsivity in THF–water mixtures in in Britton–
Robinson (B–R) buffer in the pH range of 7.5 to 1.0. These studies
underline the advantage of our dyad sensor revealing three
spectroscopically distinguishable emission bands at 351 nm,
500 nm, and 582 nm upon excitation at a single wavelength of
315 nm, originating from the phenanthrene monomer, phenan-
threne excimer and the rhodamine. In comparison to a simple
OFF–ON sensor molecule like a fluorescent probe exploiting a
photoinduced electron transfer (PET), utilization of the phenan-
threne monomer emission or the combined monomer and
excimer emission as the pH-inert reference signal enables not
only ratiometric measurements for analyte quantification but
also the visualization of the dyad at all pH values, also at pH
values Z7.5 where the rhodamine is in its ring-closed non-
emissive spirolactam form. The use of two phenanthrene units
provides more versatility regarding the choice of the reference
signal, namely either the blue phenanthrene monomer emission
or combined blue and green phenanthrene fluorescence. Screening
studies with a series of biologically relevant metal ions underline the
excellent selectivity of our pH-responsive dyad.
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