OFF-ON-OFF Red-Emitting Fluorescent Indicators for a Narrow pH Window

Article abstract of DOI:10.1002/chem.201604978

A unique combination of two independent mechanisms of fluorescence quenching, namely intramolecular charge transfer (ICT) from a peripheral donor and protonation of azomethine nitrogen atoms in zinc tetrapyrazinoporphyrazines (TPyzPz), provides a new possibility for sensing pH in a specific range. The pH selectivity was controlled by the different basicities of the donor for ICT (dimethylaminoaryl), which was connected to the macrocycle by π-extended linkers of different lengths. ICT and protonation have been studied in detail by photophysical, spectral (UV/Vis and MCD spectra), and electrochemical measurements, and further supported by theoretical calculations (DFT, TDDFT). The pH-sensing properties of the TPyzPzs have been investigated in THF and in water after anchoring the TPyzPzs to liposomes. The salient pK_avalues were around 1.3 (azomethine nitrogen) and 2.29–4.76 (donor for ICT). The lead indicators (sensing over a pH range of 1.0–2.5) with fairly steep sensing profiles exhibited increases in fluorescence between the OFF/ON states of more than 20-fold and strong absorption in the red region (Q-band maximum >650 nm, ?≈2×10⁶m^?1cm^?1).

Full text of DOI:10.1002/chem.201604978

A Journal of
Accepted Article
Title: OFF-ON-OFF red-emitting fluorescence indicators for a narrow
pH window
Authors: Antonin Cidlina, Miroslav Miletin, Mahtab Fathi-Rasekh, Victor
Nemykin, Petr Zimcik, and Veronika Novakova
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To be cited as: Chem. Eur. J. 10.1002/chem.201604978
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FULL PAPER
OFF-ON-OFF red-emitting fluorescence indicators for a narrow
pH window
Antonin Cidlina,[a] Miroslav Miletin,[a] Mahtab Fathi-Rasekh,[b] Victor Nemykin,*[b,c] Petr Zimcik,* [a] and
Veronika Novakova,* [d]
Abstract: A unique combination of two independent mechanisms of
fluorescence quenching, intramolecular charge transfer (ICT) from a
peripheral donor and protonation of azomethine nitrogens in zinc
tetrapyrazinoporphyrazines (TPyzPz) provides a new possibility for
sensing pH in a specific range. The pH selectivity was controlled by
the different basicities of the donor for ICT (dimethylaminoaryl),
connected to the macrocycle by π-extended linkers of different
lengths. ICT and protonation were studied in detail by photophysical,
spectral (UV-Vis and MCD spectra) and electrochemical
measurements that were further supported by theoretical calculations
(DFT, TDDFT). The pH-sensing properties of the TPyzPzs were
investigated in THF and in water after anchoring the TPyzPzs to
liposomes. pKa values were of ∼1.3 (azomethine nitrogen) and 2.29-
4.76 (donor for ICT). The lead indicators (sensing over a pH range of
1.0-2.5) with fairly steep sensing profiles exhibited increases in
fluorescence between the OFF/ON states of more than 20 times and
strong absorption in the red region (Q-band maximum > 650 nm, ε ~
2×106 M-1 cm-1).
well-established electrochemical pH sensor, which suffers from a
susceptibility to electrical interference, an unsuitability for long-
term measurements due to electrode signal drift and the necessity
of a separate reference sensor.[2] Thus, the development of
structurally-new pH indicators for optodes with improved
properties in relation to the generally used fluorescein and
coumarin derivatives is highly appreciated.
Tetrapyrazinoporphyrazines (TPyzPzs), aza-analogues of
4]
phthalocyanines (Pcs)[3,
with an extended 18 π-conjugated
macrocyclic system absorb and emit light in the red region (above
650 nm, ε ~ 2×106 M-1 cm-1). TPyzPzs possess many properties
common to Pcs, such as strong fluorescence, chemical stability
and fine tuning of optical and electrochemical properties by
peripheral substitution.[5] On the other hand, the unique strongly
electron deficient[6] character of the TPyzPz core predestines
them for specific applications where Pcs cannot be used. In recent
years, TPyzPzs have become effective fluorescence indicators
for either metal cations[7] or pH[8-10] based on the ultrafast
intramolecular charge transfer (ICT) or photo-induced electron
transfer (PET) between a donor (e.g., peripheral amine or
phenolate) and acceptor (i.e., macrocyclic core).[11] For detailed
descriptions of the principles of ICT- or PET-based indicators,
Introduction
readers are encouraged to read the excellent reviews[12]
.
Since the establishment of the pH scale by Sørensen and the
discovery of the first pH electrode in the beginning of the 20th
century,[1] pH sensors have become essential in a wide range of
applications in various fields such as medical diagnosis,
biochemical and environmental analysis, and industrial
applications, as well as in different research areas. Current trends
involving miniaturization down to submicrometer dimensions, low
cost and compatibility with mass-production methods make
optical chemical pH sensors (i.e., optodes) more useful than the
An interesting strategy that may result in higher selectivity at
particular pH values involves the development of sensors
operating on the OFF-ON-OFF principle, i.e., using pH indicators
turned on at only selected pH values. Reports of only few such
indicators have been published to date, with the principles based
mostly on the presence of two different donors for PET[13] or on
changing the overall charge using various micelles[14]. However,
all these published molecular switches suffer from emission in
only the UV area. To the best of our knowledge, only two
examples of OFF-ON-OFF indicators emitting in the red region of
the spectrum have been published, and only recently. One
describes indicators lacking a narrow pH-sensitive window (4.0-
15.0, experiments made in DMSO)[15], and the second project
focused on indicators suitable over a basic pH range, with rather
low fluorescence enhancement when the indicators are switched
ON[10]. In this project, we designed pH-driven OFF-ON-OFF
indicators for an acidic pH range using two completely different
switching mechanisms available in TPyzPzs, i.e., ICT switching at
the peripheral donor amine and protonation of the azomethine
nitrogen. The idea is that the fluorescence switches ON when the
proton concentration is increased but switches OFF again as the
acidity increases further (Figure 1). Thus, strong fluorescence is
expected at only intermediate pH values, leading to a narrow pH-
sensing window. Notably, the protonation of the azomethine
nitrogen has been mentioned in the literature to lead to a
substantial decrease in the fluorescence quantum yields of both
[a]
A. Cidlina, M. Miletin, P. Zimcik
Department of Pharmaceutical Chemistry and Pharmaceutical
Analysis, Faculty of Pharmacy in Hradec Kralove, Charles University
in Prague, Heyrovskeho 1203, Hradec Kralove, 500 05, Czech
Republic, E-mail: petr.zimcik@faf.cuni.cz
[b]
[c]
[d]
M. Fathi-Rasekh, V. Nemykin
Department of Chemistry and Biochemistry, University of Minnesota,
Duluth 1039 University Drive, Duluth, MN 55812, USA,
V. Nemykin
Department of Chemistry, University of Manitoba, Winnipeg, MB
R3T 2N2, Canada. E-mail: victor.nemykin@umanitoba.ca
V. Novakova
Department of Biophysics and Physical Chemistry, Faculty of
Pharmacy in Hradec Kralove, Charles University in Prague,
Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic E-
mail: veronika.novakova@faf.cuni.cz, web:
http://portal.faf.cuni.cz/Groups/Azaphthalocyanine-group/
Supporting information for this article is given via a link at the end of the
document.
This article is protected by copyright. All rights reserved.

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Pcs and TPyzPzs[16-19] but has never been used as a concept in
molecular switches or logic gates.
because of the rapid decomposition typical of similar precursors
containing aliphatic tertiary amines.[22]
The Suzuki-Miyaura cross-coupling reaction of halides 13 and 14
with boronic esters 8-10 was used for the synthesis of precursors
with peripheral substituents connected via C-C bonds (Scheme
2). Synthon 13 was prepared by condensation of glyoxylic acid
with diaminomaleonitrile (DAMN) in acetic acid with subsequent
chlorination with POCl3 according to a previously published
procedure.[23] Similarly, compound 14 was obtained as a product
of the condensation of DAMN with 2-(4-iodophenyl)-2-
oxoacetaldehyde, which was prepared by partial oxidation of 4-
iodoacetophenone with selenium dioxide. The optimization of the
reaction conditions of the Suzuki-Miyaura cross-coupling
reactions is summarized in Table 1.
Interestingly, the use of a bidentate ligand catalyst, Pd(dppf)Cl2,
was found to be crucial in the coupling reaction of pyrazine 13
with pinacol boronate 8 to obtain precursor 15 in a reasonable
yield of 59%. A similar observation has been reported by McKillop
et al.[24] using 2-chloropyrazine. On the other hand, lower yields
were always obtained in the synthesis of 16-18 using this catalyst
in comparison with Pd(PPh3)2Cl2 in the reaction of the iodo-
substituted synthon 14 with the appropriate pinacol boronates 8-
10 (Table 1).
The synthesis of unsymmetrical TPyzPz was accomplished by
cyclotetramerization of two different precursors (A and B) in a ratio
of 3:1 under Linstead conditions (Scheme 3).[3] The statistical
condensation of precursors A (20) and B (14-16 or 19), initiated
by magnesium butoxide, led to the formation of a mixture of six
congeners (AAAA, AAAB, AABB, ABAB, BBBA and BBBB), of
which the AAAB type was of interest. The mixture of magnesium
complexes was hardly separable from each other due to strong
tailing on silica. Therefore, the mixture was directly converted to
the corresponding metal-free derivatives upon treatment with p-
TsOH in THF, and the desired metal-free AAAB congeners were
chromatographically isolated and obtained in reasonable yields of
8 - 13%.
Figure 1. Proposed principle of OFF-ON-OFF switching of fluorescence
Hence, a series of TPyzPzs 1 – 4 with an N,N-dimethylamino
group attached to π-conjugated linkers of different lengths was
designed (Scheme 1). Compound 5 with a donor connected
through a three-atom aliphatic linker was included in the study for
comparison of ICT and PET. The peripheral substitution (tert-
butylsulfanyl substituents) of the TPyzPz macrocycle as a whole
series was chosen to suppress the undesirable formation of H-
aggregates[20] and, together with the central zinc cation, to ensure
good photophysical properties after activation of the indicator to
the ON state.
Table 1. Conditions of Suzuki-Miyaura cross-coupling reactions[a]
Starting
Product
Catalyst
Yield
material
13
13
14
15
15
16
Pd(PPh3)2Cl2
Pd(dppf)Cl2
Pd(PPh3)2Cl2
0 %
59 %
53 %
Scheme 1: Structures of the target indicators 1-5 and controls 6 and 7.
14
14
14
14
14
16
17
17
18
18
Pd(dppf)Cl2
Pd(PPh3)2Cl2
Pd(dppf)Cl2
Pd(PPh3)2Cl2
Pd(dppf)Cl2
25 %
86 %
79 %
43 %
35 %
Results and Discussion
Synthesis
Generally, the synthesis of TPyzPzs is performed by
cyclotetramerization of precursors, typically 5,6-disubstituted
pyrazine-2,3-dicarbonitriles. Based on the atom connecting the
peripheral substituent, different synthetic approaches are used for
the synthesis of the desired precursors.[4] Thus, alkylheteroatom-
substituted 19 and 20[21] were prepared via nucleophilic
substitution with N,N-dimethylaminoethanethiolate and tert-
butanethiolate, respectively (Scheme 2). Compound 19 was
converted directly to its hydrochloride and stored in this form
[a] Reaction conditions: 13 or 14 (1 eq.), appropriate derivative of pinacol
boronate 8 – 10 (1.3 eq.), K2CO3 (2 eq.), Pd catalyst (5 mol%), 80 °C, THF/H2O
(4:1) (v/v), 7 h, under an argon atmosphere.
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Scheme 2: Synthetic pathways for the precursors. Reaction conditions: i) Pd(dppf)Cl2 (0.05 equiv.), K2CO3 (2 equiv.), THF/H2O (4:1) (v/v), 7 h, 80 °C, Ar atmosphere.
ii) SeO2, dioxane/H2O (2:1) (v/v), reflux, 12 h. iii) DAMN, AcOH, 3 h, reflux. iv) Pd(PPh3)2Cl2 (0.05 equiv.), K2CO3 (2 equiv.), THF/H2O (4:1) (v/v), 7 h, 80 °C. v) 2-
(dimethylamino)ethanethiol hydrochloride, K2CO3, DMSO, 30 min, rt. vi) 2-methyl-2-propanethiol, NaOH, THF, 15 min, rt.
UV-Vis and MCD spectra
First, the UV-Vis spectra of compounds 1–7 were measured in
dichloromethane (DCM). It was found that the majority of new
compounds form a significant amount of aggregates in this
solvent, as indicated by the appearance of a broad band at the
lower-energy side of the Q-band. However, The UV-Vis and MCD
spectra of the same compounds in THF, pyridine or DCM/pyridine
(99.5:0.5 v/v) systems have band widths in the Q-band region that
are typical of monomeric phthalocyanines and their analogues. All
prepared compounds of the series exhibited characteristic
absorption bands in their UV-Vis and MCD spectra (Figures 2 and
S1) typical of TPyzPzs (i.e., a high-energy B-band at 377 - 379
Scheme 3: Synthetic pathways for TPyzPzs. Reaction conditions: i) Mg(BuO)2,
nm and a low-energy Q-band at 647 - 656 nm, Table 2). It is
interesting to note that the addition of the conjugated peripheral
π-system to compounds 1-4 did not affect the energy of the Q-
band in these systems compared to the reference, symmetric
TPyzPz 7, which indicates that the electronic effects of the new
peripheral substituents in 1–4 are close to the cumulative effects
of the alkylsulfanyl groups in 7. Moreover, in all asymmetric zinc
TPyzPzs 1-6, the Q-band remains unsplit, which is indicative of
their effective four-fold symmetry. In agreement with this
observation, a very strong Faraday MCD A-term associated with
the corresponding Q-bands was observed in the UV-Vis spectra
of 1-6. Another Faraday MCD A-term was observed in the B-band
region of all target compounds and is typical of the symmetric
TPyzPzs.[27] Finally, a rather weak and broad negative MCD
signal in 1-6 is associated with the broad unstructured absorption
in the 420 - 500 nm region, which will be discussed in detail in the
computational section. Thus, the UV-Vis and MCD spectra of the
target asymmetric TPyzPzs 1-4 suggest that the influence of the
dimethylaminoaryl-containing substituent on the energies of the
TPyzPz-core centered * transitions is rather small, which
correlates well with the electrochemical and DFT data discussed
below.
BuOH, reflux, 8 h. ii) p-TsOH, THF, rt, 1 h. iii) Zn(OAc)2, pyridine, reflux, 1 h. iv)
appropriate acetylene 11 or 12 (2 equiv.), Pd(PPh3)2Cl2 (0.03 equiv.), CuI (0.03
equiv.), PPh3 (0.02 equiv.), triethylamine (4 equiv.), 48 h, room temperature,
anhydr. THF, Ar atmosphere.
Finally, zinc complexes 1, 2, 5 and 6 were prepared from the
corresponding metal-free derivatives by their heating with
anhydrous zinc acetate in pyridine, with yields of the final
conversion step between 74 - 92%.
The corresponding cyclotetramerization of precursors 20 (A) with
17 or 18 (B) failed due to the extremely low solubility of precursors
17 and 18 in all common solvents, and a different approach had
to be employed for the synthesis of TPyzPzs bearing longer π-
conjugated linkers. Thus, 4´-iodophenyl-substituted TPyzPz 6
was chosen as
a suitable intermediate for further post-
cyclotetramerization modification by the cross-coupling reaction
(Scheme 3). However, the Suzuki-Miyaura cross-coupling
reactions of 6 with synthons 8 or 9 gave only traces of the desired
products using either Pd(dppf)Cl2 or Pd(PPh3)2Cl2 as the catalysts.
For this reason, the structures of the target TPyzPzs with longer
π-conjugated linkers were modified to enable the use of
Sonogashira instead of Suzuki-Miyaura cross-coupling, and the
appropriate TPyzPzs 3 and 4 were prepared by coupling TPyzPz
6 with ethynyl derivatives 11 or 12 in yields of 40% and 72%,
respectively. Notably, successful post-cyclotetramerization
modifications of the Pc[25] or TPyzPz[26] core under Sonogashira
cross-coupling conditions have rarely been described in the
literature.
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Table 2. Absorption, photophysical and electrochemical properties of the investigated TPyzPzs in THF[a]
TPyzPz
λmax
λem
ΦΔ
ΦF
ΦΔ + ΦF
E1ox (V)
E2ox (V)
E1red (V)
E2red (V)
1
2
3
4
5
6
7
656
652
651
650
647
649
650
663
660
659
658
653
655
656
0.029
0.037
0.038
0.079
0.32
0.006
0.016
0.019
0.041
0.19
0.035
0.053
0.057
0.12
1.01
0.92
1.00
0.89
1.19
1.32
1.33
1.35
1.31
1.38
1.33
1.31
-
-0.56
-0.54
-0.58
-0.54
-0.55
-0.51
-0.69
-0.73
-0.72
-0.76
-0.66
-0.69
-0.66
-
0.51
0.62
0.31
0.92
0.56
0.34
0.89
-
[a] Absorption maximum at the Q-band (λmax), fluorescence emission maximum (λem), fluorescence quantum yield (ΦF), singlet oxygen quantum yield (ΦΔ), half-
wave reduction potential (Ered), and half-wave oxidation potential (Eox). ΦF and ΦΔ are expressed as the mean of three independent measurements; estimated error
±15%. Potentials Ered and Eox are expressed as E1/2 (in V vs. SCE) with Fc/Fc+ as an internal standard.
Figure 2. MCD spectra (green, upper graph) in a DCM/pyridine system; experimental UV-Vis spectra (red, middle graph) and TDDFT-PCM (blue, lower graph) of
asymmetric compounds 1–6 in a pyridine system.
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and reference compound 7 is given in Figure 4, and the orbital
compositions of all target compounds are shown in Figure S3.
DFT
LUMO
HOMO
HOMO-1
1
2
3
4
Figure 4. DFT-predicted orbital energy diagram for asymmetric compounds 1-
6 and reference compound 7. (A) Frontier orbital energies; (B) selected orbital
energies for the most important orbitals.
predicted that the LUMO and LUMO+1 in all compounds are
centered at the aromatic macrocycle and are nearly degenerated
in all amino group-containing systems (1–5), as well as in
reference compound 7. Indeed, the energy difference between
the LUMO and the LUMO+1 in asymmetric compounds 1-5 does
not exceed 0.02 eV, which explains the presence of a single
unsplit Q-band in these compounds (see TDDFT discussion
below). The macrocycle-centered nature of the LUMO also
explains the similarity between the spectroscopic signatures of
the single-electron reduced compounds 1, 2, 3, and 5 obtained
under spectroelectrochemical conditions. The energy difference
between the LUMOs of the individual compounds 1-5 (~0.05 eV)
is also rather small and correlates well with the small difference
(40 mV) in their first reduction potential. The DFT-predicted nature
of the HOMO in asymmetric compounds 1–6, as well as in
reference system 7, allows explanation of their photophysical
properties. Indeed, the HOMO in 1 has a large (~30%)
contribution, whereas the HOMO in 2-4 is dominated (~75 - 90%)
by a contribution from the dimethylaminoaryl fragment. The
HOMO in TPyzPz 1 is ~0.3 eV higher in energy than the
predominantly peripheral group-centered HOMO-1. In contrast,
the HOMO in asymmetric systems 5 and 6 as well as reference
compound 7 should resemble the classic Gouterman's a1u orbital
expected for a normal tetraazaporphyrin core. DFT also predicted
that such a Gouterman's a1u orbital in TPyzPzs 2–4 is the HOMO-
1 (Figures 3 and S3). The presence of the redox-active,
diaminoaryl-centered HOMO in 2–4 and closely spaced HOMO
and HOMO-1 in 1 creates an opportunity for ICT process in these
compounds but not in systems 5-7. As a result, one might expect
that the fluorescence should be severely suppressed only in
systems 1-4, in excellent agreement with the experimental
photophysical data (see below).
5
6
7
Figure 3. Selected DFT-predicted orbitals of compounds 1-7.
DFT and TDDFT calculations
In order to explain the observed optical, photophysical, and redox
properties of the new asymmetric compounds 1-5 in comparison
to the symmetric reference compounds 6 and 7, their electronic
structures and the nature of the excited states were probed by
DFT and TDDFT methods coupled with the polarized continuum
model (PCM) approach to account for the solvent effect. CAM-
B3LYP was used to account for the long-range ICT transitions in
the TDDFT calculations. The DFT-predicted frontier orbitals are
shown in Figure 3, the energy diagram for asymmetric systems 1-
6
Moreover, DFT predicted that the first oxidation in compounds 1-
4 should be centered at the peripheral substituents but the first
oxidation process in systems 6-7 should be macrocycle-centered,
in agreement with the electrochemical data. Another interesting
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observation is that the increase in the -NMe2 group-to-macrocycle
distance results in a higher degree of localization of the HOMO at
the peripheral substituent, which correlates with a minor increase
in the fluorescence quantum yield. Indeed, the HOMO of 1
resembles Gouterman's a1u orbital with high (32%) contamination
from the dimethylaminophenyl group. This orbital is closely
followed by HOMO-1, which is slightly (56%) dominated by a
contribution from the peripheral substituent. The HOMO in 2-4 is
predominantly localized on the peripheral substituent, whereas
the closely spaced (0.17 - 0.22 eV) HOMO-1 is almost purely a
Gouterman's type macrocycle-centered a1u. The lack of
conjugation between the -NMe2 group and an aromatic
macrocycle in asymmetric system 5 results in a situation where
the localization on this fragment MO was predicted to be the well-
stabilized HOMO-4, although CAM-B3LYP calculations might
underestimate its energy. The DFT-predicted energies of the
HOMOs in all tested compounds correlate with their first oxidation
potential determined by electrochemical methods. Taking into
consideration that the nearly degenerate LUMO and LUMO+1 are
macrocycle-centered, one might expect that some of the ICT band
be present in the low-energy region of the UV-Vis spectra of
compounds 1-4 but not system 5. The TDDFT calculations of all
target compounds in comparison with their experimental spectra
are shown in Figure 2. Indeed, the TDDFT-predicted ICT energy
increases in the following order: 1 < 2 ~ 3 < 4 << 5 (453, 430, 435,
411, and 294 nm, respectively), and this order correlates very well
with the experimentally observed fluorescence quantum yields
and the singlet oxygen generation capability of compounds 1-5
(Figure 5). Thus, one might speculate that, in the simplified
approach, an energy of the ICT transition would dictate the
efficiency of the fluorescence quenching and the singlet oxygen
production (i.e., the lower the ICT energy, the stronger
fluorescence quenching and the lower singlet oxygen production
ability that specific compound would possess).
The electrochemical data were obtained from cyclic voltammetry
and square wave voltammetry in THF at room temperature.
Appropriate Ered and Eox were determined from square wave
voltammetry (Table 2) using ferrocene (Fc/Fc+) as an internal
standard and tetrabutylammonium hexafluorophosphate as a
supporting electrolyte.
The aminogroup-containing TPyzPzs 1–5 underwent two
reductions (Figure S2). The E1 and E2 were almost equal in
red
red
1–5 (~ -0.55 V and ~ -0.70 V vs. SCE). These values correlate
well with the values of the structurally close control 6 (-0.51 and -
0.66 V vs. SCE) but were slightly different from E1
of
red
symmetrical 7 (-0.69 V vs. SCE, similar to the value published
previously[27]). This result may indicate spreading of the π-
conjugated system on the peripheral aryl moiety. However, due to
the small difference from the value of 7, the presence of a π-
extended peripheral substituent is not believed to significantly
affect the electron-accepting properties of the TPyzPz core.
Two irreversible oxidations were observed for 1-5. The E2ox values
of 1–5 in the range of 1.31 – 1.38 V vs. SCE can be assigned to
the oxidation of the TPyzPz core because of the similar values
observed for control compounds 7 and 6 (1.33 and 1.32 V vs. SCE,
respectively). In addition to the oxidation of the core, another
oxidation process with E1ox = 0.89-1.01 V vs. SCE was detected
for aminoaryl-bearing compounds 1–4 as a consequence of
oxidation of the amine moiety, which is in accordance with the
DFT predictions. This oxidation did not occur for controls 6 and 7.
The E1ox values differed only slightly within the series, suggesting
a comparable electron-donating ability of the amine for ICT. A
similar trend in Eox values was recently described by Bures et al.
for
dialkylamino-substituted
pyrazine-2,3-dicarbonitriles
substituted with various π-extended linkers.[28] Noticeably, a
different value of E1ox for the amino group was observed only for
TPyzPz 5, i.e., a model compound of the PET process, which is
simply explained by the aliphatic character of the donor amine.
To gain insight into the nature of the redox-generated species in
In addition to the ICT bands in compounds 1-5, TDDFT predicted
classic macrocyclic * transitions, which are responsible for the
most intense bands in the Q- and B-regions of all studied
symmetric and asymmetric macrocyclic systems. Indeed, in all
TPyzPzs 1-7, the first two excited states are dominated by the
Gouterman's macrocycle-centered a1u (HOMO or HOMO-1) →
LUMO, LUMO+1 single electron excitations and are
predominantly * in nature. Similarly, the most intense
transitions in the B-band region predicted by TDDFT are
dominated by the macrocycle-centered * single-electron
excitations from the HOMO-n to LUMO, LUMO+1.
asymmetric systems 1-5,
a set of spectroelectrochemical
experiments was conducted in a pyridine/tetrabutylammonium
perchlorate (TBAP) system. Because the first oxidation process
in all systems and the first reduction process in 4 were found to
be irreversible, only the first reduction processes were studied for
compounds 1, 2, 3, and 5. In addition, spectroelectrochemical
reduction of reference compound 7 was previously discussed by
our group[27]. The choice of the pyridine/0.3 M TBAP system was
dictated by an increased stability of the electrochemically
generated anion-radicals of the target compounds in pyridine.
Similar experiments conducted in THF, DMF, and DCM/pyridine
systems resulted in a fast decomposition of the reduced species
under spectroelectrochemical conditions. Since an influence of
the dimethylamino group on the energies of * transitions in
asymmetric systems 1-5 was found to be negligible and, as was
shown by the DFT calculations, the LUMO in 1-5 is macrocycle-
centered. Therefore, it is not surprising that the reduction of
compounds 1, 2, 3, and
5 under spectroelectrochemical
conditions led to a very similar transformation of their UV-Vis
spectra dominated by the TPyzPz-core-centered * excitations
(Figure 6). In particular, during reduction of compounds 1, 2, 3,
and 5 under spectroelectrochemical conditions, the Q-band
disappeared; the B-band was reduced in intensity; and two new
absorption bands formed at approximately 440 and 550 nm, along
with a very characteristic and broad new band in the NIR region
Figure 5. Correlation of the singlet oxygen (A) and fluorescence quantum yields
(B) of 1-5 with the TDDFT predictions.
Electrochemistry and spectroelectrochemistry
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between 750 and 1000 nm. Because of the broad nature of the
NIR band, a peak at ~830 nm was identified for all compounds,
while a structural peak at ~900 nm was also observed in the case
of reduced compounds 3 and 5. The transformations of the UV-
Vis spectra of asymmetric systems 1, 2, 3, and 5 were very close
to the spectroscopic signatures of reference compound 7 reduced
under spectroelectrochemical conditions and are clearly
indicative of the single-electron reduction of the TPyzPz core.
The extremely different photophysical data of these two
compounds therefore emphasizes the importance of conjugation
between the donor and acceptor on the quenching efficiency.
Both controls 6 and 7 reached high values of both quantum yields
(Table 2), with sums of ΦΔ + ΦF close to 1. The slightly higher ΦΔ
and lower ΦF of 6 containing an iodo substituent may be clearly
explained by the heavy atom effect, according to which the
introduction of heavy atoms into the molecule supports the
relaxation of excited states through intersystem crossing.[31]
Generally, the feasibility of ICT is improved in a polar medium that
stabilizes the charge-separated state and, conversely, is
suppressed in a non-polar medium.[32] To further characterize the
impact of ICT in the investigated compounds, the ΦF of 2 was
determined in a series of solvents of different polarity. In some
cases, 1% pyridine was added to the solution to suppress
undesirable aggregation. Such an amount of pyridine was
believed not to influence the final polarity of the investigated
solvent. The obtained ΦF values were plotted as a function of the
relative permittivity (εr) of the solvent (Figure S6, Table S1). The
lowest ΦF of 0.004 was observed in DMSO (εr = 48.9). On the
contrary, the ICT was not as effective in non-polar benzene (εr =
2.2), in which ΦF reached a value of 0.19. The ΦF of 2 clearly
correlated with the polarity of the medium, as the feasibility of ICT
increased in more polar solvents. The significant differences in ΦF
demonstrate the high sensitivity of ICT in the tested TPyzPz
systems and the possibility of its control in a desired manner.
Figure 6. Spectroelectrochemical transformation of the neutral compounds 1-3
and 5 during the first reduction in the pyridine/0.3 M TBAP system.
pH sensing – OFF-ON-OFF fluorescence switching
Contrary to all routinely used indicators that operate on a simple
ON-OFF principle, sensing in a narrow pH window requires two
quenching mechanisms that must closely cooperate and for which
switching must occur at approximately similar pH values. In this
work, we chose the ICT process from a peripheral donor and
quenching based on protonation of azomethine nitrogens.
Since all target indicators 1-5 possess the same macrocyclic core,
quenching by protonation of the azomethine nitrogens proceeds
at identical pH (or at identical cTFA, see below) for the whole
series, and the sigmoidal branches at lower pH are virtually
coincident. Therefore, the pH sensing range may be tuned by the
use of different donors for ICT. The ICT efficiency and the basicity
of the donor amine are the two most important parameters to
consider to acquire fluorescence indicators with high sensitivity
and selectivity. The ICT efficiency determines the difference in
fluorescence intensity between ON/OFF states (i.e., signal-to-
noise ratio), while the donor basicity controls the pH range in
which the switching to the ON state occurs. Both the sensitivity
and selectivity of TPyzPzs 1-5 to pH and acid concentration in
organic solvent (including controls 6 and 7) were investigated by
the mean of the fluorescence measurements, firstly in THF upon
stepwise addition of trifluoroacetic acid (TFA) and then in water in
buffers of different pH. The results are summarized in Table 3 and
depicted in Figure 7.
Photophysical measurements and ICT efficiency
Singlet oxygen quantum yields (ΦΔ) were determined in THF
using a singlet oxygen quencher, 1,3-diphenylisobenzofuran, and
unsubstituted zinc Pc as a reference (ΦΔ(THF) = 0.53[29]).
Fluorescence quantum yields (ΦF) were determined in THF after
excitation in the Q-band region (λexc = 595 nm) with zinc Pc as a
reference (ΦF(THF) = 0.32[5]). These data are summarized in Table
2. The emission spectra featured a shape typical of TPyzPzs,
mirroring the appropriate absorption spectra with a typical small
Stokes shift (~ 7 nm). The excitation spectra perfectly matched
the absorption spectra, which confirmed the presence of only
monomeric species; therefore, the determined quantum yields
were not affected by undesirable aggregation (Figure S4).
Because ICT and PET are competitive relaxation pathways to
singlet oxygen production and fluorescence emission, the sum of
the quantum yields (ΦΔ + ΦF) may give reliable information about
the efficiency of ICT or PET. Obviously, the values of ΦΔ and ΦF
of 1 – 4 are one order of magnitude lower than those of the
controls 6 and 7, not exceeding 0.08 and 0.04 for ΦΔ and ΦF,
respectively (Figure S5). These results prove the excellent
efficiency of ICT, which increased in a series of 4 << 3 ~ 2 < 1,
which is in accordance with the TDDFT predictions (Figure 5). As
the oxidation ability of the donors in TPyzPzs 1-4 is comparable
to the measured E1ox values (see Table 2), the increase in the ICT
efficiency can be clearly attributed to the shortening of the π-
conjugated linker.
The addition of TFA into THF solutions of indicators 1-5 led to
protonation of the peripheral donor amine, being the most basic
center in the studied TPyzPzs. Protonation of the dimethylamino
group did not affect the absorption spectra but was accompanied
by a significant increase in fluorescence intensity (up to 19 times)
due to suppression of ICT. The progress of the protonation driven
by the basicity of the particular donor was characterized by the
equilibrium constants for association of TFA with N,N-
The particular quantum yields of 5 were less suppressed, giving
the sum (ΦΔ+ΦF) = 0.51 and fully in accordance with the TDF
prediction and values determined for similar compounds[30]. Of
note is that the distance between the dimethylamino group (donor
for ICT) and the macrocycle (acceptor) is comparable in 1 and 5.
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Figure 7. Study of the pH-sensitive properties of the investigated compounds: a-c) changes in ΦF of 1-7 in THF upon addition of TFA; d) changes in the absorption
spectra of 2 in THF upon addition of TFA (cTFA = 0-5 mol/L); and e-f) changes in the fluorescence intensity of 2, 4, 6 and 7 after their incorporation into the DOPC
liposomes in buffers of different pH (λexc = 375 nm). The mean of three independent measurements.
dimethylamino group of TPyzPz in THF (KDon). Considering the
values of KDon, the basicity of the donors decreased in the series
5 >> 4 > 2 ~ 3 > 1. The fluorescence enhancement factor (FEF,
i.e., ratio between the fluorescence in the OFF/ON states) of ICT-
based indicators 1-4 showed promising values, ranging from 5.8
- 19.0 when going from 0 to approx. 2.5 mol/L TFA in solution.
The difference in FEF was caused primarily by the dissimilar
quenching efficiency of ICT in the OFF states (see above).
Expectedly, 5 bearing a strongly basic aliphatic alkylamino group
reached a maximum of ΦF, even under slightly acidic conditions,
but with only a weak increase in fluorescence intensity (only 1.2×).
Therefore, PET does not appear to be a very suitable process for
the development of TPyzPz-based pH indicators.
Even if the FEF was quite high for 1-4, the ΦF in the ON state
(0.11-0.20) did not reach the possible maximum values
determined for controls 6 and 7 without possible ICT/PET (ΦF of
0.31 and 0.34, respectively). This result can be clearly explained
by the concomitant protonation of the second basic center at
higher TFA concentrations, the azomethine nitrogens, i.e., the
appearance of the second quenching mechanism involved in
these OFF-ON-OFF indicators.
In accordance with the literature, protonation of the azomethine
nitrogens was accompanied by
a significant decrease in
fluorescence.[11, 16-18] The ΦF values of the azomethine-protonated
species dropped to zero, indicating that this quenching process is
extremely efficient. Although the exact mechanism of this
quenching has not yet been described, some authors have
suggested that it might be attributed to an increased internal
conversion pathway.[18] In addition to its effect on fluorescence
intensity, the protonation of azomethine nitrogens can also be
monitored by the characteristic decrease in absorbance in the Q-
band area with the formation of a new red-shifted band[33] (Figure
7d, S7). The effect of protonation of the azomethine nitrogens on
the fluorescence intensity is simply documented by the controls 6
and 7 (Figure 7c), which also enabled its full characterization by
the association constants of TFA with azomethine nitrogen of
TPyzPz (KAz = 0.26 and 0.32 M-1 in THF, respectively). The KAz
values of the other compounds (1-5) in this series were found to
be very similar (Table 3) and corresponded well with the values of
KAz determined from changes in the absorption spectra (Table S2).
The obtained data are in good agreement with the results of the
electrochemical measurements and TDDFT calculations that
suggested a limited influence of amino group-bearing substitution
on the electron density of the core and consequently on the
Table 3. pH sensing properties of indicators 1-5 and comparison to controls 6
and 7 [a]
Az
Don
Cpd.
ΦF max
KAz [M-1]
KDon [M-1]
pKA
pKA
THF
THF
THF
H2O
H2O
[b]
1
2
3
4
5
6
7
0.11
0.18
0.11
0.20
0.23
0.31
0.34
0.26
0.26
0.28
0.31
0.28[c]
0.26
0.32
0.55
0.90
0.81
1.09
-
2.29
2.89
3.18
2.57
4.76
1.02
[b]
-
1.11
1.61
1.37
1.63
[b)
-
Don
Az
[a]
values for protonation of the peripheral donor amine;
values for
protonation of azomethine nitrogens; [b] not possible to determine; [c]
determined from the decrease in the Q-band maxima in the absorption
spectrum.
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basicity of the azomethine nitrogens. Of note is that a slightly
higher KAz value was determined previously for analogous zinc
octakis(tert-butylsulfanyl)phthalocyanine (0.53 M-1 in THF[16]),
which is in accordance with its less electron-deficient core.
Photophysical, spectral and electrochemical measurements,
which were further supported and explained by theoretical
calculations, described in detail the ICT phenomenon of TPyzPzs
1-5. In summary, the first oxidation in 1-4 proceeds at the
peripheral donor but is macrocycle-centered in 6-7. Further, the
HOMO is less delocalized (more centered) at the peripheral donor
group with the increasing length of the π-extended linker, which
results in their lower ICT efficiency. This is in accordance with the
TDDFT-predicted increase in the ICT band energy in the series 1
< 2 ~ 3 < 4 << 5. The longer π-linker between the peripheral donor
amine and TPyzPz core serving as an acceptor was undoubtedly
found to decrease the ICT efficiency, which was clearly evident in
the sum of ΦΔ+ΦF; TPyzPz 1 was completely quenched by ICT,
whereas 4 showed significantly retained fluorescence and singlet
oxygen production. PET quenching (in TPyzPz 5) was found to
be less suitable for the development of fluorescence indicators
based on TPyzPzs.
ICT-quenched TPyzPzs 1-4, especially 1 and 2, behaved as
interesting pH indicators at acidic pH, operating with the OFF-ON-
OFF principle by employing a combination of ICT quenching from
the dimethylamino group and quenching by protonation of the
azomethine nitrogens, which was clearly demonstrated in both
organic solvent and water. The apparent K (using TFA in THF
solutions of 1-7) and pKA (in water) values were determined for
the protonation of the azomethine nitrogens in the TPyzPzs and
the donor moieties.
Indicators 1 and 2, sensitive to a pH range of 1.0-2.5 with fairly
steep profiles and FEF higher than 20, are considered the
promising candidates for further usage in fluorescence sensor
devices. Their high impact in this area is also supported by the
fact that these TPyzPz indicators possess strong absorption in the
red region (Q-band maximum > 650 nm) with high extinction
coefficients (ε ~ 2×106 M-1 cm-1), resulting in high brightness of the
sensors in the ON state, which is a particularly critical parameter
in the case of fast-responding pH sensor devices. Moreover, the
fluorescence emission in the red region (in contrast to the majority
of available sensors operating only in the UV region) offers
significant advantages in biological applications due to low light
The abovementioned experiments in organic solvent prove that
the combination of ICT blocking from a peripheral donor together
with the protonation of azomethine nitrogens occurring with rather
similar K values (compare KAz and KDon, Table 3) may lead to the
development of a new class of indicators that are sensitive to a
specific amount of acid in organic solvents or to specific pH range,
i.e., operating on the OFF-ON-OFF principle. It is interesting to
note that the compounds can be used as fluorogenic indicators
for acids in organic solvents, in a manner similar to recently
reported multicolorimetric indicator for acid-base equilibrium in
DCM.[34] Despite the fact that the abovementioned OFF-ON-OFF
switching in THF showed quite promising results, pH sensing is
most often applied in biological-friendly environments; therefore,
the behavior of the studied TPyzPzs had to be demonstrated in
aqueous conditions. Hence, compounds 1–5 and controls 6 and
7 were anchored to liposomes, which are considered a simple
model of biological membranes[35], and the fluorescence intensity
was studied in buffers with pH ranging from 0.6 to 5.4 (or up to
7.8 for the more basic compound 5). Liposomes have been
demonstrated to be suitable delivery systems that maintain tert-
butylsulfanyl-substituted TPyzPzs in monomeric form, i.e., the
photophysically active form.[9] The results are shown in Figures
7e, f and S8. A similar biphasic character of the fluorescence
dependence on pH to that displayed in THF was observed in the
buffers for indicators 1-5, whereas an increase in fluorescence
reaching a plateau at ~pH > 3 was detected only for 6 and 7. The
Az
acidity constants of the conjugated acids pKA (azomethine
nitrogen) and pKADon (dimethylamino group) could be determined
in most cases. pKAAz values of approximately 1.5 (Table 3) were
calculated for the azomethine nitrogens in 5-7, whereas lower
values were observed for 2 and 4, for which the determination
most likely suffers from concomitant donor protonation. To the
best of our knowledge, this is the first report of pKA values for the
azomethine nitrogens in Pc and analogous compounds to date.
Don
The apparent pKA
values of the donor indicate the following
scattering,
limited
autofluorescence
of
endogenous
order of donor basicity: 1 < 4 ~ 2 ~ 3 << 5, with pKA values ranging
from 2.29 - 4.76. Determination of the pKA values for the least
basic compounds may, however, suffer from concomitant
protonation of the azomethine nitrogens, which is why these
values cannot be considered absolute. However, it is clear that all
the sensors 1-5 were selectively sensitive to a narrow pH range
(see Figures 7d and S7) with a promisingly high difference
between the ON and OFF states, particularly for compounds 1
and 2 (FEF higher than 25 when going from neutral to pH~1.5 and
2.0, respectively).
chromophores, and high light penetration depth in tissues. As a
consequence, indicators 1 and 2 belong among the candidates of
pH indicators that may be further used in fluorescence sensor
devices.
Acknowledgements
The work was supported by Czech Science Foundation (14-
02165P), Charles University in Prague (SVV 260 291), Minnesota
Supercomputing Institute and U of M Grant-in-Aid. Authors are
grateful to Jiří Kuneš for NMR measurements, to Juraj Lenčo for
measurements of MALDI-TOF MS and to Lucie Novakova for
HRMS measurements.
Conclusions
The aim of this project was to develop pH indicators operating with
the OFF-ON-OFF principle for specific pH ranges. Hence, a series
of target pH indicators 1-4 operating on the ICT principle was
prepared together with a PET-based indicator 5 and two controls
6 and 7.
Keywords: phthalocyanine • sensor • azomethine nitrogen •
fluorescence • intramolecular charge transfer
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Entry for the Table of Contents
FULL PAPER
Selective pH sensing: A unique
strategy of sensing pH with a fairly
steep sensing profile using red-
Antonin Cidlina, Miroslav Miletin, Mahtab Fathi-
Rasekh, Victor Nemykin,* Petr Zimcik,* Veronika
Novakova,*
emitting
indicators
from
phthalocyanine family is described.
Intramolecular charge transfer and
protonation of azomethine nitrogens
were shown to drive the sensing pH
range. High brightness was achieved
by strong absorption of indicator in
the red region (Q-band maximum >
650 nm, ε ~ 2×106 M-1 cm-1).
Page No. – Page No.
OFF-ON-OFF red-emitting fluorescence
indicators for a narrow pH window
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