Azaphthalocyanines: Red fluorescent probes for cations

Article abstract of DOI:10.1002/chem.201300079

The work was financially supported by the Czech Science Foundation (P207/11/1200, P208/10/1678) and by Charles University in Prague (SVV 265 001). Authors thank to Antonn Cidlina for photodocumentation, Dr. Ivo Jakubec for HRTEM images of NPs, Dr. Vclav S tengl for allowing us to use a DLS instrument, and Dr. Jir Kunes and Zdenek Novk for NMR measurements.

Full text of DOI:10.1002/chem.201300079

COMMUNICATION
DOI: 10.1002/chem.201300079
Azaphthalocyanines: Red Fluorescent Probes for Cations
Veronika Novakova,*[a] Lukꢀsˇ Lochman,[b] Ivana Zajꢁcovꢀ,[b] Kamil Kopecky,[b]
Miroslav Miletin,[b] Kamil Lang,[c] Kaplan Kirakci,[c] and Petr Zimcik*[b]
Fluorescent indicators are important tools for the analysis
of biological cations. For example, these probes are useful
for the quantification of blood electrolytes,[1] assaying ion-
transport channels,[2] and monitoring of lithium-drug
levels.[3] While the interactions of metal cations with phtha-
locyanines (Pcs) have previously been studied using absorp-
tion, magnetic circular dichroism, and NMR spectrosco-
py,[4,5] none of these methods is suitable for in vitro and in
vivo studies, owing primarily to a lack of sensitivity, and/or
to the incompatibility of the instrumentation with biological
systems. In this context, we describe the development of
new aza analogues of Pc, azaphthalocyanines (AzaPcs),
which upon binding to cations fluoresce in the red region of
the visible spectrum. The development of probes that
absorb and emit red light at wavelengths greater than
630 nm is highly desirable, especially for in vivo applications,
because this increases the depth that the excitation light
penetrates, and reduces the effects of light scattering and
the autofluorescence of endogenous chromophores that
often occurs at shorter wavelengths.
based on the coordination of cations to the donor center. In
a proof of concept study, an aza[15]crown-5 moiety, which is
used as the recognition element, was attached to the periph-
ery of the AzaPc macrocycle. This molecule was further
functionalized with bulky tert-butylsulfanyl substituents to
eliminate AzaPc aggregation. During the synthesis of the
sensors, we observed that their fluorescence quantum yields
(FF) are sensitive to substituent groups in the ortho position
to the crown ether. Therefore, a series of model compounds
with a diethylamino donor center (1a–f, Scheme 1) was syn-
Recently, we confirmed that alkylamino-functionalized
AzaPcs undergo an ultrafast intramolecular charge transfer
(ICT) upon excitation. The ICT relaxation pathway of the
excited states results in the quenching of the AzaPc fluores-
cence (i.e., produces the OFF state).[6] Their fluorescence
can be switched ON by protonation of the donor center
(i.e., of the amino substituent), if it is sufficiently basic.[7]
This property led us to develop a new type of AzaPc sensor
[a] Dr. V. Novakova
Department of Biophysics and Physical Chemistry
Faculty of Pharmacy in Hradec Kralove
Charles University in Prague
Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic)
E-mail: veronika.novakova@faf.cuni.cz
[b] L. Lochman, I. Zajꢀcovꢁ, Dr. K. Kopecky, Dr. M. Miletin,
Prof. P. Zimcik
Scheme 1. Reaction conditions: i): 1) Mg butoxide, butanol, reflux, 6 h; 2)
p-toluenesulfonic acid, THF, RT, 2 h; 3) ZnACTHNUGTRNEG(UN CH3COO)2, pyridine, reflux,
2 h.
Department of Pharmaceutical Chemistry and Drug Control
Faculty of Pharmacy in Hradec Kralove
Charles University in Prague
Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic)
E-mail: petr.zimcik@faf.cuni.cz
thesized to assess the effects of ortho substituents on
quenching ability and to select the most suitable substituents
for incorporation into the sensors (2a–c, Scheme 1).
Both the model compounds 1a–f and the sensors 2a–c
were synthesized from two different precursors, 5,6-bis(tert-
butylsulfanyl)pyrazine-2,3-dicarbonitrile and a substituted
[c] Dr. K. Lang, Dr. K. Kirakci
Institute of Inorganic Chemistry
Academy of Sciences of the Czech Republic, v.v.i.
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Husinec-Rez1001, 250 68 Rez(Czech Republic)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300079.
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pyrazine-2,3-dicarbonitrile bearing a donor center by mixed
cyclotetramerization using magnesium butoxide as the initia-
tor (for details of their synthesis, see the Supporting Infor-
mation).
pound 3, which lacks a donor center (Table S1, in the Sup-
porting Information). Therefore, the substituents in 1c–f
induce only a limited fluorescence interval between the
OFF and ON states and are not suitable as sensitive probes.
The data from steady-state fluorescence measurements were
corroborated by fluorescence lifetime (tF) results (Table 1,
The absorption spectra of compounds 1 and 2 in THF
were typical of monomeric species in solution and the pres-
ence of aggregates that could reduce the sensitivity of the
sensor was not detected (Figure 1a, b). Typical Q- and B-
bands were observed at approximately 655 and 375 nm, re-
spectively, and another band with characteristics typical of
charge-transfer (CT) bands occurred at approximately
480 nm. The CT band confirms the conjugation of the donor
substituent (i.e., the lone electron pair on the peripheral ni-
trogen) with the macrocycle. The fluorescence emission
spectra of 1 and 2 exhibited typical shapes for these com-
pounds (lem ca. 658 nm, Figure 1c, d). However, the FF
values varied with the substituent in the ortho position to
the donor center. While the FF values of 1a and 1b were
very low and indicated that ICT was highly feasible in these
cases, the FF values for 1c–f were similar to those of com-
Table 1. Properties of 1a–c, 2a–c, and 3 in THF.
FF
tF [ns]
FD
FD +FF
1a
1b
1c
2a
2b
2c
3[a]
0.006
0.021
0.135
0.010
0.029
0.133
0.35
<0.5 (70%), 1.3 (30%)
<0.5 (80%), 1.7 (20%)
1.54
<0.5 (73%), 1.4 (27%)
<0.5 (44%), 2.1 (56%)
1.49
0.11
0.19
0.62
0.13
0.26
0.55
0.55
0.12
0.21
0.76
0.14
0.29
0.69
0.90
2.66
[a] Data from reference [8].
Table S1 in the Supporting Information). The fluorescence
decay curves for 1c–f were monoexponential on a nanosec-
ond timescale, which is typical of Zn–AzaPcs. The fluores-
cence lifetimes of 1a and 1b were shorter due to ultrafast
ICT, and these decay curves were well characterized by a
biexponential function.[6] The singlet oxygen quantum yields
(FD) of 1a and 1b, which were employed as indicators of in-
tersystem crossing, were also low (Table 1). The sum FF +
FD is less than 0.21, which is much less than the value for
control compound 3 (i.e., 0.90), which does not contain a
donor center. It confirms that efficient ICT occurs in com-
pounds 1a and 1b (1a being more efficiently quenched).
This result suggests that compounds similar to 1a and 1b
may provide high signal-to-noise ratios in sensing applica-
tions. Therefore, three sensors, 2a–c, were prepared and in-
vestigated in cation binding studies. The FF, tF, and FD
values obtained for the sensor molecules were in agreement
with the results for the corresponding model compounds
1a–c (Table 1).
Both sodium and potassium cations exhibit a strong bind-
ing with aza[15]crown-5.[9] The addition of NaSCN or KSCN
to sensors 2a–c in THF caused increases in both FF and tF
due to chelation of the recognition moiety (Table 2). The in-
crease in fluorescence intensity can be attributed to the co-
Table 2. Photophysical and photochemical data of 2a–c in THF after
complete binding of cation.[11]
[a]
FF
tF [ns][a]
KD [mm][b]
2a+Na+
2a+K+
2b+Na+
2b+K+
2c+Na+
2c+K+
0.115
0.105
0.210
0.205
0.188
0.195
0.8 (22%), 2.2 (78%)
0.8 (30%), 2.0 (70%)
1.1 (12%), 2.3 (88%)
1.1 (17%), 2.2 (83%)
2.06
1.03 (0.83)
0.38 (0.37)
0.61 (0.77)
2.01
Figure 1. Interactions of 2a with Na+ (a, c, e) and K+ (b, d, f): a), b) Ab-
sorption spectra in THF (1 mm) during titration with corresponding
SCNÀ. c), d) Fluorescence emission spectra (1 mm, lexc =600 nm). Inset:
Dependence of FF on the concentration of the added cation. e), f) Jobꢃs
plot with highlighted stoichiometries (c(M +2a) =10 mm). g) Schematic il-
lustration of 2a–cation complexes.
[a] After the addition of NaSCN or KSCN in MeOH (c(M+) =20 mm). The
fluorescence lifetime for these compounds is fitted by biexponential func-
tion. The numbers in parentheses represent the fractional intensity of
each decay time. [b] c(AzaPc) =1 mm. KD were determined from FF, values
in parentheses were determined by global fitting of the absorption spec-
tra. Std. error= Æ15%.
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Azaphthalocyanines
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ordination of the lone pair of the donor nitrogen by the
cation, which blocks ICT. The fluorescence enhancement
factor (FEF) reached values of 11, 7 and 1.5 for 2a–c, re-
spectively (Table S2 in Supporting Information). As predict-
ed, compound 2a had the highest signal-to-noise ratio, be-
cause it had the most efficient ICT. Compound 2c is the
least sensitive, which is in agreement with the analysis of
model compound 1c.[10]
It has been noted that the cavity of [15]crown-5 is a good
fit for the size of Na+, but is too small for K+. Therefore,
K+ and the [15]crown-5 can form complexes with a 1:2 stoi-
chiometry.[5] Jobꢃs method of continuous variation revealed
that this is the case for sensor molecule 2a, which was
chosen as a leading compound (Figure 1e, f). The results in-
dicate that 2a forms 1:1 complexes with Na+, but complexes
with K+ in a 1:2 stoichiometry (Figure 1g).
Titration of sensors 2a–c (1 mm solution in THF) with
NaSCN and KSCN (both in MeOH) resulted in small
changes in the absorption spectra of these species (Fig-
ure 1a, b). The notable change was the decrease in the in-
tensity of the CT band at 480 nm, which is in agreement
with the coordination of the metal cation to the donor
center.[11] The fluorescence intensity increased substantially
after the addition of the cation solutions (Figure 1c, d and
the Supporting Information). There was negligible change in
the shape of the emission spectra for 2b and 2c; however, a
small but significant blue shift was observed in both the
emission and absorption spectra of 2a. This indicates that
the oxygen atom in the butoxy moiety of 2a may also be in-
volved in the coordination of the cation. Based on a nonlin-
ear regression analysis of the fluorescence data, the dissocia-
tion constants (KD) were found to be in the range of hun-
dreds of mm (Table 2), which is in line with previous data ob-
tained for the complexation of the [15]crown-5 moiety to
Na+.[11,12] In addition, a global fitting of the absorption spec-
tra yielded valid values for KD, which were of comparable
magnitude to those obtained from the fluorescence assay
(Table 2 and the Supporting Information). As mentioned
above, the binding of cations may also be observed in
changes to tF, which can be exploited in lifetime-based sens-
ing techniques. As an example, the titration data for 2a ex-
hibited very good response in the percentage of the slow
component (tF =2.1 ns) upon the addition of Na+ (see the
Supporting Information).
Figure 2. a) FEF of 2a at complete binding for different cations used as
SCNÀ salts. The same for model compounds 1c and 3 and Na+ (c(AzaPc)
=
1 mm). b) Photo of the solutions used for FEF calculations (lexc =366 nm).
of ICT in the more polar solvent mixture. Exclusive recogni-
tion of the cations by the aza[15]crown-5 moiety was con-
firmed by the limited interaction of model compound 1c or
3 with Na+ (Figure 2).
Azacrown-based sensors are limited by the basicity of
their donor center,[14] which precludes their use under even
slightly acidic conditions because protonation of the donor
switches on the fluorescence signal.[13] However, the sensors
examined in this study do not suffer any interference in an
acidic environment, because of the very low basicity of their
donor center.[15] This fact and the transferability of the re-
sults obtained in THF to aqueous media were examined for
sensor 2a. In general, AzaPcs and Pcs are too hydrophobic
to be used in water without losing their monomeric charac-
ter and photoactivity. To overcome these limitations, com-
pound 2a was imbedded into porous silica nanoparticles
(NPs) that were prepared using an oil-in-water microemul-
Because the aza[15]crown-5 moiety is known to bind cati-
ons other than K+ and Na+,[9,13] the change in the fluores-
cence intensity (expressed as FEF) of 2a was also tested in
the presence of other salts. The data from Figure 2 indicate
that the inhibition of ICT occurring after binding to other
cations is smaller. This is due to limited coordination to the
donor center as reported, for example, for lithium.[13] Some
of the cations tested did not bind to the recognition moiety
at all (e.g., Pb2+, Co2+, Hg2+), and the fluorescence of 2a
decreased after the addition of solutions containing their thi-
ocyanates in methanol. A similar decrease in fluorescence
intensity was observed after the addition of a corresponding
volume of methanol only and is explained by a promotion
Figure 3. a) Incorporation of 2a into silica NPs. b) Absorption spectra of
2a@NPs in water and at pH 3 (0.5% acetic acid). c) Changes of fluores-
cence intensity of 2a@NPs at 668 nm (pH 3, c(AzaPc) ca. 1 mm) after the ad-
dition of NaSCN.
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V. Novakova, P. Zimcik et al.
sion (Figure 3a, see Supporting Information for details).[16]
Compound 2a remained in a monomeric form, as indicated
by the absorption spectra in water (Figure 3b). The absorp-
tion spectra did not change, and the fluorescence intensity
of 2a@NPs actually decreased slightly at pH 3 (0.5% acetic
acid in water) indicating that low pH has little influence on
ICT in this system. The access of the analyte cations to 2a
in the NPs was preserved, and the addition of NaSCN led to
a fivefold increase in the fluorescence intensity (Figure 3c
and Supporting Information).
In conclusion, the concept of sensing metal cations using
fluorescence changes in non-aggregating AzaPc molecules
that exhibit significant ICT in the OFF state was demon-
strated by binding Na+ and K+ to a aza[15]crown-5 recogni-
tion moiety. The data obtained in this study may be easily
extrapolated to other ICT-based recognition moieties that
are selective for different cations. It is important to note
that this concept works not only in organic solvents, but also
in water after the sensor molecules are incorporated into
silica NPs. Aqueous sensing is typically extremely problem-
atic for dyes based on hydrophobic AzaPc and Pc cores. The
substantial advantages of AzaPcs over other types of sensors
are the absorbance and emission in the red region of the
visible spectrum and the presence of a very non-basic donor
center that permits sensing in acidic conditions.
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[2] D. W. Beacham, T. Blackmer, M. OꢃGrady, G. T. Hanson, J. Biomol.
Screening 2010, 15, 441.
[3] D. Citterio, J. Takeda, M. Kosugi, H. Hisamoto, S.-i. Sasaki, H. Ko-
matsu, K. Suzuki, Anal. Chem. 2007, 79, 1237.
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[4] a) M. KoÅak, A. Cihan, A. I. Okur, A. Gꢄl, O. Bekaroglu, Dyes
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Tanaka, Angew. Chem. 2012, 124, 733.
[5] T. Fukuda, I. Sugita, N. Kobayashi, Chem. Commun. 2009, 3449.
[6] V. Novakova, P. Zimcik, M. Miletin, L. Vachova, K. Kopecky, K.
Lang, P. Chꢁbera, T. Polꢀvka, Phys. Chem. Chem. Phys. 2010, 12,
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E. Svandrlikova, L. Vꢁchovꢁ, K. Lang, Inorg. Chem. 2012, 51, 4215.
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[10] All photophysical experiments with macrocycles 1a–f and 2a–c
were performed simultaneously with magnesium complexed into the
center of AzaPc instead of zinc. The data confirmed validity of the
results obtained for zinc derivatives (see the Supporting Informa-
tion); however, no improvement in sensitivity was brought by mag-
nesium derivatives.
[11] The stepwise formation of 1:1 and 1:2 complexes of the sensors with
K+ was not detected in either absorption or fluorescence spectra
due to very small spectral differences between these two forms.
Therefore, the KD values for K+ could not be properly determined.
[12] J. R. Lakowicz, Principles of fluorescence spectroscopy, 3rd ed.,
Springer, New York, 2006.
[13] M. Kollmannsberger, K. Rurack, U. Resch-Genger, J. Daub, J. Phys.
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Acknowledgements
The work was financially supported by the Czech Science Foundation
(P207/11/1200, P208/10/1678) and by Charles University in Prague (SVV
265 001). Authors thank to Antonꢀn Cidlina for photodocumentation, Dr.
[15] pKa =À2.5 was estimated by calculation using Advanced Chemistry
ˇ
Ivo Jakubec for HRTEM images of NPs, Dr. Vꢁclav Stengl for allowing
Development (ACD/Labs) Software V8.14.
ˇ
ˇ
ˇ
us to use a DLS instrument, and Dr. Jirꢀ Kunes and Zdenek Novꢁk for
[16] R. Lin, L. Zhou, Y. Lin, A. Wang, J. H. Zhou, S. H. Wei, Spectrosco-
py 2011, 26, 179.
NMR measurements.
Keywords: crown compounds
·
fluorescent probes
·
Received: January 9, 2013
phthalocyanines · potassium · sodium
Published online: && &&, 0000
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Azaphthalocyanines
COMMUNICATION
Light up a red crown: Chelation of
sodium and potassium cations by
aza[15]crown-5 switches on strong red
fluorescence in azaphthalocyanines. It
is due to an inhibition of ultrafast
intramolecular charge transfer by coor-
dination of the cations to donor center.
Sodium cations fit well into a cavity of
the recognition moiety, while potas-
sium forms supramolecular assemblies
of azaphthalocyanines with 1:2 stoichi-
ometry.
Crown Compounds
V. Novakova,* L. Lochman,
I. Zajꢀcovꢁ, K. Kopecky, M. Miletin,
K. Lang, K. Kirakci,
P. Zimcik* ..................... &&&& — &&&&
Azaphthalocyanines: Red Fluorescent
Probes for Cations
The concept of sensing metal
cations…
…by using fluorescence changes in
non-aggregating AzaPc molecules
that exhibit significant intramolec-
ular charge transfer (ICT) in the
OFF state was demonstrated by
binding Na+ and K+ to a
aza[15]crown-5 recognition moiety.
It is important to note that this
concept works not only in organic
solvents but also in water after the
sensor molecules are incorporated
into silica nanoparticles. For more
details see the Communication by
V. Novakova, P. Zimcik et al. on
page && ff.
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