Integration of the 1,2,3-triazole "click" motif as a potent signalling element in metal ion responsive fluorescent probes

Article abstract of DOI:10.1002/chem.201201575

In a systematic approach we synthesized a new series of fluorescent probes incorporating donor-acceptor (D-A) substituted 1,2,3-triazoles as conjugative π-linkers between the alkali metal ion receptor N-phenylaza-[18]crown-6 and different fluorophoric gro

Full text of DOI:10.1002/chem.201201575

FULL PAPER
DOI: 10.1002/chem.201201575
Integration of the 1,2,3-Triazole “Click” Motif as a Potent Signalling Element
in Metal Ion Responsive Fluorescent Probes
Sandra Ast,[a] Tobias Fischer,[b] Holger Mꢀller,[a] Wulfhard Mickler,[a]
Mathias Schwichtenberg,[a] Knut Rurack,*[b] and Hans-Jꢀrgen Holdt*[a]
Abstract: In a systematic approach we
synthesized a new series of fluorescent
probes incorporating donor–acceptor
(D-A) substituted 1,2,3-triazoles as
conjugative p-linkers between the
alkali metal ion receptor N-phenylaza-
[18]crown-6 and different fluorophoric
groups with different electron-acceptor
properties (4-naphthalimide, meso-
phenyl-BODIPY and 9-anthracene)
and investigated their performance in
organic and aqueous environments
(physiological conditions). In the
charge-transfer (CT) type probes 1, 2
and 7, the fluorescence is almost com-
pletely quenched by intramolecular CT
(ICT) processes involving charge-sepa-
rated states. In the presence of Na+
and K+ ICT is interrupted, which re-
sulted in a lighting-up of the fluores-
cence in acetonitrile. Among the inves-
tigated fluoroionophores, compound 7,
which contains a 9-anthracenyl moiety
as the electron-accepting fluorophore,
is the only probe which retains light-up
features in water and works as a highly
K+/Na+-selective probe under simulat-
ed physiological conditions. Virtually
decoupled BODIPY-based 6 and pho-
toinduced electron transfer (PET) type
probes 3–5, where the 10-substituted
anthracen-9-yl fluorophores are con-
nected to the 1,2,3-triazole through a
methylene spacer, show strong ion-in-
duced fluorescence enhancement in
acetonitrile, but not under physiologi-
cal conditions. Electrochemical studies
and theoretical calculations were used
to assess and support the underlying
mechanisms for the new ICT and PET
1,2,3-triazole fluoroionophores.
Keywords: charge
transfer
·
click chemistry · electron transfer ·
fluorescent probes · metal ions
Introduction
classical approaches proposed recently,[18,19] the design strat-
egies commonly follow three major routes: i) intramolecular
charge-transfer (CT or ICT) active probes with a binding
site integrated into a p-conjugated electron donor–acceptor-
substituted fluorophore, ii) photoinduced electron transfer
(PET) active probes with alkyl spacer-separated binding site
and fluorescent group and iii) excimer probes with specific
integration of binding site and polycyclic aromatic hydrocar-
bon fluorophores.[20–22] However, a large number of such
probes reported so far harbors several drawbacks such as
the requirement of an organic (co-) solvent for operation of
most ICT probes,[23] the lack of spectral discrimination be-
tween bound and unbound form for PET probes[29] and the
restrictions in system design for excimer probes. The quest
for potent yet straightforward design strategies thus remains
highly topical.[32,33]
Fluorometric techniques are indispensable tools to assess
the quantity of analytes at site, in situ or in ever smaller
sample volumes in the laboratory and to image the topology
of objects at nanometric dimensions.[1–11] Despite the devel-
opment of new types of powerful labels and probes such as
fluorescent proteins or conjugated polymers,[12,13] Fçrster (or
as frequently termed: fluorescence) resonance energy trans-
fer (FRET) ensembles or optical switch probes,[14,15] semi-
conductor nanocrystals or up-converting nanoparticles,[16,17]
indicator dyes continue to be the most abundant form of flu-
orescent reporter employed. Accordingly, their development
receives unquenched attention. Besides a number of non-
Traditionally, one of the key areas of fluorescent probe
development is the detection and tracking of metal ions in a
large variety of environmental and biomedical samples and
situations.[34,35] Targets range from heavy metal ions through
dia- and paramagnetic transition metal ions to alkaline-
earth and alkali metal ions.[36–40] The molecular toolbox com-
prises classical indicator dyes as well as aptamers,[20–22,41] and
techniques include conventional one-photon and spatially
higher resolving two-photon excited fluorescence spectros-
copies.[42,43]
[a] S. Ast, H. Mꢀller, Dr. W. Mickler, M. Schwichtenberg,
Prof. Dr. H.-J. Holdt
Institut fꢀr Chemie, Anorganische Chemie, Universitꢁt Potsdam
Karl-Liebknecht Str. 24–25, 14467 Golm (Germany)
Fax : (+49)331-977-5055
E-mail: holdt@uni-potsdam.de
[b] T. Fischer, Dr. K. Rurack
Fachbereich 1.9 Sensormaterialien
BAM Bundesanstalt fꢀr Materialforschung und -prꢀfung
Richard-Willstꢁtter Str. 11, 12489 Berlin (Germany)
E-mail: knut.rurack@bam.de
A continuing challenge in small-molecule probe design
for metal ion analysis under physiological conditions is the
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201575.
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quest to obtain pronounced spectroscopic responses upon
binding of monovalent “hard” metal ions,[44] that is, non-
aminophilic alkali metal ions with a comparatively low
charge density such as Na+ and K+.[20–22] Even under ideal
conditions such as in (mixed) organic solvents, ICT probes
commonly show only moderate shifts of a few tens of nano-
metres and fluorescence enhancement or quenching factors
for PET probes are often around or below 30 in the pres-
ence of such analytes. Few examples of more efficient indi-
cation have been reported[47–52] so that the search for alter-
native signalling strategies has received strong attention in
the last decades.[53]
ing in the presence of open-shell cations such as, Ni2+ and
[65,67]
Cu2+[64,66,69] or heavy metal ions as for example, Hg2+
.
The latter is basically due to the fact that the 1,2,3-triazole
unit is electronically not an ideal partner for ICT or PET
processes. As Diederichꢃs group has shown recently, this sit-
uation changes when the triazole unit is coupled with strong
electron donor and acceptor moieties.[70] We thus strived to
exploit this option more deeply in the context of functional
dye design and report here on a series of fluorescent probes
incorporating donor–acceptor- (D-A) substituted 1,2,3-tria-
zoles as conjugative p-linkers between receptor and fluoro-
phore units. The responsive donor unit is N-phenylaza-
[18]crown-6 as a model receptor for K+ and popular fluoro-
phores such as anthracene, naphthalimide and BODIPY
comprise the acceptor moiety (Figure1).[71] N-Phenylaza-
For several years, we are particularly interested in inte-
grating efficient signal transduction pathways in small probe
molecules. For instance, we have succeeded in establishing
the concept of “virtual decou-
pling” for the fluorescence
light-up detection of notorious-
ly quenching heavy and transi-
tion metal ions and weakly co-
ordinating
alkali
metal
ions[47,48,54] and the tuning of the
interplay of two intramolecular
ICT and PET processes through
guest binding for the amplified
fluorescence
detection
of
PdII.[55,56] However, one of the
major challenges in conceiving
and testing uncommon or inter-
acting signalling pathways with
various fluorophores such as for
instance popular anthracene,
boron–dipyrromethene
(BODIPY) or coumarin dyes
often is synthetic realization.
When screening the recent liter-
ature on fluorescent probe de-
Figure 1. “Click”-type fluorescent probes investigated in this work.
velopment, however, it seems
that the versatility of the rather
young yet extremely popular CuI-catalyzed azide–alkyne cy-
cloaddition (CuAAC) or “click” chemistry which has been
widely successfully used as a bioconjugation method,[57]
might offer more opportunities for system design.[58] Since
the first report on a fluorescent probe incorporating the typ-
ical “click” motif,[59] a large number of examples utilizing
different fluorophores—from coumarin and rhodamine
through pyrene and anthracene to dansyl and naphthali-
mide—for a number of metal ions—mainly heavy and tran-
sition metal ions though—have been reported.[60] However,
in these ensembles the 1,2,3-triazol-1,4-diyl moiety has been
basically used as a chelating site, either isolated from[61–65] or
conjugated to the chromophore,[59,66–68] or as a synthetic link
in a traditional, decoupled PET type of architecture.[69] All
of these approaches thus do not lead to uncommon signal-
ling pathways or behaviour, that is, they show the common
features of fluorescence enhancement in the presence of
closed-shell cations as for example, Zn2+[62–64,68] and quench-
[18]crown-6 and K+ (and Na+) were primarily chosen to
assess the generality of the approach for a weakly coordinat-
ing analyte and to contrast it also with the majority of metal
ion “click” probes that target transition metal ions because
of the coordination chemistry of triazoles.[73]
Our present aim is to assess the potential of providing the
triazole unit in “click” probes with a decisive signalling func-
tion. Thus, it is essential to systematically study its perform-
ance in conventional ICT as well as PET architectures. Com-
pounds 1 and 2 were thus prepared to elucidate whether the
triazolyl–naphthalimide moiety with its favourable fea-
tures[62,74,75] can also be employed in a p-conjugated manner
when coupled to an electron-rich receptor. Moreover, inves-
tigation of these two probes should provide more informa-
tion about the role of the regioisomeric integration of the
triazolyl moiety. Compounds 3–5 were synthesized as model
PET probes, especially to be able to judge whether our ulti-
mate approach of a CT/LE (locally excited)[76] state-reversal
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Metal Ion Responsive Fluorescent Probes
FULL PAPER
probe realized in 7 is superior to a conventional PET probe
design with the same subunits. meso-Phenyl-BODIPY-incor-
porating 6 was attempted to get an idea whether the D–A
triazole relay retains its performance in combination with a
chromophore that absorbs in the visible spectral range in-
stead of the UV as anthracene, naphthalimide and coumarin
do. Finally, 7 was conceived as a first example of a probe
containing a typical LE fluorophore such as anthracene in
an all-p-conjugated architecture with the ICT-type triazole-
aniline moiety, to allow for a unique interplay of LE and CT
processes, potentially integrating features of the most popu-
lar signalling mechanisms.
probes with two superimposed, directional ICT processes.
Whereas 2 contains the more common 4-NI nitrogen atom,
regioisomer 1 is connected through a carbon atom.
Table 1 lists the spectroscopic properties of both com-
pounds in a highly polar model solvent such as acetonitrile.
In comparison to for instance 8 (Figure 2), which absorbs
and emits at 415 and 528 nm in acetonitrile and fluoresces
with a reasonable quantum yield of 0.05,[89,90] both 1 and 2
show dramatically reduced fluorescence quantum yields and
hypsochromically shifted spectra, with a broad absorption
band of considerable low intensity stretching out to about
500 nm in the case of 2 (see also Figure 3). The behaviour of
1 and 2 is also distinctly different from model compound 9,
carrying only the triazole unit without any additional donor
group at the 4-position (Table 1).
Results and Discussion
To assess the photophysical mechanisms active in 1 and 2,
it is helpful to consider the electronic transitions and the re-
spective states and molecular orbitals involved as obtained
by quantum chemical calculations (Table 2, Figure 4). The
With respect to the synthesis of the compounds, CuAAC of
azido-functionalized N-phenylaza-[18]crown-6[72,77,78] with N-
(n-butyl)-4-ethinyl-1,8-naphthalimide,[79]
4,4-difluoro-8-(4-
!
ethinylphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-inda-
cene[80–83] and 9-ethinyl-anthracene[84] afforded 1, 6 and 7.
The respective reaction of ethinyl-functionalized N-phenyl-
typical naphthalimide-centred, oscillator-strong S1 S0 tran-
sition, involving HOMO and LUMO, has a certain CT char-
acter for 4-amino-substituted NI dyes and is for instance
found at 385 nm in the gas phase for 8a (imido N-ethyl ana-
logue of 8, Figure 2, Chart S1).[92,94] If this 4-N atom is inte-
grated in the weaker electron-donating triazole unit such as
in 9, the features of 8a are largely preserved yet the CT
aza-[18]crown-6[72,85] with
6-azido-N-ethyl-1,8-naphthal-
imide,[62] 9-azidomethyl-anthracene,[86] 9-azidomethyl-10-cya-
noanthracene,[86,87] and 9-azidomethyl-10-methylanthra-
cene[86,88] yielded 2–5. Details of the synthetic procedures
and characterization are given
in the Experimental Section
and the Supporting Informa-
tion, along with the synthetic
details on the model com-
pounds
investigated
here
(Figure 2).
Spectroscopic properties of 1
and 2: For 4-(4-R-1H-1,2,3-tri-
azol-1-yl)-anilines as well as 4-
(1-R-1H-1,2,3-triazol-4-yl)-ani-
lines (with R=substituent), Jar-
owski et al. have shown that the
ICT process proceeds from the
electron-rich aniline moiety to
the electron-poorer triazole,
carrying a further substituent
either in the 4- or 1-position.[70]
On the other hand, for naph-
thalimide (NI) dyes it is well
known that an ICT process can
best be installed when a strong
electron donor moiety such as a
dimethylamino group is attach-
ed to the 4-position of the NI
skeleton (as in 8).[89] As a con-
sequence, we introduced the tri-
azole-aniline unit in a vectorial
fashion at the 4-position of the
Figure 2. Model compounds experimentally investigated in this work; Theoretically investigated model com-
pounds comprise 1a, 2a and 6a as the N,N-dimethylamino analogues of 1, 2 and 6, 8a as the imido N-ethyl an-
alogue of 8 and 10a as the N,N-dimethyl-3-ethyl analogue of 10b and its regioisomer 10b (see Chart S1, Table
NI core, to arrive at composite S5).
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K. Rurack, H.-J. Holdt et al.
Table 2. Calculated properties for the vertical excitation of the energy-
minimized ground-state geometries of 1a and 2a and model compounds
8a and 9 by TD-DFT (for calculation details, see Experimental Section).
f[b]
DmS
Orbitals (coefficients)[d]
!
!
(n)
S0
lS
(n)
S0
n
n
[nm][a]
[D][c]
1a
2a
422.0 (1)
358.0 (2)
340.2 (3)
570.4 (1)
349.9 (2)
0.130 40.1
0.441 7.0
0.003 ꢀ4.4
0.079 39.9
0.390 5.2
HOMO–LUMO
HOMO–1ꢀLUMO
HOMO–2ꢀLUMO
HOMO–LUMO
HOMOꢀ1–LUMO (0.63)
HOMOꢀ4–LUMO
(ꢀ0.15)
343.2 (3)
0.026 0.4
HOMOꢀ1–LUMO (0.14)
HOMOꢀ4–LUMO (0.67)
HOMO–LUMO
8a[e] 385.5 (1)
0.215 9.2
334.7 (2)
0.001 ꢀ0.7
HOMOꢀ1–LUMO (0.55)
HOMOꢀ2–LUMO (0.41)
mixed (HOMOꢀ3–
LUMO)
306.1 (3)
0.002 ꢀ0.6
9
352.8 (1)
0.348 6.0
HOMO–LUMO (0.64)
HOMOꢀ1–LUMO (0.10)
HOMOꢀ2–LUMO
HOMO–LUMO (0.66)
HOMOꢀ2–LUMO
(ꢀ0.13)
344.1 (2)
316.6 (3)
0.001 5.4
0.007 14.6
[a] Wavelength of the transition. [b] Oscillator strength of the transition.
[c] Dipole moment difference between ground (m0) and respective excited
(mn) state. [d] MOs involved in the transitions, mixed: more than four or-
bitals are involved. [e] The calculated structure of 8a contains an ethyl
instead of the n-butyl group at the imido nitrogen, which however has no
Figure 3. Absorption (cprobe =5ꢄ10ꢀ5 m) and fluorescence spectra (cprobe
=
5ꢄ10ꢀ6 m) of 1 (top, black lines) and 2 (bottom, black lines) in acetoni-
trile and corresponding titration spectra upon addition of Na+ (red, se-
lected steps between 0.25–13.0 mm; spectra of 1ꢁNa+ and 2ꢁNa+ as
blue lines).
significant influence on the findings.[90]
a minor contribution from HOMOꢀ4–LUMO in the latter
!
case. Thus, the typical NI S1 S0 transitions in 8a/8 and 9
!
become S2 S0 in 1a/1 and 2a/2.
Table 2 and Figure 4 further reveal that the HOMO–
LUMO transitions in the title dyes, which for both are
equivalent to the S1 S0 transitions, lie at lower energies and
Table 1. Spectroscopic properties of 1, 2, their Na+ and K+ complexes as
well as model compounds 8–10b and Na+ and K+ complexes of 10a.
!
have significantly smaller oscillator strengths. Moreover,
these HOMO–LUMO transitions have a pronounced CT
character with virtually complete charge localization in both
molecular orbitals, also stressed by the large dipole moment
differences between first (Dm~40 D) as well as second (Dm~
6 D) excited and the ground state (Table 2). For 1a, the
Species
labs [nm]
lem [nm]
(500)[b]
434
444
ff
10ꢀ4[b]
0.10
tf [ns]
1
239, 297, 347, 357
239, 281, 360
240, 281, 361,
235, 296, 331, 345, 393
237, 253, 336, 344
236 (263), 333, 344
415
236, 334, 344
293
273
G
<0.01[c]
3.38
1ꢁNa+
1ꢁK+
2
6ꢄ10ꢀ4
10ꢀ4[b]
5ꢄ10ꢀ3
2ꢄ10ꢀ4
0.05
<0.01[c]
<0.01[c]
0.24
A
2ꢁNa+
2ꢁK+
8[d]
436
E
0.01[c]
!
S1 S0 transition is found at 422 nm and for 2a even at
528
417
0.8 (98)[e]
570 nm. Although the absolute positions as obtained by the
calculations seem to be overestimated, they support the ap-
pearance of a red-shifted band of low intensity visible in the
tailing of the long-wavelength band in 1 and the broad
shoulder in 2, stretching out to 500 nm. If one further con-
siders that the electronic p-system is neither planar in 1a
nor in 2a but significantly twisted by overall ꢀ<(qNT+qTP)
9
10
0.21
1.12
[f]
393
0.034
10ꢁNa+
[f]
[f]
[f]
[f]
10ꢁK+
276
393
0.048
[a] In acetonitrile solution. [b] Too weak/low to be determined with ac-
ceptable accuracy with the instrument employed. [c] Within the temporal
resolution of the instrument employed. Traces of a slow component are
also found which are most likely due to traces of water/protons in the sol-
vent used. [d] From ref. [89]. [e] Major component (98%) as given in ref.
[89]. [f] Not determined.
!
> 458 (Table 3), the low intensity of the S1 S0 transition,
Table 3. Intramolecular torsion angles
q
of the geometry-optimized
ground state structures of 1a and 2a.[a]
character is weakened and the respective transition is shifted
to shorter wavelengths (353 nm, Table 2). The corresponding
NI-centred transitions in 1a and 2a (N,N-dimethylamino an-
alogues of 1 and 2, Figure 2, Chart S1) are, however, not the
lowest transitions in these compounds, but only the second
lowest S2 S0 transitions at about 358 and 350 nm. They in-
volve HOMOꢀ1 and LUMO in the case of 1a and 2a, with
Species
qNT [8][b]
qTP [8][c]
1a
2a
29.4
45.4
30.2
0.9
[a] Naphthalimide plane defined by the 13 atoms of the NI core skeleton,
triazole plane defined by the five ring atoms, phenyl plane defined by the
six ring atoms. [b] Angle between NI and triazole planes. [c] Angle be-
tween triazole and phenyl planes.
!
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Metal Ion Responsive Fluorescent Probes
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tems published in the litera-
ture.[97,98] For such compounds,
a twisting of the molecular sub-
units in the excited state can be
a prominent non-radiative de-
activation pathway especially in
highly polar solvents, stabilizing
charge separation and leading
to low fluorescence quantum
yields. We thus tentatively as-
cribe the pronounced quench-
ing in the present case to such
behaviour. In addition, closer
inspection of the quantum
chemical data provides a fur-
ther hint. Whereas the singlet–
triplet energy gap for the
lowest or second-lowest excited
states is 0.27 eV or higher in 8a
and 9, it amounts to only 0.03
and 0.14 eV for 1a and 2a, po-
tentially opening another effi-
cient non-radiative deactivation
pathway through intersystem
crossing (Table S7). Summariz-
ing the theoretical results it can
Figure 4. Frontier molecular orbitals HOMOꢀ4, ꢀ3, ꢀ2, ꢀ1, HOMO, LUMO and LUMO+1, +2 of 1a and
2a and HOMOꢀ3, ꢀ2, ꢀ1, HOMO, LUMO and LUMO+1 of 8a and 9.
which would be significantly broadened in a highly polar
solvent such as acetonitrile compared to the gas phase calcu-
lations, also correlates well. Regarding the mismatch of the
absolute position of that transition in the sense that TD-
DFT calculations predict such charge-separated states to lie
at rather low energies, our results are in line with similar
findings by Diederichꢃs group.[70,95]
be said that in 1a/1 and 2a/2 a strong ICT process leading
to pronounced charge separation between the molecular NI
and triazole–aniline subunits and a very weakly emissive
state masks the typical and usually highly emissive 4-amino
naphthalimide-type CT process, entailing
a
strongly
quenched fluorescence. The fact that 10 shows a moderate
fluorescence (Table 1), yet 1 and 2 as well as the dyes re-
ported in reference [70] are virtually non-emissive suggests
that a D–A substitution pattern is mandatory to achieve
quenching in a 1,2,3-triazole-based ICT architecture.
As is evident from Table 2, for both 1a and 2a, a series of
weak to moderate transitions is found at l<350 nm, which
are apparently responsible for the rich spectra in that region
in Figure 3. Comparison of the absorption maxima of 1 with
the relay-receptor model 10 (Figure 2) let us assign the in-
tense band at about 295 nm to a transition located on the
aniline–triazole fragment (Table 1), which is supported by
Electrochemical properties of 1 and 2: To facilitate the un-
derstanding of the processes at play, it is helpful to consider
the redox properties of the title compounds. We thus investi-
gated 1, 2 and model compounds 9–11 by cyclic voltammetry
in acetonitrile. Phenylazacrown 11 is irreversibly oxidized at
+730 mV (Table 4). Whereas 2 shows a similar oxidation
wave at +760 mV, only slightly anodically shifted compared
with 11, the corresponding potential is significantly shifted
!
the theoretical results listed in Table S5, that is, S5 S0 oc-
curring at 295 nm and undergoing
a moderate dipole
moment change of Dm=8 D for 1a. This aniline-triazole-
centred transition lies at slightly shorter wavelengths for 2a,
that is, 275.5 nm and possess a significantly higher oscillator
strength (Table S5) which is in line with the ratio of the 295
to the 360 nm bands in 1 and 2 in Figure 3.
!
Table 4. Selected redox potentials (peak, in mV) of 1, 2, 9–11 in the ab-
sence and presence of NaPF6/KPF6 in acetonitrile (for complete data, see
Supporting Information, Table S9).[a]
Although the oscillator strengths of the S1 S0 CT transi-
tion in 1a and 2a are reduced compared with the NI-centred
!
S1 S0 transition with CT character in 8a and 9, this differ-
1
2
9
10b
11
ence does not explain why the fluorescence quantum yields
of 1 and 2 are more than two orders of magnitude lower
than those of 8 and 9. However, in contrast to 8a and 9 for
which the delocalization of HOMO and LUMO is largely
identical, the strong charge localization and negligible orbi-
tal overlap of the frontier MOs in 1a and 2a is more remi-
niscent of strongly donor–acceptor substituted triaryl sys-
free
+900r
+760r
–
+860r
+730ir
ꢀ1260r
+1020r
ꢀ1270r
+1020r
ꢀ1260r
ꢀ1140r
+950r
ꢀ1050r
+960r
ꢀ1050r
ꢀ1150r
–
–
+Na+
+K+
–
–
–
–
+940r
–
+950r
–
+770ir
–
+760ir
–
[a] Data against Fc/Fc+. Subscripts: r=reversible, ir=irreversible.
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K. Rurack, H.-J. Holdt et al.
to +900 mV in 1. However, when considering the respective
oxidation at +860 mV in 10, which contains the same
formal phenylenediamine fragment as 1 does, it becomes
evident that the regioisomer obtained from the p-azido-sub-
stituted phenylazacrown is more difficult to oxidize, presum-
ably because of the electron-withdrawing effect of the N
atom connecting the aniline moiety to the triazole ring.
(Note that the sum of the Mulliken charges of this fragment
obtained from the quantum chemical calculations amounts
to 0.414 in 1a yet only to 0.104 in 2a.) In both 1 and 2, the
aniline-centred oxidation is reversible. Model compound 9,
representing the triazole–NI composite and here especially
2, exhibits a reversible reduction at ꢀ1150 mV, which is very
similar to the ꢀ1140 mV found for 2. This is consistent with
literature data on simple l,8-naphthalimide derivatives, for
instance, a reversible reduction at ꢀ1000 mV for 2-ethyl–NI
and ꢀ1110 mV for 2-ethyl-6-methoxy–NI.[99] A cathodic
shift towards ꢀ1260 mV can be noted for 1 with the re-
versed triazole pattern. The other redox potentials which
are not primarily important for the signaling process are
given in the Supporting Information.
combinations all obey to a 1:1 model and the binding con-
stants have been determined to logK
(1ꢁK+)=2.7, logK(2ꢁNa+)=2.8 and logK
example, logK
ACHTUNGTRENNUNG
G
E
G
AHCTUNGTRENNUNG
stronger complexes than Na+ agrees well with the fact that
[18]crown-6 better accommodates the larger K+ ion.[103,104]
Furthermore, the difference seen between the complexation
constants of 1 and 2 also correlates well with the sum of the
charges on the dimethylamino group as calculated for the
dimethylamino analogues 1a and 2a, ꢀ0.135 and ꢀ0.148,
such a correlation being a general design aid for ICT
probes.[94]
Assessing the ion response in acetonitrile by cyclic vol-
tammetry results in an anodic shift of about 120 mV (for 1)
and about 200 mV (for 2) of the oxidation of the receptor
moiety for both Na+ and K+ (Table 4). The aniline-centred
oxidation also becomes reversible, showing the stabilization
of the ligand in the complexed state which has been report-
ed before in the literature.[78] Again, 10 which is regioiso-
merically similar to 1 shows virtually identical shifts in the
presence of Na+ and K+ (Table 4), while the ion-induced
effect is smaller for unsubstituted 11. The minor differences
seen for Na+ and K+ and 1 and 2 reflect well the minor dif-
ferences seen in the cation-induced absorption shifts
(Table 1). If we now consider the following observations
that for both 1 and 2, i) Na+ and K+ influence the electronic
structure in the ground state to a comparable degree (ab-
sorption spectral and redox shifts), ii) Na+ leads to a much
more pronounced fluorescence enhancement and iii) K+
forms the stronger ground-state complexes, it seems reason-
able to assume that K+ is more tightly bound through the
oxygen donor atoms of the crown, facilitating additionally a
more efficient excited-state de-coordination from the
crownꢃs nitrogen atom.
Response of 1 and 2 to Na+ and K+ under model condi-
tions: Following the design outlined above, that is, the mask-
ing of a highly emissive by a strong and non-emissive ICT
process, the latter of which is generated especially by the
bridgehead atom between electronic p-system and receptor
moiety, the binding of a positively charged species such as a
metal cation at this electron-rich aniline-N should redefine
forces in the probe molecules, diminishing the quenching
process. Indeed, as can be seen from Table 1 and Figure 3,
addition of Na+ and K+ salts to an acetonitrile solution of 1
and 2 entails moderate changes in absorption, in particular
the vanishing of the low-energy bands/tails of low intensity
and the aniline–triazole-centred bands, and a large increase
in fluorescence, especially for Na+. Comparison of for in-
stance the spectral endpoint data in absorption of 2ꢁNa+
and 2ꢁK+ with those of 9, the NI–triazole structural ana-
logue of 2, reveals a good agreement, suggesting that al-
ready the monovalent, non-aminophilic alkali metal ions can
efficiently switch off the quenching ICT process in an aprot-
ic organic solvent. The fluorescence of 2ꢁNa+ and 2ꢁK+ is
still slightly red-shifted compared with 9, which is most
likely due to the fact that in such ICT probes the coordina-
tive bond is weakened in the excited state, leading to elec-
trostatic repulsion and partial de-coordination.[100–102] An in-
teresting conclusion that can be drawn from a comparison
of the cation-induced effects on 1 and 2 is the fact that the
spectroscopic response is much stronger for the regioisomer
connected through the 4-C atom to the triazole than for the
classic 4-N naphthalimide connection: 1ꢁNa+ shows a much
higher fluorescence enhancement than 2ꢁNa+ and a 20-
times brighter overall fluorescence (ff data in Table 1). Es-
pecially the impressive fluorescence enhancement factor of
>100 for 1ꢁNa+ is noteworthy, because such fluorescence
amplification is rather uncommon for ICT-type probes. Re-
garding complex formation, the stoichiometries for the four
Response of 1 and 2 to Na+ and K+ under simulated phys-
iological conditions: For a fluorescent probe that shows
promising behaviour in model environments, the move to
media for realistic analytical applications such as simulated
physiological conditions (aqueous solution, buffered at
pH 7.2 with 10 mm Tris and ꢂ1500 mm choline chloride for
maintaining constant ionic strength) is usually the crucial
step. Although 1 and 2 are only weakly soluble under such
conditions, absorption measurements reveal that the CT
transitions are significantly shifted. 1 shows now a broad
band at about 440 nm while the weak band at 408 nm seen
for 2 in acetonitrile is now still reduced in intensity with its
maximum not being reliably detectable anymore (Figure 5).
The fluorescence band of both compounds is also strongly
shifted, to about 530 for 2 and 586 nm for 1, which is in line
with general findings on the fluorescence behavior of 4-
donor-substituted NI dyes in water.[105,106] In the case of 2,
the fluorescence is still very weak, hampering any more reli-
able determination of the fluorescence data. Interaction of
the protic solvent with the anilineꢃs nitrogen atom, which is
expected to take place in this environment, is presumably
not strong enough to lead to a significant increase in fluores-
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Metal Ion Responsive Fluorescent Probes
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Table 5. Spectroscopic properties of 3–5, their Na+ and K+ complexes,
and model compound 12.[a]
Species labs [nm]
lem [nm]
ff
tf [ns]
3
254, 292, 333, 349, 367, 387 391, 413, 438, 465 0.005 0.09[d]
3ꢁNa+ 254, 333, 349, 367, 387
391, 413, 438, 465 74[c]
5.77
2.47
3ꢁK+
16[c]
[b]
[b]
4
257, 292, 352, 369, 386, 408 419, 431 (468)
0.002 0.09[d]
365[c] 10.61
4ꢁNa+ 257, 352, 369, 386, 408
418, 444, 468
4ꢁK+
2.5[c]
0.1[d]
[b]
[b]
5
259, 292, 339, 357, 376, 397 403, 427, 448
0.004 0.05[d]
120[c] 8.45
5ꢁNa+ 258, 339, 357, 376, 397
403, 427, 448
5ꢁK+
35[c]
4.07
4.91
[b]
[b]
12
254, 333, 348, 366, 386
390, 413, 437, 464 0.29
[a] In acetonitrile solution, c1 = 5ꢄ10ꢀ6 m. [b] The spectral features of
XꢁK+ are virtually identical to XꢁNa+, X=3, 4 or 5. [c] Fluorescence
enhancement with respect to unbound probe. [d] Traces of a slow compo-
nent are also found which are most likely due to traces of water/protons
in the solvent used. [e] Not determined.
Figure 5. Absorption (cprobe =1ꢄ10ꢀ5 m) spectra of 1 (black) and 2 (green)
under physiological conditions (inset: corresponding titration spectra for
1 upon addition of K+ (red, 5–160 mm; spectra of 1 and 1ꢁK+ as black
and blue lines).
cence quantum yield. On the other hand, for 1, the fluores-
cence quantum yield is slightly increased to ff = 2ꢄ10ꢀ3.
Addition of Na+ and K+ at concentrations between 5 and
160 mm, which represents the relevant physiological concen-
tration range for these cations and especially for K+,[30,31]
leads to negligible effects in the case of 2 and to a 1.4- and
1.5-fold fluorescence enhancement for Na+ and K+ and 1
(Figure 5). Although this enhancement is analytically not
very exploitable, it shows that the design concept is rather
powerful. Regioisomeric attachment of a triazole–aniline re-
ceptor unit through the triazole C4 atom to the acceptor of
an ICT probe, like in 1, thus seems to yield more powerful
reporter molecules also in a realistic environment like we
have observed in the case of a coumarin chromophore.[72]
ized.[110] Reduction of the electron density at the aniline-N
through protonation or complexation then revives emission.
The situation is similar in 3–5. If one compares the data of
10 and 12 in Tables 1 and 5 with those of 3, it is evident that
the absorption spectrum of 3 is a linear combination of ani-
line–triazole- and anthracene-centred transitions. (The tran-
sition that is localized exclusively on the isolated triazole
fragment in 12 presumably lies outside of the wavelength
range considered here, that is, at higher energies. Moreover,
regardless of regioisomeric substitution pattern, the aniline–
triazole-centred transition is found at about 300 nm, see
[70].) The quenching process is also obvious if one takes a
look at the MOs of 3, which are exemplarily shown in Fig-
ure S2, Supporting Information. Anthracene-centred excita-
tion predominantly involves HOMOꢀ1 and LUMO, and fa-
cilitates ET from the triazole–aniline-localized HOMO
(Tables S6, S7).
Spectroscopic properties of 3–5: The installation of a
second, parallel ICT process by introduction of a “click” ele-
ment has proven to be rather beneficial for creating a pro-
nounced fluorescence enhancement for ICT-type probes.
Compounds 3–5 were prepared to obtain further informa-
tion in how far the donor-substituted 1,2,3-triazole unit is
also a potent partner in a typical PET probe architecture.
The absorption spectrum of 3 shows the typical anthra-
cene bands at 348, 367 and 387 nm, the strong anthracene
band at 254 nm and a broad, intense band at 290 nm in ace-
tonitrile (Figure 6). In addition, a weak anthracene-like fluo-
rescence band is found. The features of 4 and 5 are rather
similar, the slight spectral shifts being related to the influ-
ence of the substituent at the 10-position of the anthryl
moiety (Table 5). Such features are typical for PET probe
architectures consisting of anthracene, a methylene bridge
and a substituted aniline receptor.[107–109] For such com-
pounds, quenching is commonly due to an electron transfer
(ET) from the HOMO, which is localized entirely on the
electron-rich aniline fragment, to the excited anthracene, on
which fragment commonly HOMOꢀ1 and LUMO are local-
Electrochemical properties of 3–5: Further support for the
mechanisms involved is provided by an electrochemical in-
vestigation of the PET-type probes, together with a series of
model compounds. The oxidation at about +730 mV given
for 11 in Table 4 agrees well with the oxidation of the re-
spective fragments in 3–5 (Table 6). Although not p-conju-
gated, it can be noted that the nature of the substituent at-
tached to the anthracene at the 10-position has an influence
on the stability of the free probe as well as on the stability
of the complexes, because only the aniline-centred oxida-
tions in 4, 5 and 4ꢁK+ are reversible. The redox potentials
obtained for the anthracene moieties are in agreement with
redox potentials of substituted anthracenes from the litera-
ture. The lowest reduction potentials at about ꢀ2000 mV in
3, 5 and 12 and ꢀ1500 mV in 4 are most likely anthracene-
based,[111] whereas the second, more positive reduction at
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K. Rurack, H.-J. Holdt et al.
which shifts from 293 to about 275 nm for 10 upon binding
to the alkali metal ions (Table 1), is well reflected by the
spectral changes in that region displayed by 5 (Figure 6),
where it is only seen as a shoulder on the low-energy side of
the intense anthracene absorption band at 254 nm in 5ꢁK+.
The behaviour is similar for 3 and 4. The binding constants
for 1:1 complexation have been determined to logK-
A
E
ACHTUNGTRENNUNG
logK
3.5.
E
U
G
With regard to the electrochemical behaviour, in general,
complexation with Na+ or K+ leads to a positive shift of the
oxidation potential and is slightly more pronounced for the
Na+ complexes than for the K+ complexes (Table 6).
Response of 3–5 to Na+ and K+ under simulated physiologi-
cal conditions: Although the ion-induced effects in a model
environment are favorable with respect to fluorescence
switching (up to >350), upon moving to physiological condi-
tions and assessing the sensing performance in the important
concentration range for Na+ and K+, 3–5 show only negligi-
ble changes, hence, are not qualified for signalling purposes.
The PET probes are still largely quenched in the unbound
state in water and complexation presumably does not modu-
late PET efficiently.
Figure 6. Absorption (c5 =5ꢄ10ꢀ5 m) and fluorescence spectra (inset, c3 =
5ꢄ10ꢀ6 m) of 5 and 3 in acetonitrile and corresponding titration spectra
upon addition of K+ (red, 0.25–2.83 mm; spectra of 5ꢁK+ in absorption
and 3ꢁK+ in fluorescence as blue lines).
Table 6. Selected redox potentials (peak potentials, in mV) of (3–5) and
12 in the absence and presence of NaPF6/KPF6 in acetonitrile (for com-
plete data, see Supporting Information, Table S10).[a]
3
4
5
12
free
+1360
+1660
+1280r
+740r
ꢀ1770
ꢀ2030
+1270
+940
+1470r
–
+720
ꢀ1750
ꢀ2030
+1360
+890
+750r
ꢀ1280
ꢀ1500
+1690
+910r
+1700
+850r
ꢀ1780
Spectroscopic properties of 6: Having established the validi-
ty of the “click” concept for a typical PET probe architec-
ture, our interest was to elucidate the potential of the tria-
zole-aniline unit for integration with a fluorophore that ab-
sorbs and emits in the commonly more preferred visible
spectral range. For this purpose, we chose a boron-dipyrro-
methene (BODIPY) fluorophore, which is one of the most
popular fluorophores today because of various beneficial
properties.[116,117] In addition, integration of potentially
strongly quenching analyte-responsive units in a virtually de-
coupled manner[118] through the meso-position as in 6
(Figure 1) has been shown by others and us to yield power-
ful metal ion probes.[47,48,54,119] These facts together with syn-
thetic feasibility and accessibility of the precursor com-
pounds let us pursue the design realized in 6. The quest
here was to judge whether the architecture that proved most
promising for the NI dyes also performs well with this fluo-
rophore. Table 7 shows that 6 is distinctly quenched in ace-
ꢀ2040
+Na+
+K+
–
–
–
–
+1360
+850
+1270
+880
[a] Data against Fc/Fc+. Subscript r denotes reversible reactions.
about ꢀ1760 mV for 3, 5 and 12 and ꢀ1280 mV for 4 origi-
nates from the triazole unit; such a reduction wave is lack-
ing in anthracene.[111] Considering the oxidation of anthra-
cene, it is found at about 200 mV more positive values in
the ligands than in the analogous substituted anthracenes
(see Table 6 and ref. [111]). For example, the reversible oxi-
dation of 9,10-dimethylanthracene (+1070 mV) occurs at
+1280 mV in 5, with the reversibility of the potential being
retained.[112]
Combination of the spectroscopic and the electrochemical
data allows calculation of the PET driving forces according
to the Rehm–Weller equation[113] and yields values of ꢀ0.50,
ꢀ0.84 and ꢀ0.41 eV for 3, 4 and 5, respectively, reflecting
well the distinctly stronger quenching and ion-induced reviv-
al of the fluorescence in 4 compared with 3 and 5.
Table 7. Spectroscopic properties of 6 its Na+ and K+ complexes as well
as 13 and 14.[a]
Species
labs [nm]
lem [nm]
ff
tf [ns]
6
248, 308, 498
255, 498
508
508
508
505
505
0.06
0.58[c]
2.83
2.54
3.17
<0.01
6ꢁNa+
6ꢁK+
13[d]
5.0[b]
4.5[b]
0.60
Response to Na+ and K+ under model conditions: As ex-
pected for PET probes of the present type, addition of Na+
and K+ to acetonitrile solutions of 3–5 leads to a disappear-
ance of the triazole-aniline CT band at 290 nm and a revival
of the typical anthracene emission (Figure 6), which is virtu-
ally identical for 3ꢁNa+/K+ and model compound 12. The
behaviour of the broad CT-type band at about 280 nm,
255, 498
229, 497[e]
229, 265, 496[e]
14[d]
2ꢄ10ꢀ4
[a] In acetonitrile solution. [b] Fluorescence enhancement with respect to
unbound probe. [c] Traces of a slow component are also found which are
most likely due to traces of water/protons in the solvent used. [d] From
ref. [47]. [e] Several weak BODIPY-centred bands between 285 and
420 nm not listed; 265 nm=aniline band in 14.
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Metal Ion Responsive Fluorescent Probes
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tonitrile compared to the highly fluorescent model dye 13
(Figure 2), yet it is still much better fluorescent than BODI-
PYs with a directly meso-fused phenyl-aza-crown unit like
14 (Table 7). Evaluation of the optical transitions and MO
energetics of model analogue 6a (N,N-dimethylamino ana-
logue of 6, Figure 2, Chart S1) as obtained by quantum
chemical calculations revealed that HOMO and LUMO are
both localized on the BODIPY fragment, unlike the differ-
ent localizations of the frontier MOs in 1a, 2a and 3 (Fig-
ure S2, Tables S6, S7). The lowest transition, however, is
again an oscillator-weak CT transition with a large dipole
moment difference, involving HOMOꢀ1 and LUMO. More-
over, whereas the CT state in 1a, 2a and 3 lies >0.50 eV at
lower energies in the gas phase,
gating the unit responsible for the strongly quenching ICT
process with an intrinsically highly emissive fluorophore
such as anthracene, however, not in a virtually decoupled
scenario such as in 6, but in a p-conjugated manner as in 1.
Recently, anthracene–metal complex receptor p-conjugates
were used for the detection of phosphate anions and non-co-
valent protein labelling in aqueous solution.[121] Staying with
the terminology of ICT and PET dyes, anthracene is a typi-
cal so-called locally excited or LE fluorophore, that is, a flu-
orophore for which the MOs involved in the self-centred
transitions are distributed over the entire molecular frag-
ment and dipole moment changes are usually small with Dm
ꢃ ꢄ1 D. The UV/Vis absorption spectrum of 7 in Figure 7
this difference amounts to only
0.13 eV in 6a. Thus, when at-
tached to longer wavelengths
absorbing chromophores such
as BODIPY, the quenching po-
tential of the aniline-triazole
unit seems to be gradually di-
minished.
Response to Na+ and K+: In
acetonitrile model solutions, the
response of 6 toward Na+ and
K+ is as would be expected,
that is, the fluorescence increas-
es without significant spectral
fluorescence shifts to be no-
ticed.[47,48] This is a well-known
feature of such virtually decou-
Figure 7. Absorption (c7 =5ꢄ10ꢀ5 m) and fluorescence spectra (c7 =5ꢄ10ꢀ6 m) of 7 (black lines) in acetonitrile
and corresponding titration spectra upon addition of Na+ (red, 0.05–2.80 mm; spectra of 7ꢁNa+ as blue lines).
Arrows indicate changes upon cation addition.
pled probes. Likewise, only the CT band stemming from the
triazole-aniline unit at about 295 nm, which overlaps entire-
ly with higher transitions centred on the BODIPY fragment,
is noticeably reduced while the typical BODIPY band at
498 nm is virtually unchanged (Figure S3). Since the fluores-
cence enhancement factors are much smaller than for other
virtually decoupled BODIPY probes, for example, 14 it is
not surprising that the move to physiological conditions
does not entail any pronounced effects. In addition, 6 is only
weakly soluble in water, shows signs of aggregation[120] and a
fluorescence with two maxima at about 535 and 640 nm
which is still reduced compared with MeCN. Like 3–5, the
donor-substituted 1,2,3-triazole unit does not seem to qualify
for a virtually decoupled probe architecture like in 6 under
realistic conditions.
reveals an intense band at 254 nm, a broad band with a max-
imum at 282 nm (after deconvolution) and a structured
band of moderate intensity with maxima at 349, 368, and
387 nm that partly overlaps with a broader band of compa-
rable intensity centred at 418 nm. The fluorescence of 7
shows two broad bands of low intensity, the integral fluores-
cence quantum yield ff amounting to 0.005 (Table 8, Fig-
Table 8. Spectroscopic properties of 7 and its Na+ and K+ complexes.[a]
Species
labs [nm]
lem [nm]
ff
7
254, 282, 349, 368, 387, 418
254, 352, 369, 388
254, 350, 369, 388, 414
254, 332, 348, 365, 385
415, 539[b]
402, 423, 446
402, 420, 445
403, 422
0.005
125[c]
13[c]
7ꢁNa+
7ꢁK+
15
0.45
[a] In acetonitrile, c7 =5ꢄ10ꢀ6 m. [b] For spectrum, see Figure S4. [c] Flu-
orescence enhancement with respect to unbound probe.
Spectroscopic properties of 7 and response to Na+ and K+
under model conditions: So far, the introduction of the tria-
zole–aniline-receptor moiety into a typical ICT, PET and vir-
tually decoupled CT probe architecture revealed good to ex-
cellent performance under ideal conditions. Under more re-
alistic conditions, however, only the performance of 1 was
promising yet cation-induced effects were still not ideal to
qualify the probe for actual applications in alkali metal ion
indication. We thus strived to improve signalling by p-conju-
ure S4). Such unstructured dual bands of reduced intensity
are well-known for donor-substituted anthracenes, the blue-
shifted band representing the residual LE fluorescence and
the red-shifted band stemming from a weakly emissive CT
state.[122]
Before we attempt an assignment of the absorption bands
to illustrate design, it is helpful to inspect the spectroscopic
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K. Rurack, H.-J. Holdt et al.
Table 9. Calculated properties of the six lowest vertical transitions of the
optimized ground-state geometry of 7 by TD-DFT (for additional data,
see Table S8).
changes induced by the complexation of alkali metal ions.
As is evident from Figure 7, only the intense band at 254 nm
and the structured bands in the 350–400 nm region remain
unchanged upon addition of a metal ion in MeCN. The
broad bands at 418 and 282 nm are significantly diminished
and blue-shifted. The fluorescence titration spectra indicate
that the emission of 7 is strongly enhanced. The band at
539 nm vanishes and the band shape of the spectrum of
7ꢁM+ is reminiscent of the structured anthracene emission.
Moreover, the fluorescence enhancement is larger for Na+
(125-fold) compared with K+ (13-fold). In contrast, the
complex stability constant is higher for K+ (logK=3.0 vs
logK=2.6 for 7ꢁNa+). The latter behaviour can be rational-
ized in terms of the better fit-in-size of K+ into the 18-mem-
bered macrocycle[103,104] and the stronger enhancement in-
duced by Na+ is presumably due to the higher charge densi-
ty and stronger ion–dipole interaction of this ion with the ni-
trogen bridgehead atom, especially in a solvent like acetoni-
trile that does not coordinate strongly with alkali metal ions
(see also discussion for 1 and 2 above). Both cation-induced
changes, however, suggest that the bands at 282 and 418 nm
are typical CT bands the intensity of which is strongly re-
duced upon engagement of the nitrogen atom of the N-phe-
nylaza-[18]crown-6 in complexation.
!
!
S0
Sn lS
(n)
f[b]
DmS
n
[D][c]
Orbitals (coeffients)[d]
S0
n
[nm][a]
S1 396.8
0.181 +3.4
HOMO–LUMO (0.64)
HOMOꢀ2–LUMO+3 (ꢀ0.11)
HOMOꢀ1–LUMO (0.70)
6 mixed (mainly A)
6 mixed (incl. HOMO–
LUMO+1)
S2 367.4
S3 322.2
S4 315.2
0.001 +34.9
0.002 ꢀ1.3
0.033 +12.8
S5 288.8
S6 288.4
0.157 ꢀ1.0
0.493 +6.7
6 mixed (mainly AT)
6 mixed (mainly AT)
[a] Wavelength of the transition. [b] Oscillator strength of the transition.
[c] Dipole moment difference between ground (m0) and respective excited
(mn)state. [d] MOs involved in the transitions. 6 mixed=six different
MOs involved in mixed transition; mainly A/AT=mainly localized on
anthracene/aniline-triazole fragment.
ed by the HOMO–LUMO transition with some admixture
of a HOMOꢀ2–LUMO+3 transition, all MOs being mainly
localized on the anthracene moiety; oscillator strength f and
dipole moment change Dm are moderate to weak. According
to Figure 8 and Table 9, the next lowest HOMOꢀ1–LUMO
transition shows all characteristics of a strong CT process
with significant charge separation between two twisted frag-
ments, that is, a large Dm=35 D and a low f=0.001. The two
next higher transitions are mixed transitions, largely involv-
ing MOs localized on the anthracene fragment with some
The unperturbed bands at 254 nm and 355–395 nm, to-
gether with the characteristic fluorescence spectrum, can
thus be ascribed to the typical anthracene-localized transi-
tions. These findings stress the previously outlined consider-
ations that the anthracene fluorophore is the LE partner.
!
CT character (anthracene to triazole–aniline) for S4. S5 S0
!
and in particular S6 S0 with moderate to high f are also
Signalling process in 7: To theoretically underpin the signal-
ling mechanism, quantum chemical calculations were also
performed for 7. The optimized geometry of 7 reveals that
the phenyl and triazole rings are twisted by 29.78 and the tri-
azole and anthryl moieties by 58.58.[123] Figure 8 collects the
frontier and next closest molecular orbitals (MOs) as de-
rived from TD-DFT calculations. The corresponding data of
the six lowest transitions are included in Table 9. For the iso-
lated molecule in the gas phase, excitation to S1 is dominat-
mixed transitions, now with a certain CT character within
the triazole-aniline fragment.[124] With respect to the absorp-
tion spectra in Figure 7, the transitions to S6 (and S5) are at-
tributed to the band at 282 nm, the CT in the aniline–tria-
zole fragment, and the fourth to first lowest transitions,
except for S2, to the typical anthracene-like and other weak
bands at 300–400 nm. The S2 transition finally is assigned to
the unstructured absorption band at 418 nm. The discrepan-
cy between theory (in the gas phase) and experiment (in
acetonitrile) especially for the latter is evident when consid-
ering the stabilization that such a highly dipolar state experi-
ences in a polar solvent such as acetonitrile. The large
dipole moment difference presumably will lead to a state re-
versal and the HOMOꢀ1–LUMO transition with a strong
CT character will become the lowest transition, whereas the
former S1 will become S2.
Having established the photophysics in free 7, the proc-
esses happening upon complexation are obvious. Interaction
of the cation with the crown ether nitrogen atom influences
the electron donating character of the triazole–aniline frag-
ment, resulting in a diminution of all CT bands accompanied
by a strong, up to 125-fold enhancement of the anthracene
or LE fluorescence. The design is thus based on a combina-
tion of a non-emissive CT process with a typical LE fluoro-
phore in such a way that in the unbound state the lowest-
lying transition is the CT and the second lowest the LE tran-
sition—hence, the free probe is virtually non-emissive—and
Figure 8. Frontier molecular orbitals of geometry-optimized 7. Arrows
denote major transitions between MOs mainly responsible for the S1
(dashed), S2 (solid) and S6 (dotted) transition in the gas phase (for addi-
tional MOs, see Figure S5).
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Metal Ion Responsive Fluorescent Probes
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upon binding, the order of the states is reversed, switching
the non-fluorescent CT for the strongly emissive LE state.
Because the dye is entirely p-conjugated, the LE fluoro-
phore acts at the same time as the acceptor in the CT proc-
ess. In contrast to known CT/LE probes,[125,126] this approach
has the advantage that the LE state by design is highly emis-
sive and that absorption spectral shifts are immanent to the
system, which is for instance not the case for virtually de-
coupled “switch-on” probes.[118,127] If one reconsiders now
the picture for 1 and 2 given above, it is obvious that the an-
seems very promising for the construction of potent fluores-
cent probes under realistic conditions.
Conclusion
The present report has shown how the different ways of in-
corporating a donor-substituted 1,2,3-triazole unit into con-
ventional and newly conceived fluorescent probe architec-
tures can lead to functional dye molecules that respond to
metal ions with different combinations of absorption and
fluorescence spectroscopic effects. Whereas the installation
of two vectorial ICT processes in 1 and 2 led to absorption
spectral shifts and a potent switching-on of the fluorescence
in organic environments, aqueous solvents only gave a negli-
gible response. Regioisomeric integration clearly seems to
have an effect on the complex stability and fluorescence en-
hancement of these p-conjugated fluoroionophores.[72] It
should be noted that even for organic solvents, fluorescence
light-up factors of >100 are very rarely found for ICT-type
probes. The concept of integration in actually or virtually
decoupled architectures gave varying light-up factors in ace-
tonitrile but failed under physiological conditions, most
likely because the photophysical processes are not directly
coupled. However, especially for BODIPY dyes with their
many attachment points for functional units, 3,5-integration
might open up more promising possibilities. Newly designed
7, however, showed favorable K+-sensing features under re-
alistic conditions, even though it carries only a rather
weakly binding receptor unit such as aza-[18]crown-6. The
lighting-up mechanism proved to be especially interesting:
Upon ion binding, the CT state is energetically shifted up-
wards and LE becomes S1, that is, state reversal occurs re-
sulting in a fluorescence lighting-up. Moreover, the concept
of using the donor-acceptor-substituted 1,2,3-triazole unit as
relay elaborates our earlier approaches toward probes with
more than one active photophysical process[131] in the sense
!
alytically addressable process (S2 S0 in the gas phase and
!
most likely S1 S0 in the condensed phase) is much better
fine-tuned with regard to the highly emissive process in 7.
Response to Na+ and K+ under physiological conditions:
Based on the favorable features reported for 7 in model
media, the next step was to test the validity of the design
concept under realistic conditions. Under simulated physio-
logical conditions, the spectral features of 7 are not signifi-
cantly changed with respect to acetonitrile—absorption
bands at 255, 286, 351, 369, 388 and 423 nm and fluores-
cence at 427 nm—and the probe is also only weakly fluores-
cent (ff = 6ꢄ10ꢀ3). Moreover, in the more competitive sol-
vent, Na+ is barely bound and only K+ modulates the fluo-
rescence to a sizeable degree, that is, leads to a 12-fold in-
crease of the typical anthracene emission (Figure 9); for
comparison: Na+ only leads to a two-fold increase. If we
further approach realistic conditions and add 2 mm of poten-
tially competing Ca2+ and Mg2+ ions to the Tris-buffered
and choline-containing medium and assess the relevant con-
centration range for K+ sensing (0–160 mm K+), the fluores-
cence increase is certainly lower but still amounts to a 3-fold
increase for 160 mmK+. In addition to the analytical silence
of Na+, it is important to note that 2 mm of Ca2+ and Mg2+
also do not interfere with K+ indication. Although the
crown unit chosen as model in our studies is not the ideal
receptor for targeting alkali metal ions in water, the concept
Figure 9. Left: Fluorescence spectrum of 7 (thick black line, c7 =7ꢄ10ꢀ6 m) in aqueous solution (buffered at pH 7.2, 10 mm Tris, ꢂ 1500 mm choline chlor-
ide for maintaining constant ionic strength) and corresponding titration spectra upon addition of K+ (5–1500 mm; spectrum of 7ꢁK+ as blue line).
Right: Titration curve plotted according to a formalism for weak complexes F /
metal ion concentration cM =0, F=fluorescence intensity for steps of metal ion addition)[130] for 7 and K+ (black squares, r2 =0.98) and Na+
similar conditions.
(F0ꢀF) vs. cꢀ1 m (F0 =fluorescence intensity of unbound 7 at 435 nm and
*
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K. Rurack, H.-J. Holdt et al.
porting Information)[79] and the 6-azido-N-ethyl-1,8-naphthalimide (21,
Supporting Information)[62,144] followed reported procedures and referen-
ces therein.
that 7 actually performs under realistic conditions. This im-
portant practical implication becomes possible by the choice
of a suitable LE fluorophore that fluoresces strongly in
water. Current research in our groups focuses on the inte-
gration of specific and more potent alkali and alkaline-earth
metal ion receptors, to possibly improve the performance of
established ion indication strategies, the application of the
probes for K+, Na+ and Ca2+ in biological samples and the
further elucidation whether the combination of the aniline–
triazole unit with other long wavelength-absorbing fluoro-
phores also allows to construct powerful probes or whether
an electronic modification of the relay unit might be neces-
sary. In our view, especially the modularity of the approach
is very appealing for approximating other potent fluoro-
phore-receptor combinations.
9-Azidomethyl-anthracene (22): The synthesis followed the literature ac-
cording to a modified procedure.[86] A suspension of 9-chloromethylan-
thracene (1.00 g, 4.41 mmol) and sodium azide (0.43 g, 6.62 mmol,
1.5 equiv) in acetonitrile (30 mL) was refluxed for 5 h. After the reaction
was complete (monitoring via DC) the reaction mixture was cooled to
room temperature and the resulting solid was filtered off. The solution
was concentrated in vacuo and the resulting yellow solid was purified by
column chromatography (silica gel, n-hexane/ethyl acetate 3:1). Yield:
1
0.92 g (90%); m.p. 78–808C (lit.: 80–828C); H NMR (CDCl3, 500 MHz):
d = 8.52 (s, 1H, H5), 8.29 (d, 2H, J=8.85 Hz, H1), 8.05 (d, 2H, J=
8.40 Hz, H4), 7.60 (m, 2H, H2), 7.52 (m, 2H, H3), 5.31 ppm (s, 2H, H5);
13C NMR (CDCl3, 125 MHz): d = 131.45, 130.79, 129.39, 129.09, 126.94,
125.87, 125.30, 123.31, 46.42 ppm; FT-IR (KBr): n=2095 (s), 1444 cmꢀ1
(m); MS (EI): m/z (%): 233 (100) [M+].
9-Azidomethyl-10-cyanoanthracene (23):[86,87] The synthesis followed the
procedure for the preparation of 22 starting from 9-cyano-10-methylan-
thracene (23b, Supporting Information). The crude product was re-crys-
tallized from CH2Cl2/MeOH to give the product as yellow fluffy needles.
Yield: 0.10 g, 39%; 1H NMR (CDCl3, 300 MHz): d = 8.48 (d, 2H, J=
8.84 Hz, H1), 8.35 (d, 2H, J=8.13 Hz, H4), 7.79–7.65 (m, 4H, H2, H3),
5.33 ppm (s, 2H, H5); 13C NMR (CDCl3, 75 MHz): d = 133.04, 132.90,
129.94, 128.85, 127.84, 126.52, 124.48, 117.07, 108.08, 46.29 ppm; FT-IR
(KBr): n=2213 (s), 2100 (s), 1444 cmꢀ1 (m); MS (+ESI): m/z: calcd for
C16H11N4: 259.10; found: 259.15.
Experimental Section
General methods and procedures: All commercially available chemicals
were used without further purification. Solvents were distilled prior use.
Air/water-sensitive reactions were performed in oven-dried glassware
under an argon atmosphere. Column chromatography was performed
with SiO2 (Merck Silica Gel 60 (0.04–0.063 mesh)).
9-Azidomethyl-10-methylanthracene (24): The synthesis followed the
procedure for the preparation of 22 using 9-bromomethyl-10-methylan-
thracene (24a, Supporting Information).[86, 88] Yield: 0.98 g, 90%;
1H NMR (CDCl3, 300 MHz): d = 8.39–8.31 (m, 4H, H2, H5), 7.63–7.53
(m, 4H, H3, H4), 5.34 (s, 2H, H1), 3.14 ppm (s, 3H, H6); 13C NMR
(CDCl3, 75 MHz): d = 132.91, 130.62, 130.03, 126.44, 125.74, 125.22,
124.29, 46.69, 14.64 ppm; FT-IR (KBr): n=2214 (s), 2114 (s), 1444 cmꢀ1
(m); MS (EI): m/z (%): 247 (100) [M+].
Optical spectroscopy: UV/Vis measurements were recorded on a Perkin–
Elmer Lambda 950 spectrophotometer using 1 cm path length quartz
cuvettes. Titrations of the compounds 1–7 (c=5ꢄ10ꢀ5 m) were carried out
in acetonitrile and recorded 5 min after the addition of 0.01 mL of volu-
metric standard solutions of respectively NaPF6 or KPF6 (c=5ꢄ10ꢀ4–5ꢄ
10ꢀ2 m) in acetonitrile. To ensure complete reaction towards the corre-
sponding complex, titration was continued until no change in the UV/Vis
absorption spectra was observed. Fluorescence titration spectra of the
compounds 1–7 (c=5ꢄ10ꢀ6 m) in acetonitrile were recorded 5 min after
the addition of 0.02 mL of volumetric standard solution of NaPF6 or
KPF6 (c=5ꢄ10ꢀ5–5ꢄ10ꢀ2 m), respectively. Titration was continued until
no change in fluorescence enhancement was observed. Fluorescence
quantum yields were determined using a PL Quantum Yield measure-
ment System C9920-2 from Hamamatsu and with the relative approach
according to Parker and Rees[136] on a Fluoromax 4 from Horiba Jobin–
Yvon using quinine sulfate in 0.105m perchloric acid (ff =0.60)[137] or
coumarin 153 in ethanol (ff =0.54)[138] as the standard. Fluorescence life-
times (t) were determined with a FL920 fluorescence lifetime spectrome-
ter (Edinburgh Instruments) with a Ti/Sa laser system in the frequency-
doubled or tripled mode and with a unique customized laser impulse flu-
orometer with picosecond time resolution described in.[52,139] The fluores-
cence decays were recorded with modular single photon timing units. The
fluorescence lifetime profiles were analyzed with a PC using the software
package Global Unlimited V2.2 (Laboratory for Fluorescence Dynamics,
University of Illinois). The goodness of the fit of the single decays as
judged by reduced chi-squared (cR2) and the autocorrelation function
C(j) of the residuals was always below cR2 <1.2.
4,4-Difluoro-8-(4-trimethylsilylethinylphenyl)-1,3,5,7-tetramethyl-4-bora-
3a,4a-diaza-s-indacene (25b): The synthesis of 4-iodophenyl derivative
25a (see also Supporting Information) followed the literature according
to a modified procedure.[80,81] The reaction towards (25b) followed a liter-
ature procedure for
a 2ACHTNUTGNRUEGN(PPh3)2] (91.2 mg,
similar reaction.[83] [PdCl
0.13 mmol, 5.0 mol%), CuI (24.8 mg, 0.13 mmol, 5.0 mol%), TEA
(770 mL, 5.5 mmol, 2.2 equiv) and trimethylsilylacetylene (0.469 mL,
5.0 mmol, 2.0 equiv) were placed in a dry round-bottom flask before a
solution of 25a (740 mg, 2.5 mmol) in dry DMF (25 mL) was added. The
reaction mixture was stirred for 5 h at 608C, then cooled to room temper-
ature, diluted with H2O and extracted with CH2Cl2 (100 mL). The com-
bined organic phases were dried over MgSO4, concentrated and the
crude product was purified by column chromatography (silica gel,
hexane/ethyl acetate 4:1 v/v), to give 25b as a purple-red solid. Yield:
0.467 g, 67%; 1H NMR (CDCl3, 500 MHz): d = 7.60 (d, 2H, J=8.1 Hz,
H5), 7.23 (d, 2H, J=8.1 Hz, H4), 5.98 (s, 2H, H2), 2.55 (s, 6H, H3), 1.39
(s, 6H, H1), 0.28 ppm (s, 9H, H6); 13C NMR (CDCl3, 125 MHz): d =
155.89, 143.13, 140.88, 135.35, 132.86, 131.28, 128.21, 124.06, 121.49, 95.96,
29.84, 14.27, 1.17 ppm; 19F NMR (CDCl3, 300 MHz): d = ꢀ146.7 ppm (m,
2F); EI-MS: m/z (%): 420 (100) [M+].
Electrochemical measurements: Electrochemical data were determined
with the electrochemical analyzer PGSTAT302N (Metrohm, Switzerland)
and cell stand C3 (Bioanalytical Systems, USA) using a platinum disc
working electrode (2 mm2), a platinium wire auxiliary electrode and an
anhydrous Ag/Ag+-reference electrode.
4,4-Difluoro-8-(4-ethinylphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-
indacene (25): The TMS-deprotection of 25b (Supporting Information)
was followed a literature procedure according to a similar reaction.[82]
Compound 25b (100 mg, 0.239 mmol) was dissolved in a mixture of
THF/MeOH (45 mL, 4:1 v/v) before a solution of tetrabutylammonium-
fluoride (1m in THF, 45 mL, 0.357 mmol, 1.5 equiv) was added. Stirring
at room temperature was continued for 19 h. The reaction mixture was
concentrated in vacuo and purified by column chromatography (silica
gel, hexane/ethyl acetate 4:1 v/v) to give 25 as an orange solid (0.060 g,
73%). M.p. 245.5–250.18C; 1H NMR (CDCl3, 125 MHz): d = 7.85 (d,
2H, J=8.3 Hz, H5), 7.05 (d, 2H, J=8.3 Hz, H4), 5.99 (s, 2H, H2), 2.55 (s,
6H, H3), 2.17 (s, 1H, H6), 1.42 ppm (s, 6H, H1); 13C NMR (CDCl3,
500 MHz): d = 156.88, 143.88, 141.05, 139.33, 135.56, 132.13, 130.95,
Computational details: The optimization of the S0 ground state geome-
tries in the gas phase was performed with the density functional theory
(DFT) method employing the hybrid functional B3LYP with a 6-31G-
ACHTUNGTRENNUNG
(d,p) basis set and energy-minimized as implemented in Gaussian 03.[140]
Synthetic procedures: The preparation of N-phenylaza-[18]crown-6 ether
(11),[141] N-(4-ethinyl)phenylaza-[18]crown-6 ether (16),[72,85] N-(4-azido)-
phenylaza-[18]crown-6 ether (17),[72,77,78] 9-ethinylanthracene (18),[84] 1-
azidooctane (19),[142,143] N-(n-butyl)-4-ethinyl-1,8-naphthalimide (20, Sup-
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Metal Ion Responsive Fluorescent Probes
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122.43, 95.69, 15.67, 15.57 ppm; 19F NMR (CDCl3, 300 MHz):
d
=
51.22, 46.27 ppm; HRMS (+ESI): m/z: calcd for C36H40N5O5: 622.3029;
found: 622.3065.
ꢀ146.6 ppm (m, 2F); EI-MS: m/z (%): 348 (100) [M+].
General CuAAC procedure: The reaction followed the literature.[145] To
the stirred suspension of Cu/C (0.1 equiv) in THF (3 mL) was added trie-
thylamine (1.0 equiv) followed by the azide derivative (1.0 equiv) and the
acetylene derivative (1.0 equiv). The reaction mixture was stirred at 608C
for 16 h after which an additional portion of the Cu/C (0.1 equiv) was
added. It was heated for two days and cooled to room temperature
before the Cu/C was filtered off through celite. Concentration of the sol-
ution gave the crude product which was purified by column chromatogra-
phy.
10-Methyl-9-{4-[4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phen-
yl]-1H-1,2,3-triazol-1-yl}-methylanthracene (5): Compounds 16 and 24
were reacted according to the general CuAAC procedure. The crude
product was purified by column chromatography (CHCl3/MeOH 97:3 v/
v) with subsequent recrystallization from CH2Cl2/nhexane/ethyl acetate
1:1:0.25 v/v/v), to give 5 in form of brownish crystals (64 mg, 48%). M.p.
148.7–151.38C; 1H NMR (CDCl3, 500 MHz): d = 8.41 (dd, 2H, J=7.65,
0.60 Hz, H2), 8.41–8.31 (m, 2H, H5), 7.61–7.56 (m, 4H, H3, H4), 7.44 (d,
2H, J=8.9 Hz, H8), 7.11 (s, 1H, H7), 6.57 (d, 2H, J=8.9 HZ, H9), 6.55 (s,
2H, H6), 3.66–3.56 (m, 24H, H10–15), 3.19 ppm (s, 3H, H1); 13C NMR
(CDCl3, 125 MHz): d = 148.29, 147.73, 133.90, 130.73, 130.14, 127.22,
126.84, 126.15, 125.89, 125.44, 123.88, 122.50, 118.39, 117.71, 111.67, 70.97,
70.92, 70.87, 70.82, 68.74, 51.35, 46.79, 14.78 ppm; HRMS (+ESI): m/z
calcd for C36H43N4O5: 611.3233; found: 611.3279.
2-Butyl-6-{1-[4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-
1H-1,2,3-triazol-4-yl}-1H-benzo[de]isoquinoline-1,3(2H)-dione (1): Com-
pounds 17 and 20 (Supporting Information) were reacted according to
the general CuAAC procedure. The crude product was purified by
column chromatography (silica gel, CHCl3/MeOH 98:2 v/v) to yield 1 as
a yellow solid (0.086 g, 49%). M.p. 136.1–139.08C; 1H NMR (CDCl3,
600 MHz): d = 9.1 (dd, 1H, J=8.55, 1.05 Hz, H9), 8.63 (dd, 1H, J=7.02,
1.02 Hz, H7), 8.61 (d, 1H, J=8.55 Hz, H5), 8.26 (s, 1H, H10), 7.99 (d, 1H,
J=7.56 Hz, H6), 7.79 (dd, 1H, J=8.52, 7.26 Hz, H8), 7.61 (d, 2H, J=
9.12 Hz, H11), 6.81 (d, 2H, J=9.12 Hz, H12), 4.18 (t, 2H, J=7.62 Hz, H4),
3.74–3.64 (m, 24H, H13–18), 1.76–1.69 (m, 2H, H3), 1.45 (sx, 2H, J=
7.62 Hz, H2), 0.98 ppm (t, 3H, J=7.38 Hz, H1); 13C NMR (CDCl3,
150 MHz): d = 164.34, 164.06, 148.68, 146.15, 145.66, 134.32, 132.92,
131.52, 130.81, 129.40, 128.95, 127.48, 127.35, 125.96, 122.94, 122.56,
121.52, 111.99, 70.98–70.82,68.8, 68.60, 51.59, 40.41, 30.32, 20.52,
13.99 ppm; HRMS (+ESI): m/z: calcd for C36H44N5O7: 658.3242; found:
658.3220.
4,4-Difluoro-8-(4-{1-[4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)-
phenyl]-1H-1,2,3-triazol-4-yl}phenyl)-1,3,7,9-tetramethyl-4-bora-3a,4a-
diaza-s-indacene (6): Compounds 17 and 25 were reacted according to
the general CuAAC procedure. Crude 6 was purified by column chroma-
tography (silica gel, CHCl3/MeOH/hexane 80:7:13 v/v/v) to give 6 in the
form of reddish crystals (0.040 g, 28%). M.p. 197.2199.48C; 1H NMR
(CDCl3, 600 MHz): d = 8.14 (s, 1H, H6), 8.04 (d, 2H, J=8.3 Hz, H5), 7.56
(d, 2H, J=8.9 Hz, H7), 7.38 (d, 2H, J=8.3 Hz, H4), 6.79 (d, 2H, J=
8.9 Hz, H8), 5.99 (s, 2H, H3), 3.75 (t, 4H, J=5.5 Hz, H9), 7.70–3.76 (m,
20H, H10–14), 2.57 (s, 6H, H2), 1.46 ppm (s, 6H, H1); 13C NMR (CDCl3,
150 MHz): d = 155.72, 147.24, 143.27, 141.41, 134.90, 131.54, 128.77,
126.52, 122.36, 121.42, 118.28, 111.99, 71.02, 70.97, 70.93, 68.65, 51.63,
29.85, 14.79 ppm; 19F NMR (CDCl3, 300 MHz): d = ꢀ146.7 ppm (m, 2F);
HRMS (+ESI): m/z calcd for C39H48BF2N6O5: 729.3747; found: 729.3746.
2-Ethyl-6-{4-[4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-
1H-1,2,3-triazol-1-yl}-1H-benzo[de]isoquinoline-1,3(2H)-dione (2): Com-
pounds 16 and 21 (Supporting Information) were reacted according to
the general CuAAC procedure. The crude product was purified by
column chromatography (silica gel, CHCl3/MeOH 95:5 v/v) to yield 2 as
an orange solid (0.024 g, 19%). M.p. 213.6–216.48C; 1H NMR (CDCl3,
600 MHz): d = 8.71 (t, 2H, J=7.47 Hz, H3, H5), 8.36 (d, 1H, J=8.1 Hz,
H7), 8.09 (s, 1H, H8), 7.89 (d, 1H, J=7.47 Hz, H4), 7.83 (tr, 1H, J=
5.26 Hz, H6), 7.79 (d, 2H, J=8.7 Hz, H9), 6.79 (d, 2H, J=8.82 Hz, H10),
4.28 (q, 2H, J=7.12 Hz, H2), 3.74–3.68 (m, 24H, H11–16), 1.36 ppm (t, 3H,
J=7.12 Hz, H1); 13C NMR (CDCl3, 150 MHz): d = 163.68, 163.16, 149.13,
148.48, 138.59, 132.29, 130.82, 129.87, 129.29, 128.63, 127.31, 126.71,
123.93, 123.46, 123.19, 120.20, 117.23, 112.01, 71.04–70.93, 68.82, 51.50,
35.93, 13.47 ppm; HRMS (+ESI): m/z: calcd for C34H40N5O7: 630.2928;
found: 630.2883.
9-{1-[4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1H-
1,2,3-triazol-4-yl}-anthracene (7): Compounds 17 and 18 were reacted ac-
cording to the general CuAAC procedure. The crude product was puri-
fied by column chromatography (65ꢄ2 cm, silica gel, CHCl3/MeOH 95:5
v/v) to yield 7 as a light brown oil (0.021 g, 23%). 1H NMR (CDCl3,
300 MHz): d=8.56 (s, 1H, H1), 8.11 (s, 1H, H6), 8.10–8.00 (m, 2H, H2),
7.99–7.93 (m, 2H, H5), 7.55–7.40 (m, 4H, H3, H4), 6.87 (d, 2H, J=
9.12 Hz, H7), 6.67 (d, 2H, J=9.12 Hz, H8), 3.73–3.59 ppm (m, 24H, H9–
14);13C NMR (CDCl3, 75 MHz):d= 145.62, 148.02, 131.34, 131.02, 128.64,
126.89, 126.07, 124.54, 125.48, 125.21, 122.07, 119.95, 111.24, 70.85, 68.37,
51.16 ppm; MS (+ESI): m/z calcd for C34H39N4O5: 583.29; found: 583.34.
2-Ethyl-6-(4-octyl-1H-1,2,3-triazol-1-yl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (9): 1-Decyne (0.021 g, 0.015) and 21 (0.04 g, 0.15 mmol)
were reacted according to the general CuAAC procedure. The crude
product was purified by column chromatography (silica gel, CHCl3/
MeOH 250:1 v/v) to give 9 as an orange semi-solid (38 mg, 63%).
1H NMR (CDCl3, 600 MHz): d = 8.68 (d, 2H, J=7.44 Hz, H9, H5), 8.27
(d, 1H, J=8.58 Hz, H8), 7.83–7.80 (m, 2H, H8,H6), 7.78 (s, 1H, H10), 4.26
(q, 2H, J=7.08 Hz, H2), 2.8 8(t, 2H, J=7.77 Hz, H11), 1.80 (qu, 2H, H12),
1.30 (m, 2H, H13), 1.39–1.24 (m, 11H, H1, H14–18), 0.88 ppm (t, 3H, J=
7.0 Hz, H18); 13C NMR (CDCl3, 150 MHz): d = 163.63, 163.12, 149.41,
138.62, 132.24, 130.77, 129.76, 129.22, 128.58, 126.66, 123.87, 123.16,
123.14, 35.90, 31.99, 29.50, 29.47, 29.37, 25.78, 22.80, 14.24, 13.44 ppm;
MS (EI): m/z (%): 404 (100) [M+].
9-4-[4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1H-
1,2,3-triazol-1-yl}-methylanthracene (3): Compounds 16 and 22 were re-
acted according to the general CuAAC procedure. The crude product
was purified by column chromatography (silica gel, CHCl3/MeOH 97:3 v/
v) to give
3 as a
yellow solid (0.029 g, 22%). 1H NMR (CDCl3,
600 MHz): d = 8.59 (s, 1H, H1), 8.36 (d, 2H, J=8.70 Hz, H5), 8.09 (d,
2H, J=8.2 Hz, H2), 7.74–7.44 (m, 6H, J=8.2 Hz, H4,H3, H8, H2, H3), 7.15
(s, 1H, H7), 6.59 (d, 2H, J=8.65 Hz, H9), 6.55(s, 2H, H6) 3.72–3.49 ppm
(m, 24H, H10–15); 13C NMR (CDCl3, 150 MHz):
d = 148.40, 147.78,
131.66, 131.06, 129.91, 129.58, 127.77, 126.87, 125.57, 124.18, 123.27,
118.37, 117.66, 111.70, 70.98, 70.94, 70.89, 70.84, 68.76, 51.39 ppm; HRMS
(+ESI): m/z calcd for C35H41N4O5: 597.3070; found: 597.3120.
1-1-[4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1H-
1,2,3-triazol-4-yl}-octan (10): Compound 17 (0.05 g, 0.13 mmol) and 1-
decyne (0.018 g, 0.13 mmol) were dissolved in THF/water (3:1 v/v, 4 mL)
and CuSO4 (1.7 mg, 0.007 mmol, 5 mol%) and sodium ascorbate (2.6 mg,
0.013 mmol, 10 mol%) were added.[66] The reaction mixture was stirred
at 608C for 16 h. Aqueous NH4Cl (10%, 4 mL) was added and the THF
was removed under reduced pressure. The aqueous layer was extracted
with dichloromethane (3ꢄ7 mL), the organic layer was dried with
MgSO4 and concentrated. The crude product was purified by column
chromatography (silica gel, ethyl acetate/MeOH 7:3) to give 11 as a
greenish oil (0.072 g, >99%). 1H NMR (CDCl3, 300 MHz): d = 7.56 (s,
1H, H9), 7.58 (d, 2H, J=9.04 Hz, H10), 6.76 (d, 2H, J=7.35 Hz, H11),
3.74–3.66 (m, 24H, H12–17), 2.76 (tr, 2H, J=7.53 Hz, H8), 1.71 (qu, 2H,
J=7.72 Hz, H7), 1.41–1.22 (m, 10H, H2–6), 0.87 ppm (tr, 3H, J=6.97 Hz,
10-Cyano-9-{4-[4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phen-
yl]-1H-1,2,3-triazol-1-yl}-methylanthracene (4): Compounds 16 and 23
were reacted according to the general CuAAC procedure. Crude 4 was
purified by column chromatography (silica gel, CHCl3/MeOH 97:3 v/v)
with subsequent recrystallization from CH2Cl2/petrol ether (50–708C) 1:1
v/v, to give yellow crystals (0.028 g, 20%). M.p. 199.5–203.98C; 1H NMR
(CDCl3, 600 MHz): d = 8.53 (d, 2H, J=8.64 Hz, H1), 8.49 (d, 2H, J=
8.76 Hz, H4), 7.77 (m, 2H, H2), 7.72 (m, 2H, H3), 7.45 (d, 2H, J=9.0 Hz,
H7), 7.18 (s, 1H, H6), 6.59 (d, 2H, J=8.8 Hz, H8), 6.57 (s, 2H, H5), 3.65–
3.56 ppm (m, 24H, H9–15); 13C NMR (CDCl3, 150 MHz): d = 148.68,
147.83, 133.00, 130.78, 130.05, 128.94, 128.52, 126.77, 126.59, 124.02,
117.74, 117.34, 116.81, 111.57, 108.84, 70.85, 70.80, 70.75, 70.70, 68.59,
Chem. Eur. J. 2013, 00, 0 – 0
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K. Rurack, H.-J. Holdt et al.
H1); 13C NMR (CDCl3, 75 MHz): d = 149.09, 122.44 (3ꢄ), 119.24, 112.79,
70.82–70.74, 68.86, 52.26, 32.24, 29.86, 29.74, 29.68, 29.59, 26.11, 23.03,
14.44 ppm; HRMS (EI): m/z calcd for C28H47N4O5: 518.3546; found:
518.3545.
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spectral shifts, yet weak fluorescence intensity changes.[24, 25] In the
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9-[4-(Octyl)-1H-1,2,3-triazol-1-yl]-methylanthracene (12): The reaction
followed the procedure outlined for 10 using 22 (0.07 g, 0.3 mmol) and 1-
decyne (0.041 g, 0.3 mmol). The crude product was purified by column
chromatography (silica gel, ethyl acetate/hexane 1:1) to give 12 as a
yellow solid (0.081 g, 72%). M.p. 1138C, 1H NMR (CDCl3, 300 MHz): d
= 8.58 (s, 1H, H15), 8.33 (d, 2H, J=8.10 Hz, H14), 8.08 (d, 2H, J=
8.30 Hz, H11), 7.60- 7.50 (m, 4H, H12, H13), 6.82 (s, 1H, H9), 6.50 (s, 2H,
H10), 2.51 (t, 2H, J=7.72 Hz, H8), 1.48 (qu, 2H, J=7.72 Hz, H7), 1.26–
1.17 (m, 10H, H2–6), 0.83 ppm (t, 3H, J=6.59 Hz, H1); 13C NMR (CDCl3,
75 MHz): d = 138.55, 137.60, 131.53, 130.91, 129.68, 129.43, 128.09,
127.55, 125.39, 124.20, 123.77, 123.144, 120.93, 119.99, 48.72, 46.33, 31.75,
29.29, 29.17, 29.09, 25.68, 22.57, 13.99 ppm; HRMS (EI): m/z calcd for
C25H30N3: 372.2440; found: 372.2442.
9-[1-(Octyl)-1H-1,2,3-triazol-4-yl]-anthracene
(15):
Compound
18
(0.169 g, 0.084 mmol) and 19 (0.13 g, 0.084 mmol) were reacted according
to the general CuAAC procedure. The crude product was purified by
column chromatography (silica gel, CHCl3) to give 15 as a dark orange
oil (0.106 g, 35%). 1H NMR (CDCl3, 600 MHz): d = 8.53 (s, 1H, H1),
8.04 (dd, 2H, J=8.07, 0.69 Hz, H5), 7.82 (dd, 2H, J=7.92, 0.96 Hz, H2),
7.76 (s, 1H, H6), 7.46 (m, 2H, H4), 7.40 (m, 2H, H3), 4.57 (t, 2H, J=
7.23 Hz, H7), 2.09 (qu, 2H, J=7.35 Hz, H8), 1.5–1.3 (m, 10H, H),
0.90 ppm (t, 3H, J=6.96 Hz, H14); 13C NMR (CDCl3, 150 MHz): d =
144.04, 131.45, 128.61, 128.35, 126.16, 125.36, 124.661, 50.78, 31.85, 30.59,
29.27, 29.15, 26.79, 22.77, 14.24 ppm; MS (+ESI): m/z: calcd for: 358.23;
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Acknowledgements
Financial support from the DFG GRK 837 is gratefully acknowledged.
We thank Roman Flehr for time-resolved fluorescence measurements of
compounds 3–5 and Dr. S. Czapla for providing the Cu/C catalyst for the
“click” reactions.
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Metal Ion Responsive Fluorescent Probes
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Received: May 6, 2012
Published online: && &&, 0000
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ÝÝ
These are not the final page numbers!

Metal Ion Responsive Fluorescent Probes
FULL PAPER
Light-Up: Combination of a typical
alkali metal ion receptor with various
popular fluorophores in different sig-
nalling architectures through simple
clicking of the modular precursors
yields probes that integrate a novel
donor–acceptor-substituted 1,2,3-tria-
zole motif which gives rise to interest-
ing absorption and fluorescence
Fluorescent Probes
S. Ast, T. Fischer, H. Mꢀller,
W. Mickler, M. Schwichtenberg,
K. Rurack,* H.-J. Holdt* ...... &&& — &&&
Integration of the 1,2,3-Triazole
“Click” Motif as a Potent Signalling
Element in Metal Ion Responsive
Fluorescent Probes
responses upon target binding.
Chem. Eur. J. 2013, 00, 0 – 0
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
17
&
ÞÞ
These are not the final page numbers!