Synthesis and spectroscopic characterization of fluorophore-labeled oligospiroketal rods

Article abstract of DOI:10.1002/hlca.201200616

Fluorescence probes consisting of well-established fluorophores in combination with rigid molecular rods based on spirane-type structures were investigated with respect to their fluorescence properties under different solvent conditions. The attachment of the dyes was accomplished by 1,3-dipolar cycloaddition between alkynes and azides ('click' reaction) and is a prime example for a novel class of sensor constructs. Especially, the attachment of two (different) fluorophores on opposite sides of the molecular rods paves the way to new sensor systems with less bulky (compared to the conventional DNA- or protein-based concepts), nevertheless rigid spacer constructs, e.g., for FRET-based sensing applications. A detailed photophysical characterization was performed in MeOH (and in basic H₂O/MeOH mixtures) for i) rod constructs containing carboxyfluorescein, ii) rod constructs containing carboxyrhodamine, iii) rod constructs containing both carboxyfluorescein and carboxyrhodamine, and iv) rod constructs containing both pyrene and perylene parts. For each dye (pair), two rod lengths with different numbers of spirane units were synthesized and investigated. The rod constructs were characterized in ensemble as well as single-molecule fluorescence experiments with respect to i) specific rod-dye and ii) dye-dye interactions. In addition to MeOH and MeOH/NaOH, the rod constructs were also investigated in micellar systems, which were chosen as a simplified model for membranes.

Full text of DOI:10.1002/hlca.201200616

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Helvetica Chimica Acta – Vol. 96 (2013)
Synthesis and Spectroscopic Characterization of Fluorophore-Labeled
Oligospiroketal Rods
by Anne Techena), Sylvia Czaplab), Kristian Mçllnitzb), Dennis Budachb), Pablo Wessig*b), and
Michael U. Kumke*a)
a) Universitꢀt Potsdam, Institut fꢁr Chemie, Physical Chemistry, Karl-Liebknecht-Strasse 24 – 25,
D-14476 Potsdam (e-mail: kumke@uni-potsdam.de)
b) Universitꢀt Potsdam, Institut fꢁr Chemie, Bioorganic Chemistry, Karl-Liebknecht-Strasse 24 – 25,
D-14476 Potsdam (e-mail: wessig@uni-potsdam.de)
Fluorescence probes consisting of well-established fluorophores in combination with rigid molecular
rods based on spirane-type structures were investigated with respect to their fluorescence properties
under different solvent conditions. The attachment of the dyes was accomplished by 1,3-dipolar
cycloaddition between alkynes and azides (ꢂclickꢃ reaction) and is a prime example for a novel class of
sensor constructs. Especially, the attachment of two (different) fluorophores on opposite sides of the
molecular rods paves the way to new sensor systems with less bulky (compared to the conventional DNA-
or protein-based concepts), nevertheless rigid spacer constructs, e.g., for FRET-based sensing
applications. A detailed photophysical characterization was performed in MeOH (and in basic H2O/
MeOH mixtures) for i) rod constructs containing carboxyfluorescein, ii) rod constructs containing
carboxyrhodamine, iii) rod constructs containing both carboxyfluorescein and carboxyrhodamine, and
iv) rod constructs containing both pyrene and perylene parts. For each dye (pair), two rod lengths with
different numbers of spirane units were synthesized and investigated. The rod constructs were
characterized in ensemble as well as single-molecule fluorescence experiments with respect to i) specific
rodꢀdye and ii) dyeꢀdye interactions. In addition to MeOH and MeOH/NaOH, the rod constructs were
also investigated in micellar systems, which were chosen as a simplified model for membranes.
Introduction. – In fluorescence-based applications, improved sensor dyes are
needed steadily. Especially, the sensing of distances on the sub-nm scale using Fçrster
resonance energy transfer (FRET) as the fundamental principle calls for novel, tailor-
made fluorescence systems.
Specific interactions between the dye molecule and biochemical spacers such as
DNA or polypeptides can be i) intercalation into the major grove of the DNA, ii)
specific interaction with a nucleic acids or amino acids, iii) incorporation into
polypeptides upon formation of the ternary structure, and many more [1]. Compared to
conventional biochemical spacers, the oligospiroketal (OSK) rod [2] (see Scheme 1) is
an attractive alternative, because i) the OSK rod is much smaller in diameter, making it
less bulky and subsequently more suitable for incorporation into 2D structures such as
membranes, ii) the functionalities defining the hydrophobicity of the rod (but also the
possible interactions with the dyes) can be tailor-made for the particular application,
and iii) no formation of super-structures, e.g., as in case of polypeptides (cf. ternary and
quaternary structures) will additionally influence the spatial orientation of dyes
attached to the OSK rods [1c][1d][3][4]. Due to its ꢂpure chemicalꢃ character,
ꢄ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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additional modifications, i.e., increasing (or decreasing) the solubility of the OSK
construct or disabling (enabling) specific (bio)chemical properties of the OSK rod, can
be envisaged (e.g., no biodegradation by enzymes; hydrophobic structure that can be
used to anchor molecules, which may serve as a recognition elements in membranes)
[2c][2d].
In the present interdisciplinary work, the synthesis of the dye-labeled OSK rod
constructs (labeled with carboxyfluorescein (FLU), carboxyrhodamine (RHO),
pyrene (PYR), and perylene (PER) structures) as well as their fundamental
photophysical characterization are reported. Special emphasis is given to OSK rod
constructs labeled with the dye pairs FLU/RHO or PYR/PER, respectively. Here, the
potential of OSK rods as specifically designed spacers in FRET applications is
investigated. The rotation of the fluorescence probe, the properties of the linker
attaching the dye to the OSK rod, as well as the rigidity of the OSK rod itself are major
parameters determining the performance of the OSK rod constructs as FRET spacers
and were characterized in detail. In a first set of experiments, the dye-labeled OSK rods
were introduced to CTAB (cetyltrimethylammonium bromide) micelles as simple
biomimetic model, and the photophysics was investigated as well.
Results and Discussion. – Synthesis of the Rod Constructs. The synthesis of dye-
labeled oligospiroketal (DOSK) rods is based on stepwise Cu-catalyzed alkyneꢀazide
cycloaddition (CuAAC, ꢂclickꢃ reaction) of w,wꢃ-diazido rods 1 with ethynyl-substituted
dyes 2 [5]. To evaluate the influence of the rod length on the photophysical behavior of
DOSK rods, two diazido rods, 1a and 1b, have been chosen with different lengths. The
long rod (LR) 1b was previously described [2b], for the short rod (SR) 1a, the reader is
referred to the Exper. Part. The general approach and the formulae of the reactants 1
and 2 are depicted in Scheme 1.
For the preparation of single-labeled rods 3, the diazido rods 1a or 1b were
subjected to the CuAAC with 1.1 equiv. of alkynes 2a/2b, whereas the double-labeled
rods 4 were obtained by treatment of 1a or 1b with 1:1 mixtures 2a/2b or 2c/2d, and
separation of the resulting product mixture. In both cases, the previously reported
catalyst Cu/C was employed [6]. The formulae of the eight DOSK rods obtained by this
procedure are given in Fig. 1. Compounds 3e and 3f were used as reference for the
elucidation of the photophysical processes.
Investigation of Fçrster Resonance Energy Transfer (FRET) in Double-Labeled
DOSK Rods. In the course of designing novel probes for sensing applications, different
dye molecules were attached to OSK rods to obtain double-labeled DOSK rods by the
ꢂclickꢃ reaction.
As a first sample, PYR and PER were attached to the opposite ends of the OSK
rods (see Scheme 1). PYR is a well-known fluorescence probe for monitoring its
molecular environment (e.g., in membranes) [7]. Depending on the polarity, the ratio
of fluorescence bands as well as its fluorescence decay time are altered. In combination
with PER, an intriguing FRET pair is created. In this donorꢀacceptor pair, the PYR
with its outstandingly long fluorescence decay time acts as the energy donor (D) and
should stamp its decay kinetics on PER (acceptor (A)), which subsequently should be
of great benefit with respect to an advanced two-color time-gated detection scheme.
Based on the fundamental photophysical parameters of the PYR and PER compounds,

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Fig. 1. Single-labeled DOSK rods 3a – 3d, investigated reference compounds 3e – 3g, and double-labeled
DOSK rods 4a – 4d
a Fçrster distance, R0, of 2.2 nm was calculated. In combination with the theoretical
length of the OSK rods, energy-transfer efficiencies, EC, of 0.59 and 0.03 for the short
(SR) and long (LR) OSK rod, respectively, were expected (see Table 1).

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Table 1. FRET Efficiencies of the Samples Determined from Time-Resolved Fluorescence Spectroscopy
(Et) and Calculated for R0 ¼ 2.2 nm, and the Distances R between Donor and Acceptor (R ¼ 2.1 and R ¼
4.0 nm for 4c and 4d, resp.)
Sample
Ec
Et
PYR-SR-PER (4c)
PYR-LR-PER (4d)
0.59
0.03
0.36
0.2
In the stationary emission measurements, the typical behavior expected for a
successful FRET process was observed: i) upon excitation of the PYR (D), an
enhanced PER (A) emission was detected, ii) in the fluorescence excitation spectrum
with the detection wavelength set to a PER emission wavelength, the spectral features
of PYR were measured, and iii) the fluorescence-decay time of PYR was quenched.
But the most convincing observation was the increased fluorescence decay time of PER
in the double-labeled DOSK rods. In Fig. 2, the fluorescence-decay curves of 2c, and
the double-labeled DOSK rods 4c and 4d are shown.
Fig. 2. a) Fluorescence-decay curves of PER derivative 2c (black, lexc 447 nm) and PYR-SR-PER (4c;
grey, lexc 254 nm) in CH2Cl2. b) Fluorescence-decay curves of 2c (black, lexc 447 nm) and PYR-LR-PER
(4d; grey, lexc 254 nm) in CH2Cl2. The emission was detected at lem 536 nm. The IRF is indicated as black
line.
Measuring the fluorescence decay of PER under indirect excitation conditions
(after energy transfer from PYR), a considerable increase in the fluorescence-decay
time of the PER was observed, which directly points toward a successful FRET process
(see Fig. 2). The effect was strongest for the short OSK rod. Based on the time-resolved
fluorescence measurements, the experimentally apparent FRET efficiencies, Et, were
calculated. In Table 1, the calculated and experimental FRET efficiencies are
compared. Calculated and experimental values do not match at all. The values of EC
were calculated on the assumption that the DOSK rods are present in an extended
linear configuration. However, because of solubility constraints the dye molecules
could fold back and interact with the OSK rod. In simple molecular-dynamics (MD)
simulations1), a possible contribution of such folding was found for 4c and 4d. The
1
)
MD Simulations were performed with the MMFF94x using the program MOE 2010.

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observed differences can be a result of such unexpected conformations, e.g., in case of
4d, a folded conformation would lead to much smaller distances between the pyrene
and perylene moiety, and subsequently, compared to the theoretically expected, a
higher energy-transfer efficiency would be observed. On the other hand, due to
intramolecular interactions, a limitation with respect to the dyeꢀdye orientation could
result in an altered k2 value, which in turn could be responsible for the low efficiency
observed for 4c.
Since the aim is to use the DOSK rods in life-science applications, the solubility of
the constructs in aqueous solutions was a major concern. Moreover, for the application
in microscopy-based methods the use of well-established xanthene dyes such as FLU
and RHO is beneficial. Both dyes have been used as FRET pair successfully before [8].
Therefore, in the course of developing novel constructs as FRET probes for life-science
applications, double-labeled DOSK rods containing FLU and RHO at the opposite
ends of the rod were constructed. As in the case of the PYR/PER FRET construct, the
DOSK rods FLU-SR-RHO (4a) and FLU-LR-RHO (4b) were characterized in steady-
state and time-resolved fluorescence measurements, and in addition by single-molecule
spectroscopy with respect to their FRET performance. Based on the results obtained
for 4c and 4d, the measurements were carried out in different solvents: i) MeOH, ii)
MeOH/NaOH, and iii) in CTAB micelles as a first simple biomimetic model system for
membranes.
In Fig. 3,a, the absorption spectra of single (i.e., 3a and 3c) and double-labeled
DOSK rods (i.e., 4a) are compared.
From Table 2, it can be seen that the absorption and emission maxima of the
double-labeled DOSK rods 4a and 4b, compared to the corresponding single-labeled
DOSK rods, are not altered. The absorption spectrum of the double-labeled DOSK
compound 4a (and 4b) comprises of the absorption spectra of FLU and of RHO,
respectively (see Fig. 3,a).
Based on the spectroscopic properties of the FLU- and RHO-labeled DOSK rods
(quantum efficiency of FLU, extinction coefficient of RHO, spectral overlap integral,
Fig. 3. a) Normalized absorption spectra and b) steady-state fluorescence emission spectra of labeled
DOSK rods in 1 mm NaOH/MeOH.

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Table 3. Calculated Fçrster Distance (R0) and Overlap Integral (J) Values for the Samples in Different
Solvents (FF,D, fluorescence quantum yield of the donor determined with single-labeled constructs). The
Fçrster distance, R0, was calculated with the orientation factor k2 ¼ 2/3. FRET Efficiencies [%] of the
samples in different solvents determined with steady-state (EE ), time-resolved (Et), single molecule
(ES ) fluorescence spectroscopy, and calculated with the values for R0 and the distances between donor
and acceptor (R ¼ 1.9 and R ¼ 3.8 nm for 4a and 4b, resp.).
FLU-SR-RHO (4a)
FLU-LR-RHO (4b)
MeOH
NaOH/MeOH
CTAB
MeOH
NaOH/MeOH
CTAB
n
1.33345
44
3.63
5.8
1.34348
77
4.06
6.2
26
n.m.b)
30
1.34014
79
5.6
6.6 (6.2)a)
24
1.33345
39
4.36
5.6
13
5
n.d.c)
91
1.34348
76
4.38
6.3
13
6
10
95
1.34014
73
6.21
6.6 (6.2)a)
14
FF,D [%]
J [1015 mꢀ1 cm3]
R0 [nm]
EE
22
Et
n.m.b)
n.d.c)
100
8
8
ES
85 ꢁ 10
67 ꢁ 7
EC
100
100 (100)a)
97 (95)a)
a) R0 in CTAB was calculated with k2 ¼ 0.475 according to [11]. b) n.m., No difference between single-
c
and double-labeled constructs in the observed FLU decay time. ) n.d., Not determined for MeOH due
to experimental constrains.
and based on the anisotropy data set k2 ¼ 2/3, vide infra), the critical Fçrster distance,
R0, was calculated (see Table 3). The calculated values of R0 for 4a and 4b were found
to be well within the range of reported values for derivatives of this donorꢀacceptor
pair (carboxyfluoresceinꢀtetramethylrhodamine: R0 ¼ 5.1 nm [7a]; carboxyfluores-
ceinꢀsulforhodamine B: R0 ¼ 5.8 nm [9]; 5-(N-hexadecanoylamino)fluoresceinꢀ
octadecylrhodamine B: R0 ¼ 5.4 nm [10]). Depending on the solvent used, a critical
Fçrster distance of 5.6 nm < R0 < 6.6 nm was calculated for the double-labeled DOSK
rods. Under the assumption of a rigid OSK rod (with the dye linkers in a stretched
conformation), the distance between FLU and RHO moiety was calculated, and a
theoretical FRET efficiency EC was determined. From Table 3, it can be seen that, for
FLU-SR-RHO (4a), almost 100% transfer efficiency is expected, while for the FLU-
LR-RHO (4b) ca. 80% are predicted.
A first experimental indication for successful FRET between FLU and RHO in 4a
and 4b was obtained from the fluorescence excitation spectra. Here, with the detection
wavelength set on an acceptor emission wavelength, the excitation spectrum was found
to be a combination of the spectral features of RHO and FLU (see Fig. 4,a). Also, for
4a and 4b a distinct reduction of the FLU-related fluorescence intensity and a
simultaneous enhancement of the RHO-related emission were observed when
compared to the single-labeled DOSK rods (see Fig. 4, left and right, for the different
OSK rods of SR and LR length, resp.). However, independent on the solvent, FRET
efficiencies of only ca. 24 and 13% were calculated for 4a and 4b, respectively, from the
evaluation of the steady-state fluorescence measurements. Moreover, no quenching of
the FLU fluorescence-decay time was found for FLU-SR-RHO (4a), and for FLU-LR-
RHO (4b) only a slight decrease in the fluorescence-decay time of FLU was found. In
that case, based on the decrease in the fluorescence-decay time, a FRET efficiency, Et,

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Fig. 4. a) Steady-state fluorescence excitation spectra (lem 650 nm) and b) fluorescence emission spectra
(lexc 470 nm) of the single- and double-labeled DOSK Rods in 1 mm NaOH/MeOH
of only ca. 5% was determined. Likewise, for the PYR/PER-labeled DOSK rods the
experimentally determined energy-transfer efficiencies (Et, EE) were considerably
different from the theoretically predicted (EC; see Table 3). In addition to the
ensemble fluorescence measurements, single-molecule fluorescence experiments were
performed in NaOH/MeOH, and the data fit well with the ensemble measurements
(see Table 3 and Fig. 5).
The results strongly point towards a strong contribution of a static-like (proximity)
quenching between FLU and RHO in the doubled-labeled rods, as observed before
[11]. In the double-labeled DOSK rods, even though FLU and RHO show a decent
water solubility a folding of the dye molecules towards the OSK rod moiety has been
found in MD simulations. From this folding process, two subpopulations (unfolded vs.
folded) may arise (Fig. 6).
The proximity quenching would be observed preferentially for the folded
subpopulation and should be especially effective in FLU-SR-RHO (4a) and slightly
less operative in FLU-LR-RHO (4b), due to the increased distance between the two
dyes. From the finding that the reduction in the FLU fluorescence intensity is
accompanied by an increase in the RHO fluorescence intensity, it may be concluded
that the underlying mechanism of the observed fluorescence quenching is still an
energy-transfer process. Using the calculated R0 value and Eqn. 1 to obtain an estimate
for the rate constant of the energy transfer step, a kET > 1010 sꢀ1 is found for 4a. This is

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Fig. 5. FRET Histogram extracted from single-molecule experiments for a) FLU-SR-RHO (4a) and
b) FLU-LR-RHO (4b) in 1 mm NaOH/MeOH (lexc 470 nm). The data were fitted according to a
Gaussian distribution (grey line).
Fig. 6. Two examples of possible subpopulations (folded and unfolded). The OSK rod is shown as a black
rectangle, and the dyes are symbolized by grey and black circles.
about an order of magnitude faster than the rate constant of the corresponding
fluorescence process.
ꢀ
ꢁ
6
1
tD0
R0
R
(1)
kET
¼
ꢂ
In such a case, large differences between steady-state and time-resolved data can be
found. The time-resolved measurements ꢂseeꢃ only a less-quenched fraction (which is
present, e.g., due to conformational changes a negative influence of k2 could reduce the
effective rate constant kET for that fraction of molecules). A second possibility is that an
additional quenching mechanism such as photoinduced electron transfer becomes
operative, when the two fluorophores are in closer proximity. Here, the presence of the
triazole moiety of the DOSK needs to be taken into account. A rough estimation based
on the redox properties of FLU (oxidation potential EOx ¼ ꢀ1.19 V) and RHO
(reduction potential ERed ¼ ꢀ0.95 V) showed that it could be feasible (yielding a free
enthalpy DG ca. ꢀ 2.7 V according to RehmꢀWeller) [3d][12][13].
To further explore the distinct differences between calculated and experimentally
obtained energy-transfer efficiencies for the double-labeled DOSK rods, a set of
reference compounds, 3e and 3f, was investigated (Fig. 1). Especially, the influence of
additional intramolecular interactions (e.g., triazole ring) on the fluorescence proper-
ties was investigated in more detail.
Photophysical Characterization of the Single-Labeled DOSK Rod Constructs. In the
Huisgen [3 þ 2] cycloaddition reaction, a 1,2,3-triazole ring is formed, which could

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introduce ꢂextraꢃ (photophysical) properties interfering with the wanted FRET process
in the double-labeled DOSK rods. The N-atoms can actively participate in complex-
ation reactions (e.g., with solvent molecules, etc.) via their lone electron pairs, e.g.,
acting as electron donor or as partners in H-bonding. Moreover, the CꢀH group has a
decent acidic character, which further makes the 1,2,3-triazole ring a possible partner in
intra- and intermolecular interactions [14].
The absorption and emission spectra of the parent fluorescence dyes as well as of
the DOSK rods 3a – 3d (see Fig. 1) were recorded in MeOH, NaOH/MeOH (see
Fig. 3), and in CTAB/PBS (phosphate buffered saline). The fundamental photo-
physical parameters of the samples are compiled in Table 2.
Upon coupling to the OSK rods (SR or LR), only weak-to-moderate alterations in
the fundamental photophysical parameters of the dyes were observed compared to the
respective parent compounds (see Table 2). In going from MeOH to NaOH/MeOH,
slight changes in the absorption and emission properties of all DOSK samples, i.e., 3a –
3d, were induced. In general, a bathochromic shift of the related absorption and
emission maxima as well as a slight increase of the fluorescence decay times were
observed. In MeOH and MeOH/NaOH, the FLU-labeled DOSK rods, 3a and 3c,
showed a decay time of t ¼ (4.1 ꢁ 0.1) ns, and for the RHO labeled DOSK rods, 3b and
3d, a decay time t ¼ (2.0 ꢁ 0.3) ns was determined, which were slightly increased
compared to those of the parent compounds, FLU and RHO, under similar solvent
conditions. The fluorescence quantum yields of the dyes in the single-labeled DOSK
rods were slightly decreased in the case of FLU and not changed for RHO. A distinct
alteration of the spectra was found for the RHO-labeled DOSK rods. In the
fluorescence spectrum of the RHO-SR-N3 (3b) and RHO-LR-N3 (3d), a weak new
fluorescence band at shorter emission wavelengths (lexc 470, lem 520 nm) was observed.
To further evaluate this alteration in the fluorescence spectrum of the samples 3b and
3d, the influence of unbound 2-(4-isopropyl-1H-1,2,3-triazol-1-yl)acetic acid (ITS; 3g;
as model for the 1,2,3-triazole ring) on the photophysical properties of the parent
compounds, FLU and RHO, was investigated. It was found that the fluorescence-decay
time of FLU slightly decreased with increasing 3g concentration, and that in the
fluorescence spectra of RHO an additional emission signal arose upon addition of 3g
(lem ꢃ 520 nm). This is comparable to the changes observed for the RHO-labeled
DOSK rods, 3b and 3d. From these observations, it can already be concluded that the
observed slight modifications observed for the photophysical properties of the FLU-
(i.e., 3a and 3c) and RHO-labeled DOSK rods (i.e., 3b and 3d) are connected to the
presence of the 1,2,3-triazole ring in the DOSK rods.
To further investigate possible intramolecular interactions, such as stacking of the
dyes on the OSK rod, time-resolved fluorescence depolarization measurements were
performed. For MeOH and NaOH/MeOH solutions, the anisotropy decays of the
different samples were analyzed using a single exponential decay law (see Eqn. 3). The
results are collected in Table 4. It can be seen that the obtained rotational correlation
times f for FLU and RHO are only slightly increased upon linkage to the OSK rod. No
significant change in the anisotropy-decay kinetics was observed, probably due to the
fact that the differences in size of the rotational volume between the parent molecules
(FLU and RHO) and DOSK rods 3a – 3d were too small to cause a significant change
in the rotational correlation time f. Consequently, no further conclusions, e.g.,

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Table 4. Rotational Correlation Times f of the Samples in Different Solvents. For the CTAB solution, the
two-step and wobbling-in-cone model parameters (structure parameter S, wobbling angle q) obtained
from the analysis of time-resolved anisotropy experiments are shown. Fluorescence-anisotropy
measurements for FLU and FLU-labeled DOSK rods were performed at lexc 470 and lem 529 nm,
while for RHO and RHO-labeled DOSK rods lexc 550 and lem 620 nm were used, respectively. For the
calculation of fL Eqn. 5 was applied using a rotational correlation time of the micelle, fM of 21 ns [15],
which was calculated based on the hydrodynamic radius determined in fluorescence correlation
spectroscopy experiments.
Sample
MeOH NaOH/MeOH CTAB
f [ns]
f [ns]
ffast [ns] fslow [ns] fL [ns] fw [ns]
S
q [8]
FLU
FLU-SR-N3 (3a)
FLU-LR-N3 (3c)
0.3
0.4
0.3
0.3
0.6
0.5
0.7
1.1
1.2
3.9
5.3
4.6
4.8
7.1
5.9
0.8
1.4
1.5
0.6 46
0.7 38
0.7 38
RHO
RHO-SR-N3 (3b)
RHO-LR-N3 (3d)
0.3
0.4
0.4
0.4
0.6
0.7
1.1
0.7
0.7
8.5
7.3
7.5
14.4
11.2
11.7
1.2
0.8
0.7
0.6 49
0.8 31
0.8 31
FLU-SR-RHO (4a)a) 0.5
0.5
FLU-LR-RHO (4b)a) 0.4
0.4
0.6
0.7
0.7
0.6
0.9
0.6
0.9
0.8
6.4
6.7
5.7
7.7
9.2
9.9
7.8
1.0
0.7
1.1
1.0
0.8 31
0.9 26
0.7 38
0.8 33
12.2
a) First line: lexc 470, lem 529 nm; second line: lexc 550, lem 620 nm.
regarding possible stacking between dye molecule and OSK rod, can be drawn from
these data.
DOSK Rods as Membrane Probes. The DOSK rods were investigated in the
presence of CTAB micelles, which served as a first simple robust model for membranes.
In the CTAB solution, all DOSK rods were readily incorporated into the micelles. The
addition of the cationic detergent CTAB induced a slight alteration in the spectroscopic
properties of the FLU-labeled DOSK rods 3a and 3c. A bathochromic shift in the
absorption and fluorescence spectrum was found for the FLU fluorophore, and can be
attributed to the specific interaction of the FLU fluorophore with the CTAB micelles
(inclusion of the FLU probes into the micelle). Comparable spectral shifts were
reported before for the parent compound FLU and other FLU probes such as 5-
(hexadecanoylamino)fluorescein (HAF) in micellar CTAB solutions [15]. In addition,
also an increase in the fluorescence-decay times of the single-labeled DOSK rods 3a –
3d was found upon incorporation in CTAB micelles (see Table 2).
To further investigate the incorporation of DOSK rods in micelles, time-resolved
fluorescence anisotropy measurements were carried out. In Fig. 7, the fluorescence
anisotropy decay of the double-labeled DOSK rods is shown. The anisotropy decay was
measured for the FLU and RHO part of the double-labeled DOSK rods under selective
excitation and emission conditions, respectively. The comparison reveals only slight
differences between the rotational correlation times f of the single-labeled DOSK rods
and the dyes in the respective double-labeled DOSK rods (see Table 4).
In CTAB micelles, the fluorescence anisotropy decay of the parent compounds as
well as of the DOSK rods became connected to the overall motion of the micelle. The

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Fig. 7. Anisotropy decay of the double-labeled DOSK rods. The anisotropy was measured for FLU as well
as for RHO. Shown are the anisotropy decays of both dyes under selective excitation (grey: lexc 470, lem
529 nm; black: lexc 550, lem 620 nm) in 1 mm CTAB/PBS solution at 294 K. a) FLU-SR-RHO (4a); b)
FLU-LR-RHO (4b).
finding, that complex anisotropy decay kinetics is observed, already indicates that the
DOSK rods are not rigidly attached/incorporated to/in the micelles. Applying the
wobble-in-cone model (see Eqns. 4 – 6 in the Exper. Part), the rotational and
translational diffusion of the DOSK rod in a micellar environment was analyzed
[16]. For the determination of the contributions of the different rotational and
translational motions as well as of the order parameters (S, q), and to reduce the
number of free parameters in the evaluation of the anisotropy data, the overall
rotational correlation time of the micelle, fM, was determined based on fluorescence
correlation spectroscopy experiments (using the size of the CTAB micelles to calculate
fM). The analysis of the anisotropy decay of the parent compounds yielded rotational
correlation times and order parameters, which were in good agreement with literature
values of the same or similar compounds (see Table 4) [16]. For the FLU-labeled
DOSK rods, an increase of the fast and slow rotational motion was found, while for the
RHO-labeled DOSK rods a slight decrease was observed. The differences in the
corresponding rotational correlation times between the short (SR) and long (LR)
DOSK rods were small. The calculated order parameters (and the corresponding
wobble-in-cone angle) indicate that the rotational freedom of the dyes decreased upon
labeling to the OSK rod. The fluorophores may still reside in the interfacial layer
between micelle and bulk solution, but the rod can act like an anchor reducing the
overall rotational freedom of the labeled dyes. Moreover, for the double-labeled
DOSK rods 4a and 4b, the observed energy-transfer efficiency, ES, increased, especially,
when measured in single-molecule fluorescence experiments. Here, ES nearly
approached the calculated value EC. Steady-state fluorescence as well as single-
molecule fluorescence measurements (SMS) are intensity-related methods, but the
latter technique has the advantage that the averaging-out due to observation of a

Helvetica Chimica Acta – Vol. 96 (2013)
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fluorophore ensemble can be avoided. Thus, SMS enables the resolution and detection
of subpopulations. In CTAB micellar solutions, the equilibrium between the
subpopulations seems to be shifted towards the unfolded configuration due to an
improved solubility of the doubled-labeled DOSK rods in the micelles. Subsequently,
the proximity quenching is relaxed and the ꢂexpectedꢃ energy transfer between the
donorꢀacceptor pair is now observed.
Conclusions. – The combination of OSK rods and fluorescence probes enables
design of novel tailor-made FRET probes with scalable distances between donor and
acceptor without the necessity of using bulky biomolecules (e.g., proteins or DNA).
Especially, the relatively low molecular weight of the rod is very attractive for the
construction of standards for FRET investigation or for novel probe systems applicable
in competitive fluorescence assays. Compared to bimolecular spacers, the dimensions
of the OSK rods are smaller, and moreover, the ꢂchemical surfaceꢃ of the rod can be
adapted (e.g., with respect to polarity), which can be a further benefit for the design of
novel probes for life sciences. The DOSK rods can be envisaged for the investigation of
membranes and transmembrane processes. Attaching different reporters to the
opposite ends of the OSK rods, transmembrane processes involving a first messenger
situated outside the cell and stimulating a second messenger inside the cell can be
studied. As a first confirmation of the principle, the induced FRET of the double-
labeled DOSK rods in micelles was discussed (here, the first messenger is a photon
absorbed by the donor, and the second messenger is also a photon (of different color),
which is emitted by the acceptor.
The specific influences of structural elements arising for dye-labeling chemistry
need to be controlled very carefully. FLU and RHO are frequently used fluorescence
probes in life-science applications. However, due to their chemical nature, intra- and
intermolecular interactions with parts of the OSK rod are possible. For the OSK rods
designed in the present study, depending on the properties of the dyes attached to the
rod moiety, specific interactions with 1,2,3-triazole ring or the OSK rod itself were
shown to be of major influence. For the FLU-RHO dye pair, the combination of
ensemble and single-molecule fluorescence experiments revealed that, in the double-
labeled DOSK rods, a combination of proximity and dynamic quenching is present,
which underlines the necessity i) to perform case-to-case studies for the probes used in
combination with the specific OSK rod architecture and ii) to apply complementary
experimental techniques in the characterization of the FRET probes.
The financial support of this work by the Deutsche Forschungsgemeinschaft (We1850-7/1-2) is
gratefully acknowledged. M. U. K. is grateful to J. von der Bever for discussions on lifetime evaluations.
Experimental Part
General. (Hexadecyl)(trimethyl)ammonium bromide (CTAB; CAS: 57-09-0; ꢄ 99.0%) and 5(6)-
carboxyrhodamine (RHO; CAS: 81-88-9; ꢄ 97.0%) were purchased from Sigma-Aldrich (DE-
Deisenhofen), 5(6)-carboxyfluorescein (FLU; CAS: 72088-94-9) was obtained from Invitrogen (DE-
Karlsruhe). Stock solns. of the samples were prepared in MeOH, in a soln. of 1 mm NaOH/MeOH, 3 :1,
and in 1 mm CTAB/PBS (physiological buffered saline, pH 7.4). Samples investigated were i) free dyes
(FLU and RHO), ii) molecular rods containing either FLU or RHO (FLU-SR-N3 (3a), RHO-SR-N3

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(3b), FLU-LR-N3 (3c), RHO-LR-N3 (3d)), iii) molecular rods containing FLU as well as RHO (FLU-
SR-RHO (4a) and FLU-LR-RHO (4b)), and iv) molecular rods containing PYR as well as PER (PYR-
SR-PER (4c) and PYR-LR-PER (4d)). The refractive indices of MeOH, NaOH/MeOH, and CTAB
solns. were determined using a digital multiple-wavelength refractometer (DSR-l, Schmidt and Haensch,
DE-Berlin) at l of 470 nm (293 K). The error of the measurements was determined from repeated
measurements as ꢁ 0.0004.
OSK-Rod Construct Synthesis. All reactions using dry solvents were performed under N2 in heat gun-
dried flasks. The solvents were dried and distilled prior to use by usual laboratory methods. All reagents
obtained from commercial sources were used without further purification. TLC: silica-gel plates (Merck,
silica gel 60 F254). Flash chromatography (FC): silica gel (Merck Geduran Si 60, 40 – 63 mm). Prep. NP-
HPLC: Eurospher 100 column (Knauer, 5 mm, 32 ꢅ 250 mm) in a isocratic mode over 20 min at a flow
rate of 32 ml/min with UV detection at 254; used for the products 4c and 4d. The crude products 3a – 3c
and 4a were purified by semi-prep. RP-HPLC on an Eurosil, Bioselct-300 C18 column (Knauer, 10 mm,
16 ꢅ 250 mm) with a linear gradient program from 20 – 90% B over 30 min (solvent A: H2O 0.05%
CF3COOH; solvent B: MeCN 0.05% CF3COOH) at a flow rate of 16 ml/min. The purification of
product 4b was carried out on a Sephadex LH 20 (GE Healthcare, UK-Buckinghamshire) column (15 ꢅ
60 mm) in CH2Cl2/MeOH 1:1. For the purification of products 3e and 3f, the same method was used with
Sephadex LH 20 and MeOH. M.p.: Elektrothermal 9100; uncorrected. IR Spectra: as KBr pellets or as
films; Perkin Elmer IR-881 spectrometer. 1H- and 13C-NMR spectra: Bruker AVANCE 300 and Bruker
AVANCE 500 spectrometer; chemical shifts d in ppm with TMS as internal standard, coupling constants
(J) in Hz. ESI-MS: Bruker maXis QTOF instrument. MALDI-TOF-MS: AXIMATM Assurance
(Shimadzu Biotech, Kratos Analytical Limited, Manchester, UK) time-of-flight mass spectrometer,
fitted with 337-nm pulsed nitrogen laser; all spectra were obtained in a positive-ion linear mode, data
were processed using the Launchpad 2.8. software package (Shimadzu Biotech, Kratos Analytical
Limited, Manchester, UK); a sat. soln. of sinapinic acid (Fluka) in MeCN/H2O 50 :50 þ 0.1% CF3COOH
was used as matrix for all samples.
1,1’-(7,11,18,21-Tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]henicosane-3,15-diyl)bis(2-chloroetha-
none) (7). Compound 5 (498 mg, 2.84 mmol, 1.95 equiv.) [2b] and 6 (617 mg, 1.45 mmol) were added to
dry Et2O (25 ml). Trimethylsilyl triflate (¼trimethylsilyl trifluoromethanesulfonate (TMSOTf); three
drops, 20 ml, 70 mmol, 0.05 equiv.) was added, and the mixture was stirred at r.t. overnight (Scheme 2).
Monitoring with TLC showed still small amounts of reactants. Dry CH2Cl2 (4 ml) and TMSOTf (three
drops, 20 ml, 70 mmol, 0.05 equiv.) were added, and the mixture was stirred until TLC showed complete
conversion. Brine was added, and the aq. layer was extracted three times with CH2Cl2. The combined org.
layers were dried (MgSO4), filtered, and evaporated. The resulting residue was purified by FC (CH2Cl2/
MeOH 100 :3) to yield 7 (556 mg, 1.23 mmol, 87%). White foam. Rf (CH2Cl2/MeOH 10 :1) 0.54. M.p.
172 – 1738. IR: 3452, 2969, 2939, 2867, 1655, 1444, 1362, 1339, 1269, 1240, 1220, 1166, 1150, 1088, 1059,
1037, 937, 886, 787, 729, 659. 1H-NMR (300 MHz, CDCl3): 1.82 – 1.91 (m, 4 CH2); 3.46 – 3.51 (m, 2 CH2);
3.58 – 3.63 (m, 2 CH2); 3.71 (s, 2 CH2); 3.78 (s, 2 CH2); 4.06 (s, 2 CH2). 13C-NMR (75 MHz, CDCl3): 31.0
(CH2); 33.0 (C); 33.2 (CH2); 38.9 (CH2); 40.9 (CH2); 42.9 (CH2); 63.2 (CH2); 63.3 (CH2); 96.6 (C); 164.8
(C). HR-ESI-MS: 451.1396 ([M þ H]þ, C19H2935Cl2N2O6þ ; calc. 451.1397).
Scheme 2. Synthesis of Compound 7

Helvetica Chimica Acta – Vol. 96 (2013)
2061
1,1’-(7,11,18,21-Tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]henicosane-3,15-diyl)bis(2-azidoethanone)
(1a). Compound 7 (496 mg, 1.10 mmol) was dissolved in dry DMF (6 ml). NaN3 (159 mg, 2.42 mmol,
2.2 equiv.) was added, and the mixture was stirred 2 h at r.t. Brine was added, and the aq. soln. was
extracted three times with CH2Cl2. The combined org. layers were dried (MgSO4), filtered, and
evaporated. The resulting residue was purified by FC (CH2Cl2/MeOH 100 :4) to yield 1a (496 mg,
1.07 mmol, 97%). White solid. Rf (CH2Cl2/MeOH 10 :1) 0.57. M.p. 171 – 1738. IR: 3449, 2108, 1642, 1442,
1367, 1268, 1232, 1167, 1151, 1084, 1059, 795. 1H-NMR (300 MHz, CDCl3): 1.80 – 1.87 (m, 4 CH2); 3.32 –
3.36 (m, 2 CH2); 3.58 – 3.63 (m, 2 CH2); 3.69 (s, 2 CH2); 3.77 (s, 2 CH2); 3.92, (s, 2 CH2). 13C-NMR
(75 MHz, CDCl3): 30.9 (CH2); 32.9 (C); 33.3 (CH2); 38.7 (CH2); 41.6 (CH2); 50.5 (CH2); 63.2 (CH2); 63.3
(CH2); 96.5 (C); 165.3 (C). HR-ESI-MS: 487.2028 ([M þ Na]þ, C19H28N823NaO6þ ; calc. 487.2024).
Benzyl 4-[(2,2-Dimethylpropanoyl)oxy]piperidine-1-carboxylate (9). Compound 8 [2a] (4.5 g,
19.5 mmol) was dissolved in 100 ml of dry CH2Cl2, and 3.2 ml (33.15 mmol, 1.7 equiv.) of pyridine
were added (Scheme 3). The mixture was cooled to ꢀ 208, and 4.0 ml (33.15 mmol, 1.7 equiv.) of pivaloyl
chloride (tBuCOCl) were added slowly. The mixture was warmed to r.t. and stirred overnight. The
mixture was washed with aq. tartaric acid and sat. aq. NaHCO3 soln. The org. layer was dried (MgSO4),
filtered, and evaporated. The resulting residue was purified by FC (CH2Cl2/MeOH 100 :1) to give 10
(4.5 g, 14.09 mmol, 72%). White solid. Rf (CH2Cl2/MeOH 100 :1) 0.21. 1H-NMR (300 MHz, CDCl3): 1.20
(s, 3 Me); 1.58 – 1.70 (m, CH2); 1.78 – 1.90 (m, CH2); 3.46 (m, CH2); 3.62 – 3.70 (m, CH2); 4.94 (tt, J ¼ 7.2,
CH); 5.13 (s, CH2); 7.30 – 7.37 (m, 5 arom. H). 13C-NMR (75 MHz, CDCl3): 27.0 (Me); 30.2 (CH2); 38.7
(C); 40.8 (CH2); 67.0 (CH2); 68.5 (CH); 127.8 (arom. C); 127.9 (arom. C); 128.4 (arom. C); 136.6 (arom.
C); 155.1 (C); 177.6 (C).
Scheme 3. Synthesis of Compound 11
1-(Chloroacetyl)piperidin-4-yl 2,2-Dimethylpropanoate (10). Compound 9 (4 g, 11.8 mmol) was
dissolved in 100 ml of dry THF. Pd/C (200 mg, 10%) was added, and the suspension was stirred under H2,
until TLC showed complete conversion (Scheme 3). The H2 atmosphere was changed to a N2
atmosphere, and the mixture was cooled to 08. 4-Nitrophenyl 2-chloroacetate (2.8 g, 13.06 mmol,
1.1 equiv.) [17] was added. The mixture was warmed to r.t. and stirred for another 12 h. The mixture was
filtered through Celite, and the solvent was evaporated. The residue was purified by FC (hexane/AcOEt
5 :1) to afford 10 (2.1 g, 8.04 mmol, 68%). White solid. Rf (hexane/AcOEt 5 :1) 0.36. 1H-NMR
(300 MHz, CDCl3): 1.12 (s, 3 Me); 1.56 – 1.91 (m, 2 CH2); 3.36 – 3.64 (m, 2 CH2); 4.01 (s, CH2); 4.91 (tt,
J ¼ 3.6, 6.9, CH). 13C-NMR (75 MHz, CDCl3): 26.9 (Me); 29.7 (CH2); 30.5 (CH2); 38.5 (C); 38.9 (CH2);
40.8 (CH2); 42.9 (CH2); 67.8 (CH); 164.7 (C); 177.3 (C).
1-(Azidoacetyl)piperidin-4-yl 2,2-Dimethylpropanoate (11). To a suspension of 10 (1.75 g, 6.7 mmol)
in 20 ml of dry DMF were added 522.6 mg (8.0 mmol, 1.2 equiv.) of NaN3 (Scheme 3). The mixture was
stirred for 5 h. Brine (5 ml) was added, and the org. layer was separated. The aq. layer was extracted with
10 ml of CH2Cl2, and the combined org. layers were dried (MgSO4). The solvent was removed in vacuo.
The residue was purified by FC (CH2Cl2/MeOH 100 :1) to furnish 11 (1.63 g, 6.07 mmol, 91%). White
solid. Rf (CH2Cl2/MeOH 100 :1) 0.29. 1H-NMR (300 MHz, CDCl3): 1.17 (s, 3 Me); 1.62 – 1.91 (m, 2 CH2);
3.25 – 3.52 (m, CH2); 3.58 – 3.73 (m, CH2); 3.92 (s, CH2); 4.96 (tt, J ¼ 3.6, 6.9, CH). 13C-NMR (75 MHz,
CDCl3): 27.0 (Me); 29.9 (CH2); 30.7 (CH2); 38.7 (C); 38.9 (CH2); 41.9 (CH2); 50.6 (CH2); 67.9 (CH);
165.4 (C); 177.4 (C).

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Synthesis of Fluorescent Dyes. For the [3 þ 2] cycloaddition (ꢂclickꢃ reaction), fluorescent molecules
bearing CꢆC bonds without any spacers have been synthesized in good yields (Scheme 4).
Scheme 4. Synthesis of Compounds 2b and 2c
2-[6-(Diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl]-5-(prop-2-yn-1-ylcarbamoyl)benzoate
(2b). 5-Carboxytetraethylrhodamine (12; 150 mg, 0.31 mmol) was dissolved in anh. DMF (5 ml), and
propargylamine (25 mg, 0.46 mmol, 1.5 equiv.), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HATU; (243 mg, 0.62 mmol, 2 equiv.), 1-hydroxy-7-azabenzotriazole
(HOAt; 84 mg, 0.62 mmol, 2 equiv.) and N-methylmorpholine (NMM; 125 mg, 1.23 mmol, 4 equiv.)
were added to the soln. After stirring for 24 h at r.t., the solvent was removed via rotary evaporation in
vacuo. The crude product was dried under high vaccum for 2 h. FC (CH2Cl2/MeOH/Et3N 20 :1:0.5)
afforded 2b (117 mg, 0.22 mmol, 72%). Dark-pink solid. Rf (CH2Cl2/MeOH/Et3N 10 :1:0.5) 0.32.
1H-NMR (500 MHz; CDCl3): 1.30 (t, J ¼ 8.7, 4 Me); 2.25 (t, J ¼ 2.47, CH); 3.56 (q, J ¼ 7.18, 4 CH2); 4.28
(dd, J ¼ 2.4, 5.0, CH2N); 6.70 (d, J ¼ 2.4, 2 CH); 6.78 (dd, J ¼ 2.4, 9.5, 2 CH); 7.30 (d, J ¼ 9.5, 2 CH); 8.15
(t, J ¼ 5.0, NH); 8.28 (dd, J ¼ 1.1, 7.9, CH); 8.71 (s, CH). 13C-NMR (125 MHz, CDCl3): 12.5 (Me); 29.8
(CH2N); 45.9 (NCH2Me); 71.4 (C); 79.3 (C); 96.1 (CH); 113.6 (C); 114.0 (CH); 129.8 (CH); 130.0 (CH);
131.5 (CH); 131.6 (CH); 131.7 (CH); 135.9 (C); 136.1 (C); 155.5 (C); 157.8 (C); 159.3 (CO); 166.0 (C).
HR-ESI-MS: 524.2562 ([M þ H]þ, C32H34N3O4þ ; calc. 524.2549).
2-[2,4-Di-(tert-butyl)phenyl]-9-(prop-2-yn-1-yl)isoquinolino[4’,5’,6’:6,5,10]anthra[2,1,9-def]isoqui-
noline-1,3,8,10(2H,9H)-tetrone (2c). To a flask containing 13 (167 mg, 0.28 mmol) was added MeCN
(5 ml), followed by propargyl bromide (137 mg, 1.15 mmol, 4 equiv.) and K2CO3 (399 mg, 2.89 mmol,
10 equiv.), and the suspension was stirred at 858 overnight. The mixture was filtered and concentrated in
vacuo. The crude product was purified by FC (CH2Cl2) to yield 2c (55 mg, 0.089 mmol; 31%). Purple
solid. Rf (CH2Cl2) 0.32. 1H-NMR (300 MHz, CDCl3): 1.28 (s, tBu); 1.38 (s, tBu); 2.21 (t, J ¼ 2.4, CH); 4.97
(d, J ¼ 2.4, CH2N); 7.28 (d, J ¼ 2.1, CH); 7.49 (dd, J ¼ 2.1, 8.6, CH); 7.61 (d, J ¼ 8.6, CH); 8.54 (m, 8 H).
HR-ESI-MS: 616.2368 ([M þ H]þ, C41H32N2O4þ ; calc. 616.2362).
General Procedure 1 (GP 1): Preparation of Single-Labeled Rods 3a – 3d, and Reference Compounds
3e and 3f. The disazido rods 1a, 1b, or compound 11, fluorescent dye 2a or 2b (1.1 equiv.), and Et3N
(1.1 equiv.) were dissolved in CH2Cl2 (0.5m), and Cu/C (5 mol-%) was added. After stirring overnight,
the mixture was diluted with CH2Cl2, filtered over Celite, and the solvent was evaporated. The resulting
residue was purified by FC or semi-prep. RP-HPLC.

Helvetica Chimica Acta – Vol. 96 (2013)
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General Procedure 2 (GP 2): Preparation of Double-Labeled Rods 4c and 4d. As described in GP 1, a
soln. of 1a or 1b, both fluorescent dyes 2c and 2d simultaneously (each 1.1 equiv.), Et3N (2.2 equiv.), and
Cu/C (10 mol-%) was stirred in CH2Cl2 (0.5m) at r.t. After 7 h, the mixture was diluted with CH2Cl2,
filtered over Celite, and the solvent was evaporated. The resulting residue was purified by prep. NP-
HPLC.
5-{[(1-{2-[15-(Azidoacetyl)-7,11,18,21-tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]henicos-3-yl]-2-oxo-
ethyl}-1H-1,2,3-triazol-4-yl)methyl]carbamoyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic Acid (3a).
Compound 3a was synthesized according to GP 1 with MeOH as solvent instead of CH2Cl2. The residue
was separated by semi-prep. RP-HPLC, to give 3a (63%). Yellow solid. RP-HPLC: tR 12.5 min. MALDI-
TOF-MS: 875.81 ([M þ H]þ, C43H44N9O1þ2 ; calc. 873.31).
5-{[(1-{2-[15-(Azidoacetyl)-7,11,18,21-tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]henicos-3-yl]-2-oxo-
ethyl}-1H-1,2,3-triazol-4-yl)methyl]carbamoyl}-2-[6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl]-
benzoate (3b). Compound 3b was synthesized according to GP 1. The residue was separated by semi-
prep. RP-HPLC, to give 3b (74%). Pink solid. RP-HPLC: tR 14.5 min. MALDI-TOF-MS: 988.26 ([M þ
H]þ, C51H62N11O1þ0 ; calc. 988.47).
5-{[(1-{2-[1’’’’’’’’-(Azidoacetyl)-4’’’’,8’’’’-dibutyl-1H-octaspiro[piperidine-4,2’-[1,3]dioxane-5’,5’’-
[1,3]dioxane-2’’,1’’’-cyclohexane-4’’’,2’’’’-[1,3]dioxolo[4,5-f][1,3]benzodioxole-6’’’’,1’’’’’-cyclohexane-
4’’’’’,2’’’’’’-[1,3]dioxane-5’’’’’’,5’’’’’’’-[1,3]dioxane-2’’’’’’’,4’’’’’’’’-piperidin]-1-yl]-2-oxoethyl}-1H-1,2,3-triazol-4-
yl)methyl]carbamoyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (3c). The Compound 3c was
synthesized according to GP 1 with MeOH/CH2Cl2 1:1 as solvent. The yellow residue was purified by
semi-prep. RP-HPLC to give 3c (52%). RP-HPLC: tR 26.8 min. MALDI-TOF-MS: 1421.53 ([M þ H]þ,
C74H86N9Oþ20 ; calc. 1420.60).
5-{[(1-{2-[1’’’’’’’’-(Azidoacetyl)-4’’’’,8’’’’-dibutyl-1H-octaspiro[piperidine-4,2’-[1,3]dioxane-5’,5’’-
[1,3]dioxane-2’’,1’’’-cyclohexane-4’’’,2’’’’-[1,3]dioxolo[4,5-f][1,3]benzodioxole-6’’’’,1’’’’’-cyclohexane-
4’’’’’,2’’’’’’-[1,3]dioxane-5’’’’’’,5’’’’’’’-[1,3]dioxane-2’’’’’’’,4’’’’’’’’-piperidin]-1-yl]-2-oxoethyl}-1H-1,2,3-triazol-4-
yl)methyl]carbamoyl}-2-[6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl]benzoate (3d). Com-
pound 3d was synthesized according to GP 1. The resulting residue was purified by FC (CH2Cl2/
MeOH/Et3N 20 :1:0.5) to give 3d (65%). Pink solid. Rf (CH2Cl2/MeOH/Et3N 20 :1:0.5) 0.18. MALDI-
TOF-MS: 1531.52 ([M þ H]þ, C82H104N11O8þ ; calc. 1530.76).
5-({[1-(2-{4-[(2,2-Dimethylpropanoyl)oxy]piperidin-1-yl}-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}-
carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic Acid (3e). Compound 3e was synthesized
according to GP 1 with MeOH as solvent instead of CH2Cl2. The residue was purified on a Sephadex LH
20 column with MeOH and yielded 3e (63%). Yellow solid. Rf (CH2Cl2/MeOH 100 :5) 0.38. MALDI-
TOF-MS: 706.37 ([M þ Na]þ, C36H35N523NaO9þ ; calc. 704.23).
2-[6-(Diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl]-5-({[1-(2-{4-[(2,2-dimethylpropanoyl)-
oxy]piperidin-1-yl}-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}carbamoyl)benzoate (3f). Compound 3f
was synthesized as described for 3e. The residue was separated by using a Sephadex LH 20 column to
give 3f (53%). Purple solid. Rf (CH2Cl2/MeOH 10 :1) 0.42. MALDI-TOF-MS: 794.36 ([M þ H]þ,
C44H54N7Oþ9 ; calc. 792.41).
5-[({1-[2-(15-{[4-({[3-Carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]amino}methyl)-1H-
1,2,3-triazol-1-yl]acetyl}-7,11,18,21-tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]henicos-3-yl)-2-oxoethyl]-
1H-1,2,3-triazol-4-yl}methyl)carbamoyl]-2-[6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl]ben-
zoate (4a). A suspension of 3b (5 mg, 5.06 mmol, 1 equiv.), 2a (2.3 mg, 5.57 mmol, 1.1 equiv.), Et3N
(0.8 ml, 5.57 mmol, 1.1 equiv.) and 1 mg of Cu/C was stirred in MeOH (150 ml) at r.t., and the reaction was
monitored by TLC. After 7 h, the mixture was diluted with MeOH, filtered over Celite, and the solvent
was evaporated. The purple residue was purified by semi-prep. RP-HPLC to give 4a (2.8 mg, 2.0 mmol,
39%). tR 13.4 min. MALDI-TOF-MS: 1402.09 ([M þ H]þ, C75H77N12O1þ6 ; calc. 1401.56).
5-[({1-[2-(4’’’’,8’’’’-Dibutyl-1’’’’’’’’-{[4-({[3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]-
amino}methyl)-1H-1,2,3-triazol-1-yl]acetyl}-1H-octaspiro[piperidine-4,2’-[1,3]dioxane-5’,5’’-[1,3]diox-
ane-2’’,1’’’-cyclohexane-4’’’,2’’’’-[1,3]dioxolo[4,5-f][1,3]benzodioxole-6’’’’,1’’’’’-cyclohexane-4’’’’’,2’’’’’’-
[1,3]dioxane-5’’’’’’,5’’’’’’’-[1,3]dioxane-2’’’’’’’,4’’’’’’’’-piperidin]-1-yl)-2-oxoethyl]-1H-1,2,3-triazol-4-yl}me-
thyl)carbamoyl]-2-[6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl]benzoate (4b). A suspension of
3d (3.6 mg, 2.35 mmol, 1 equiv.), 2a (1.1 mg, 2.59 mmol, 1.1 equiv.), Et3N (0.4 ml, 2.59 mmol, 1.1 equiv.)

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Helvetica Chimica Acta – Vol. 96 (2013)
and 1 mg of Cu/C was stirred in CH2Cl2/MeOH 1:1 (200 ml) at r.t., and the reaction was monitored by
TLC. After 8 h, the mixture was diluted with CH2Cl2 and filtered over Celite. The solvent was evaporated,
and the resulting residue was purified by size exclusion chromatography (Sephadex LH 20; CH2Cl2/
MeOH 1:1) to yield 4b (2.5 mg, 1.29 mmol, 55%). Purple solid. MALDI-TOF-MS: 1944.41 ([M þ H]þ,
C106H119N12O24; calc. 1943.85).
2-[2,4-Di-(tert)-butylphenyl]-9-[(1-{2-oxo-2-[15-({4-[(pyren-1-ylmethoxy)methyl]-1H-1,2,3-triazol-
1-yl}acetyl)-7,11,18,21-tetraoxa-3,15-diazatrispiro[5.2.2.512.29.26]henicos-3-yl]ethyl}-1H-1,2,3-triazol-4-
yl)methyl]isoquinolino[4’,5’,6’:6,5,10]anthra[2,1,9-def]isoquinoline-1,3,8,10(2H,9H)-tetrone (4c). Com-
pound 4c was synthesized according GP 2, by simultaneous ꢂclickꢃ reaction of 1a, and fluorescent dyes 2c
and 2d. NP-HPLC (CH2Cl2/MeOH 100 :5) afforded 4c (38%). Purple solid. tR 7.8 min. MALDI-TOF-
MS: 1352.25 ([M þ H]þ, C80H75N10O11; calc. 1351.56), 1375.23 ([M þ Na]þ, C85H74N1023NaO1þ1 ; calc.
1373.54).
2-(2,4-Di-(tert)-butylphenyl)-9-[(1-{2-[4’’’’,8’’’’-dibutyl-1’’’’’’’’-({4-[(pyren-1-ylmethoxy)methyl]-1H-
1,2,3-triazol-1-yl}acetyl)-1H-octaspiro[piperidine-4,2’-[1,3]dioxane-5’,5’’-[1,3]dioxane-2’’,1’’’-cyclohex-
ane-4’’’,2’’’’-[1,3]dioxolo[4,5-f][1,3]benzodioxole-6’’’’,1’’’’’-cyclohexane-4’’’’’,2’’’’’’-[1,3]dioxane-5’’’’’’,5’’’’’’’-
[1,3]dioxane-2’’’’’’’,4’’’’’’’’-piperidin]-1-yl]-2-oxoethyl}-1H-1,2,3-triazol-4-yl)methyl]isoquinolino[4’,5’,6’:
6,5,10]anthra[2,1,9-def]isoquinoline-1,3,8,10(2H,9H)-tetrone (4d). Compound 4d was synthesized ac-
cording to GP 2, by simultaneous ꢂclickꢃ reaction of 1b and fluorescent dyes 2c and 2d . The resulting
residue was purified by NP-HPLC (CHCl3/iPrOH 100 :4) to afford 4d (32%). Purple solid. tR 6.5 min.
MALDI-TOF-MS: 1895.27 ([M þ H]þ, C111H117N10O1þ9
;
calc. 1893.85), 1917.38 ([M þ Na]þ,
C111H116N1023NaO1þ9 ; calc. 1915.83).
Absorption and Steady-State Fluorescence Spectroscopy. The absorption spectra were recorded with
a Cary 500 UV/VIS spectrometer (Varian, DE-Darmstadt) in the spectral range of 400 nm < labs
<
700 nm, and the background-corrected steady-state fluorescence spectra were recorded on a Fluoro-
max-3 spectrofluorometer (HORIBA Jobin Yvon GmbH, DE-Munich). The fluorescence quantum
yields were determined on a quantum yield fluorescence spectrometer using an integrating sphere (PL
Quantum Yield Measurement System C9920-02, Hamamatsu Photonics, Japan).
Time-Resolved Fluorescence Spectroscopy and Depolarization Measurements. The fluorescence-
decay times were determined with a FLS920 fluorescence lifetime spectrophotometer (Edinburgh
Instruments, UK-Livingston) operated in the time-correlated single photon-counting (TCSPC) mode.
Samples were excited with a vertically polarized supercontinuum white light source (SC400-PP, Fianium
Ltd., UK-Southhampton) at a repetition rate of 20 MHz and a pulse width (FWHM) of 30 ps. For the
detection under magic angle conditions (54.88), a multichannel plate (ELDY EM1-132/300 MCP-PMT,
Europhoton, DE-Berlin) was used (see Table 1 for specific information on excitation and emission
wavelengths).
For the determination of the time-resolved anisotropy (including the respective G factor), the
fluorescence-decay curves were recorded with an emission polarizer placed in vertical and horizontal
position, resp. The experimental data were evaluated using Eqns. 2 and 3 [18a]:
IvvðtÞ ꢀ IvhðtÞ ꢂ G
(2)
rðtÞ ¼
IvvðtÞ þ 2 ꢂ IvhðtÞ ꢂ G
Ihv
Ihh
(3)
G ¼
For small molecules, the anisotropy decay, r(t), can be analyzed according to Eqn. 4 [18a]:
ꢀ
ꢁ
t
ꢀ
(4)
rðtÞ ¼ r0 ꢂ exp
ꢀ
From the evaluation of r(t), the fundamental anisotropy, r0, at the time t ¼ 0, and the rotational
correlation time f of the molecule were obtained. The fundamental anisotropy, r0, was also determined in
a reference experiment measuring the steady-state anisotropy at 77 K.

Helvetica Chimica Acta – Vol. 96 (2013)
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For the analysis of the fluorescence-anisotropy data obtained for micellar samples, the ꢂtwo-stepꢃ
model as an extension of the ꢂwobbling-in-coneꢃ model was applied [18b]. For dyes incorporated in
micelles, two motions causing depolarization are considered: i) a fast hindered rotation of the dye
molecules within the micellar environment, resulting in an anisotropy loss, and ii) a contribution of the
slow overall motion of the micelle itself. In such a case, the time-resolved anisotropy is a product of the
two anisotropy functions describing the different motions (see Eqn. 5) [18b]:
ꢀ
ꢁ
ꢀ
ꢁ
ꢂ
ꢃ
ꢀt
ꢀt
(5)
rðtÞ ¼ rfastðtÞ ꢂ rslowðtÞ ¼ 1 ꢀ S2 ꢂ exp
þ S2exp
ꢀfast
ꢀslow
with fslow and ffast describing the rotational correlation time of the slow and fast motion, resp. Here, the
slow rotational correlation time, fslow, is a sum of the rotational motion of the micelle (fM) and the lateral
diffusion of the dye in the micelle (fL) [18b]:
1
ꢀslow ꢀM ꢀL
1
1
(6)
¼
þ
The fast correlation time ffast is defined according to [18b]:
1
ꢀfast ꢀw ꢀslow
1
1
(7)
¼
þ
with the effective correlation time fw (reorientation time for the wobbling motion).
The relationship between the anisotropy decay, r(t), and the structure of the micelles can be followed
using the order parameter S. In case the motion is fast and isotropic, S ¼ 0, and in case the motion is
completely restricted, S ¼ 1. The simple ꢂwobbling-in-coneꢃ model describes a restricted motion for a
symmetrical ellipsoid with its transition dipole moment parallel to the symmetry axis and the molecule
diffusing freely in a cone with an angle q. The wobbling cone angle q can be calculated using S [18b]:
ꢄ
ꢅ
2
1
2
S2 ¼
ꢂ ðcosqÞ ꢂ ð1 þ cosqÞ for 0 ꢇ q ꢇ 90ꢈ
(8)
For the analysis, the weighting wi of the anisotropy data was carried out according to Eqn. 9 [18c]:
3 ꢂ ½IvvðtiÞ þ 2 ꢂ IvhðtiÞꢉ
(9)
wi ¼
with i ¼ 1:::2
2
3
2 þ rðtiÞ þ 5 ꢂ ½rðtiÞꢉ ꢀ2 ꢂ ½rðtiÞꢉ
Single-Molecule Fluorescence Measurements. Single-molecule fluorescence experiments were
performed on an inverse time-resolved fluorescence confocal microscope system MicroTime 200 driven
by a PicoHarp 300 PC-board (PicoQuant, DE-Berlin). Excitation at lexc 470 nm was carried out with a
supercontinuum white light source (SC400-2, Fianium Ltd., UK-Southhampton) operated at a repetition
rate of 20 MHz. The excitation light was coupled via a single mode fibre into the body of the inverted
microscope (Olympus IX71). The excitation power of (70 ꢁ 5) mW was measured at the focus of the
microscope objective. The data were collected for 20 min at T¼ (294 ꢁ 1) K. After reflection by a
dichroic mirror, the collimated laser beam was focused by an oil immersion objective (Olympus,
PlanApo, 100x/NA1.4). The collected fluorescence light was transmitted by the dichroic mirror, passed a
long-pass 500-nm filter for complete excitation light rejection and a 50-mm pinhole. To distinguish
between the donor and acceptor emission, a band-pass filter of 522 nm (BP 522/15, Semrock, Inc., USA)
and of 589 nm (BP 589/15, Semrock, Inc., USA) was placed in front of the avalanche photodiodes
(SPCM-AQR-13 & SPCM-CD-2801, Perkin-Elmer, Waltham, USA), resp. The signals of the donor and
acceptor channel were separately but simultaneously detected as a function of time and binned in 1-ms
time intervals. After removing the background noise by setting a threshold to filter out the significant
bursts of photons, the FRET efficiency was calculated according to Eqn. 10, using the software
SymPhoTime 5.1.31 (PicoQuant, DE-Berlin) [19][20]:

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Helvetica Chimica Acta – Vol. 96 (2013)
nA ꢀ nD ꢂ LAD
E ¼
(10)
nA ꢂ ð1 ꢀ LDAÞ þ nD ꢂ ðg ꢀ LAD
Þ
In Eqn. 10, nA and nD denote the number of photons in the burst of the acceptor and donor channel,
resp., g is the compensation for the quantum yields of the dyes and detection efficiency differences
between donor and acceptor channel. Due to the difficulties in the determination of the experimental
parameter, g was assumed to be 1 [19]. Compensation for the donor/acceptor and acceptor/donor bleed
through, LDA and LAD, was experimentally determined with the single dye-labeled samples. No
photobleaching of the dyes was observed during the experiments.
Ensemble FRET Experiments. In ensemble measurements, the FRET efficiencies were calculated
from the fluorescence intensities and decay times of the donor in the absence (ID and tD) and presence
(IDA and tDA ) of acceptor molecules according to Eqn. 11 [18a]:
IDA
ID
tDA
tD
EE ¼ 1 ꢀ
andEt ¼ 1 ꢀ
(11)
The calculated FRET efficiency (EC) was derived from [18a]:
R06
R60 þ R6
EC
¼
(12)
with the interchromophoric distance R, and the critical Fçrster distance R0 that was calculated according
to Eqn. 13 [21]:
1
Z
9 ꢂ lnð10Þ ꢂ k2 ꢂ FF;D
(13)
R60 ¼
ꢂ
IDðlÞ ꢂ eAðlÞ ꢂ l4 ꢂ dl
128 ꢂ p5 ꢂ NA ꢂ n4
0
In Eqn. 13, the Avogadro constant is given by NA and the refractive index by n. The orientation factor
is denoted by k2, and the quantum yield of the donor in the absence of energy transfer is FF,D. The spectral
overlap integral J was calculated from the normalized fluorescence spectrum of the donor ID(l) and the
molar absorption coefficient eA(l) of the acceptor.
REFERENCES
[1] a) D. M. Togashi, B. Szczupak, A. G. Ryder, A. Calvet, M. OꢃLoughlin, J. Phys. Chem. A 2009, 113,
´
2757; b) N. Marme, J.-P. Knemeyer, M. Sauer, J. Wolfrum, Bioconjugate Chem. 2003, 14, 1133; c) X.
Li, R. Zhu, A. Yu, X. S. Zhao, J. Phys. Chem. B 2011, 115, 6265; d) J. R. Unruh, G. Gokulrangan,
G. H. Lushington, C. K. Johnson, G. S. Wilson, Biophys. J. 2005, 88, 3455; e) E. Dolghih, A. E.
Roitberg, J. L. Krause, J. Photochem. Photobiol., A 2007, 190, 321; f) A. Iqbal, S. Arslan, B. Okumus,
T. J. Wilson, G. Giraud, D. G. Norman, T. Ha, D. M. J. Lilley, Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
11176.
[2] a) P. Wessig, K. Mçllnitz, C. Eiserbeck, Chem. – Eur. J. 2007, 13, 4859; b) P. Wessig, K. Mçllnitz, J.
Org. Chem. 2008, 73, 4452; c) P. Mꢁller, J. Nikolaus, S. Schiller, A. Herrmann, K. Mçllnitz, S. Czapla,
P. Wessig, Angew. Chem. 2009, 121, 4497; d) J. Nikolaus, S. Czapla, K. Mçllnitz, C. T. Hçfer, A.
Herrmann, P. Wessig, P. Mꢁller, Biochim. Biophys. Acta, Biomembr. 2011, 1808, 2781; e) P. F. H.
Schwab, M. D. Levin, J. Michl, Chem. Rev. 1999, 99, 1863.
[3] a) M. Sauer, K. H. Drexhage, U. Lieberwirth, R. Mꢁller, S. Nord, C. Zander, Chem. Phys. Lett. 1998,
284, 153; b) M. Sauer, K.-T. Han, R. Mꢁller, S. Nord, A. Schulz, S. Seeger, J. Wolfrum, J. Arden-
Jacob, G. Deltau, N. J. Marx, C. Zander, K. H. Drexhage, J. Fluoresc. 1995, 5, 247; c) F. F. Lewis, T.
Wu, Y. Zhang, R. L. Letsinger, S. R. Greenfield, M. R. Wasielewski, Science 1997, 277, 673; d) A.
Dietrich, V. Buschmann, C. Mꢁller, M. Sauer, Rev. Mol. Biotechnol. 2002, 82, 211.

Helvetica Chimica Acta – Vol. 96 (2013)
2067
[4] D. Bose, D. Sarkar, N. Chattopadhyay, Photochem. Photobiol. 2010, 86, 538; M. E. Sanborn, B. K.
´
Connolly, K. Gurunathan, M. Levitus, J. Phys. Chem. B 2007, 111, 11064; G. Vamosi, C. Gohike,
R. M. Clegg, Biophys. J. 1996, 71, 972.
[5] P. Wu, V. V. Fokin, Aldrichim. Acta 2007, 40, 7; H. C. Kolb, H. G. Finn, K. B. Sharpless, Angew.
Chem. 2001, 113, 2056; Angew. Chem., Int. Ed. 2001, 40, 2004; M. G. Finn, V. V. Fokin, Chem. Soc.
Rev. 2010, 39, 1231.
[6] B. H. Lipshutz, B. R. Taft, Angew. Chem., Int. Ed. 2006, 45, 8235.
[7] a) M. Dominska, G. J. Blanchard, Langmuir 2010, 26, 1043; b) L. You, G. W. Gokel, Chem. – Eur. J.
2008, 14, 5861; c) C. E. Bunker, B. Ma, K. J. Simmons, H. W. Rollins, J. T. Liu, J. J. Ma, C. W. Martin,
D. D. DesMarteau, Y. P. Sun, J. Electroanal. Chem. 1998, 459, 15; d) E. Szajdzinska-Pietek, M.
Wolszczak, A. Plonka, S. Schlick, J. Am. Chem. Soc. 1998, 120, 4215.
[8] S. Voss, R. Fischer, G. Jung, K.-H. Wiesmueller, R. Brock, J. Am. Chem. Soc. 2007, 129, 554; F.
Caruso, E. Donath, H. Mohwald, J. Phys. Chem. B 1998, 102, 2011; T. Tuschl, C. Gohlke, T. M. Jovin,
E. Westhof, F. Eckstein, Science 1994, 266, 785.
[9] R. F. Chen, J. R. Knutson, Anal. Biochem. 1988, 172, 61.
[10] D. A. Holowka, B. Baird, Biochemistry 1983, 22, 3466.
[11] I. Z. Steinberg, J. Chem. Phys. 1968, 48, 2411.
[12] Y. O. Posokhov, M. Merzlyakov, K. Hristova, A. S. Ladokhin, Anal. Biochem. 2008, 380, 134.
[13] B. A. Coles, R. G. Compton, J. Electroanal. Chem. 1983, 144, 87.
[14] Y. Hua, A. H. Flood, Chem. Soc. Rev. 2010, 39, 1262.
[15] A. Techen, C. Hille, C. Dosche, M. U. Kumke, J. Colloid Interface Sci. 2012, 377, 251.
[16] N. C. Maiti, M. M. G. Krishna, P. J. Britto, N. Periasamy, J. Phys. Chem. 1997, 101, 11051; G. B. Dutt,
J. Phys. Chem. 2002, 106, 7398.
[17] D. Behar, P. Neta, J. Phys. Chem. 1981, 85, 690.
[18] a) J. R. Lakowicz, ꢂPrinciples of Fluorescence Spectroscopyꢃ, 3rd edn., Springer Science þ Business
Media, New York, 2006; b) E. L. Quitevis, A. H. Marcus, M. D. Fayer, J. Phys. Chem. 1993, 97, 5762;
c) D. V. OꢃConnor, D. Phillips, ꢂTime-correlated Single Photon Countingꢃ, Academic Press, London,
1984, Chapt. 8.
[19] B. Schuler, E. A. Lipman, W. A. Eaton, Nature 2002, 419, 743.
[20] M. Dahan, A. A. Deniz, T. Ha, D. S. Chemla, P. G. Schultz, S. Weiss, Chem. Phys. 1999, 247, 85.
[21] A. Kupstat, T. Ritschel, M. U. Kumke, Bioconjugate Chem. 2011, 22, 2546.
Received November 21, 2012