Synthesis and optical properties of cyanine dyes as fluorescent DNA base substitutions for live cell imaging

Article abstract of DOI:10.1002/ejoc.200901423

The chromophore of thiazole orange (TO) and its derivative TO3 were incorporated synthetically as base surrogates into oligonucleotides using automated phosphoramidite chemistry. In comparison to TO, the TO3 chromophore contains an extended carbomethine b

Full text of DOI:10.1002/ejoc.200901423

FULL PAPER
DOI: 10.1002/ejoc.200901423
Synthesis and Optical Properties of Cyanine Dyes as Fluorescent DNA Base
Substitutions for Live Cell Imaging
Carolin Holzhauser,[a] Sina Berndl,[a] Florian Menacher,[a] Miriam Breunig,[b]
Achim Göpferich,[b] and Hans-Achim Wagenknecht*[a]
Keywords: Thiazole orange / Nucleic acids / Oligonucleotides / Fluorescence / Energy transfer / Dyes/pigments
The chromophore of thiazole orange (TO) and its derivative
TO3 were incorporated synthetically as base surrogates into
oligonucleotides using automated phosphoramidite chemis-
try. In comparison to TO, the TO3 chromophore contains an
extended carbomethine bridge that shifts the absorption and
emission significantly to the red. (S)-1-Aminopropane-2,3-
diol served as an acyclic linker between the phosphodiesters.
This linker was attached either to the quinoline or the thi-
azole nitrogen of the TO and TO3 dyes. The optical proper-
ties of TO and TO3 were studied in different DNA base envi-
ronments and with different opposite bases. Both dyes as
fluorescent DNA base substitutions show a brightness that is
sufficient for bioanalytic and imaging applications. Addition-
ally the TO3 dyes were combined as interstrand dimers, but
in contrast to TO dimers, a red-shifted fluorescence was not
observed. However, TO and TO3 can be combined to an in-
terstrand chromophore pair and a DNA hybridization-de-
pendent energy transfer process can be obtained between
the modifications. As a result, the emission is shifted from the
TO-typical value of 530 nm to 670 nm. This concept can be
applied for fluorescence imaging to monitor DNA delivery as
well as processing inside living cells by confocal microscopy.
In contrast to the non-covalently binding TO dyes, the TO-
and TO3-modified oligonucleotides are cell-permeable.
(Ͼ450 nm) in combination with an energy acceptor (second
label) that emits bright fluorescence in the visible spectrum
due to a high FRET efficiency remains a challenge.
Introduction
Bioanalysis and imaging of nucleic acids in chemical cell
biology require bright and tailor-made fluorophores that
can be linked covalently to DNA (or RNA). A variety of
fluorescent dyes has been synthetically attached to oligonu-
cleotides in order to detect the complementary sequential
information and single base variations by enhanced emis-
sion intensity or quenching.[1–5] Especially molecular bea-
cons have become powerful tools in a variety of DNA/RNA
based applications.[6–9] However, simple changes of the fluo-
rescence intensity at a given wavelength carry the risk of
side effects causing artifacts in the fluorescence readout.
Undesired emission quenching is a significant problem es-
pecially in cell imaging leading to false positive signals or
reducing the sensitivity (S/B ratio). Hence, dual fluorescent
oligonucleotide probes for imaging nucleic acids are needed
which are bright as a single label and have the potential to
change their emission maximum (= color) in the presence of
the target. This has been realized e.g. as wavelength-shifting
molecular beacons based on fluorescence resonance energy
transfer processes (FRET).[10] However, the design of dual-
labeled oligonucleotide probes which carry an energy donor
(first label) that can be excited selectively beyond the UV
Currently, the excimer-induced red-shifting of the emis-
sion wavelength undergoes a renaissance with respect to
DNA analytics. DNA with intrastrand and interstrand di-
mers mainly of pyrene and perylene exhibit strong excimer
fluorescence[11–15] and can also be applied in molecular bea-
cons.[16–19] However, the excitation of pyrene requires high-
energy UV light, a circumstance that limits the bioanalytic
and imaging applications significantly. Recently, we re-
ported that the excitation of perylene bisimide pairs at
505 nm yields an excimer-type shift of the fluorescence by
ca. 100 nm in DNA, and we applied it to quantify single
base mismatches.[20] One of the major disadvantages of per-
ylene bisimide in DNA, however, is the low quantum yield
in certain DNA sequences due to fluorescence quenching
by charge transfer processes especially with guanines.[21,22]
In this context, thiazole orange (TO) represents a promising
alternative. Among the brightly emitting and broadly ap-
plied cyanine dyes, such as Cy3 and Cy5,[23] thiazole orange
exhibits an oxidation potential (TO+/TO·2+) of 1.4 eV (vs.
NHE).[24] Together with E00 = 2.4 eV for TO, the excitation
is not sufficient for the photooxidation of DNA which
stands in contrast to the perylene bisimide dye. The known
bioconjugation chemistry of TO is versatile. TO was linked
covalently to the phosphodiester bridges[25] or to 5Ј-ter-
minal[26] positions of oligonucleotides. Moreover, TO was
attached synthetically at the terminus of DNA-binding pep-
[a] University of Regensburg, Institute for Organic Chemistry,
93040 Regensburg, Germany
Fax: +49-941-943-4617
E-mail: achim.wagenknecht@chemie.uni-regensburg.de
[b] University of Regensburg, Institute for Pharmacy,
93040 Regensburg, Germany
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tides for staining in cell biology and as chemical-biological azole, compound 5 was linked by a carbamate function to
tools for the investigation of DNA damages that are caused the enantiomerically pure derivative of (S)-1-amino-
by singlet oxygen.[27–29] TO as a base surrogate in PNA was propane-2,3-diol 6 carrying already the 4,4Ј-dimethoxytrityl
studied extensively in order to detect single base variations (DMT) protecting group that is needed for the automated
in a very sensitive way.[3,30,31] We reported that the emission DNA phosphoramidite chemistry. The coupling of the
intensity of TO as an artificial DNA base can be modulated phosphiteamide group to the free hydroxy group of 7 was
by short-range electron transfer.[32] Moreover, we described performed using standard procedures in CH2Cl2 yielding
recently interstrand TO dimers in DNA which show a shift the phosphoramidite 8.
of the fluorescence color from the TO-typical green
(530 nm) to orange (580 nm). This can be used as fluores-
cence readout for DNA hybridization and for correspond-
ing molecular beacons.[33]
Herein, we describe and compare the optical properties
of the TO dye and additionally for the derivative TO3,[34]
as synthetic and fluorescent DNA bases for cell imaging.
In comparison to TO, the TO3 chromophore contains an
extended carbomethine bridge that shifts the absorption
and emission significantly to the red. Both dyes should
combine to an interstrand FRET couple that allows moni-
toring DNA hybridization by an emission color change. In
order to study the stacking interactions and the fluores-
cence properties of the TO and the TO3 dye in a given
DNA base sequence we chose our base surrogate approach.
The 2Ј-deoxyribofuranoside moiety of natural nucleotides
was replaced by an acyclic linker system that is tethered to
the nitrogen either of the thiazole ring or the quinoline ring
of the dyes. As previously, (S)-1-aminopropane-2,3-diol
serves as an acyclic linker between the phosphodiesters.
Similar propanediol linkers have been used by others in or-
der to prepare glycol nucleic acids (GNA),[35] twisted inter-
calating nucleic acids (TINA),[36] alkyne-modified oligonu-
cleotides for the click-type postsynthetic modification,[37–39]
Scheme 1. Synthesis of DNA building block 8 for the TO-b modifi-
cation: a) MeCN, 110 °C, 91 h; 90%; b) NEt3 (2.5 equiv.), CH2Cl2/
and by our group for fluorescent DNA base substitutions
MeOH = 1:1, room temp., 15 h; 31%; c) 1,1Ј-carbonyldiimidazole
(1.1 equiv.), DMF, room temp., 48 h; 56%; d) DIPEA, 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite, CH2Cl2, room temp.,
by ethidium,[40] indole,[41] perylene bisimide,[20,21] phenothi-
azine[42] as DNA base surrogates. Avoiding the acid/base
labile glycosidic bond of natural nucleosides,[43] the applica-
15 h; quant.
tion of a flexible acyclic linker provides a high chemical
stability during the preparation of chromophore-DNA con- Similar to TO, the TO3 chromophore was linked either
jugates via automated phosphoramidite chemistry, and al- by the (i) quinoline nitrogen or (ii) the thiazole nitrogen to
lows the chromophore to intercalate.
the linker and the oligonucleotides. For the first structural
alternative (TO3-a) the key step is the reaction of hydroxy-
propylated 4-methylquinoline 9[32] with the hemicyanine 10
(Scheme 2). Best yields (75%) of the dye 11 were only ob-
tained if 3 equiv. of 9 and 6 equiv. of Et3N were treated with
Results and Discussion
For the synthetic incorporation of TO into oligonucleo- 10. The remaining reaction steps were similar as described
tides the (S)-1-aminopropane-2,3-diol linker can be at- previously for the building block 8. The coupling with the
tached either to the (i) quinoline nitrogen or (ii) the thiazole linker 6 via a carbamate function and subsequent phos-
nitrogen. The synthesis of the DNA building block and the phitylation of 12 gave DNA building block 13.
protocol for automated DNA synthesis for the first alterna-
The attachment of the TO3 chromophore to oligonucleo-
tive modification (TO-a) was described recently.[32] The syn- tides through its thiazole nitrogen started with the reaction
thesis of the DNA building block for the second alternative of the hydroxypropylated thiazole 3 with N,NЈ-diphenyl-
modification (TO-b)[33] (Scheme 1) starts with the alkyl- formamidine. The hemicyanine 14 was obtained in 62%
ation of the 2-methylbenzotriazole (1) with 3-iodopropan- yield and subsequently converted with 1,4-dimethylquinol-
1-ol (2) to compound 3. The subsequent condensation with inium iodide (18) to the cyanine 15. The remaining reaction
1-methylquinolinium iodide (4) yields the TO derivative 5 steps were again similar as described previously for the
in 31%. The reaction proceeds selectively to the para posi- building blocks 8 and 13: The coupling with the linker 6
tion of the methylated quinoline as confirmed by 2D via a carbamate function and subsequent phosphitylation
NOESY NMR measurements. Using 1,1Ј-carbonyldiimid- of 16 gave DNA building block 17 (Scheme 3).
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Cyanine Dyes as Fluorescent DNA Base Substitutions
tervening step after 30.8 min for washing and refreshing the
activator/phosphoramidite solution in the CPG vial.[40] The
corresponding TO-a-modified oligonucleotides were pre-
pared according to the literature.[32] The modified single-
stranded oligonucleotides were identified by ESI mass spec-
trometry and quantified by their absorbance at 260 nm
using an extinction coefficient of 9.400 –1 cm–1 for the TO-
a and TO-b modifications.[3] Duplexes were formed by heat-
ing of the modified oligonucleotides to 90 °C (15 min) in
the presence of 1.2 equiv. of the corresponding complemen-
tary unmodified oligonucleotide strand. For each modifica-
tion, two duplex sets were synthesized representatively
(Scheme 4) in order to study the optical properties of the
dyes as artificial DNA base substitutions. Each pair of du-
plex sets contain the TO or TO3 chromophore in two dif-
ferent environments, either T-A base pairs (e.g. DNA1C-
DNA1G for the TO-a modification) or G-C base pairs (e.g.
DNA4C-DNA4G for the TO3-b modification). Addition-
ally, in each set of duplexes, the base in the complementary
strand that sits opposite to the artificial DNA base substi-
tution was varied.
Scheme 2. Synthesis of DNA building block 13 for the TO3-a
modification: a) Et3N (6.0 equiv.), CH2Cl2, room temp., 48 h; 75%;
b) 1,1Ј-carbonyldiimidazole (2.0 equiv.), DMF, room temp., 13 d,
87%; c) DIPEA, 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite, CH2Cl2, room temp., 15 h; quant.
Scheme 4. Sequences of TO- and TO3-modified DNA duplexes for
the investigation of optical properties.
The UV/Vis absorption properties of all TO-modified
duplexes in the sets DNA1Y–DNA4Y are remarkably sim-
ilar showing the presence of the TO base surrogate by the
characteristic absorption in a range between 450 and
550 nm (Figure 1). The comparison of each TO absorption
maxima around 505 nm reveals only minor differences in
the extinction coefficient. In contrast, a dramatic depen-
dence of the TO absorption on the sequential context was
described in PNA-DNA hybrids and was interpreted as re-
sult of the forced intercalation of the TO dye.[3] Hence, the
observation of small differences with our TO-modified
DNA duplexes indicate that the TO dyes are not forced to
intercalate and tend to bind in the groove. Similar observa-
tions were made for the absorption of the TO3-type DNA
Scheme 3. Synthesis of DNA building block 17 for the TO3-b
modification: a) N,NЈ-diphenylformamidine (1.0 equiv.), Ac2O,
140 °C, 1 h, 62%; b) 1) Et3N (6.0 equiv.), CH2Cl2, room temp.,
72 h, 58%; then 2) MeOH/NH3(32%) = 2:3, 74 °C, 4 h, 87 %; c)
1,1Ј-carbonyldiimidazole (2.0 equiv.), DMF, room temp., 7 d; 90%;
d) DIPEA, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite,
CH2Cl2, room temp., overnight.
Using DNA building blocks 8, 13, or 17, the cyanine- base substitutions in the sets DNA5Y–DNA8Y. The TO3
modified single-stranded (ss) oligonucleotides were pre- chromophore shows an absorption in a range between 570
pared using the DNA synthesizer. The coupling time was and 680 nm with slight variations in the extinction coeffi-
extended from 1.6 min (standard) to 61.6 min with an in- cients at the maximum around 645 nm.
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Figure 1. UV/Vis absorption of the duplex sets 2.5 µ DNA1Y–DNA8Y in 10 m Na-Pi buffer, 250 m NaCl, pH 7, ss = single stranded.
In contrast to the UV/Vis absorption spectra, the fluores-
cence spectra of the TO-modified DNA duplexes DNA1Y–
DNA4Y exhibit small differences in the emission quantities
depending on the adjacent base pairs (T-A vs. G-C) but
significant differences depending on the type of opposite
base (C, G, A or T) (Figure 2). Interestingly, the single
strands that bear the TO-a or TO-b modification exhibit
similarly strong fluorescence intensities as the correspond-
ing duplexes. This observation indicates a structural preor-
ganization of the TO dye with the adjacent bases that exists
in the single strand. These interactions reduce the structural
flexibility along the methine bridge and hence prevent the
radiationless decay of the TO dye by internal conversion.[3]
Similar results were obtained with the TO3-modified du-
plex sets DNA5Y–DNA8Y. However, in case of the TO3 dye
both structural variations (adjacent base pairs and opposite
base) influence the mode of interaction resulting in different
emission quantum yields. This sensitivity of TO and the
TO3 chromophores towards its sequential context repre-
sents an important observation that can be potentially ap-
plied e.g. in time-resolved experiments to study DNA dy-
namics on short timescales.[44,45]
The melting temperatures (Tm) of the TO-modified du-
plexes DNA1Y–DNA4Y (Table 1) are remarkably similar
within each duplex set, except DNA2A and DNA4A (which
we cannot explain). Representatively, we compared the
melting temperatures of the TO-a-modified duplexes
Figure 2. Fluorescence spectra of the duplex sets 2.5 µ DNA1Y–
DNA1C and DNA2C as well as the TO-b-modified duplexes
DNA8Y in 10 m Na-Pi buffer, 250 m NaCl, pH 7, ss = single
DNA3C and DNA4C with the corresponding unmodified
stranded.
DNA sequences[41] that bear a standard G instead of the
TO modifications. This comparison reveals a destabilization
in the range of 2.1–5.5 °C, respectively. These values are teractions of the hydrophobic TO chromophore with the
remarkably small since the incorporation of indole as a base adjacent bases and base pairs regain some of the lost ther-
surrogate into oligonucleotides via a similar acyclic linker mal stability due the glycol linker. This supports the idea
system resulted in a strong destabilization of the DNA du- that the strong fluorescence that has been observed with
plexes (approx. –12 °C).[41] Such strong destabilization is all TO-modified single strands is the result of a structural
typical for single glycol modifications.[35] Obviously, the in- preorganization by hydrophobic interactions of TO with
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Cyanine Dyes as Fluorescent DNA Base Substitutions
the adjacent base pairs. The destabilization of the TO3-a fer process. We synthesized DNA10–DNA14 identical to the
and TO3-b base substitutions in the duplex sets DNA5Y– published sequences[33] but with the TO3-a or TO3-b modi-
DNA8Y are stronger than the corresponding TO-modified fications instead of TO-b. Additionally, we prepared the
counterparts but still lower than typical single glycol modi- TO-b-modified DNA9 and DNA10 in order to combine
fications. The decrease of the melting temperatures of the them with the TO3-a- or TO3-b-modified DNA11-DNA14
representative duplexes DNA5C–DNA8C are in the range (Scheme 5).
of 3.8–9.1 °C. Obviously, the larger chromophore size in
comparison with the TO dye influences the duplex stability.
Table 1. Melting temperatures (Tm) and quantum yields φF of the
DNA duplexes, λ = 260 nm, 10–90 °C, interval: 0.7 °C/min, 2.5 µ
duplex in 10 m Na-Pi buffer, 250 m NaCl, pH 7.
Duplex
ΦF
Tm (∆Tm)[a]
[°C]
Duplex ΦF
Tm (∆Tm)[a]
[°C]
DNA1C 12.4
DNA1T
60.3 (–5.5)
59.0
58.0
DNA2C 7.2
DNA2T
68.4 (–2.3)
67.5
52.2
Scheme 5. TO and TO3-modified single-strands DNA9-DNA14
that combine to double modified duplexes (for structures of TO-
b, TO3-a and TO3-b see Scheme 4). DNA15 serves as a reference
counterstrand.
DNA1A
DNA2A
DNA1G
58.5
DNA2G
66.8
DNA3C 16.8
DNA3T
62.0 (–3.8)
58.3
58.4
DNA4C 9.7
DNA4T
68.6 (–2.1)
67.5
48.7
The duplexes that are formed of DNA11 and DNA12
(DNA11–12) and of DNA13 and DNA14 (DNA13–14) force
the two TO3-a or TO3-b modifications, respectively, into an
interstrand interaction similar to the previously published
interstrand TO-b dimers (DNA9–10). The UV/Vis absorp-
tion spectra indicate for both duplexes strong excitonic in-
teractions between the TO3 dyes by an additional absorp-
tion band at ca. 585 nm that is not present in the corre-
sponding single TO3-a- or TO3-b-modified duplexes
DNA11–15 and DNA13–15, respectively (Figure 3, left).
Such excitonic interactions were also observed in case of the
TO-b-dimer in DNA that yielded a red-shifted fluorescence
maximum.[33] Interestingly, the excitonic interactions be-
tween the TO3 dyes are not reflected by the melting tem-
peratures of the corresponding duplexes (Table 2). The Tm
value of both duplexes DNA11–12 and DNA13–14 is
60.1 °C which is significantly lower than the corresponding
duplex that contains the TO-b-dimer (65.5 °C).[33]
DNA3A
DNA4A
DNA3G
60.5
DNA4G
65.9
DNA5C 12.7
DNA5T
DNA5A
DNA5G
56.7 (–9.1)
57.7
57.2
DNA6C 30.5 66.2 (–3.8)
DNA6T
DNA6A
DNA6G
64.2
64.5
64.7
57.1
DNA7C 26.2
DNA7T
DNA7A
DNA7G
59.2 (–6.6)
58.7
56.9
DNA8C 16.1 65.7 (–5.0)
DNA8T
DNA8A
DNA8G
64.7
64.7
63.7
59.9
[a] Reference duplexes with G instead of TO-a, TO-b, TO3-a or
TO3-b.
We showed recently that photophysical interaction of
two adjacent TO-b chromophores as artificial DNA base
surrogates alter their optical properties significantly.[33] In-
trastrand TO-b dimers show strong excitonic interactions
that result mainly in fluorescence quenching whereas in-
terstrand TO-b dimers exhibit strong excitonic interactions
that yield a red-shifted emission. Such results were not ob-
tained with the TO-a modification. Hence, interstrand TO-
b dimers could be regarded as hydrophobically interacting
base pairs that stabilize the duplex and exhibit fluorescence
color readout for DNA hybridization. The large apparent
Stokes’ shift of nearly 100 nm together with a brightness
that is comparable to a single TO label in DNA make the
TO-b pairs powerful fluorescent labels for live-cell imaging
by confocal fluorescence microscopy. Accordingly, we trans-
ferred this concept from DNA to RNA, and reported about
the optical properties of interstrand TO-b dimers in RNA
duplexes, and their preliminary application to monitor de-
livery of RNA to cells.[46]
However, one of the major disadvantages of the optical
properties of the TO-b dimer in DNA is that the red-shifted
fluorescence (580 nm/orange in DNA and 570 nm/yellow in
RNA) overlays with parts of the TO-b monomer emission
(530 nm/green). In the following paragraphs, we describe
the interactions of interstrand TO3 dimers in DNA and
Figure 3. UV/Vis absorption spectra (left) and fluorescence spectra
(right) of the double TO3-modified duplexes DNA11–12 and
DNA13–14 in comparison with the single TO3-modified DNA11–
15 and DNA13–15; 2.5 µ DNA in 10 m Na-Pi buffer, 250 m
NaCl, pH 7.
However, ground state interactions between two cyanine
how the red-shift can be enhanced by combining the TO3 chromophores do not automatically yield a red-shifted fluo-
modifications with the TO-b dye based on an energy trans- rescence. Otherwise, such changes of fluorescence would
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Table 2. Melting temperatures 2.5 µ DNA in 10 m Na-Pi buffer, rescence intensity of the TO3 dye that is smaller than the
250 m NaCl, pH 7.
remaining TO emission (ratio I668nm/I531nm = 0.8 or 0.9,
Duplex
Tm [°C]
Duplex
Tm (∆Tm) [°C][a]
65.5 (–2.0)[33]
62.5
respectively) indicating a low FRET efficiency (Figure 5),
right. On the other hand, in case of the duplexes that bear
the TO3-b chromophore in combination with the TO-b dye
(DNA9–14 and DNA13–10) the FRET efficiency is high
DNA9–10
DNA11–12
DNA13–14
69.4[33]
60.1
60.1
DNA9–15
DNA11–15
DNA13–15
62.2 (–5.5)
and the TO emission is significantly reduced (ratio I668nm
/
DNA11–10
DNA9–12
57.5
58.7
DNA13–10
DNA9–14
66.7
67.7
I531nm = 4.1 and 6.7, respectively). It is important to note
that the observed FRET efficiencies correlate with the melt-
ing temperatures (Table 2). For instance, if the
counterstrands of DNA9–12 or DNA9–14, respectively,
would exchange inside the cells by unmodified counter-
parts, duplex DNA9–15 would be the result (Tm of 65.5 °C).
That means, DNA9–12 would exchange to DNA9–15
whereas DNA9–14 would not. Obviously, the interstrand
chromophore orientation of TO-b in the TO3-b-modified
duplexes DNA9–14 and DNA13–10 is more suitable for an
efficient FRET compared to the TO3-a-modified duplexes
DNA9–12 and DNA11–10. In both duplexes, DNA9–14 and
DNA13–10, the melting temperature can be also obtained
by temperature-dependent fluorescence spectroscopy (Fig-
ure 5, left) as the FRET efficiency drops at the same tem-
perature as the cooperative dehybridization.
[a] The corresponding unmodified DNA duplex that contains G
instead of TO-b, TO3-a or TO3-b shows Tm = 67.5 °C.
have been detected previously with TO dimer conjugates
(like TOTO).[47,48] Unfortunately, this is also true for the
TO3-a dimer in DNA11–12 as well as the TO3-b dimer in
DNA13–14 (Figure 3, right): When excited at 620 nm, both
duplexes exhibit a significant fluorescence quenching com-
pared to the single-labeled references DNA11–15 and
DNA13–15, a typical result of chromophore aggregation.
The emission of the TO dye (520–600 nm) overlaps pretty
well with the absorption of the TO3 dye (550–650). By com-
bining the TO and the TO3 chromophores that are placed
in two different strands a fluorescence resonance energy
transfer (FRET) process could only occur in the hybridized
duplex. Accordingly we combined the TO-b modification
of DNA9 and DNA10 with the TO3-a modification yielding
DNA9–12 and DNA11–10, and with the TO3-b modifica-
tion yielding DNA9–14 and DNA13–10. These double
modified duplexes show all the typical absorption band of
both, the TO and the TO3 dyes, excluding a groundstate
interaction between them (Figure 4, top). When excited at
490 nm (the TO-typical wavelength) all duplexes exhibit an
additional emission at 660–670 nm (the TO3-typical range)
which is the result of a FRET-type process (Figure 4, bot-
tom).
Figure 5. Temperature-dependent fluorescence (left) and fluores-
cence intensity ratios at 20 °C (right) of TO/TO3-modified duplexes
DNA9–12, DNA9–14, DNA11–10 and DNA 13–10; 2.5 µ DNA in
10 m Na-Pi buffer, 250 m NaCl, pH 7, excitation at 490 nm.
To evaluate these DNA duplexes for potential biological
applications, CHO-K1 cells were microinjected representa-
tively with DNA13–10, the duplex that exhibits the best
FRET efficiency between TO and TO3. The fluorescence
emission of DNA inside cells was recorded with a bandpass
filter from 530 to 600 nm (green channel) and a longpass
Figure 4. UV/Vis absorption spectra (top) and fluorescence spectra
(bottom) of the double TO/TO3-modified duplexes DNA9–12,
DNA9–14, DNA11–10 and DNA 13–10; 2.5 µM DNA in 10 m filter of 650 nm (red channel) (Figure 6, top). Remarkably,
Na-Pi buffer, 250 m NaCl, pH 7, excitation at 490 nm.
the difference in the fluorescence emission persisted even
inside cells. The ratio of the fluorescence intensity of the
Interestingly, the FRET efficiency differs significantly be- red to the green channel was 1.85Ϯ0.23 (in comparison
tween the duplexes. Both dye combinations with the TO3- to 0.34Ϯ0.09 for the single TO modification in the single
a modification (DNA9–12 and DNA11–10) exhibit a fluo- stranded DNA9 in Figure 6, bottom).
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Cyanine Dyes as Fluorescent DNA Base Substitutions
A white solid was yielded (90.0%). 1H NMR (300 MHz, [D6]-
DMSO): δ = 8.46–8.29 (m, 2 H, arom.), 7.93–7.78 (m, 2 H, arom.),
4.83 (m, 1 H, OH), 4.78 (t, J = 7.4 Hz, 2 H, NCH2-), 3.53 (m, 2
H, -CH2-O), 3.22 (s, 3 H, Me), 2.06 (m, 2 H, -CH2-) ppm. 13C
NMR (125.8 MHz, [D6]DMSO): δ = 177.2, 140.8, 129.3, 129.0,
128.0, 124.6, 116.7 (benzothiazole arom.), 57.28, 45.78, 30.35 (pro-
pyl), 16.70 (2-Me) ppm. MS (ESI): m/z (%) = 208.1 (100) [M]+.
HR-MS (FAB): found: m/z = 208.0801 [M]+, calcd. 208.0791.
Synthesis of Compound 5: Under nitrogen 3 (1.17 g, 3.5 mmol) and
N-methylquinoline (4, 1.14 g, 4.2 mmol) were dissolved in 20 mL
CH2Cl2/MeOH (1:1 v:v). Dry Et3N (1.2 mL, 8.6 mmol) was added
yielding in an immediate deep red color. The reaction mixture was
stirred under reflux overnight, then cooled to room temp. The
product was collected and washed three times with 15 mL cold
Et2O and dried under reduced pressure (31%). 1H NMR
(300 MHz, [D6]DMSO): δ = 8.75 (m, 1 H, arom.), 8.43 (m, 1 H,
arom.), 8.09–7.99 (m, 3 H, arom.), 7.79–7.74 (m, 2 H, arom.) 7.64–
7.58 (m, 1 H, arom.), 7.44–7.37 (m, 2 H, arom.), 7.05 (s, 1 H, olefin-
H), 5.05 (t, J = 4.67 Hz, 1 H, OH), 4.63 (m, 2 H, N-CH2-), 4.18
(s, 3 H, N-Me), 3.58 (m, 2 H, -CH2-O), 2.00 (m, 2 H, -CH2-) ppm.
13C NMR (125.8 MHz, [D6]DMSO): δ = 159.2, 148.6, 145.0, 139.9,
138.0, 133.1, 128.1, 126.9, 125.2, 124.4, 124.0, 123.8, 122.9, 118.2,
112.7, 107.8, 87.4 (thiazole orange), 57.3 (propyl), 45.6 (N-Me),
42.3, 30.1 (propyl) ppm. MS (ESI): m/z (%) = 349.0 (100) [M]+.
HR-MS (FAB): found: m/z = 349.1380 [M]+, calcd. 349.1375.
Figure 6. CHO-K1 cells were microinjected with DNA13–10 (top)
and DNA9 (bottom) and thereafter imaged for fluorescence emis-
sion from 530–600 nm (green channel) and Ͼ650 nm (red channel)
by confocal laser scanning microscopy. Bars indicate 10 µm.
Conclusions
The TO and the TO3 dyes as fluorescent DNA base sub-
stitutions show a brightness that is sufficient for bioanalytic
and imaging applications. In contrast to the non-covalently
binding TO-PRO3 TOTO and TOTO3 dyes,[49] the TO-
and TO3-modified oligonucleotides should potentially be
cell-permeable. Thus, TO and TO3 represent promising in-
ternal fluorescence labels for DNA and RNA. A DNA hy-
bridization-dependent energy transfer process can be ob-
tained between the TO and TO3 modifications if they are
placed in two complementary strands. As a result, the emis-
sion is shifted from the TO-typical value of 530 nm to
670 nm. This concept can be applied for fluorescence im-
aging to monitor DNA and RNA delivery as well as pro-
cessing inside living cells by confocal microscopy.
Synthesis of Compound 7: 5 (434 mg, 0.911 mmol) was dissolved in
5 mL DMF and 1,1Ј-carbonyldiimidazole (195 mg, 1 mmol) was
added. The solution was stirred at room temp. for 3 h. [3-(4,4Ј-
Dimethoxytrityl)-2-hydroxypropyl]amine (6, 395 mg, 1 mmol) was
added and the solution was stirred for 48 h and the solvents evapo-
rated to dryness. The crude product was purified by flash
chromatography (silica gel) with a gradient of MeOH in CH2Cl2
(0–5%) yielding a dark red solid (56%). T.l.c. (CH2Cl2/MeOH, 9:1)
Rf = 0.4. 1H NMR (300 MHz, [D6]DMSO): δ = 8.75 (m, 1 H, arom
ppm. TO), 8.64 (m, 1 H, arom. TO), 8.09–7.96 (m, 3 H, arom.
TO), 7.80–7.68 (m, 2 H, arom. TO), 7.59 (m, 1 H, arom. TO), 7.37
(m, 2 H, arom. TO), 7.34 (m, 2 H, arom. DMT), 7.23 (m, 7 H,
arom. DMT), 6.92–6.84 (m, 4 H, arom. DMT), 4.98 (d, 1 H, J =
5.22, OH), 4.64 (m, 2 H, N-CH2-), 4.19 (s, 3 H, N-Me), 4.10 (m, 2
H, -CH2-O), 3.73 (s, 1 H, -CH-OH), 3.70 (s, 6 H, 2ϫOMe), 3.33
(s, 1 H, NH), 3.30 (m, 1 H, NH-CH2-), 3.16 (m, 1 H, NH-CH2-),
2.96 (m, 1 H, -CH2-ODMT), 2.86 (m, 1 H, -CH2-ODMT), 2.09
(m, 2 H, -CH2-) ppm. 13C NMR (125.8 MHz, [D6]DMSO): δ =
166.8, 157.9, 148.8, 145.0, 139.8, 137.9, 135.6, 133.0, 131.5, 130.2,
129.6, 128.6, 128.3, 127.6, 126.9, 126.4, 123.8, 112.9, 85.1 [O-C-
(PhOMe)2Ph], 68.6, 65.6 (CH2O), 54.9 (OMe), 42.4 (propyl), 28.8
(propyl) ppm. MS (ESI): m/z (%) = 768.3 (100) [M]+. HR-MS
(FAB): found: m/z = 768.3096 [M]+, calcd. 768.3107. MS (MALDI-
TOF): found: m/z = 768.2 [M]+, calcd. 768.3.
Experimental Section
Materials and Methods: Chemicals were purchased from Aldrich,
Alfa Aesar and Merck. Unmodified oligonucleotides were pur-
chased from Metabion. T.l.c. was performed on Fluka silica gel 60
F254 coated aluminum foil. Flash chromatography was carried out
with silica gel 60 from Aldrich (60–43 µm). Spectroscopic measure-
ments were recorded in Na-Pi buffer solution (10 m) using quartz-
glass cuvettes (10 mm). Absorption spectra and the melting tem-
peratures (2.5 µ DNA, 250 m NaCl, 10–90 °C, 0.7 °C/min, step
width 1 °C) were recorded with a Varian Cary 100 spectrometer
equipped with a 6ϫ6 cell changer unit. Fluorescence was measured
with a Jobin–Yvon Fluoromax 3 fluorimeter with a step width of
1 nm and an integration time of 0.2 s. All spectra were recorded
with an excitation and emission bandpass of 2 nm and are cor-
rected for Raman emission from the buffer solution.
Synthesis of DNA Building Block 8: Under nitrogen 7 (60 mg,
0.067 mmol) was dissolved in 4 mL dry CH2Cl2. Dry diisopropyl-
amine (35 µL, 0.2 mmol) and 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite (21 µL, 0.094 mmol) was added. The reaction
mixture was stirred overnight at room temp. and subsequently
washed by freshly prepared satd. aq. NaHCO3, dried with Na2SO4,
and the solvents evaporated to dryness. The resulting dark red solid
was solved in dry MeCN (0.6 mL) and applied directly for auto-
mated DNA synthesis. T.l.c. Rf (CH2Cl2/MeOH = 9:1) = 0.5. 31P
NMR (121 MHz, DMSO): δ = 149.59, 149.06 ppm. MS (ESI): m/z
(%) = 968.6 (100) [M]+.
Synthesis of Compound 3: Under nitrogen, 2-methylbenzothiazole
(1, 13.7 mL, 107.5 mmol) and 3-iodo-1-propanol (2, 10.0 g,
53.8 mmol) were dissolved in 30 mL acetonitrile. The mixture was
stirred under reflux for 91 h, then cooled to room temp. and kept
in the fridge overnight. The precipitate was collected and washed
three times with 15 mL cold Et2O, dried under reduced pressure.
Synthesis of Compound 11: Under nitrogen, 10 (1.75 g, 4 mmol) and
9 (3.95 g, 12 mmol) were dissolved in dry DCM (25 mL) and
Eur. J. Org. Chem. 2010, 1239–1248
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1245

H.-A. Wagenknecht et al.
3.30 mL dry triethylamine (24 mmol) were added slowly. After the acetyl-CH3), 1.92–1.98 (m, 2 H, -CH2), 2.06 (s, 3 H, N-acetyl-CH3),
FULL PAPER
mixture was stirred for 2 d at room temp., the resulting precipitate
was collected by filtration under reduced pressure and washed with
diethyl ether (3ϫ10 mL) and methanol (3ϫ5 mL). The obtained
solid was recrystallized from DCM/MeOH = 10:1 to afford 11 as
a green solid product (1.55 g, 75%). 1H NMR (600 MHz; [D6]-
DMSO): δ = 1.96 (quint, 2 H, -CH2-), 3.69 (s, 3 H, N-Me), 3.48
(q, 2 H, -CH2-OH), 4.56 (t, 2 H, N-CH2-), 4.77 (t, 1 H, OH), 6.46
(d, 1 H, trimethine-H), 7.06 (d, 1 H, trimethine-H), 7.24 (t, 1 H,
arom.), 7.42 (t, 1 H, arom.), 7.52 (d, 1 H, arom.), 7.66 (t, 1 H,
arom.), 7.78 (d, 1 H, arom.), 7.83, (d, 1 H, arom.), 7.91 (t, 1 H,
arom.), 8.02 (d, 1 H, arom.), 8.09 (t, 1 H, trimethine-H), 8.32 (d,
1 H, arom.), 8.41 (d, 1 H, arom.) ppm. 13C NMR (600 MHz; [D6]-
DMSO): δ = 31.6 (propyl), 32.8 (N-Me6), 51.4 (propyl), 57.6 (pro-
pyl),98.7, 109.2, 109.5, 112.4, 117.7, 122.5, 124.0, 124.3, 124.6,
125.1, 126.5, 127.5, 133.2, 137.8, 141.9, 142.3, 143.7, 150.3, 161.6
ppm. ESI-MS: m/z (%) = 375.0 (100) [M+]. HR-MS (FAB): found:
m/z = 375.1532 [M]+, calcd. 375.1526.
3.93 (t, 2 H, O-CH2-), 4.49 (t, 2 H, N-CH2-), 5.67 (d, 1 H, methin-
H), 7.52–7.57 (m, 2 H, benzothiazol arom.), 7.63–7.81 (m, 5 H,
arom.), 8.17 (d, 1 H, benzothiazol arom.), 8.33 (dd, 1 H, benzothia-
zol arom.), 8.84 (d, 1 H, methine-H) ppm. ESI-MS: m/z (%) =
394.9 (84) [M+].
Synthesis of Compound 15: 1) Under nitrogen, 14 (1.0 g, 2 mmol)
and 4 (0.57 g, 2 mmol) were dissolved in 20 mL dry DCM and
1.66 mL dry triethylamine (12 mmol) were added slowly. After the
mixture was stirred for 3 d at room temperature, the resulting pre-
cipitate was collected by filtration under reduced pressure to afford
a dark blue solid (0.63 g, 58%). 2) The obtained blue solid was
dissolved in 25 mL of MeOH/NM3 (32%) = 2:3 and refluxed for
4 h. The resulting product was collected by filtration under reduced
pressure and washed with dry DCM (2ϫ5 mL) to afford 15 as a
1
dark green solid (0.51 g, 87%). H NMR (600 MHz; [D6]DMSO):
δ = 1.88 (quint, 2 H, -CH2-), 3.54 (q, 2 H, -CH2-O), 4.13 (s, 3 H,
N-Me), 4.25 (t, 2 H, N-CH2), 4.83 (t, 1 H, OH), 6.49 (d, 1 H,
trimethine-H), 7.09 (d, 1 H, trimethine-H), 7.27 (t, 1 H, arom.),
7.45 (t, 1 H, arom.), 7.52 (d, 1 H, arom.), 7.70 (m, 1 H, arom.),
7.82 (m, 1 H, arom.), 7.83 (d, 1 H, arom.), 7.96 (m, 1 H, arom.),
7.97 (m, 1 H, arom.), 8.11 (t, 1 H, trimethine-H), 8.38 (d, 1 H,
arom.), 8.45 (d, 1 H, arom.) ppm. 13C NMR (600 MHz; [D6]-
DMSO): δ = 30.0 (propyl), 42.2 (TO3), 42.9 (propyl), 57.6 (propyl),
98.2, 109.5, 109.3, 112.2, 117.9, 122.4, 123.9, 124.0, 124.6, 124.8,
126.7, 127.5, 133.2, 138.8, 141.5, 143.2, 143.6, 150.4, 160.7 ppm.
ESI-MS: m/z (%) = 375.0 (100) [M+]. HR-MS (FAB): found: m/z
= 375.1524 [M]+, calcd. 375.1526.
Synthesis of Compound 12: Under nitrogen, 11 (0.5 g, 1.0 mmol)
was dissolved in 40 mL dry DMF and 1,1Ј-carbodiimidazole
(0.33 g, 2 mmol) was added. After stirring at room temperature for
6 d, 6 (0.78 g, 2 mmol) was added and again stirred for 7 d at room
temp. The solvent was then removed under reduced pressure and
the resulting solid purified by flash-column-chromatography (SiO2,
DCM/MeOH, 10:1 + 10% pyridine). Compound 12 was obtained
as a blue solid (0.80 g, 87%). T.l.c. (DCM/MeOH, 10:1) Rf = 0.38.
1H NMR (300 MHz; [D6]DMSO): δ = 2.08–2.12 (m, 2 H, -CH2-),
2.83–2.86 and 2.88–2.92 (m, 1 H, -CH2-ODMT), 2.95–2.99 and
3.15–3.24 (m 1 H, NH-CH2-), 3.70 (s, 6 H, 2ϫOMe), 3.74 (s, 1
H, -CH-OH), 3.97–4.01 (t, 2 H, -CH2-O), 4.55–4.59 (t, 2 H, N-
CH2-), 4.98 (d, 1 H, OH), 6.51 (d, 1 H, TO-3), 6.86 (d, 4 H, arom.
DMT), 7.01 (t, 1 H, TO-3), 7.11 (m, 1 H, TO-3), 7.23–7.26 (m, 7
H, arom. DMT), 7.50 (t, 1 H, TO-3 arom.), 7.61 (d, 1 H, TO-3
arom.), 7.70 (t, 1 H, TO-3 arom.), 7.84–7-89 (m, 2 H, TO-3 arom.),
7.95–7.96 (m, 1 H, TO-3 arom.), 8.02–8.05 (m, 1 H, TO-3 arom.),
8.18 (t, 1 H, TO-3), 8.33 (d, 1 H, TO-3 arom.), 8.47 (d, 1 H, TO-
3 arom.) ppm. 13C NMR (600 MHz; [D6]DMSO): δ = 28.3, 30.7,
32.9, 35.7, 44.2, 54.9, 60.5, 65.6, 68.7, 85.1, 99.1, 108.2, 109.2,
112.6, 113.0, 114.9, 115.9, 117.6, 122.5, 123.8, 124.2, 124.6, 125.2,
126.5, 127.6, 129.6, 133.4, 135.7, 137.8, 141.9, 145.0, 156.0, 157.9,
162.2 ppm. ESI-MS: m/z (%) = 794.2 (100) [M+]. HR-MS (FAB):
found: m/z = 794.3270 [M]+, calcd. 794.3258.
Synthesis of Compound 16: Under nitrogen, 15 (0.5 g, 1.0 mmol)
was dissolved in 40 mL dry DMF and 0.33 g 1,1Ј-carbodiimidazole
(2 mmol) were added. After stirring at room temp. for 2 d, 6
(0.78 g, 2 mmol) was added and again stirred for 2 d at room temp.
The solvent was then removed under reduced pressure and the re-
sulting solid purified by flash-column chromatography (SiO2,
DCM/MeOH, 10:1 + 10% pyridine). Compound 16 was obtained
as a blue solid (0.83 g, 90%). T.l.c. (DCM/MeOH, 10:1) Rf = 0.38.
1H NMR (300 MHz; [D6]DMSO): δ = 1.99–2.03 (m, 2 H, -CH2-),
2.88–2.99 (m, 2 H, CH2-ODMT), 3.18–3.26 (m, 2 H, NH-CH2),
3.33 (s, 1 H, NH), 3.70 (s, 6 H, 2ϫOMe), 3.73 (s, 1 H, -CH-OH),
5.02 (d, 1 H, OH), 6.47 (d, 1 H, methine-H), 6.84 (m, 4 H, arom.
DMT), 7.08–7.13 (m 1 H, TO-3), 7.20–7.24 (m, 7 H, arom. DMT),
7.26–7.32 (m 2 H, TO-3 arom.), 7.43–7.56 (m, 2 H, TO-3 arom.),
7.64–7.69 (m, 1 H, TO-3 arom.), 7.85–7.90 (m, 2 H, TO-3 arom.),
7.93–8.02 (m, 3 H, TO-3 arom.), 8.10–8.18 (m 1 H, TO-3) ppm.
13C NMR (400 MHz; [D6]DMSO): δ = 26.5, 30.7, 35.7, 42.2, 44.3,
54.9, 65.6, 68.7, 85.1, 98.0, 109.5, 109.8, 112.1, 113.0, 118.1, 122.5,
123.8, 124.0, 124.6, 126.4, 127.6, 129.6, 133.3, 135.7, 138.1, 141.3,
143.4, 145.0, 157.9, 162.2 ppm. ESI-MS: m/z (%) = 794.2 (100)
[M+]. HR-MS (FAB): found: m/z = 794.3242 [M]+, calcd. 794.3258.
Synthesis of DNA Building Block 13: Under argon, 12 (0.3 g,
0.33 mmol) and dry 166 µL DIPEA (0.97 mmol) were dissolved in
10 mL dry DCM. Then, 125 µL 2-cyanoethyl N,N-diisopropylchlo-
rophosphoramidite (0.56 mmol) were added, the mixture was
stirred at room temperature for 16 h. After complete reaction, the
organic phase was washed with a satd. aq. NaHCO3, dried with
Na2SO4 and the solvents evaporated to dryness. The resulting dark
blue solid was solved in dry MeCN (3.5 mL) and applied directly
for automated DNA synthesis. T.l.c. Rf (CH2Cl2/acetone = 2:1) =
0.3. 31P NMR (121 MHz, DMSO): δ = 150.06, 149.70 ppm.
Synthesis of DNA Building Block 17: Under argon, 16 (0.3 g,
0.33 mmol) and 166 µL dry DIPEA (0.97 mmol) were dissolved in
10 mL dry DCM. After 125 µL 2-cyanoethyl N,N-diisopropylchlor-
ophosphoramidite (0.56 mmol) were added, the reaction mixture
was stirred at room temp. for 16 h. After complete reaction, the
organic phase was washed with satd. aq. NaHCO3, dried with
Na2SO4 and the solvents evaporated to dryness. The resulting dark
blue solid was solved in dry MeCN (3.5 mL) and applied directly
for automated DNA synthesis. T.l.c. Rf (CH2Cl2/acetone = 2:1) =
0.3. 31P NMR (121 MHz, DMSO): δ = 150.06, 149.70 ppm.
Synthesis of Compound 14: Compound 3 (3.31 g, 9.8 mmol) in dry
acetic anhydride (30 mL) was refluxed until completely dissolved.
Afterwards, N,N-diphenylformamidine (1.93 g, 9.8 mmol) was
added and refluxed again for 1 h. The mixture was cooled to room
temp. and 40 mL diethyl ether were added slowly. After the re-
sulting oil has been separated and dried under reduced pressure for
24 h, it was resolved in DCM and washed with water (4ϫ). 2.9 g
of a dark red oil 14 were obtained (62%). Unfortunatly, the purifi-
cation of this compound was never sufficient to provide all analyti-
Preparation of DNA: Oligonucleotides were prepared with an Expe-
dite 8909 Synthesizer from Applied Biosystems (ABI) using stan-
dard phosphoramidite chemistry. Reagents and CPG (1 µmol) were
1
cal data. H NMR (300 MHz; [D6]DMSO): δ = 1.79 (m, 3 H, O-
1246
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 1239–1248

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purchased from ABI and Glen Research. The synthesis of thiazole
orange-modified as well as trimethine thiazole orange-modified
DNA oligonucleotides was performed using a modified protocol.
Activator solution (0.45  tetrazole in acetonitrile) was pumped
simultaneously with the building block (0.1  in acetonitrile). The
coupling time was extended to 61 min with an intervening step after
30.8 min for washing and refreshing the activator/phosphoramidite
solution in the CPG vial. The CPG vial was flushed with dry aceto-
nitrile after the coupling. After preparation, the trityl-off oligonu-
cleotide was cleaved from the resin and deprotected by treatment
with conc. NH4OH at room temperature overnight. The modified
oligonucleotides were purified by HPLC on a semipreparative RP-
C18 column (300 Å, Supelco) using the following conditions: A =
NH4OAc buffer (50 m), pH = 6.5; B = acetonitrile; gradient 0–
20% B over 70 min for TO and 50 min for TO3, flow rate 2.5 mL/
min, UV/Vis detection at 260 and 512 nm for TO-modified oligo-
nucleotides, 260 and 600 nm for TO3-modified oligonucleotides.
The oligonucleotides were lyophilized and quantified by their ab-
sorbance in 10 m NaPi buffer at 260 nm on a Varian Cary 100
spectrometer. The following total yields range from 3% to 16%
depending on the sequential context. Duplexes were formed by
heating to 90 °C (15 min.) followed by slow cooling.
Microinjection and Confocal Laser Scanning Microscopy: CHO-K1
cells were seeded one day prior microinjection on glass coverslips.
Glass capillaries were loaded with 100 µ DNA solutions. The mi-
croinjection was performed using an InjectMan in combination
with a microinjector FemtoJet (Eppendorf) coupled to a Axiovert
200 fluorescence microscope (Carl Zeiss). The injection pressure
was ca. 120 hPa and the injection time per cell was 0.3 s. After
injection, the culture medium (Ham’s F12 containing 10% serum)
was changed and the cells were imaged using a Zeiss Axiovert 200 
microscope coupled to a Zeiss LSM 510 scanning device (Carl
Zeiss). After excitation with 488 and 514 nm the fluorescence was
detected using a 530–600 nm band-pass filter (green channel) and
a 560 nm longpass filter (red channel). The ratio of the red to green
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) (Wa 1386/9) and the University of Regensburg.
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