Thiazole orange and Cy3: Improvement of fluorescent DNA probes with use of short range electron transfer

Article abstract of DOI:10.1021/jo8004793

(Chemical Equation Presented) Thiazole orange was synthetically incorporated into oligonucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoinduced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.

Full text of DOI:10.1021/jo8004793

technique,3 for energy transfer systems,4 or for the single base
mismatch analysis.5,6 On the other hand, a variety of organic
fluorescent dyes have been synthetically incorporated at specific
positions within the oligonucleotide sequence (e.g., cyanine
dyes,7 acridine,8 coumarin,9 ethidium,10 flavin,11 perylene bi-
simide,12 pyrene,13 and other fluorosides14). Among the powerful
Thiazole Orange and Cy3: Improvement of
Fluorescent DNA Probes with Use of Short
Range Electron Transfer
Florian Menacher, Moritz Rubner, Sina Berndl, and
Hans-Achim Wagenknecht*
7
and broadly applied cyanine dyes, thiazole orange (TO)
represents a bright fluorescent probe that should be of potential
interest for DNA analytical problems. TO was never incorpo-
rated synthetically as a base surrogate into DNA or RNA.
However, TO was linked to internucleotidic15 or terminal16
positions of oligonucleotides and incorporated synthetically into
DNA-binding peptides for cellular staining17,18 and as a base
surrogate into peptide nucleic acids.19,20 The latter TO-modified
PNAs were studied extensively by the group of Seitz et al.
Remarkably, they were able to detect single nucleotide poly-
morphisms in a very sensitive and reliable way using TO-PNA-
conjugates in a homogeneous assay.20,21
UniVersity of Regensburg, Institute for Organic Chemistry,
UniVersita¨tsstrasse 31, D-93053 Regensburg, Germany
achim.wagenknecht@chemie.uni-regensburg.de
ReceiVed March 3, 2008
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Thiazole orange was synthetically incorporated into oligo-
nucleotides by using the corresponding phosphoramidite as
the building block for automated DNA synthesis. Due to the
covalent fixation of the TO dye as a DNA base surrogate,
the TO-modified oligonucleotides do not exhibit a significant
increase of fluorescence upon hybridization with the coun-
terstrand. However, if 5-nitroindole (NI) is present as a
second artificial DNA base (two base pairs away from the
TO dye) a fluorescence increase upon DNA hybridization
can be observed. That suggests that a short-range photoin-
duced electron transfer causes the fluorescence quenching
in the single strand. The latter result represents a concept
that can be transferred to the commercially available Cy3
label. It enables the Cy3 fluorophore to display the DNA
hybridization by a fluorescence increase that is normally not
observed with this dye.
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The analytical problems in biomedicinal diagnostics, molec-
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powerful and bright fluorescent probes and markers for DNA.1
Organic chromophores as fluorescent probes that have been
attached covalently either to the end of the oligonucleotide or
to the DNA bases represent important tools, e.g., for genetic
analysis on DNA microarrays,2 for the molecular beacon
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10.1021/jo8004793 CCC: $40.75
Published on Web 04/29/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 4263–4266 4263

SCHEME 1. Synthesis of DNA Building Block 8
SCHEME 2. Sequences of Duplexes DNA1-DNA8
phosphoramidite chemistry. The S-configuration of this linker
has been used to mimic the stereochemical situation of the 3′-
position of natural 2′-deoxyribofuranosides. The TO-chro-
mophore is connected to the linker via a carbamate function
whose N-H group is of low nucleophilicity and thus need not
be protected for the phosphoramidite chemistry. The coupling
of the phosphiteamide group to the free hydroxy group of 7
was performed by using standard procedures in CH2Cl2 yielding
the phosphoramidite 8.
Herein, we present the synthesis and application of the TO
dye as a covalently bound, internal fluorescent probe for DNA.
Moreover, to be able to reference the elucidated fluorescence
properties we compare TO with Cy3 as a representative
fluorophore that is commercially available as a DNA building
block and exhibits similar excitation and emission wavelengths.
We synthetically incorporated the TO dye into oligonucle-
otides using a structural approach that has been previously
described for ethidium-,10 indole-,22 phenothiazine-,23 and
perylene bisimide-modified12 oligonucleotides. The 2′-deoxyri-
bofuranoside moiety of natural nucleotides was replaced by an
acyclic linker system that is tethered to the nitrogen of the
quinoline ring as part of the TO dye. Avoiding the acid/base
By using 8 as a DNA building block, TO-modified oligo-
nucleotides were prepared with use of standard automated DNA
synthesis. In analogy to charge transfer experiments with pyrene-
modified G25 and ethidium,26 we placed cytosine as the
counterbase for TO. For the sequence design, it is important to
note that the emission should compete with a fluorescence
quenching caused by a photoinducable electron transfer. We
recently introduced the “DETEQ” concept (detection by electron
transfer-controlled emission quenching) for DNA analytics,
which is based on the defined quenching of fluorescence by
electron transfer processes.27 Using combinations of pyrene-
modified guanosine25 or ethidium26 as the fluorescent charge
donors and indole or 7-deazaguanine as quenchers and charge
acceptors, we were able to improve the fluorescent discrimina-
tion of DNA duplexes vs single strands and to detect single
base variations. Similar to ethidium, the TO chromophore
exhibits as an oxidation potential (TO+/TO• 2+) of 1.4 V (vs
NHE).28 Together with E00 ) 2.4 V for TO, the excitation
potential is not sufficient for the photoreduction of any of the
DNA bases and thus prohibits electron hopping through the
DNA. Together with 5-nitroindole (NI) as the charge acceptor
that has a reduction potential of -0.3 V (NI/NI•-)29 the electron
transfer should follow a strongly distance-dependent superex-
change mechanism.30 Hence, in the duplexes DNA1 and DNA3
we placed the TO chromophore with a distance of one or two
intervening T-A base pairs away from the NI moiety. The
labile glycosidic bond of natural nucleosides, especially in
24
combination with a cationic dye,
this linker provides the
chemical stability of the DNA building block that is crucial for
the preparation of DNA-chromophore conjugates via automated
phosphoramidite chemistry.
The synthesis of the TO-DNA building block (Scheme 1)
starts with the alkylation of quinoline (1), using 3-iodopropane-
1-ol (2) to give the quinoline 3. The subsequent condensation
of 3 with 1-methylbenzothiazolium iodide (4) yields the TO
chromophore 5. The reaction proceeds selectively to the para
position of the methylated quinoline moiety as confirmed by
2D NMR measurements (HSQC, HMBC, and ROESY). Treat-
ment with 4-nitrophenylchloroformiate furnished an active ester
of compound 5 in situ that was subsequently coupled to the
enantiomerically pure linker 610 as a derivative of (S)-1-
aminopropane-2,3-diol that carries already the 4,4′-dimethoxy-
trityl (DMT) protecting group needed for the automated DNA
(25) Wanninger-Weiss, C.; Valis, L.; Wagenknecht, H.-A. Bioorg. Med.
Chem. 2008, 16, 100–106.
(26) Valis, L.; Amann, N.; Wagenknecht, H.-A. Org. Biomol. Chem. 2005,
3, 36–38.
(27) Wagenknecht, H.-A. Ann. N.Y. Acad. Sci. 2008, . in press.
(28) Hosoi, K.; Hirano, A.; Tani, T J. Appl. Phys. 2001, 90, 6197–6204.
(29) Kokkinidis, G.; Kelaidopolou, A. J. Electroanal. Chem. 1996, 414, 197–
208.
(22) Wanninger, C.; Wagenknecht, H.-A. Synlett 2006, 2051–2054.
(23) Wagner, C.; Wagenknecht, H.-A. Org. Biomol. Chem. 2008, 6, 48–50.
(24) Amann, N.; Wagenknecht, H.-A. Tetrahedron Lett. 2003, 44, 1685–
1690.
(30) Valis, L.; Wang, Q.; Raytchev, M.; Buchvarov, I.; Wagenknecht, H.-
A.; Fiebig, T. Proc. Natl. Acad. Sci. 2006, 103, 10192–10195.
4264 J. Org. Chem. Vol. 73, No. 11, 2008

TABLE 1. Melting Temperatures (Tm), Quantum Yields (Φ),
Extinction Coefficients (E), Brightness (B ) ΦE), and Ids/Iss of
Fluorescence Intensities of DNA1-DNA8
c
duplex
Tm (°C)
Φ
ꢀ (M-1 cm-1
)
B
Ids/Iss
DNA1
DNA2
DNA3
DNA4
DNA5
DNA6
DNA7
DNA8
61.0
66.2
57.0
65.3
50.5
57.5
46.7
55.0
0.23a
0.10a
0.15a
0.10a
0.28b
0.25b
0.30b
0.29b
35800a
35200a
40900a
39900a
64300b
86400b
59500b
57600b
8300
3600
6100
6.6
1.0
2.9
1.2
1.9
1.0
1.6
1.7
4100
18000
21600
17900
16700
a At 490 nm. b At 518 nm. c I ) integrated fluorescence intensities.
It is important to note that in case of the TO-modified
duplexes without the NI modification, the relative fluorescence
intensities of the single strands (ss-DNA2, ss-DNA4) are nearly
the same as that of the corresponding duplexes (DNA2, DNA4).
Due to the covalent fixation of the TO dye as a DNA base
surrogate, the TO-modified oligonucleotides do not exhibit the
TO-typical increase of fluorescence upon hybridization with the
counterstrand. However, if NI is present as a second artificial
DNA base (two or three base pairs away from the TO dye)
a fluorescence increase upon DNA hybridization can be
observed. In fact, the relative fluorescence intensities of the
TO-modified single strands that contain the NI charge
acceptor (ss-DNA1, ss-DNA3) are significantly reduced
compared to those of the corresponding reference oligo-
nucleotides (ss-DNA2, ss-DNA4). This result suggests that
a photoinduced electron transfer could take place exclusively
in the random-coil single strands causing a significant
fluorescence quenching. This is a remarkable result since it
clearly improves the optical discrimination of duplexes vs
single strand by the fluorescent TO base surrogate (Table
1).
FIGURE 1. Fluorescence spectra of DNA1-DNA4 (2.5 µM), 10 mM
Na-Pi-buffer (pH 7.0), 250 mM NaCl, 20 °C, λexc ) 490 nm.
FIGURE 2. Fluorescence spectra of DNA5-DNA8 (2.5 µM), 10 mM
Na-Pi-buffer (pH 7.0), 250 mM NaCl, 20 °C, λexc ) 530 nm.
A similar result was obtained with the Cy3-modified duplex
pair DNA5/DNA6. This is even more remarkable since the
placement of NI as an electron acceptor at a distance of one
intervening base pair enables the Cy3 fluorophore to display
the DNA hybridization by a fluorescence increase (DNA5)
compared to the duplex without NI (DNA6).
duplexes DNA2 and DNA4 lack the charge acceptor and serve
as reference duplexes without electron transfer. In the duplex
set DNA5-DNA8, the Cy3 dye replaces the TO chromophore
(Scheme 2).
Comparing the NI-modified duplexes with the reference
duplexes two major observations could be made: (i) the relative
fluorescence intensities of the TO-modified duplexes DNA1 and
DNA3 are increased in comparison with those of the reference
duplexes DNA2 and DNA4 (Figure 1) and (ii) the relative
fluorescence intensities of the Cy3-modified duplexes DNA5
and DNA7 are nearly the same (within experimental error) as
those of DNA6 and DNA8 (Figure 2). The absence of
fluorescence quenching in all four cases does not reveal that
TO or Cy3 is able to photoinduce an electron transfer to NI
through the duplex. It is known from the studies with the
ethidium-NI DNA system that intercalation of both charge
donor and acceptor in the base stack is crucial to provide the
electronic coupling for an efficient charge transfer.30 Thus, it
can be concluded that at least the Cy3 chromophore is not
sufficiently intercalated in the corresponding modified duplexes
since it tends to bind in the minor groove. The fluorescence
increase of the TO-modified DNA1 and DNA3 in comparison
with DNA2 and DNA4, respectively, occurs concomitantly with
the decreased stability of the duplexes (see the melting tem-
peratures Tm in Table 1). That indicates that the gained
conformational flexibility of DNA1 and DNA3 allows the TO
chromophore to intercalate better in the duplex yielding an
intensified emission.
The photochemical data (Table 1) display the quantification
of the steady-state fluorescence spectra. The quantum yields of
the TO-modified duplexes lie in the range between 10% and
23% and the brightness in the range of 4000-9000 M-1 cm-1
.
These values make the TO-modified oligonucleotides clearly
suitable for fluorescence applications in chemical biology and
fluorescent diagnostic technologies. Especially DNA1 shows a
high quantum yield (23%) that is comparable to that of the Cy3-
modified DNA5 (28%), and a significant fluorescence discrimi-
nation ratio of 6.6 between duplex and single strand due to the
presence of the NI moiety. In comparison, the Cy3-modified
duplexes are brighter (17000-22000 M-1 cm-1) and exhibit
higher quantum yields (25-30%). However the fluorescent
duplex discrimination ratio in DNA5 is lower (1.9, compared
to DNA1). Please note that the quantum yields were not
determined at the absorption maxima. That means that the
brightness of all the duplexes is significantly higher if excited
at the extinction maxima.
Taken together, there are two major results from these studies:
(i) TO represents a bright fluorescent DNA base surrogate and
thus a promising alternative to the commercially available
cyanine dyes for applications in fluorescent DNA analytics and
J. Org. Chem. Vol. 73, No. 11, 2008 4265

pyl)-4-(3-methyl-3H-benzothiazol-2-ylidenmethyl)quinolinuim
Iodide (8). To a solution of 7 (306 mg, 0.34 mmol) in 10 mL of
CH2Cl2 were added ethyldiisopropylamine (178.5 µL 1.03 mmol)
and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (111 µL,
0.5 mmol). The mixture was stirred for 45 min at rt and was washed
with saturated aq NaHCO3, dried over Na2SO4, and concentrated
under vacuum. Due to the hydrolytic lability the resulting red solid
compound was solved in MeCN and applied directly for automated
DNA synthesis. Quantitative conversion was assumed based on tlc;
Rf 0.50 (9:1 CH2Cl2/MeOH); for 31P NMR, see the Supporting
Information.
cell biology. Due to the short methine bridge in TO we expect
a better photostability of this dye compared to Cy3. (ii) The
fluorescence increase of TO and Cy3 upon DNA hybridization
can be improved by a short-range electron transfer in the single
strand. The latter result could represent a concept that is
potentially applicable also for other fluorescent labels, which
is currently under investigation.
Experimental Section
1-(3-Hydroxypropyl)quinolinium Iodide (3). 2 (2.45 mL, 25.5
mmol) was added to a solution of 1 (1.01 mL, 8.5 mmol) in 9 mL
of dioxane. The solution was stirred under reflux for 2.5 h. After
cooling to rt the product was precipitated by addition of 6 mL of
acetone. The residue was separated by filtration and washed with
acetone yielding 2.28 g (85%) of 3 as a yellow solid compound:
for 1H and 13C NMR, see the Supporting Information. FAB-HRMS:
M+ calcd for C12H14NO 188.1075, found 188.1076.
Oligonucleotides (DNA1-DNA8). Oligonucleotides were pre-
pared on a synthesizer by using standard phosphoramidite chemistry.
For the TO-phosphoramidite the coupling time was enhanced from
96 to 3700 s. Commercially available reagents and CPG (1 µmol)
were used. After preparation the trityl-off oligonucleotide was
cleaved off the resin and deprotected by treatment with concd
NH4OH at rt for 24 h. The oligonucleotide was dried and purified
by HPLC on a RP-C18 column, using the following conditions: A
) NH4OAc buffer (50 mM; pH 6.5); B ) MeCN. The oligonucle-
otides were lyophilizied and quantified by their absorbance at 260
nm on a photometer. Duplexes were formed by heating to 90 °C
(15 min) followed by slow cooling. UV/vis: ꢀ260 (M-1 cm-1)/yield
(%) 161000/13.7 (ss-DNA1), 155500/7.1 (ss-DNA2), 161000/15.7
(ss-DNA3), 155500/6.0 (ss-DNA4), 145000/ 21.9 (ss-DNA5),
155000 /11.7 (ss-DNA6), 145000/18.5 (ss-DNA7), 155000/24.3 (ss-
DNA8). ESI-MS: M+ calcd for ss-DNA1 5418, found 1356.2
[M/4]4+; M+ calcd for ss-DNA2 5407, found 1353.3 [M/4]4+; M+
calcd for ss-DNA3 5418, found 1356.1 [M/4]4+; M+ calcd for ss-
DNA4 5407, found 1353.4 [M/4]4+; M+ calcd for ss-DNA5 5398,
found 1350.8 [M/4]4+; M+ calcd for ss-DNA6 5387, found 1348.1
[M/4]4+; M+ calcd for ss-DNA7 5398, found 1350.7 [M/4]4+; M+
1-(3-Hydroxypropyl)-4-(3-methyl-3H-benzothiazol-2-yliden-
methyl)quinolinium Iodide (5). 3 (2.28 g, 7.2 mmol) and 4 (2.11
g, 7.2 mmol) were dissolved in 40 mL of EtOH. Dry NEt3 (3.01
mL) was added and the mixture was stirred for 2.5 h at 65 °C. The
solution was cooled to rt and stirred for 1 h at rt. The product was
precipitated by addition of 80 mL of Et2O at -18 °C. After
filtration, the product was suspended in a mixture of 30 mL of
MeOH and 42 mL of Et2O for 1 h. The product was collected by
filtration and dried under vacuum yielding 0.68 g (20%) of 5 as a
1
red solid compound: Rf 0.21 (9:1 CH2Cl2/MeOH); for H and 13C
NMR, see the Supporting Information. FAB-HRMS: M+calcd for
C21H21N2OS+ 349.1375, found 349.1431.
1-(3-{3-[Bis(4-methoxyphenyl)phenylmethoxy]-2-hydroxy-
propylcarbamoyloxy}propyl)-4-(3-methyl-3H-benzothiazol-2-
ylidenmethyl)quinolinuim Iodide (7). To a mixture of 5 (400 mg,
0.84 mmol) and NEt3 (0.41 mL, 2.94 mmol) in 40 mL of DMF
was added a solution of 4-nitrophenylchloroformiate (559 mg, 2.77
mmol) in 160 mL of CH2Cl2 and the solution was stirred for 23 h
at rt. Subsequently, 610 (495 mg, 1.26 mmol) was added and the
mixture was stirred for 2 h at rt. The solvents were removed under
vacuum and the residue was purified via silica chromatography with
CH2Cl2/MeOH 96:4 +0.1% NEt3 as an eluent to afford 306 mg
calcd for ss-DNA8 5387, found 1346 [M/4]4+
.
Acknowledgment.. This work was supported by the Deutsche
Forschungsgemeinschaft (Wa 1386/9), the Fonds der Chemis-
chen Industrie, and the University of Regensburg.
1
Supporting Information Available: H NMR spectra, 13C
1
NMR spectra, and MS spectra for 3, 5, and 7, and an 31P NMR
spectrum for 8. This material is available free of charge via the
Internet at http://pubs.acs.org.
(41%) of 7 as a red powder: Rf 0.38 (9:1 CH2Cl2/MeOH); for H
and 13C NMR, see the Supporting Information. FAB-HRMS: M+
calcd for C46H46N3O6S+ 768.3107, found 768.3106.
1-(3-{3-[Bis(4-methoxyphenyl)phenylmethoxy]-2-[(2-cyanoeth-
oxy)diisopropylaminophosphanyloxy]propylcarbamoyloxy}pro-
JO8004793
4266 J. Org. Chem. Vol. 73, No. 11, 2008