Synthesis of a photostable energy-transfer pair for dNA traffic lights

Article abstract of DOI:10.1002/ejoc.201403119

A new cyano-substituted thiazole red derivative as a red emitter and a novel green fluorescent donor dye of the cyanine styryl type were synthesized in good yields. Characterization of their optical properties revealed excellent photostabilities and large apparent Stokes' shifts. Both dyes can be incorporated into oligonucleotides through postsynthetic click -type chemistry and combined in a diagonal arrangement in double-stranded DNA. As a result, both dyes combine to an energy-transfer pair in DNA that shows remarkable optical properties such as significant emission wavelength shift from green to red upon hybridization owing to high energy-transfer efficiency between the two dyes and remarkable emission red/green color contrast ratio. The combination of these dyes as an energy-transfer pair according to our concept of DNA traffic lights has high potential for applications in molecular imaging.

Full text of DOI:10.1002/ejoc.201403119

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201403119
Synthesis of a Photostable Energy-Transfer Pair for “DNA Traffic Lights”
[a]
[a]
Peggy R. Bohländer and Hans-Achim Wagenknecht*
Keywords: DNA / Oligonucleotides / Fluorescence / Photochemistry / Energy transfer / Dyes/pigments
A new cyano-substituted thiazole red derivative as a red
bine to an energy-transfer pair in DNA that shows remark-
emitter and a novel green fluorescent donor dye of the cyan- able optical properties such as significant emission wave-
ine styryl type were synthesized in good yields. Characteri- length shift from green to red upon hybridization owing to
zation of their optical properties revealed excellent photosta-
bilities and large apparent Stokes’ shifts. Both dyes can be
incorporated into oligonucleotides through postsynthetic
high energy-transfer efficiency between the two dyes and
remarkable emission red/green color contrast ratio. The com-
bination of these dyes as an energy-transfer pair according
“
click”-type chemistry and combined in a diagonal arrange- to our concept of “DNA traffic lights” has high potential for
ment in double-stranded DNA. As a result, both dyes com-
applications in molecular imaging.
the phosphodiester bridges.[11] However, every single DNA
building block requires a substantial amount of time and
Introduction
[
1]
Molecular cell imaging and single molecule spec-
synthetic efforts. Given that the Huisgen–Sharpless–Meldal
[
2]
troscopy represent important methodologies that allow
visualization of biological structures and processes in vivo
[12]
cycloaddition “click”-type chemistry
is well established
[13,14]
for postsynthetic oligonucleotide modification,
we cur-
[
3–6]
but require powerful fluorescent nucleic acid probes.
rently try to transfer the concept of “DNA/RNA traffic
lights” to this methodology. The first combination of TO
as energy donor incorporated through postsynthetic 2Ј-
modification of uridine with TR as energy acceptor and
DNA base substitution (as described previously) gave dual-
emitting DNA probes with good fluorescence properties
The challenges that researchers face in this area include pri-
marily high quantum yields, brightness, large (apparent)
Stokes’ shifts, and excitation beyond λ = 400 nm. We cur-
rently focus our research on the synthesis of photostable
[
7]
fluorophores and their implementation in wavelength-
shifting nucleic acid probes.[ A fluorescence color change
allows a dual readout that is not possible with conventional
fluorophore-quencher or exciton-based dye combinations.
Recently, we reported our concept of “DNA traffic lights”,
primarily as a combination of thiazole orange (TO) and
thiazole red (TR), both incorporated as base substitutions
8]
[15]
but only moderate emission color contrast ratios. More-
over, we showed that a styryl dye of the cyanine indole
quinoline (CyIQ) type represents an important photostable
alternative to photolabile TO. However, the emission color
contrast ratio of this dye combined with TR is also moder-
ate owing to the fact that the CyIQ fluorescence color is
[
8,9]
into DNA.
A fluorescence color readout red/green of up
[16]
yellow and not green, as is that of TO. Furthermore, the
to 20:1 and a large wavelength shift of 140 nm were ob-
tained on the basis of efficient energy transfer between both
dyes owing to the proximity of the dyes embedded in the
double helical architecture. We demonstrated that this con-
cept can be transferred to siRNA to monitor RNA pro-
cessing and integrity in living cells in real time with a re-
markable dynamic range, excellent contrast ratios in cellular
media, and maintained knockdown properties.[
photolability of TR is a major drawback in any application.
Armitage et al. showed that electron-withdrawing groups
such as cyano substituents at the methine bridge are able to
[17]
significantly enhance the photostability of cyanine dyes.
This can be explained by the reduced electron density that
lowers the reactivity towards singlet oxygen as one of the
identified major pathways for photochemical dye bleach-
10]
[18]
ing. Accordingly, the cyano-substituted derivative of TO
Initially, TO and TR were incorporated into DNA by the
corresponding DNA building blocks that were structurally
based on (S)-3-amino-1,2-propandiol as a linker between
showed excellent photostability but dramatically reduced
quantum yield that does not meet at all the requirements
[19]
for bright and powerful fluorescent nucleic acid probes.
As alternatives, several cyanine dyes of the styryl type (such
as CyIQ) show enhanced photostabilities. Herein, we
present the synthesis of a new photostable energy-transfer
pair that combines cyano-substituted TR derivative 1 as a
red emitter and an acceptor dye with novel green fluores-
cent donor dye 2 as the donor based on a structure related
[
a] Institute of Organic Chemistry, Karlsruhe Institute of
[7]
Technology (KIT),
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
E-mail: Wagenknecht@kit.edu
www.ioc.kit.edu/wagenknecht/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403119.
Eur. J. Org. Chem. 2014, 7547–7551
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7547

P. R. Bohländer, H.-A. Wagenknecht
SHORT COMMUNICATION
to that of CyIQ. Additionally, we report the elucidation of
their improved photostabilities, optical properties, and
fluorescence ratios in their combination as an energy-trans-
fer pair. For the latter purpose, 1 and 2 were converted into
the corresponding azides and attached to the 2Ј-position of
uridine in oligonucleotides by using postsynthetic “click”-
[
14]
type methodology.
Scheme 2. Synthesis of dye 2 and the corresponding azide 13. Rea-
gents and conditions: (a) 3-iodo-1-propanol, 1,4-dioxane, 101 °C,
Results and Discussion
16 h, quant. (b) Piperidine, ethanol, 80 °C, 20 h, 66%. (c) 1. PPh
3
,
CBr , r.t., 2 h; 2) NaI, acetone/methanol, 50 °C, 71 h, 69%.
4
New dye 1 bears a cyano substituent at the methine (d) NaN , NaI, DMF, r.t., 19 h, 79%.
3
bridge next to the benzothiazole part to improve the photo-
[
17]
stability
and a propyl linker carrying a terminal iodo
The photostabilities of dyes 1 and 2 were determined by
irradiation with a 75 W Xe lamp equipped with a 305 nm
cut-off filter to avoid excitation of the DNA components,
The photodegradations of dyes 1 and 2, in comparison to
those of TR and TO (for structures, see the Supporting In-
formation), were measured by the loss in fluorescence inten-
sity in the presence of a random sequence double-stranded
DNA (for the sequence, see the Supporting Information)
and were quantified by half-life times t1/2 (Figure 1). TO
exhibited t1/2 = 32 min, and hence, it is significantly more
stable than TR (t1/2 = 7 min). Relative to that of TR, the
group for later postsynthetic attachment to DNA (or
RNA). The published synthesis of the TR chromophore
starts with the reaction of the already propylated thiazole
[9]
part with N,NЈ-dimethylformamidine.
This synthetic
route failed for 1, as the strongly electron-withdrawing
cyano group lowers the nucleophilicity of intermediate 3
such that alkylation at the benzothiazole nitrogen atom be-
comes impossible. Thus, the synthetic strategy was changed
(Scheme 1): Chromophore 7 was first assembled and then
iodopropylated to dye 1. Accordingly, commercial available
precursor 3 was condensed with N,NЈ-dimethylformamidine
to hemicyanine 4 in 84% yield. Subsequently, 4 was acetyl-
ated at elevated temperature to 5 in 87% yield. Hemicyan-
photostability of cyano-substituted red-emitting dye 1 (t1/2
=
66 min) was significantly improved. Even more remark-
able was the improvement in the photostability of dye 2
t1/2 = 636 min) relative to that of TO.
ine 5 was condensed with 6[
9a]
in the presence of triethyl-
(
amine to cyanine 7 (93% yield). Finally, the benzothiazole
nitrogen atom of 7 was alkylated by 1,3-diiodopropane to
obtain dye 1 in 53% yield. The conversion into azide 8 by
treatment with NaN followed standard procedures.
3
Scheme 1. Synthesis of dye 1 and corresponding azide 8. Reagents
and conditions: (a) N,NЈ-diphenylformamidine, triethyl ortho-
formate, EtOH, reflux, 3 h, 84%; (b) Ac
c) NEt , CH Cl , r.t., 22 h, 93%; (d) 1,3-diiodopropane, 90 °C, 4 d,
3%; (e) NaN , NaI, DMF, r.t., 20 h, 86%.
2
O, reflux, 19 h, 87%;
(
3
2
2
5
3
Figure 1. Photostabilities of dyes 1, 2, TO, and TR measured by
the loss in fluorescence intensity in the presence of DNA (10 μm
The synthesis of dye 2 and corresponding azide 13 dye, 2.5 μm DNA, 10 mm Na-P buffer, 250 mm NaCl, 5% ethanol).
i
(
Scheme 2) started with alkylation of 2-methylpyridine (9)
with 3-iodo-1-propanol to pyridinium 10 in quantitative
yield. Compound 11 was obtained in 89% yield by methyl- portant prerequisite to apply these dyes in dual-emitting or
As mentioned above, improved photostability is an im-
[7]
ation of indole-3-carbaldehyde. Condensation of 10 and wavelength-shifting nucleic acid probes. Accordingly, it
1 in the presence of piperidine gave dye 2 in 66% yield. In looked promising to postsynthetically incorporate not only
1
the following Appel and Finkelstein reactions, the hydroxy newly synthesized dye 1 but also dye 2 as modifications to
group of dye 1 was replaced by iodide (product 12 in 69% 2Ј-uridine in oligonucleotides and to combine them in such
yield) and further by azide (product 13 in 79% yield).
a diagonal arrangement (Figure 2) that was successfully
7548
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7547–7551

A Photostable Energy-Transfer Pair for “DNA Traffic Lights”
applied for our previous DNA “traffic lights” based on TO
and TR. The reactions were performed according to the
[
14]
literature,
tified by MALDI mass spectrometry and quantified by
their absorbance at λ = 260 nm by using an ε
and the modified oligonucleotides were iden-
=
260 nm
–
1
–1
–1
–1
2
980 m cm (for 1) and 8430 m cm (for 2).
Figure 2. Sequences of DNA1–DNA6.
The UV/Vis absorption spectra of DNA1–DNA4 exhibit
maxima shifts of 9 nm for 1 (559 to 550 nm) and only 1 nm
for 2 (449 to 450 nm) upon annealing from the single strand
to the double strand, but only minor differences in the ex-
tinction coefficient were observed. Similar fluorescence
Figure 3. UV/Vis absorption (top) and fluorescence (bottom) spec-
tra of DNA1–DNA4 (2.5 μm in 10 mm Na-Pi buffer, pH 7, 250 mm
maxima shifts of 6 nm for 1 (614 to 608 nm) and 2 nm for NaCl).
(527 to 529 nm) were observed (see Figure 3 and the op-
2
tical data listed in Table 1). Remarkably, the cyano moiety
Table 1. Summary of optical properties, quantum yields (Φ
F
), and
of dye 1 induced an increased apparent Stokes’ shift of
melting temperatures (T ) of DNA1–DNA6, λ = 260 nm, 10–
m
9a]
6
0 nm (compared to 25 nm for TR).[ Together with the
–1
9
2
0 °C, interval: 0.7 °Cmin , 2.5 μm duplex in 10 mm Na-P
50 mm NaCl, pH 7.
i
buffer,
enhanced Stokes’ shift for dye 2 of 79 nm (compared to
[
16]
[9a]
70 nm for CyIQ
and to 25 nm for TO ) these optical
[a]
[b]
[c]
DNA
λ
max
λ
max
λ
Stokes
Φ
F
T
m
properties provide an additional advantage of both dyes as
nucleic acid probes.
[nm]
[nm]
[nm]
[%]
[°C]
DNA1 559
DNA2 449
DNA3 550
614
527
608
529
612
–
55
78
58
79
74
–
59.9
9.75
66.8
–
–
The fluorescence spectrum of DNA5 that combines dyes
1
and 2 as an energy-transfer pair (see titration of DNA2
65.8
64.5
67.1
65.0
with DNA1 in Figure 4) reveals an excellent red-to-green DNA4 450
contrast ratio of the fluorescence intensities of 77:1 (ac-
ceptor/donor, λexc = 430 nm) and a remarkable energy-
transfer efficiency that can be calculated by the quantum [a] UV/Vis absorption. [b] Fluorescence. [c] λexc (DNA1) = 549 nm
9.40 [
34.2/40.7
–
d]
DNA5 455
DNA6
–
(
5
5
dye 1); λexc (DNA2, DNA4) = 450 nm (dye 2); λexc (DNA3) =
40 nm (dye 1); λexc (DNA5) = 430 nm (dye 2). [d] λexc (DNA5) =
40 nm (dye 1).
yields (see below). Furthermore, this dye pair exhibits an
apparent Stokes’ shift of 182 nm, which is comparable to
[
9]
that of the previously published TO/TR combination.
Spectroscopic measurements revealed that it is optimal to
choose an excitation wavelength of 430 nm (or lower), as Dye 1 exhibits remarkably high quantum yields of 59.9%
excitation at higher wavelength yields partial direct exci- in single-strand DNA1 and 66.8% in double strand DNA,
tation of the acceptor (see comparison of emission color whereas the quantum yields of TR were found in the range
[
9a]
contrast ratios in the Supporting Information).
of 26 to 30%.
This is an important result, as it shows
Finally, quantum yields (Φ ) and melting temperatures that the introduction of a cyano substituent into the meth-
F
(
T ) were determined (Table 1). The quantum yield of ine bridge to improve the photostability works better with
m
[
9a]
DNA2 modified with dye 2 is comparable to that of TO.
longer bridges. As already mentioned in the Introduction,
Eur. J. Org. Chem. 2014, 7547–7551
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7549

P. R. Bohländer, H.-A. Wagenknecht
SHORT COMMUNICATION
2; HPLC traces and MALDI mass spectra of modified oligonucleo-
tides; photobleaching experiments; and detailed spectroscopic data
of DNA5 (variation of excitation wavelength and fluorescence in-
tensity ratio).
Acknowledgments
Financial support by the Deutsche Forschungsgemeinschaft
(
DFG) (grant numbers Wa 1386/13-1 and Wa 1386/17-1) and
Karlsruhe Institute of Technology (KIT) is gratefully acknowl-
edged.
[
1] For reviews, see: a) H. Kobayashi, M. Ogawa, R. Alford, P. L.
Choydke, Y. Urano, Chem. Rev. 2010, 110, 2620–2640, and ref-
erences cited therein; b) M. Y. Berezin, S. Achilefu, Chem. Rev.
Figure 4. UV/Vis absorption (top left), fluorescence (top right), and
images of fluorescence color change (bottom) of DNA2 during a
2
010, 110, 2641–2684, and references cited therein; c) B. A. Ar-
mitage, Curr. Opin. Chem. Biol. 2011, 15, 806–812, and refer-
ences cited therein; d) P. V. Chang, C. R. Bertozzi, Chem. Com-
mun. 2012, 48, 8864–8879, and references cited therein;
E. M. S. Stennett, M. A. Ciuba, M. Levitus, Chem. Soc. Rev.
titration with DNA1 in 0.1 equiv. steps (2.5 μm in 10 mm Na-P
i
buffer, pH 7, 250 mm NaCl, λexc = 430 nm); DNA5 represents the
duplex consisting of DNA1 and DNA2.
2014, 43, 1057–1075, and references cited therein.
the insertion of a cyano substituent into the smaller TO dye [2] For reviews, see: a) G. Patterson, M. Davidson, S. Manley, J.
Lippincott-Schwartz, Annu. Rev. Phys. Chem. 2010, 61, 345–
significantly altered its optical properties and dramatically
367, and references cited therein; b) Q. Zheng, M. F. Juette,
lowered the fluorescence quantum yields.[ The quantum
yield of DNA5 is 34.2% if dye 2 is excited at λ = 430 nm,
and the energy transfer takes place. If dye 1 in DNA5 is
excited directly at λ = 540 nm, the quantum yield is 40.7%;
hence, the energy-transfer efficiency in DNA5 can be calcu-
lated by the ratio of both quantum yields and gives the
remarkable value of 0.84. The melting temperatures of
singly modified DNA double strands DNA1 and DNA2
were not altered significantly relative to that of unmodified
DNA6. Doubly modified DNA5 showed slight stabiliza-
tion, probably as a result of dye–dye interactions.
19]
S. Jockusch, M. R. Wasserman, Z. Zhou, R. B. Altman, S. C.
Blanchard, Chem. Soc. Rev. 2014, 43, 1044–1056, and refer-
ences cited therein.
[
3] For reviews, see: a) A. A. Marti, S. Jockusch, N. Stevens, J. Ju,
N. J. Turro, Acc. Chem. Res. 2007, 40, 402–409, and references
cited therein; b) K. Wang, Z. Tang, J. Y. Chaoyong, Y. Kim,
X. Fang, W. Li, Y. Wu, C. D. Medley, Z. Cao, J. Li, P. Colon,
H. Lin, W. Tan, Angew. Chem. Int. Ed. 2009, 48, 856–870; An-
gew. Chem. 2009, 121, 870–885, and references cited therein; c)
J. Stambasky, M. Hocek, P. Kocovsky, Chem. Rev. 2009, 109,
6729–6764, and references cited therein; d) V. L. Malinowski,
D. Wenger, R. Häner, Chem. Soc. Rev. 2010, 39, 410–422, and
references cited therein; e) R. W. Sinkeldam, N. J. Greco, Y.
Tor, Chem. Rev. 2010, 110, 2579–2619, and references cited
therein; f) Y. N. Teo, E. T. Kool, Chem. Rev. 2012, 112, 4221–
4
245, and references cited therein.
4] S. Preis, L. M. Wilhelmsson, ChemBioChem 2012, 13, 1990–
001.
5] a) E. Socher, A. Knoll, O. Seitz, Org. Biomol. Chem. 2012, 10,
363–7371; b) F. Hövelmann, I. Gaspar, A. Ephrussi, O. Seitz,
J. Am. Chem. Soc. 2013, 135, 2072–2081.
Conclusions
[
[
2
Compared to our previously applied TR dye, cyano-
modified red-emitting dye 1 showed significantly improved
photostability, larger apparent Stokes’ shift, and enhanced
7
quantum yield. Moreover, in comparison to TO, green- [6] For a review, see: A. Okamoto, Chem. Soc. Rev. 2011, 40, 5815–
emitting dye 2 also exhibited improved photostability, larger
Stokes’ shift, and comparable quantum yield. Both dyes
were synthesized in good yields and were incorporated into
oligonucleotides through postsynthetic “click”-type chemis-
5828, and references cited therein.
[
[
7] P. Bohländer, H.-A. Wagenknecht, Org. Biomol. Chem. 2013,
11, 7458–7462.
8] For a review, see: C. Holzhauser, H.-A. Wagenknecht, J. Org.
Chem. 2013, 78, 7373–7379, and references cited therein.
try that facilitated the preparation of modified DNA. Both [9] a) C. Holzhauser, S. Berndl, F. Menacher, M. Breunig, A.
Göpferich, H.-A. Wagenknecht, Eur. J. Org. Chem. 2010, 1239–
photostable dyes, 1 and 2, were combined in a diagonal ar-
1248; b) C. Holzhauser, H.-A. Wagenknecht, Angew. Chem.
rangement in double-stranded DNA to give an energy-
transfer pair that provided better optical properties than the
TO/TR combination, including comparable apparent Sto-
Int. Ed. 2011, 50, 7268–7272; Angew. Chem. 2011, 123, 7406–
410; c) C. Holzhauser, H.-A. Wagenknecht, ChemBioChem
2012, 13, 1136–1138.
7
kes’ shift (182 nm), high energy-transfer efficiency (0.84), [10] C. Holzhauser, R. Liebl, A. Goepferich, H.-A. Wagenknecht,
M. Breunig, ACS Chem. Biol. 2013, 8, 890–894.
and a remarkable emission color contrast ratio (red/green =
[
11] For a review, see: W. Schmucker, H.-A. Wagenknecht, Synlett
77:1). It is evident that new cyanine dyes 1 and 2 represent
2012, 23, 2435–2448, and references cited therein.
promising candidates for photostable dual-emitting and
wavelength-shifting nucleic acid probes, such as our “DNA
traffic lights”.
[
12] a) R. Huisgen, Angew. Chem. Int. Ed. Engl. 1963, 2, 565–598;
Angew. Chem. 1963, 75, 604–637; b) Q. Wang, T. R. Chan, R.
Hilgraf, V. V. Fokin, K. B. Sharpless, M. G. Finn, J. Am. Chem.
Soc. 2003, 125, 3192–3193; c) M. Meldal, C. W. Tornoe, Chem.
Rev. 2008, 108, 2952–3015.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental details and syntheses; IR spectra, NMR spectra,
and mass spectra of each compound; elemental analyses of 1 and
[13] For reviews, see: a) P. M. Gramlich, C. T. Wirges, A. Manetto,
T. Carell, Angew. Chem. Int. Ed. 2008, 47, 8350–8358; Angew.
7550
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7547–7551

A Photostable Energy-Transfer Pair for “DNA Traffic Lights”
Chem. 2008, 120, 8478–8487, and references cited therein; b)
M. Grotli, M. Douglas, R. Eritja, B. S. Sproat, Tetrahedron
[16] M. M. Rubner, C. Holzhauser, P. R. Bohländer, H.-A. Wagen-
knecht, Chem. Eur. J. 2012, 18, 1299–1302.
[17] N. I. Shank, K. J. Zanotti, F. Lanni, P. B. Berget, B. A. Armit-
age, J. Am. Chem. Soc. 2009, 131, 12960–12969.
[18] A. Toutchkine, D.-V. Nguyen, K. M. Hahn, Org. Lett. 2007, 9,
2775–2777.
1998, 54, 5899–5914, and references cited therein; c) A. H. El-
Sagheer, T. Brown, Chem. Soc. Rev. 2010, 39, 1388–1405, and
references cited therein.
[
14] S. Berndl, N. Herzig, P. Kele, D. Lachmann, X. Li, O. S. Wolf-
beis, H.-A. Wagenknecht, Bioconjugate Chem. 2009, 20, 558–
[19] N. I. Shank, H. H. Pham, A. S. Waggoner, B. A. Armitage, J.
Am. Chem. Soc. 2013, 135, 242–251.
564.
[
15] C. Holzhauser, M. M. Rubner, H.-A. Wagenknecht, Pho-
tochem. Photobiol. Sci. 2013, 12, 722–724.
Received: August 21, 2014
Published Online: October 6, 2014
Eur. J. Org. Chem. 2014, 7547–7551
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7551