Dialkynylpyrenes: Strongly fluorescent, environment-sensitive DNA building blocks

Article abstract of DOI:10.1021/ja806747h

Two isomeric dialkynylpyrene phosphoramidites and their incorporation into oligonucleotides are described. The pyrene units closely resemble the well-known perylene bisimide dye PDI with regard to the ability to self-organize within a DNA duplex. In addition, dialkynylpyrenes exhibit significant monomer and remarkably strong excimer fluorescence. The dialkynylpyrene building blocks are promising candidates for applications in diagnostic tools, such as excimer-based molecular beacons, as well as for novel DNA-based materials with special optical properties. Copyright

Full text of DOI:10.1021/ja806747h

Published on Web 10/25/2008
Dialkynylpyrenes: Strongly Fluorescent, Environment-Sensitive DNA Building
Blocks
Holger Bittermann, Doreen Siegemund, Vladimir L. Malinovskii, and Robert Ha¨ner*
Department of Chemistry and Biochemistry, UniVersity of Bern, Freiestrasse 3, Bern, CH-3012, Switzerland
Received August 29, 2008; E-mail: robert.haener@ioc.unibe.ch
Perylenetetracarboxylic acid diimide (PDI; Scheme 1) represents
a polyaromatic compound that has been extensively studied in the
context of pigment research and optical as well as molecular
electronic devices.1-3 The compound shows a remarkable ability
to self-organize into defined structures,4-7 which has been applied
to the construction of different DNA-based hybrids adopting stable
hairpin and triplex structures.8-13 Characteristic changes in the
absorption vibronic band pattern are commonly used for monitoring
PDI aggregation processes.1,2,4-6,9-11,14,15 Relative orientation of
multiple PDI units is assessable via characteristic CD signals and
the occurrence of exciton coupled CD upon formation of twisted
aggregates.10,11 The fluorescence properties of PDI derivatives1,2,16-18
prompted their use in the field of DNA-based diagnostics. Unfor-
tunately however, in combination with DNA a dramatic loss of
the quantum yield is observed.8,10,12,19,20 In this study we dem-
onstrate that dialkynyl pyrenes (DAP; 1,6- and 1,8-isomers) in
conjugation with oligonucleotides (ONs) show very attractive
properties similar to PDI-DNA hybrids, including a well-defined
structural organization of hybrids and the possibility to use UV-vis
and CD spectroscopy as signal readouts. Furthermore, in contrast
to PDI-DNA conjugates, DAP-DNA conjugates are strongly
fluorescent.
Extension of the pyrenyl system by two triple bonds was
investigated for several reasons. First, advanced optical properties
upon such extension were demonstrated for several classes of
compounds, such as anthracene,21 porphyrine,22 and pyrene.23-26
Moreover, extension of the π-system should increase the hydro-
phobic character, thus facilitating stacking interactions within the
DNA conjugates and hybrids. Finally, it was interesting to see
whether the introduction of the triple bond would still allow the
pronounced self-organizing properties as in the case of pyrenedi-
carboxamide27-34 or triazolyl derivatives35 described previously.
converted to cyanoethyl phosphoramidites 5 and 6 by standard
procedures (Scheme 1). Incorporation of the phosphoramidites into
ONs 7-14 by solid-phase synthesis was straightforward (Table 1).
It is noteworthy that, in contrast to the analogous PDI phosphora-
midite,20 5 and 6 are readily soluble in acetonitrile, which
significantly facilitates ON synthesis.
Table 1. Sequences of Oligonucleotides (ONs) Containing
Dialkynylpyrene Building Blocks X and Y (for Structures See
Scheme 1)
ON
sequence
7
8
9
10
11
12
13
14
5′-AGC TCG GTC AXC GAG AGT GCA-3′
3′-TCG AGC CAG TXG CTC TCA CGT-5′
5′-AGC TCG GTC XXC GAG AGT GCA-3′
3′-TCG AGC CAG XXG CTC TCA CGT-5′
5′-AGC TCG GTC AYC GAG AGT GCA-3′
3′-TCG AGC CAG TYG CTC TCA CGT-5′
5′-AGC TCG GTC YYC GAG AGT GCA-3′
3′-TCG AGC CAG YYG CTC TCA CGT-5′
UV-vis absorption spectra of the monomeric building blocks 1
and 2 were recorded in THF and water (Table 2 and Supporting
Informaton, SI), revealing well-resolved vibronic bands in the
320-420 nm range. Comparison of absorption and fluorescence
emission spectra of the corresponding ONs 7 and 11 showed nearly
perfect mirror-image behavior as well as significant overlap of the
most intense signals (SI), implying assignment of the longest
wavelength absorption band to the S1rS0 transition; consequently,
the vibronic bands were assigned to the (0f0) and (0f1) transition
for the longest and second-longest wavelength band, respectively.
In agreement with other alkynylpyrene derivatives,38,39 the high
absorptivities (Table 2) indicate that the S1rS0 transition is strongly
allowed.
Scheme 1. Preparation of Phosphoramidites 5 and 6; Structure of
PDI
Table 2. UV-vis Absorption Data of Diols 1 and 2 (Concentration:
3 µM)
Sν1 )0 r Sν0 )0
Sν1 )1 r Sν0 )0
λmax (nm)
εa
λmax (nm)
εa
ratiob
1
2
THF
386
384
385
382
8.6
6.2
6.5
5.1
364
363
364
363
5.0
4.4
4.1
3.7
1.7
1.4
1.6
1.4
waterc
THF
waterc
a
×104 M-1 cm-1
.
b Intensity ratio of the two vibronic bands.
c Containing 1.5% THF, measured at 90 °C.
UV-vis spectra were recorded both for single strands and for
hybrids containing either one or two pyrene units per strand. For
ONs 7 and 11, an absorption pattern similar to those of the
monomeric building blocks with the maxima slightly shifted to
longer wavelengths can be observed. The relative intensities of the
absorption bands change, and signals are broadened when two
pyrene units are present as in 9 and 13 (Figure 1; see SI for spectra
A mixture of 1,6- and 1,8-dibromopyrene35,36 was reacted with
3-butyn-1-ol under Sonogashira conditions, yielding a mixture of
diols 1 and 2 of which pure isomeric fractions were obtained by
column chromatography.37 Reaction of the two isomers with DMT
chloride furnished the monoprotected alcohols 3 and 4 which were
9
10.1021/ja806747h CCC: $40.75
2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 15285–15287 15285

C O M M U N I C A T I O N S
of the other ONs). Upon hybridization of 7 and 8, an analogous
intensity decrease of the longest wavelength band along with a red
shift can be monitored by temperature-variable UV-vis spectros-
copy (Figure 2; see SI for 11*12). The temperature-variable spectra
very closely resemble the vibronic band pattern changes observed
upon aggregation of PDI.10,14 Hyperchromicity at 390 nm exceeds
100%, underlining the high sensitivity of the vibronic bands toward
dialkynylpyrene aggregation.
CD measurements on single strands and hybrids gave insight
into the ability of dialkynylpyrenes to self-organize within a DNA
framework. For duplex 7*8, exciton coupling41,42 leads to well-
resolved signal splitting with extrema at 400 and 366 nm.
Surprisingly, the signal shape almost perfectly matches that of two
PDI units as described recently,10,20 yet at a different wavelength
(Figure 4, top). A similar pattern is found for 11*12. Even in single
stranded ONs 9 and 13 exciton coupling is observable, while, for
10 and 14 unidirectional, induced CD signals are observed (SI).
Remarkably, for duplex 13*14, which exhibits the highest quantum
yield of all hybrids containing four pyrene units (see below), the
CD spectrum is dominated by extraordinarily strong negative signals
in the 350-400 nm range (Figure 4, bottom). These findings, along
with the high fluorescence quantum yield, indicate a well-defined,
helical structure in the pyrene region.32,43
Figure 1. UV-vis spectra (300-420 nm region only) of ONs 7 (blue), 9
(green), 11 (red), and 13 (orange; ON concn: 1.5 µM; 10 mM phosphate
buffer, pH 7.0, 100 mM NaCl).
Figure 2. Temperature-dependent UV-vis spectrum of hybrid 7*8 (10
°C intervals, 20 to 90 °C; arrows indicate rising temperature (ON concn:
1.5 µM each strand; 10 mM phosphate buffer, pH 7.0, 100 mM NaCl).
Thermal denaturation experiments showed distinct heating/
cooling hysteresis for all hybrids (Figure 3), indicating a compara-
tively slow organization processes in the pyrene area. Due to the
abovementioned high sensitivity of the vibronic band intensities,
even small changes in the relative orientation of the pyrene moieties
lead to significant differences in the absorption bands.
Figure 4. Temperature-dependent CD spectra of 7*8 (top) and 13*14
(bottom), recorded in 10 °C steps from 20 to 90 °C; direction of arrows
indicates increasing temperature (ON concentration: 4.5 + 4.5 µM in 10
mM phosphate buffer pH 7.0, 100 mM NaCl).
As mentioned above, the excellent fluorescence properties of PDI
in organic solvents are completely suppressed in DNA conjugates
which require aqueous buffer systems representing a major
drawback of PDI-DNA conjugates. Thus, the fluorescence proper-
ties of DAP-modified ONs are of fundamental interest. ONs
containing one DAP fluorophore exhibit monomer emission spectra
that are red-shifted by ∼20 nm in comparison to unsubstituted
pyrene. When two pyrene units are incorporated into one strand or
when two strands each containing one unit are hybridized, the
monomer fluorescence disappears completely in favor of a strong
excimer signal with a maximum at ∼525 nm (Figure 5 and SI).
For use as diagnostic tools, fluorescent tags are expected to
exhibit reasonably high quantum yields and brightness.44,45 Quan-
tum yields were, therefore, determined for single stranded DAP-ONs
and selected hybrids (Table 3). Remarkably, for both isomers
quantum yields increase dramatically, when two single strands
bearing one pyrene (7, 8, 11 and 12) hybridize, giving rise to
interstrand excimer formation. It is noteworthy that excimer
emission of DAP-DNA is not significantly influenced by adjacent
bases, which allows for versatile applications of these tags.46 In
cases where intra- or interstrand excimers or exciplexes of two units
can be formed, high quantum yields (>0.20) are found. The highest
value of 0.39 is found for duplex 11*12. Considering furthermore
the high absorptivitiy of 11*12 (72 000 M-1 cm-1, 370 nm; see
SI for details), a remarkably high fluorescence brightness of up to
Figure 3. Heating and cooling curves of duplex 7*8 (1.5 + 1.5 µM in 10
mM phosphate buffer pH 7.0, 100 mM NaCl).
The hysteretic behavior could be attributed to the pyrene units
serving as points of interactions between the two strands. These
interactions are primarily based on hydrophobic interactions and,
thus, show different temperature dependence and dynamics than
in the case of the formation of hydrogen bonds between the natural
bases.40 Alternatively, minor conformational changes in the natural
DNA parts prior to melting of the duplex can lead to rearrangements
in the pyrene area that are reflected in a change in absorbance,
which, in turn, precludes the unambiguous determination of TM
values by means of UV-vis spectroscopy (see below).
9
15286 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

C O M M U N I C A T I O N S
28 000 M-1 cm-1 is obtained. Thus, DAP-DNA conjugates exhibit
high fluorescence quantum yields along with a general red shift,
similar to the recently reported triazolylpyrene-DNA conjugates.35
spectra. This information is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) Langhals, H. HelV. Chim. Acta 2005, 88, 1309–1343.
(2) Wu¨rthner, F. Chem. Commun. 2004, 1564–1579.
(3) Jones, B. A.; Facchetti, A.; Wasielewski, M. R.; Marks, T. J. J. Am. Chem.
Soc. 2007, 129, 15259–15278.
(4) Neuteboom, E. E.; Meskers, S. C. J.; Meijer, E. W.; Janssen, R. A. J.
Macromol. Chem. Phys. 2004, 205, 217–222.
(5) Wang, W.; Li, L. S.; Helms, G.; Zhou, H. H.; Li, A. D. Q. J. Am. Chem.
Soc. 2003, 125, 1120–1121.
(6) Ahrens, M. J.; Sinks, L. E.; Rybtchinski, B.; Liu, W. H.; Jones, B. A.;
Giaimo, J. M.; Gusev, A. V.; Goshe, A. J.; Tiede, D. M.; Wasielewski,
M. R. J. Am. Chem. Soc. 2004, 126, 8284–8294.
(7) Wang, H.; Kaiser, T. E.; Uemura, S.; Wu¨rthner, F. Chem. Commun. 2008,
1181–1183.
Figure 5. Normalized fluorescence spectra of 7 (blue), 11 (red), 7*8 (green),
and 11*12 (orange; ON concn: 1.5 µM each strand; 10 mM phosphate
buffer, pH 7.0, 100 mM NaCl; excitation wavelength: 370 nm).
(8) Bevers, S.; Schutte, S.; McLaughlin, L. W. J. Am. Chem. Soc. 2000, 122,
5905–5915.
(9) Wang, W.; Wan, W.; Zhou, H. H.; Niu, S. Q.; Li, A. D. Q. J. Am. Chem.
Soc. 2003, 125, 5248–5249.
(10) Zheng, Y.; Long, H.; Schatz, G. C.; Lewis, F. D. Chem. Commun. 2005,
4795–4797.
Table 3. Fluorescence Quantum Yields of Single Strands and
Duplexes (for Wavelengths see Supporting Information)
(11) Zheng, Y.; Long, H.; Schatz, G. C.; Lewis, F. D. Chem. Commun. 2006,
3830–3832.
ON
Φ
ON
Φ
ONs
Φ
ONs
Φ
(12) Lewis, F. D.; Zhang, L. G.; Kelley, R. F.; McCamant, D.; Wasielewski,
M. R. Tetrahedron 2007, 63, 3457–3464.
7
8
9
10
0.11
0.008
0.32
0.32
11
12
13
14
0.049
0.015
0.36
7*8
0.26
0.36
0.13
0.17
11*12
11*8
13*14
13*10
0.39
0.36
0.33
0.17
(13) Rahe, N.; Rinn, C.; Carell, T. Chem. Commun. 2003, 2119–2121.
(14) Clark, A. E.; Qin, C. Y.; Li, A. D. Q. J. Am. Chem. Soc. 2007, 129, 7586–
7595.
7*12
9*10
9*14
0.21
(15) Baumstark, D.; Wagenknecht, H. A. Angew. Chem., Int. Ed. 2008, 47, 2612–
2614.
(16) Wang, W.; Han, J. J.; Wang, L. Q.; Li, L. S.; Shaw, W. J.; Li, A. D. Q.
Nano Lett. 2003, 3, 455–458.
(17) Zhu, L. Y.; Wu, W. W.; Zhu, M. Q.; Han, J. J.; Hurst, J. K.; Li, A. D. Q.
J. Am. Chem. Soc. 2007, 129, 3524–3526.
(18) Giaimo, J. M.; Lockard, J. V.; Sinks, L. E.; Scott, A. M.; Wilson, T. M.;
Wasielewski, M. R. J. Phys. Chem. A 2008, 112, 2322–2330.
(19) Wagner, C.; Wagenknecht, H. A. Org. Lett. 2006, 8, 4191–4194.
(20) Bouquin, N.; Malinovskii, V. L.; Ha¨ner, R. Chem. Commun. 2008, 1974–
1976.
(21) Malakhov, A. D.; Skorobogatyi, M. V.; Prokhorenko, I. A.; Gontarev, S. V.;
Kozhich, D. T.; Stetsenko, D. A.; Stepanova, I. A.; Shenkarev, Z. O.; Berlin,
Y. A.; Korshun, V. A. Eur. J. Org. Chem. 2004, 1298–1307.
(22) Huang, T. H.; Chen, Y. J.; Lo, S. S.; Yen, W. N.; Mai, C. L.; Kuo, M. C.;
Yeh, C. Y. Dalton Trans. 2006, 2207–2213. (and references cited therein).
(23) Venkataramana, G.; Sankararaman, S. Eur. J. Org. Chem. 2005, 4162–
4166.
(24) Malinovskii, V. L.; Ha¨ner, R. Eur. J. Org. Chem. 2006, 3550–3553.
(25) Maeda, H.; Maeda, T.; Mizuno, K.; Fujimoto, K.; Shimizu, H.; Inouye,
M. Chem.sEur. J. 2006, 12, 824–831.
(26) Xiao, J. C.; Xu, J. L.; Cui, S.; Liu, H. B.; Wang, S.; Li, Y. L. Org. Lett.
2008, 10, 645–648.
(27) Langenegger, S. M.; Ha¨ner, R. Chem. Commun. 2004, 2792–2793.
(28) Langenegger, S. M.; Ha¨ner, R. ChemBioChem 2005, 6, 848–851.
(29) Langenegger, S. M.; Ha¨ner, R. Bioorg. Med. Chem. Lett. 2006, 16, 5062–
5065.
(30) Trkulja, I.; Ha¨ner, R. J. Am. Chem. Soc. 2007, 129, 7982–7989.
(31) Trkulja, I.; Ha¨ner, R. Bioconjugate Chem. 2007, 18, 289–292.
(32) Malinovskii, V. L.; Samain, F.; Ha¨ner, R. Angew. Chem., Int. Ed. 2007,
46, 4464–4467.
(33) Trkulja, I.; Biner, S. M.; Langenegger, S. M.; Ha¨ner, R. ChemBioChem
2007, 8, 25–27.
(34) Looser, V.; Langenegger, S. M.; Ha¨ner, R.; Hartig, J. S. Chem. Commun.
2007, 4357–4359.
(35) Werder, S.; Malinovskii, V. L.; Ha¨ner, R. Org. Lett. 2008, 10, 2011–2014.
(36) Grimshaw, J.; Trochagr, J. J. Chem. Soc., Perkin Trans. 1 1972, 1622–
1623.
(37) Recently, the isolation of the 1,6-isomer from an isomeric mixture has been
reported: Arai, R.; Uemura, S.; Irie, M.; Matsuda, K. J. Am. Chem. Soc.
2008, 130, 9371–9379.
(38) Shyamala, T.; Sankararaman, S.; Mishra, A. K. Chem. Phys. 2006, 330,
469–477.
(39) Benniston, A. C.; Harriman, A.; Howell, S. L.; Sams, C. A.; Zhi, Y. G.
Chem.sEur. J. 2007, 13, 4665–4674 (and references cited therein).
(40) For a recent example in which hysteresis is ascribed to hydrogen bonding
and hydrophobicity acting in a nonconcerted way, see: Sorrells, J. L.;
Menger, F. M. J. Am. Chem. Soc. 2008, 130, 10072–10073.
(41) Berova, N.; Nakanishi, K.; Woody, R. W. Circular Dichroism - Principles
and Applications; Wiley-VCH: New York, 2000.
Figure 6. Heating/cooling curves of hybrid 7*8, observed by fluorescence
measurement at 400 nm (monomer) and 520 nm (excimer); excitation: 385
nm; 1.5 + 1.5 µM; 10 mM phosphate buffer pH 7.0, 100 mM NaCl.
The excimer signal serves as a readout for TM determination
which is not possible by UV-vis spectroscopy as mentioned above.
This is demonstrated for hybrid 7*8, which was also analyzed by
UV-vis (Figure 3). With fluorescence detection, a TM of 74 °C is
obtained (Figure 6; see SI for 11*12 and unmodified duplex).
In conclusion, the synthesis of two isomeric dialkynylpyrene
phosphoramidites and their incorporation into oligonucleotides have
been demonstrated. The pyrene units show features that closely
resemble those of the well-known perylene bisimide dye PDI with
regard to its ability to self-organize within a DNA duplex. Thus,
the dialkynylpyrenes open the possibility to gain information on
structural details of DNA hybrids through characteristic absorption
patterns and CD signals. Similar to PDI, the alkynyl pyrenes have
high absorptivities in their S0-S1 transitions. In contrast to PDI,
however, dialkynylpyrenes exhibit significant monomer as well as
remarkably strong excimer fluorescence by formation of either intra-
or interstrand excimers. Considering the relative ease of synthesis
and the high quantum yields of the DNA conjugates, these building
blocks represent promising candidates for applications in diagnostic
tools like excimer-based molecular beacons, as well as for novel
DNA-based materials with special optical properties.
(42) Boiadjiev, S. E.; Lightner, D. A. Monatsh. Chem. 2005, 136, 489–508.
(43) Lewis, F. D.; Zhang, L. G.; Liu, X. Y.; Zuo, X. B.; Tiede, D. M.; Long,
H.; Schatz, G. C. J. Am. Chem. Soc. 2005, 127, 14445–14453.
(44) Ranasinghe, R. T.; Brown, T. Chem. Commun. 2005, 5487–5502.
(45) Cuppoletti, A.; Cho, Y. J.; Park, J. S.; Strassler, C.; Kool, E. T. Bioconjugate
Chem. 2005, 16, 528–534.
Acknowledgment. This work was supported by the Swiss
National Foundation (Grant 200020-117617).
(46) Venkatesan, N.; Seo, Y. J.; Kim, B. H. Chem. Soc. ReV. 2008, 37, 648–663.
Supporting Information Available: Synthetic procedures, analytical
details, additional melting curves, UV-vis, fluorescence and CD
JA806747H
9
J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15287