Fluorescent 1,10-phenanthroline-containing oligonucleotides distinguish between perfect and mismatched base pairing

Article abstract of DOI:10.1021/ol026043x

(Matrix Presented) A fluorescent deoxyuridine analogue is sensitive to the polarity of its environment and exhibits a distinct emission profile in single-vs double-stranded oligonucleotides. Emission-monitored denaturation curves of internally modified dU

Full text of DOI:10.1021/ol026043x

ORGANIC
LETTERS
2002
Vol. 4, No. 14
2305-2308
Fluorescent
1,10-Phenanthroline-Containing
Oligonucleotides Distinguish between
Perfect and Mismatched Base Pairing
Dennis J. Hurley, Susan E. Seaman, Jan C. Mazura, and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego,
La Jolla, California 92093-0358
ytor@ucsd.edu
Received April 18, 2002
ABSTRACT
A fluorescent deoxyuridine analogue is sensitive to the polarity of its environment and exhibits a distinct emission profile in single- vs
phen
double-stranded oligonucleotides. Emission-monitored denaturation curves of internally modified dU
base opposite dUphen and distinguish between perfect and mismatched complementary oligonucleotides.
duplexes are characteristic of the
Fluorescent nucleosides that are sensitive to the local
environment within DNA duplexes have been attracting
attention as probes for DNA hybridization and DNA-ligand
interactions.1 Various approaches have been explored, in-
cluding the following: (a) isomorphic base analogues such
as 2-aminopurine2 and 5-methylpyrimidin-2-one,3 (b) ex-
tended bases such as 1,N6-ethenoadenine4 and benzo[g]-
quinazoline-2,4-(1H,3H)-dione,5 (c) base conjugates such as
pyrene-linked pyrimidines6 or ethynyl extended deazapu-
rines,7 and (d) nucleoside analogues with fluorophores
replacing the natural heterocycles.8 Such modifications are
particularly valuable since the natural bases are practically
nonemissive9 and end labeling with fluorescent tags is not
necessarily sensitive to remote binding events. No universal
(1) For review articles, see: Wojczewski, C.; Stolze, K.; Engels, J. W.
Synlett 1999, 1667-1678. Hawkins, M. E. Cell Biochem. Biophys. 2001,
34, 257-281.
(5) Godde, F.; Toulme´, J.-J.; Moreau, S. Biochemistry 1998, 37, 13765-
13775. Arzumanov, A.; Godde, F.; Moreau, S.; Toulme´, J.-J.; Weeds, A.;
Gait, M. J. HelV. Chim. Acta 2000, 83, 1424-1436.
(6) Netzel, T. L.; Zhao, M.; Nafisik, K.; Headrick, J.; Sigman, M. S.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 9119-9128. Manoharan, M.;
Tivel, K. L.; Zhao, M.; Nafisil, K.; Netzel, T. L. J. Phys. Chem. 1995, 99,
17461-16472. Kerr, C. E.; Mitchell, C. D.; Headrick, J.; Eaton, B. E.;
Netzel, T. L. J. Phys. Chem. B 2000, 104, 1637-1650.
(2) For selected examples, see: Ward, D. C.; Reich, E.; Stryer, L. J.
Biol. Chem. 1969, 244, 1228-1237. Menger, M.; Tuschl, T.; Eckstein, F.;
Porschke, D. Biochemistry 1996, 35, 14710-14716. Lacourciere, K. A.;
Stivers, J. T.; Marino, J. P. Biochemistry 2000, 39, 5630-5641. Kawai,
M.; Lee, M. J.; Evans, K. O.; Nordlund, T. M. J. Fluoresc. 2001, 11, 23-
32. Jean, J. M.; Hall, K. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 37-41.
Rachofsky, E. L.; Osman, R.; Ross, J. B. A. Biochemistry 2001, 40, 946-
956. Kirk, S. R.; Luedtke, N. W.; Tor, Y. Bioorg. Med. Chem. 2001, 9,
2295-2301.
(7) Seela, F.; Zulauf, M.; Sauer, M.; Deimel, M. HelV. Chim. Acta 2000,
83, 910-927. Seela, F.; Feiling, E.; Gross, J.; Hillenkamp, F.; Ramzaeva,
N.; Rosemeyer, H.; Zulauf, M. J. Biotech. 2001, 86, 269-279.
(8) Paris, P. L.; Langenhan, J. M.; Kool, E. T. Nucleic Acids Res. 1998,
26, 3789-3793. Stra¨ssler, C.; Davis, N. E.; Kool, E. T. HelV. Chim. Acta
1999, 82, 2160-2171. Singh, I.; Hecker, W.; Prasad, A. K.; Parmar, V. S.;
Seitz, O. Chem. Commun. 2002, 500-501.
(3) Singleton, S. F.; Shan, F.; Kanan, M. W.; McIntosh, C. M.; Stearman,
C. J.; Helm, J. S.; Webb, K. J. Org. Lett. 2001, 3, 3919-3922.
(4) Secrist, J. A., III; Barrio, J. R.; Leonard, N. J. Science 1972, 175,
646-647. Holme´n, A.; Albinsson, Norde´n, B, J. Phys. Chem. 1994, 98,
13460-13469 and references therein.
(9) Daniels, M.; Hauswirth, W. Science 1971, 171, 675-677. Pecourt,
J.-M. L.; Peon, J.; Kohler, B. J. Am. Chem. Soc. 2000, 122, 9348-9349.
10.1021/ol026043x CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/08/2002

solutions exist, however, and additional modifications have
to be developed and empirically evaluated.
trityl chloride in the presence of 4-(dimethylamino)pyridine
provides the DMT-protected nucleoside 4, which is cross-
coupled to 3-ethynyl-1,10-phenanthroline to give 5. Phos-
phitylation with (2-cyanoethyoxy)-bis(diisopropyl-amino)-
phosphine in the presence of 1H-tetrazole yields the
corresponding phosphoramidite 6.13
Phenanthroline-containing oligonucleotides, incorporating
the modified base dUphen at various positions, have been
prepared in good yields using the novel phosphoramidite 6.14
Figure 1 shows the various oligonucleotides used in this
We have previously demonstrated that extending the
conjugation of 1,10-phenanthroline via ethynyl linkages
provides a unique family of intense and tunable fluorophores,
where changes in the electronic polarization, solvation, and
coordination are translated into dramatic spectral changes.10
These observations have prompted us to explore conjugated
1,10-phenanthroline-containing nucleosides as “reporter”
probes for the local environment of the DNA double helix.
Here, we disclose the synthesis of a novel fluorescent
nucleoside and its incorporation into oligonucleotides using
solid-phase phosphoramidite chemistry. The emission of the
fluorescent DNA oligonucleotides changes upon hybridiza-
tion, and is sensitive to the formation of a Watson-Crick
vs mismatched base pairing.
The building block for synthesizing fluorescent oligo-
nucleotides is a modified 2′-deoxyuridine 3 (dUphen), where
the 5-position on the heterocyclic base is conjugated to a
1,10-phenanthroline at the 3 position via an ethynyl linker
(Scheme 1). Its synthesis entails the preparation of 5-eth-
Figure 1. Oligonucleotides synthesized and studied.
Scheme 1. Synthesis of dUphen 3 and Its Phosphoramidite 6a
study. The incorporation of a phenanthroline-containing
nucleoside has a relatively small detrimental effect on duplex
stability as determined by thermal denaturation measure-
ments.13
Figure 2 shows the normalized absorption and emission
a Reagents and conditions: (a) (i) (CF3CO)2O; (ii) Me3SiCtCH,
(Ph3P)4Pd, CuI, DMF, Et3N, 95-98%; (iii) K2CO3, MeOH, 75-
92%; (b) 3-bromo-1,10-phenanthroline, (dppf)PdCl2‚CH2Cl2, CuI,
DMF, Et3N, 72-84%; (c) 4,4′-dimethoxytrityl chloride (DMT-Cl),
DMAP, pyridine, Et3N, 92%; (d) 3-ethynyl-1,10-phenanthroline,
(dppf)PdCl2‚CH2Cl2, CuI, DMF, Et3N, 73-76%; (e) (i-Pr2N)2-
POCH2CH2CN, 1H-tetrazole, CH3CN, 81%.
Figure 2. Normalized absorption (a) and emission (b) spectra of
dUphen in CH2Cl2 (blue) or phosphate buffer (red), single stranded
oligo 9 (black) and duplex 9‚10 in aqueous buffer (green).
ynyl-2′-deoxyuridine 2, a known modified nucleoside ob-
tained from the commercially available 5-iodo-2′-deoxyuri-
dine 1,11 followed by a palladium-catalyzed cross-coupling
reaction with 3-bromo-1,10-phenanthroline12 (Scheme 1).
Alternatively,treating5-iodo-2′-deoxyuridine1with4,4′-dimethoxy-
spectra of the dUphen nucleoside 3 in dichloromethane and
in water. While the absorption spectra show little sensitivity
to solvent polarity, the emission maxima are much more
susceptible to environmental changes. Upon excitation at 333
(10) Joshi, H. S.; Jamshidi, R.; Tor, Y. Angew. Chem., Int. Ed. 1999,
38, 2721-2725.
(11) Robins, M. J.; Barr, P. J. J. Org. Chem. 1983, 48, 1854-1862.
Hashimoto, H.; Nelson, M. G.; Switzer, C. J. Am. Chem. Soc. 1993, 115,
7128-7138.
(12) Tzalis, D.; Tor, Y.; Failla, S.; Siegel, J. S. Tetrahedron Lett. 1995,
36, 3489-3490. Tzalis, D.; Tor, Y. Tetrahedron Lett. 1995, 36, 6017-
6020. Connors, P. J., Jr.; Tzalis, D.; Dunnick, A. L.; Tor, Y. Inorg. Chem.
1998, 37, 1121-1123.
(13) See the Supporting Information for additional experimental details.
2306
Org. Lett., Vol. 4, No. 14, 2002

nm of a dilute solution in dichloromethane, nucleoside 3
displays an intense purple emission that peaks at 385 nm
and extends into the visible range. In water, on the other
hand, the emission spectrum displays a maximum at 408 nm.
As shown in Figure 3, this tendency holds for a number of
To explore whether these emission features can be utilized
to follow DNA hybridization, fluorescence-monitored ther-
mal denaturation experiments have been conducted. Figure
4 shows the steady-state emission spectra of duplex 9‚10 as
Figure 3. Emission energy vs ET(30) value for dUphen 3 (square),
O-Me 14 (triangle), and N-Me 15 (circle).
Figure 4. Emission spectra of duplex 9‚10 as a function of
temperature from 35 °C (black) to 75 °C (cyan).
solvents, where decreasing solvent polarity results in higher
emission energy. The bathochromic shift observed upon
increasing solvent polarity is suggestive of a greater stabi-
lization of the excited state. This trend is typical of polar
fluorophores that are likely to have enlarged dipoles and
charge-transfer character in their excited state.15 Similar
behavior has been observed in related conjugated phenan-
throlines.10 It is important to add that unlike 1,10-phenan-
function of temperature. It is apparent that the relative
intensity of the species emitting at longer wavelengths
increases at elevated temperatures. Plotting the ratio between
the intensity of the visible emission at 407 nm (typical of
an exposed fluorophore in a single strand) and the shorter
emission wavelength at 387 nm (characteristic of a “pro-
tected” fluorophore in a less polar duplex environment) yields
a denaturation curve that closely follows the melting curve
of the duplex as determined by UV absorption spectroscopy
(Figure 5). It is important to note that when the fluorescent
throline, which is essentially nonemissive in water (λem
)
360 nm, φF e 0.01),16 nucleoside 3 exhibits respectable
fluorescence quantum efficiency in aqueous solutions (φF )
0.16).17,18
Absorption and emission spectra of a singly modified
single-stranded oligonucleotide 9 and the corresponding
double-stranded DNA duplex 9‚10 in an aqueous buffer are
shown in Figure 2. The lower energy transitions above ca.
300 nm are practically identical to those of the free
nucleoside 3. In contrast, significant differences are observed
in the emission spectra. While the single-stranded oligo-
nucleotide 9 shows a broad emission with a maximum above
400 nm, duplex 9‚10 exhibits a shorter wavelength maximum
(ca. 385 nm), reminiscent of the emission spectrum of
nucleoside 3 in less polar environment (e.g., CH2Cl2). This
observation suggests that the fluorescent nucleoside encoun-
ters different environments in the single- vs double-stranded
structure.
(14) Removal of the completed oligonucleotides from the solid support
using concentrated ammonium hydroxide was followed by incubation at
55 °C for 8-12 h to afford the crude oligonucleotides. The fluorescent
oligonucleotides were purified by denaturing polyacrylamide gel electro-
phoresis and semipreparative HPLC.
(15) Creaser, C. S.; Sodeau J. R. In PerspectiVe in Modern Chemical
Spectroscopy; Andrews, D. L., Ed.; Springer-Verlag: Berlin, 1990; pp 103-
136.
Figure 5. Emission-monitored thermal denaturation curves perfect
complement 9‚10 (green triangle), G mismatch 9‚11 (black circle),
T mismatch 9‚12 (blue square), and C mismatch 9‚13 (red
diamond).
(16) Bandyopadhyay, B. N.; Harriman, A. J. Chem. Soc., Faraday Trans.
1 1977, 73, 663-674.
(17) As expected, the emission quantum efficiency is higher in organic
solvents such as acetonitrile (φF ) 0.22).
(18) Measured in degassed solutions.
nucleoside dUphen is incorporated at the terminus of a duplex
as in 7‚8, the ratio I407/I387 remains unchanged upon
Org. Lett., Vol. 4, No. 14, 2002
2307

denaturation.19 We therefore conclude that internally placed
dUphen residues can be used to monitor duplex formation and
denaturation.
To investigate the sensitivity of dUphen to the formation
of mismatched base pairs, duplexes 9‚11-9‚13 that contain
G, T, or C opposite dUphen, respectively, have been as-
sembled. Emission-based thermal denaturation experiments
for all DNA sequences are shown in Figure 5. Duplexes
containing a single mismatch show higher I407/I387 ratios than
the perfect duplex 9‚10 at ambient temperatures. Interest-
ingly, the emission ratio is sensitive to the nature of the
mismatch. Duplex 9‚11, containing a reasonably well-
tolerated dG-dUphen base pair, displays a I407/I387 ratio that is
close to that of the native duplex 9‚10. In contrast, duplexes
containing the destabilizing pyrimidine-pyrimidine pairs (e.g.,
9‚12, 9‚13) exhibit a much higher I407/I387 ratio at ambient
temperature. As expected, all curves converge to the same
ratio of emission intensity upon thermal denaturation,
characteristic of the corresponding single-stranded oligo-
nucleotide 9. Evidently, the emission-based melting curves
of the internally modified dUphen duplexes exhibit a distinc-
tive pattern, characteristic of the base opposite the fluorescent
base.
Figure 6. Structure of “tautomerically trapped” dUphen analogues.
denaturation experiments have been conducted with duplex
9‚13 and increasing concentrations of the mismatched
complement 13. Although diminished I407/I387 ratios are
observed, they never reach the value observed for the perfect
duplex, even in the presence of 10-fold excess of comple-
mentary oligonucleotide.13 We therefore conclude that the
distinctive I407/I387 ratios observed for mismatched duplexes
9‚11-9‚13 reflect both a characteristic local environment
and a higher equilibrium population of a single stranded
structure when compared to the perfect duplex 9‚10.
A priori, different phenomena can account for the char-
acteristic spectral features and I407/I387 ratios that distinguish
the dUphen-containing duplexes 9‚10-9‚13 both from the
corresponding modified single strand 9 and from each
other: (a) hybridization-induced tautomerization of dUphen
that alters the electronic nature of the chromophore and thus
the spectral features of the single vs the double stranded
oligonucleotide, (b) structural perturbation of the duplex upon
mismatch formation that disturbs the local environment of
dUphen thus altering the emission features of the single and
double stranded oligonucleotides, and (c) a shift in the
equilibrium composition of the double vs the single stranded
oligonucleotide. To explore the first possibility, two “trapped
tautomers” of dUphen, 14 and 15, have been synthesized and
spectrally characterized (Figure 6).13 As shown in Figure 3,
both the O-Me derivative 14 and N-Me derivative 15 show
similar emission energies and sensitivity to solvent polarity
Our results demonstrate that the fluorescent deoxyuridine
analogue dUphen 3 is a sensitive probe for its local environ-
ment within DNA duplexes. Its fluorescence emission as a
free nucleoside and within a single-stranded oligonucleotide
is different from that observed when incorporated into a DNA
duplex. Additionally, the emission-monitored denaturation
curves of internally modified dUphen duplexes are character-
istic of the nucleotide opposite the fluorescent base and allow
one to distinguish between the perfect and various mis-
matched complementary oligonucleotides. It is anticipated
that further refinement of the chromophore’s structure may
shift the emission wavelengths further into the visible range
and may enhance its sensitivity to the local environment.
Acknowledgment. We thank the National Institutes of
Health (Grant No. GM 58447) for generous support.
when compared to the parent dUphen 14
. We therefore exclude
Supporting Information Available: Synthetic procedures
and spectral data for all new derivatives, as well as thermal
denaturation and fluorescence spectroscopy experiments. This
material is available free of charge via the Internet at
http://pubs.acs.org.
the possibility that changes in the I407/I387 ratio are caused
by tautomerization of dUphen upon hybridization. To inves-
tigate the other possibilities, additional emission-based
(19) Since the terminal nucleotide within any duplex is solvent exposed,
it does not experience a significant environmental change upon duplex
melting. See the Supporting Information.
OL026043X
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Org. Lett., Vol. 4, No. 14, 2002