Simple fluorescent pyrimidine analogues detect the presence of DNA abasic sites

Article abstract of DOI:10.1021/ja052000a

A family of simple pyrimidine analogues has been synthesized, and their photophysical properties have been investigated. The most responsive of the family was incorporated in DNA. This isosteric fluorescent DNA analogue monitors denaturation of a DNA dupl

Full text of DOI:10.1021/ja052000a

Published on Web 07/14/2005
Simple Fluorescent Pyrimidine Analogues Detect the Presence of DNA Abasic
Sites
Nicholas J. Greco and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego, La Jolla, California 92093
Received March 29, 2005; E-mail: ytor@ucsd.edu
Table 1. Photophysical Data of Nucleosides 2a-da
Nucleic acids experience a variety of perturbations, including
strand cleavage, depurination and depyrimidination, local confor-
mational changes, base flipping, as well as more subtle and transient
effects induced by ligand binding. Fluorescent nucleoside analogues
that are sensitive to their local environment have become powerful
tools for investigating nucleic acid structure, dynamics, and
recognition.1 These probes can be broadly classified into the
following categories: (1) isomorphic base analogues that resemble
the natural nucleobases with respect to their dimension, hydrogen
bonding face, and ability to form isostructural Watson-Crick base
pairs (e.g., 2-aminopurine);2,3 (2) pteridines, intensely fluorescent
purine analogues;1b (3) expanded nucleobases, where the natural
bases are extended by conjugation to additional aromatic rings (e.g.,
1,N6-ethenoadenine);4,5 (4) base analogues, where the natural hetero-
cycle is replaced with a fluorescent aromatic residue;6 and (5) con-
jugated base analogues, where fluorescent aromatic moieties are
linked to the natural nucleobases.7,8 Notably, favorable photophysi-
cal characteristics (e.g., long emission wavelengths and high emis-
sion quantum efficiencies) are typically associated with significant
structural perturbation when compared to the native nucleobases.
Our research program is directed toward the design, synthesis,
and implementation of new fluorescent isosteric nucleosides that
satisfy the following criteria: (a) maintain the highest possible
structural similarity to the natural nucleobases, (b) display emission
at long wavelengths (preferably in the visible range), (c) retain
adequate emission quantum efficiency, and, importantly, (d) exhibit
emission that is sensitive to the microenvironment. Here, we
describe the synthesis and photophysical characteristics of a series
of simple and responsive thymidine analogues, where a 2′-deoxy-U
core is conjugated to aromatic five-membered heterocycles, includ-
ing furan, thiophene, oxazole, and thiazole (Scheme 1). When
incorporated into oligonucleotides, the furan analogue 2a is shown
to positively signal the presence of DNA abasic sites.
λ
max Et2O
λ
max H2O
(nm)
Φ
H2O
λ
em Et2O
λ
em H2O
I
(nm)
(nm)
(nm)
H2O/Et2O
2a
2b
2c
2d
314
320
292
318
316
314
296
316
0.03
0.01
<0.01
<0.01
395
421
390
397
431
434
400
404
5.6
1.6
1.0
2.1
a 5.0 × 10-5 M (λmax), 1.0 × 10-5 M (λem), H2O (ref 13), Φ (ref 9).
Figure 1. (a) Steady-state emission spectra of nucleoside 2a in (red)
buffer,13 (dark blue) methanol, (green) acetonitrile, (black) dichloromethane,
(light blue) ether. (b) A linear relationship between emission energy and
ET(30) values.9
Figure 2. Synthesized oligonucleotides. X ) 2a and Y ) THF residue.9
hydroxyl affords the building blocks necessary for solid-phase DNA
synthesis (Scheme 1).12
Photophysical properties of nucleosides 2a-d were examined
prior to incorporation into oligonucleotides (Table 1). To evaluate
the nucleoside’s potential to respond to polarity changes, their
photophysical characteristics have been evaluated in different
solvents. Increasing solvent polarity has little influence on the
absorption maxima of the conjugated nucleosides. In contrast, both
emission wavelength and intensity are markedly affected by solvent
polarity. In ether, the least polar solvent tested, nucleoside 2a
displays a relatively weak emission with a maximum at 395 nm
(Figure 1). In water, the most polar solvent examined, 2a exhibits
an intense emission band (Figure 1), which peaks around 430 nm
and decays deeply into the visible (>550 nm). Solvents of
intermediate polarity display an intermediate behavior with a clear
emission bathochromic and hyperchromic effects with increasing
solvent polarity (Figure 1).14 Nucleoside 2a, containing a conjugated
furan, exhibits the highest sensitivity to solvent polarity (Table 1)
and is, therefore, selected for incorporation into oligonucleotides
(Figure 2).
The one-step synthesis of the modified pyrimidines is straight-
forward (Scheme 1).9,10 It entails a palladium-mediated cross
coupling of the commercially available 5-iodo-2′-deoxyuridine11
(or 3′,5′-diTol-Iodo-dU) and the corresponding stannylated hetero-
cycles. Standard protection of the 5′-hydroxyl with 4,4′-dimethoxy-
trityl chloride followed by phosphitylation of the unprotected 3′-
Scheme 1. Synthesis of Nucleosides 2a-d and Amidite 4a
a Reagents: (a) 2a: 1a, 2-(Bu3Sn)furan, PdCl2(Ph3P)2, dioxane, 94%.
2b: 1a, 2-(Bu3Sn)thiophene, PdCl2(Ph3P)2, dioxane, 53%. 2c: (i) 1b,
2-(Bu3Sn)oxazole, Pd(Ph3P)4, toluene; (ii) K2CO3, 5% THF/methanol, 10%.
2d: (i) 1b, 2-(Bu3Sn)thiazole, PdCl2(Ph3P)2, dioxane; (ii) K2CO3, 5% THF/
methanol, 34%. (b) DMTCl, pyridine, Et3N, 71%. (c) (iPr2N)2POCH2CH2CN,
1H-tetrazole, CH3CN, 65%.
The absorption and emission spectra of the singly modified
single-stranded oligonucleotide 5 (Figure 3) are similar to those
exhibited by the free nucleoside 2a in buffer.13,15 When hybridized
9
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J. AM. CHEM. SOC. 2005, 127, 10784-10785
10.1021/ja052000a CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
and display different emission in a duplex versus single-stranded
oligonucleotides. Of particular importance is its ability to positively
report the presence of DNA abasic sites with significantly increased
emission.
Acknowledgment. We thank the National Institutes of Health
(GM 069773) for generous support, and Professor Doug Magde
for his assistance and insight.
Supporting Information Available: Synthetic details, thermal de-
naturation, absorption/fluorescence spectra, and crystal structures. This
material is available free of charge via the Internet at http://pubs.acs.org.
Figure 3. Steady-state emission of 5‚6 (blue, 1.0 × 10-6 M), 5 (green,
1.0 × 10-6 M), and 5‚7 (red, 1.0 × 10-6 M) in buffer.13,17
References
(1) For review articles, see: (a) Wojczewski, C.; Stolze, K.; Engels, J. W.
Synlett 1999, 1667-1678. (b) Hawkins, M. E. Cell Biochem. Biophys.
2001, 34, 257-281. (c) Rist, M. J.; Marino, J. P. Curr. Org. Chem. 2002,
6, 775-793.
(2) Ward, D. C.; Reich, E.; Stryer, L. J. Biol. Chem. 1969, 244, 1228-1237.
(3) 5-methylpyrimidin-2-one is another example. See: (a) Wu, P.; Nordlund,
T. M.; Gildea, B.; McLaughlin, L. W. Biochemistry 1990, 29, 6508-
6514. (b) 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) (a) Secrist, J. A., III; Barrio, J. R.; Leonard, N. J. Science 1972, 175,
646-647. (b) Holme´n, A.; Albinsson, B.; Norde´n, B. J. Phys. Chem. 1994,
98, 13460-13469.
(5) For benzo[g]quinazoline-2,4-(1H,3H)-dione, see: (a) Godde, F.; Toulme´,
J.-J.; Moreau, S. Biochemistry 1998, 37, 13765-13775. (b) Arzumanov,
A.; Godde, F.; Moreau, S.; Toulme´, J.-J.; Weeds, A.; Gait, M. J. HelV.
Chim. Acta 2000, 83, 1424-1436.
(6) (a) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney, S., IV; Kool,
E. T. J. Am. Chem. Soc. 1996, 118, 7671-7678. (b) Stra¨ssler, C.; Davis,
N. E.; Kool, E. T. HelV. Chim. Acta 1999, 82, 2160-2171.
Figure 4. Denaturation of duplex 5‚6 monitored by absorbance (red
triangles, 1.0 × 10-6 M) and fluorescence (blue squares, 2.0 × 10-6 M) in
buffer.13,16
(7) (a) Netzel, T. L.; Zhao, M.; Nafisik, K.; Headrick, J.; Sigman, M. S.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 9119-9128. (b) Kerr, C. E.;
Mitchell, C. D.; Headrick, J.; Eaton, B. E.; Netzel, T. L. J. Phys. Chem.
B 2000, 104, 1637-1650. (c) Seela, F.; Zulauf, M.; Sauer, M.; Deimel,
M. HelV. Chim. Acta 2000, 83, 910-927. (d) Seela, F.; Feiling, E.; Gross,
J.; Hillenkamp, F.; Ramzaeva, N.; Rosemeyer, H.; Zulauf, M. J.
Biotechnol. 2001, 86, 269-279.
to its perfect complement 6, a duplex (5‚6) that is as stable as the
control unmodified duplex 6‚8 is obtained (Tm ) 56 °C for both).9
Similar to other emissive nucleosides (e.g., 2-aminopurine), the
emission of the furan containing dU is significantly quenched when
found in a perfectly base-paired duplex (Figure 3). Importantly,
thermal denaturation curves (Figure 4), determined by either
absorbance at 260 nm or emission at 430 nm, yield the same melting
temperature (Tm ) 56 °C).16
Abasic sites are important DNA lesions that can be generated
either spontaneously or via enzymatic base excision of damaged
nucleosides. Several methods have been developed for detecting
the presence of these cytotoxic abasic sites, most require irreversible
modifications of isolated DNA.18 When oligo 5 is hybridized to
the tetrahydrofuran-containing oligo 7, a duplex containing an abasic
site is formed. Remarkably, the emission of duplex 5‚7 is enhanced
7-fold when compared to that of duplex 5‚6, formed upon
hybridization to the perfect complement (Figure 3). Nucleoside 2a,
when incorporated into a reporter oligonucleotide, positively signals
the presence of a DNA abasic site.
An unpaired base opposite an abasic site can be intrahelical or
extrahelical, depending on the sequence context. Our current
working hypothesis is that 2a is intrahelical, assuming a syn
conformation. This stacked conformation protects the hydrophobic
furan moiety, while projecting the hydrogen bonding face toward
the major groove. Support is offered by the following observations.
(a) Duplex 5‚7 is more stable than the control duplex 7‚8 that
contains a dT residue opposite the abasic site (Tm ) 39 and 35 °C,
respectively).9 The increased stability of the modified abasic duplex
(∆Tm ) +4 °C) suggests a favorable accommodation of the
modified nucleobase by the duplex. (b) The emission band observed
for duplex 5‚7 decays sharper (>500 nm) than when compared to
the emission exhibited by the free nucleoside in solution. This is
consistent with flattening of the chromophore that can be associated
with the restricted rotation of the conjugated furan ring upon
inclusion within the DNA duplex.19
(8) Hurley, D. J.; Seaman, S. E.; Mazura, J. C.; Tor, Y. Org. Lett. 2002, 4,
2305-2308.
(9) See Supporting Information for experimental details.
(10) Previous synthesis of 2a, 2b, and 2d: (a) Wigerinck, P.; Pannecouque,
C.; Snoeck, R.; Claes, P.; De Clercq, E.; Herdewijn, P. J. Med. Chem.
1991, 34, 2383-2389. 2d: (b) Gutierrez, A. J.; Terhorst, T. J.; Matteucci,
M. D.; Froehler, B. C. J. Am. Chem. Soc. 1994, 116, 5540-5544.
(11) Commercial availability of 5-iodouridine makes RNA analogues possible.
(12) Incorporation of 4 into oligonucleotides was accomplished using standard
protocols for solid-phase oligonucleotide synthesis (88% unoptimized
coupling efficiency). Deprotection using concentrated ammonium hy-
droxide was followed by PAGE purification. Oligonucleotides were
sequenced using MALDI-TOF MS.9
(13) Aqueous buffer ) 0.01 M sodium phosphate, 0.1 M NaCl, pH ) 7.0.
(14) This behavior typically occurs in polar molecules that are likely to have
enlarged dipoles and charge-transfer character in their excited state.
(15) The λmax values of 5‚6 and 5‚7 are identical (Figure S7.4) and similar to
that of 2a.9
(16) The emission of 2a is inherently temperature dependent and decreases as
temperature increases. See Figures S8.1-S8.3 in the Supporting Informa-
tion.9
(17) Duplex 6‚8 was subtracted for background correction.9
(18) Irreversible abasic site detection: (a) Weinfeld, M.; Liuzzi, M.; Paterson,
M. C. Biochemistry 1990, 29, 1737-1743. (b) Kubo, K.; Ide, H.; Wallace,
S. S.; Kow, Y. W. Biochemistry 1992, 31, 3703-3708. (c) Lhomme, J.;
Constant, J.-F.; Demeunynck, M. Biopolymers 2000, 52, 65-83. (d)
Atamna, H.; Cheung, I.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 686-691. (e) Bowman, K. J.; Le Pla, R.; Guichard, Y.; Farmer, P.
B.; Jones, G. D. D. Nucleic Acids Res. 2001, 29, e101. (f) Sun, H. B.;
Qian, L.; Yokota, H. Anal. Chem. 2001, 73, 2229-2232. (g) Georgakilas,
A. G.; Bennett, P. V.; Sutherland, B. M. Nucleic Acids Res. 2002, 30,
2800-2808. (h) Zhang, L.-K.; Gross, M. L. J. Am. Soc. Mass Spectrom.
2002, 13, 1418-1426. (i) Hirose, T.; Ohtani, T.; Muramatsu, H.; Tanaka,
A. Photochem. Photobiol. 2002, 76, 123-126. (j) JongMin, K.; Hiroshi,
M.; HeaYeon, L.; Tomoji, K. FEBS Lett. 2003, 555, 611-615. (k) Sato,
K.; Greenberg, M. M. J. Am. Chem. Soc. 2005, 127, 2806-2807. Other
methods: (l) Fukui, K.; Morimoto, M.; Segawa, H.; Tanaka, K.; Shimidzu,
T. Bioconjugate Chem. 1996, 7, 349-355. (m) Matray, J. T.; Kool, E. T.
Nature 1999, 399, 704-708. (n) Rachofsky, E. L.; Seibert, E.; Stivers, J.
T.; Osman, R.; Alexander Ross, J. B. Biochemistry 2001, 40, 957-967.
(o) Brotschi, C.; Ha¨berli, A.; Leumann, C. J. Angew. Chem. 2001, 40,
3012-3014. (p) Yoshimoto, K.; Nishizawa, S.; Minagawa, M.; Teramae,
N. J. Am. Chem. Soc. 2003, 125, 8982-8983. (q) Valis, L.; Amann N.;
Wagenknecht, H.-A. Org. Biomol. Chem. 2005, 3, 36-38.
(19) We are currently pursuing a high-resolution NMR structural elucidation
of the modified duplex.
In summary, an accessible and simple fluorescent dT analogue
is demonstrated to effectively probe the DNA microenvironment
JA052000A
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