A blue-to-red energy-transfer thymidine analogue that functions in DNA

Article abstract of DOI:10.1021/ol049684w

Matrix presented. The thymidine analogue 1 is a blue-to-red energy transfer cassette designed to absorb UV light In the 300 nm region and emit it as fluorescence at around 520 nm. When incorporated into DNA, the fluorescence intensity of this modified nuc

Full text of DOI:10.1021/ol049684w

ORGANIC
LETTERS
2004
Vol. 6, No. 11
1701-1704
A Blue-to-Red Energy-Transfer
Thymidine Analogue That Functions in
DNA
,†
Guan-Sheng Jiao,† Taeg Gyum Kim,‡ Michael R. Topp,‡ and Kevin Burgess*
Department of Chemistry, Texas A & M UniVersity, Box 30012,
College Station, Texas 77842-3012, and Department of Chemistry,
UniVersity of PennsylVania, Philadelphia, PennsylVania 19104
burgess@tamu.edu
Received February 23, 2004
ABSTRACT
The thymidine analogue 1 is a blue-to-red energy transfer cassette designed to absorb UV light in the 300 nm region and emit it as fluorescence
at around 520 nm. When incorporated into DNA, the fluorescence intensity of this modified nucleobase is not significantly reduced by the
surrounding structure in the oligonucleotides.
Natural DNA nucleobases have negligible fluorescence, even
as isolated nucleosides.1 Consequently, some nucleobase
analogues are interesting for their relatively enhanced
fluorescence. However, fluorescent nucleobase analogues that
absorb at short wavelengths (ca. 300 nm) exhibit dramatically
reduced fluorescence in DNA.2 This is unfortunate because
there is a need for analogues that can be incorporated into
DNA and fluoresce at relatively long wavelengths when
excited in the UV region at around 300 nm.3 This Letter
features investigations of the thymidine analogue 1 that has
potential in this regard.
theme is molecules with a donor part that absorbs light at
relatively short wavelengths, and an acceptor part that accepts
this energy and fluoresces at a much longer wavelength.5
The key structural feature of these molecules is that the donor
and acceptor parts would be electronically conjugated were
it not for a twist in the molecule that keeps them from
becoming planar. This barrier to planarity means that the
molecules do not behave as a single, flat dye system. The
fact that the molecules would otherwise be conjugated means
they can transmit energy through bonds as well as through
space, i.e., they are conceptually distinct from donor-
acceptor cassettes based solely on Fo¨rster energy transfer.6
These studies are an extension of our previous work on
donor-acceptor cassettes for biotechnology.4 The unifying
The distinctive feature of nucleoside 1 is that it is
potentially a through-bond energy transfer system where a
† Texas A & M University.
‡ University of Pennsylvania.
(1) Daniels, M.; Hauswirth, W. Science 1971, 171, 675. Pecourt, J.-M.
L.; Peon, J.; Kohler, B. J. Am. Chem. Soc. 2000, 122, 9348.
(2) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc.
2003, 125, 9296. Seela, F.; Becher, G. HelV. Chim. Acta 2000, 83, 928.
Singleton, S. F.; Shan, F.; Kanan, M. W.; McIntosh, C. M.; Stearmann, C.
J.; Helm, J. S.; Webb, K. J. Org. Lett. 2001, 3, 3919.
(3) Haugland, R. P. Handbook of Fluorescent Probes and Research
Chemicals, 6th ed.; Molecular Probes: Eugene, OR, 1996. Lee, L. G.
Method for Detecting Oligonucleotides Using UV Light Source; PE
Corporation: Foster City, CA, 2001.
(4) Burghart, A.; Thoresen, L. H.; Chen, J.; Burgess, K.; Bergstro¨m, F.;
Johansson, L. B.-A. Chem. Commun. 2000, 2203. Burgess, K.; Burghart,
A.; Chen, J.; Wan, C.-W. New Chemistry of BODIPY Dyes, and BODIPY
Dye Cassettes Featuring Through-Bond Energy Transfer: San Jose, CA,
2000. Thoresen, L. H.; Jiao, G. S.; Haaland, W. C.; Metzker, M. L.; Burgess,
K. Chem. Eur. J. 2003, 9, 4603. Jiao, G.-S.; Thoresen, L. H.; Burgess, K.
J. Am. Chem. Soc. 2003, 125, 14668.
(5) Wagner, R. W.; Lindsey, J. S. J. Am. Chem. Soc. 1994, 116, 9759.
Tour, J. M. Chem. ReV. 1996, 96, 537. McQuade, D. T.; Pullen, A. E.;
Swager, T. M. Chem. ReV. 2000, 100, 2537.
10.1021/ol049684w CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/24/2004

Figure 1. Featured cassette 1 and molecules resembling the donor
and acceptor parts of this system.
modified thymidine forms part of the donor. Figure 2a shows
UV absorption spectra for this molecule, the hydroxyxan-
thone 27 resembling the acceptor part, and a nucleoside 3
that was prepared as a control for the donor part. The
acceptor fragment 2 has a small absorption in the wavelength
range around 300 nm. At 320 nm, λmax for 3, the absorption
of 2 is around 5% of that of the donor control 3. The
absorption spectra of nucleoside 1 is essentially the sum of
the donor-acceptor parts 2 and 3 proving that this molecule
behaves as two separate conjugated systems, as required for
an energy transfer cassette.
Figure 2b shows the fluorescence of equimolar 1-3 when
excited at 320 nm. The donor control molecule 3 has almost
no fluorescence at 520 nm, a wavelength close to the λmax
emiss for 1 and 2. Similarly, when the acceptor part is excited
under the same conditions, it has negligible fluorescence.
However, cassette 1 is 2 orders of magnitude more fluores-
cent than 3 under these conditions. Thus, while the acceptor
control 2 absorbs few photons at the 320 nm and gives a
correspondingly weak fluorescence, nucleoside 1 acts as an
energy transfer cassette. Further, the fluorescence of the
donor part is almost completely quenched in cassette 1,
implying close to 100% energy transfer. This assertion is
supported by the observation of comparably high quantum
yields for cassette 1 irradiated at wavelengths corresponding
to absorption predominantly by the donor and by the acceptor
(300 and 490 nm, respectively; Table 1).8
Figure 2. (a) UV absorption spectra of 1-3, as equimolar solutions
in Tris‚HCl solution (50 mM, pH ) 7.5). (b) Fluorescence spectra
of 1-3; equimolar solutions in Tris‚HCl solution (50 mM, pH )
7.5) excited at 320 nm (uncorrected for the spectral response of
the instrument).
tend to quench the fluorescence of cassette. To explore this,
it was necessary to generate solutions of 1 and of the
oligonucleotides that were precisely equimolar. Practically,
this was a challenge because the small amounts involved
precluded accurate weighing. Moreover, the extinction coef-
ficients of 1 and the oligonucleotides containing it cannot
be assumed to be the same, so determining concentrations
via UV absorbance (or fluorescence) would not be simple.
Consequently, the following procedure was used. Stock
solutions of the oligonucleotides were prepared and diluted
with buffer for the spectroscopic studies. To obtain equimolar
solutions of compound 1, the same stock solutions were
mixed with phosphodiesterase and incubated. HPLC analyses
were used to confirm that the oligonucleotides had been
completely hydrolyzed to the constituent bases. The digested
Key issues in this project relate to the properties of the
cassette when incorporated into oligonucleotides. Conse-
quently, oligonucleotides 4-6 containing 1 were prepared
via solid-phase syntheses (Figure 3a).9 It was critical to
determine the extent to which the organized hydrogen
bonding network and base stacking effects in DNA would
(6) Speiser, S. Chem. ReV. 1996, 96, 1953.
(9) Jiao, G.-S.; Burgess, K. Bioorg. Med. Chem. Lett. 2003, 13, 2785.
This paper also reports thermal denaturation data that show 1 has a slight
destabilizing effect on annulation with complementary oligonucleotide
sequences, though not as much as A:A mismatches.
(7) Shi, J.; Zhang, X.; Neckers, D. C. J. Org. Chem. 1992, 57, 4418.
(8) Donor control compound 3 shows 114 nm of Stokes’ shift and 200
times greater quantum yield than native thymidine.
1702
Org. Lett., Vol. 6, No. 11, 2004

Compounds 7 and 8, i.e., isomers of nucleoside 1 and the
control compound 3, were also prepared and studied in
parallel. The spectroscopic data obtained from these nucleo-
sides and oligonucleotide 9 formed from them shows no
significant difference from that described above (see Sup-
porting Information).
Table 1. Selected Fluorescence Data
relative
fluorescence
compound
intensitya
quantum yield (φ)b
1
1
0.81 ( 0.04 @ 300 nm
0.85 ( 0.02 @ 490 nm
2
na
0.91 ( 0.02 @ 490 nm
3
na
0.015 ( 0.003 @ 300 nm
ss-4
d s-4
ss-5
d s-5
ss-6
d s-6
0.84c
0.75c
0.78c
0.71c
0.89c
0.76c
nd
nd
nd
nd
nd
nd
a Equimolar concentrations in Tris‚HCl buffer (50 mM, pH ) 7.5) excited
at 320 nm. b Determined in 10 mM Tris‚HCl buffer pH ) 8.0. Rhodamine
6G in the same solvent was used as a reference (φ ) 0.95) for 1, fluorescein
(φ ) 0.79) for 2, coumarin 1 (φ ) 0.73) for 3. c Compared with cassette 1;
na ) not applicable; nd ) not determined.
mixtures were then diluted to the same concentration as the
oligonucleotides.
Figure 3b shows typical data obtained by comparing the
fluorescence spectra of an oligonucleotide containing 1 (in
this case 4), the corresponding double-strand (ds) DNA
Compounds A10 and B11 are typical of numerous studies
by others in which fluorescent labels have been attached to
nucleoside bases. The compounds described here are distinct
from those in two respects. Nucleoside A has a fluorescent
group conjugated to a modified thymidine, but the molecule
can freely adopt planar conformations; hence, it does not
behave as a cassette. Instead, it represents a unified dye
system that absorbs at appreciably longer wavelengths than
1 but emits at much shorter wavelengths. The linker
connecting the fluorescein and the nucleobase in molecule
B precludes through-bond energy transfer between the label
and the base. The flexible linker in B also opens the
possibility that the fluorescein part would interact with, or
even intercalate into, the DNA strand. Indeed, the fluores-
cence of this nucleotide is dramatically reduced in oligo-
nucleotides. Conversely, the fluorescein is held away from
the DNA strand in oligonucleotides formed from 1, and we
have demonstrated that its fluorescence intensity is similar
to that in the nucleoside.
Overall, our interpretation of the data shown in this paper
is as follows. Nucleobase 1 has a donor fragment that
resembles an alkyne-modified T-residue. This part of the
molecule dominates the UV absorption of 1 in the 320 nm
region, i.e., near, but not completely overlapping with, the
intrinsic absorption of DNA bases (ca. 260 nm). Introduction
of the twisted linker-hydroxyxanthone system provided an
acceptor part and a viable pathway for through-bond energy
transfer. This was kinetically competitive with nonradiative
decay processes that ensued when the donor part was
Figure 3. (a) Oligonucleotides 4-6 where 1′ is the phosphodiester
of 1, and (b) fluorescence emission spectra of 1, 4, and ds DNA
wherein 1′ is paired with A in equimolar Tris‚HCl solution (50
mM, pH ) 7.5) excited at 320 nm.
(10) Korshun, V. A.; Manasova, E. V.; Balakin, K. V.; Malakhov, A.
D.; Perepelov, A. V.; Sokolova, T. A.; Berlin, Y. A. Nucleosides Nucleotides
1998, 17, 1809. Korshun, V. A.; Prokhorenko, I. A.; Gontarev, S. V.;
Skorobogatyi, M. V.; Balakin, K. V.; Manasova, E. V.; Malakhov, A. D.;
Berlin, Y. A. Nucleosides Nucleotides 1997, 16, 1461. Korshun, V. A.;
Manasova, E. V.; Balakin, K. V.; Prokhorenko, I. A.; Buchatskii, A. G.;
Berlin, Y. A. Bioorganicheskaya Khimiya 1996, 22, 923.
wherein 4 is paired with A, and an equimolar solution of 1
formed via the phosphodiesterase procedure. These data show
that the fluorescence intensity of 1 in single-strand or ds
DNA is at least 75% of that in the parent nucleoside. Data
for similar experiments with 5 and 6 are shown in Table 1.
(11) Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L. J.
Am. Chem. Soc. 1989, 111, 6966.
Org. Lett., Vol. 6, No. 11, 2004
1703

oligonucleotides containing it are irradiated at 320 nm, very
close to the absorption of the natural DNA bases.12
Acknowledgment. We would like to thank Dr. Lars H.
Thoresen for useful discussions, Dr. Robin Hochstrasser, and
the RLBL at the University of Pennsylvania for use of their
facilities. Use of the TAMU/LBMS-Applications Laboratory
directed by Dr. Shane Tichy is acknowledged. Support for
this work was provided by The National Institutes of Health
(HG 01745) and by The Robert A. Welch Foundation.
Supporting Information Available: Experimental pro-
cedures for the preparation of 1 and 3-9; absorption and
fluorescence spectra of 2, 7, and 8; fluorescence spectra of
DNAs containing the modified nucleoside; tabulated pho-
tophysical data of 1-3, 7, and 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL049684W
irradiated at 320 nm. Consequently, fluorescence of the
cassette could be used as a marker for events that occur when
(12) Others have noted this would be valuable. Hurley, D. J.; Seaman,
S. E.; Mazura, J. C.; Tor, Y. Org. Lett. 2002, 4, 2305.
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Org. Lett., Vol. 6, No. 11, 2004