Oligodeoxynucleotides incorporating structurally simple 5-alkynyl-2′-deoxyuridines fluorometrically respond to hybridization

Article abstract of DOI:10.1039/b705805e

Selected 5-ethynyl derivatives of 2′-deoxyuridine are shown to fluorometrically respond to hybridization and selectively base-pair to adenine whilst maintaining duplex stability. The Royal Society of Chemistry.

Full text of DOI:10.1039/b705805e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Oligodeoxynucleotides incorporating structurally simple 5-alkynyl-2ꢀ-
deoxyuridines fluorometrically respond to hybridization†
Robert H. E. Hudson* and Arash Ghorbani-Choghamarani‡
Received 17th April 2007, Accepted 8th May 2007
First published as an Advance Article on the web 16th May 2007
DOI: 10.1039/b705805e
Selected 5-ethynyl derivatives of 2ꢀ-deoxyuridine are shown
to fluorometrically respond to hybridization and selectively
base-pair to adenine whilst maintaining duplex stability.
With advances in genome sequencing, there is an increasing need
for probes and other molecular tools for clincal diagnostics,
environmental and agricultural monitoring, and research use.
Of the various techniques used for detection of hybridization
events, fluorescence-based methods are popular because of their
sensitivity, rapidity and possibility of automation.
Over the last several years, there has been much research into
fluorescent derivatives of nucleosides for use as hybridization
Fig. 1 Structure of 5-alkynyluridine and its proposed base-pairing (left)
compared to the cyclic derivative furanouridine. dR = 2ꢀ-deoxyribose.
probes. Nucleoside derivatives that remain capable of recognition
of a complementary base have been termed “base discriminating
fluorescent” nucleosides,1 or fluorescent DNA base replacements.2
Within the various and diverse structures of fluorescent nucleo-
sides possessing a modified nucleobase, two general subclasses
of fluorophores can be differentiated based on the nature of
the chromophore. One manifestation is achieved by appending
a relatively large fluorophore to the nucleobase3 while the other
approach relies on transforming the base-pairing moiety itself into
a fluorophore.4 Potential advantages of the latter approach are that
the chromophore is minimized and one can also take advantage of
the predictable change in environment of the nucleobase between
the single-stranded state and duplex states.
are fluorescent, that the fluorescence is responsive to the state
of hybridization and the modified bases maintain the fidelity of
base-pairing, Fig. 1.
Previously, we have reported that N1-unsubstituted and N1-
alkyl derivatives of 5-alkynyluracils are potential fluorophores for
use in the oligonucleotide analog peptide nucleic acid (PNA).8
Since the chemistry and use of oligodeoxynucleotides are much
better developed and more accessible to the research community,
as compared to PNA chemistry, we pursued the study of the same
modifications in DNA. These fluorophores are readily prepared
according to Scheme 1.
Recently, there has also been some interest in minimized
chromophores based on derivatization of uracil, such as the
compact 5-biaryl derivatives, used for the detection of abasic
sites.5 It has long been recognized that the bicyclic uracil derivative
furanouracil, that is prepared from 5-alkynyluracil, is fluorescent,
Fig. 1.6 However, upon formation of the fused ring, its hydrogen
bonding properties change such that it is no longer complementary
to adenine. Only quite recently has it been clearly described
that structurally simple 5-phenylethynyl-derivatives of uracil are
intrinsically fluorescent and that a large extrinsic chromophore is
not a structural requirement.7,8 Although structurally modest 5-
alkynyluracil derivatives (e.g. alkyl, phenyl) have been extensively
studied in nucleic acid analogs, their fluorescence properties and
use as fluorescent labels has been largely overlooked. Herein, we
report that simple, compact acyclic precursors to furanouracil
Scheme 1 Synthesis of the (2ꢀ-deoxy-b-D-ribo-pentofuranosyl)-5-alkynyl-
uridine derivatives suitable for oligonucleotide synthesis, via the bis-
(acetylated) nucleoside. (i) Ac2O, py, 24 h. (ii) RC≡CH, Pd(PPh3)4, CuI,
Et3N, DMF, rt, 24 h. (iii) py–EtOH (1 : 5, v/v), 1 M NaOH, 4 ◦C.
(iv) DMT-Cl, py, 5 h. (v) Cl(i-Pr2N)POCH2CH2CN, CH2Cl2, NEt3. Overall
chemical yield from the common intermediate 5-iodo-5ꢀ,3ꢀ-O-bis(acetyl)-
2ꢀ-dexoyuridine, is shown.
Department of Chemistry, The University of Western Ontario, London,
Ontario, Canada N6A 5B7. E-mail: robert.hudson@uwo.ca; Fax: +1-519-
661-3022; Tel: +1-519-661-2111 ext. 86349
† Electronic supplementary information (ESI) available: Characteriza-
tion of key compounds and additional fluorescence data. See DOI:
10.1039/b705805e
‡ Current address: Department of Chemistry, Bu-Ali Sina University,
Hamadan, Iran 65174; E-mail: aghorban@uwo.ca
Firstly, the commercially available 5-iodo-2ꢀ-deoxyuridine was
acetylated and then employed as a substrate in the Sonogashira
coupling with a small selection of terminal alkynes.9 Subsequent
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Table 2 Thermal denaturation data for DNA sequencesa
saponification gave the nucleoside derivative, which, after standard
transformations, yielded the phosphoramidite reagent.
5ꢀ Terminus modification
Target strand (3ꢀ → 5ꢀ)
AGG-TCG-CGT-TG
Although these conditions worked well, the coupling with
methyl propargyl ether gave a product with better aqueous
solubility than the corresponding 5-phenylethynyl nucleosides and
this resulted in losses during the aqueous workup. To circumvent
this difficulty, dimethoxytritylation of 2ꢀ-deoxy-5-iodouridine was
done first, followed by cross-coupling as previously reported,
Scheme 2.10 This method gave a better yield of 1a, was one step
shorter and was suitable for the other alkynes used, leading to
improved overall yields as compared to the approach in Scheme 1.§
(5ꢀ → 3ꢀ)
10 TCC-AGC-GCA-AC
11 MMEUCC-AGC-GCA-AC
12 PhUCC-AGC-GCA-AC
53.0
52.5
53.5
13 MeOPhUCC-AGC-GCA-AC 53.0
Target strand (3ꢀ → 5ꢀ)
CGG-ATT-GAA-GGC-CTC-TAC-A
3ꢀ Terminus modification
14 GCC-TAA-CTT-CCG-
GAG-ATG-T
63.0
63.0
62.0
63.0
15 GCC-TAA-CTT-CCG-
GAG-ATG-MME
U
16 GCC-TAA-CTT-CCG-
GAG-ATG-Ph
17 GCC-TAA-CTT-CCG-
GAG-ATG-MeOPh
U
U
In bold: modified nucleobase and complement.a See Table 1, footnote.
Table 3 Spectral data for modified 2ꢀ-deoxyuridines
Scheme 2 Synthesis of the (2ꢀ-deoxy-b-D-ribo-pentofuranosyl)-5-alkynyl-
uridine derivatives suitable for oligonucleotide synthesis via the 5ꢀ-di-
methoxytrityl nucleoside (i) DMT-Cl, py, 5 h. (ii) CH3OCH2C≡CH,
Pd(PPh3)4, CuI, Et3N, DMF, rt, 24 h. (iii) Cl(i-Pr2N)POCH2CH2CN,
CH2Cl2, NEt3.
ea
k/nmb
k/nm
Modification
(260 nm)
Excitation
Emission
1a
1b
1c
MMEU
PhU
4100
14 075
19 450
350
320
330
450
400
450
MeOPhU
The three modified nucleosides were incorporated into
oligodeoxynucleotides, using standard conditions with the excep-
tion of extended coupling time (3 min), Table 1. Each modification
is tolerated, having a negligible effect on the overall duplex stability
while maintaining excellent mismatch discrimination—properties
that are in accord with literature precedent.11
The base-modified nucleosides were also placed at either the
3ꢀ- or 5ꢀ-terminus and showed no deleterious effect on duplex
stability, Table 2. The thermal denaturation temperature (Tm)
data support the conclusion that the expected hydrogen bonding
is in operation and that the 5-substitution does not substantially
affect the tautomeric form of the uracil.
a Extinction coefficients were determined for the nucleoside by Beer’s plot.
b Excitation and emission data are reported for single-stranded oligomers
3, 4 and 5, representing the modifications 1a, 1b, and 1c, respectively.
The fluorescence properties of the oligomers were subsequently
examined according to the parameters shown in Table 3, under
identical conditions as the thermal denaturation studies (concen-
tration and ionic strength) to maintain the same Tm value as
observed in the thermal denaturation experiments.
From our earlier synthetic studies on cytosine nucleobase
derivatives,12 we expected the 5-methoxymethylethynyl-derivatized
uracil (1a) to demonstrate relatively poor fluorescence emission in
comparison to the phenylethynyl cogeners (1b,c). Although this
was found, oligomer 3 also demonstrated an excellent ability to
fluorometrically report hydridization. As illustrated in Fig. 2, at
2 lM strand concentration, the single-stranded species (grey line)
has little fluorescence, whereas in the presence of complementary
DNA (6), a six-fold increase in fluorescence intensity is observed.
We reasoned that we could improve on the weak fluorescence,
yet maintain a compact fluorophore by employing an unsaturated
substituent on the alkyne. Thus, compound 1b was prepared
and incorporated into an oligomer (4). Indeed, compared to 3,
the emission is more intense, yet a loss in the fluorescent-based
discrimination between the single-stranded 4 (grey line) and its
matched complex (4 + 6, black line) was observed (2-fold change),
Fig. 3.
Table 1 Thermal denaturation data for DNA sequencesa
Tm/◦Ca
Target strand (3ꢀ → 5ꢀ)
Central modification
GCG-TTA-X-ATT-GCG
DNA sequence (5ꢀ → 3ꢀ)
X = A X = G X = C X = T
6
7
8
9
2
3
2
5
CGC-AAT-T-TAA-CGC
51.5
42.5
42.0
40.0
39.5
39.5
40.0
40.5
39.5
40.5
41.5
39.0
38.5
CGC-AAT-MMEU-TAA-CGC 52.0
CGC-AAT-PhU-TAA-CGC
CGC-AAT-MeOPhU-TAA-CGC 49.5
51.5
In bold: modified nucleobase and complement.a Ionic conditions: 100 mM
NaCl, 10 mM Na2HPO4, 0.1 mM EDTA, pH 7.0. MMEU = (2ꢀ-deoxy-b-
D-ribo-pentofuranosyl)-5-methoxymethylethynyluridine. PhU = (2ꢀ-deoxy-
b-D-ribo-pentofuranosyl)-5-phenylethynyluridine. MeOPhU = 2ꢀ-deoxy-b-D-
ribo-pentofuranosyl)-5-(para-methoxy)phenylethynyluridine.
To maintain the compactness of the overall chromophore, we
were inspired to investigate the p-methoxyphenylethynyl sub-
stituent (1c). This substituent proved to impart the combined
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of hybridization. Taken altogether, the fluorescence response is
consistent with the idea that the modified base experiences less
quenching in the rigid, hydrophobic base stack environment
compared to the unstructured and solvent exposed single-strands
or terminal positions that are susceptible to end-fraying.
The fluorescence response of the centrally modified oligomers
to mismatches is interesting.† Even though the experimentally
determined thermal denaturation data (Table 1) and calculated
Tm data13 indicate roughly equal thermodynamic stability of
the mismatched hybrid duplexes, irrespective of the nature of
the mismatch, the fluorescence data infers different environments
for the chromophore. For each mismatched duplex, when the 5-
modified nucleobase is across from guanine (e.g. duplexes 3 + 7,
4 + 7, 5 + 7), the emission spectrum is more similar to the single-
strand spectrum than the duplex, that is, it suffers from an almost
equivalent amount of quenching. Whereas, for the oligomers
bearing the phenylethynyl-modified bases (4, 7), a mismatch to
thymine produces less quenching. Finally, a mismatch to cytosine
produces spectra that are more similar to the fully matched duplex
than any other mismatch or the single-strand. These results suggest
that the chromophore is sensitive to its local environment and may
be useful for examining changes in structure that are more subtle
than complete denaturation/renaturation. Currently, we present
a phenomenological description of the fluorescence response;
further studies are needed to characterize the photophysical basis
of these observations.
Fig. 2 Steady state fluorescence response of a 5-methoxymethylethyny-
luracil-containing oligonucleotide (3) in the presence of complementary
oligonucleotide 6 (black) as compared to single-strand (grey). Oligonu-
cleotides at 2.0 lM in 100 mM NaCl, 10 mM Na2HPO4, 0.1 mM EDTA,
pH 7.0, 22 ◦C.
In conclusion, we show that structurally simple and compact
5-alkynyl-derivatized 2ꢀ-deoxyuridines are fluorescent reporters
of hybridization events when placed at internal positions in
oligodeoxynucleotides. In addition, the chromophores are re-
sponsive to their local structure/environment and therefore have
potential use in the examination of nucleic acid conformation or
nucleic acid–ligand interactions.
Fig. 3 Steady state fluorescence response of a 5-phenylethynyluracil-con-
taining oligonucleotide (4) in the presence of complementary oligonu-
cleotide 6 (black) as compared to single-strand (grey). Oligonucleotides
at 2.0 lM in 100 mM NaCl, 10 mM Na2HPO4, 0.1 mM EDTA, pH 7.0,
22 ◦C.
desirable properties of relatively high emission and a large change
in fluoresence upon hybridization. In this instance, a six-fold
increase in fluorescence intensity was observed when oligomer
5 bound complementary DNA (6), Fig. 4.
Notes and references
§ Sonogashira cross-coupling was also successful using the unprotected
nucleoside, as reported in ref. 6.
1 A. Okamoto, Y. Saito and I. Saito, J. Photochem. Photobiol., C, 2005,
6, 108.
2 J. N. Wilson and E. T. Kool, Org. Biomol. Chem., 2006, 4, 4265.
3 Selected examples of chromophore-labelled nucleobases that fluo-
rometrically report hybridization or conformational changes when
incorporated into oligonucleotides: (a) M. V. Skorobogatyi, A. D.
Malakhov, A. A. Pchelintseva, A. A. Turban, S. L. Bondarev and V. A.
Korshun, ChemBioChem, 2006, 7, 810; (b) Y. J. Seo and B. H. Kim,
Chem. Commun., 2006, 150; (c) A. Okamoto, K. Tainaka, K.-I. Nishiza
and I. Saito, J. Am. Chem. Soc., 2005, 127, 13128; (d) A. Okamoto, K.
Tainaka, Y. Ochi, K. Kanatani and I. Saito, Mol. BioSyst., 2006, 2, 122;
(e) E. Mayer-Enthart and H.-A. Wagenknecht, Angew. Chem., Int. Ed.,
2006, 45, 3372; (f) S. S. Bag, Y. Saito, K. Hanawa, S. Kodate, I. Suzuka
and I. Saito, Bioorg. Med. Chem. Lett., 2006, 16, 6338.
Fig. 4 Steady state fluorescence response of a 5-(p-methoxyphenyl-
ethynyl)uracil-containing oligonucleotide (5) in the presence of comple-
mentary oligonucleotides 6 (black) as compared to single-strand (grey).
Oligonucleotides at 2.0 lM in 100 mM NaCl, 10 mM Na2HPO4, 0.1 mM
EDTA, pH 7.0, 22 ◦C.
4 Intrinsically fluorescent 5-substituted 2ꢀ-deoxyuridines have been noted
for their larger ethynyl-linked aromatic chromophores: (a) D. J. Hurley,
S. E. Seaman, J. C. Mazura and Y. Tor, Org. Lett., 2002, 4, 2305;
(b) L. H. Thoresen, G.-S. Jiao, W. C. Haaland, M. L. Metzker and K.
Burgess, Chem.–Eur. J., 2003, 9, 4603; (c) G. T. Hwang, Y. J. Seo and
B. H. Kim, J. Am. Chem. Soc., 2004, 126, 6528.
5 N. J. Greco and Y. Tor, J. Am. Chem. Soc., 2005, 127, 10784.
6 M. J. Robins and P. J. Barr, Tetrahedron Lett., 1981, 22, 421.
7 The fluorescence of 5-tolylethynyl-2ꢀ-deoxyuridine has been noted: N.
Esho, B. Davies, J. Lee and R. Dembinski, Chem. Commun., 2002,
332.
The fluorescence reporting ability of these nucleobases was
examined when placed at either 5ꢀ- or 3ꢀ-terminal positions
(oligomers 10–17). Generally, the fluorescence signal of the
terminally modified oligonucleotides was unresponsive to state
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8 (a) R. H. E. Hudson, A. K. Dambenieks and J. M. Moszynski, Proc.
SPIE-Int. Soc. Opt. Eng., 2005, 103; (b) R. H. E. Hudson and J. M.
Moszynski, Synlett, 2006, 18, 2997.
9 M. J. Robins and P. H. Barr, J. Org. Chem., 1983, 48, 1854.
10 K. A. Cruickshank and D. L. Stockwell, Tetrahedron Lett., 1988, 29,
5221.
Tetrahedron Lett., 1992, 33, 5307;(c) T. Kottysch, C. Ahlborn, F. Brotzel
and C. Richert, Chem.–Eur. J., 2004, 10, 4017.
12 R. H. E. Hudson, A. K. Dambenieks and R. D. Viirre, Synlett, 2004,
13, 2400.
13 (a) HYTHERTM version 1.0, N. Peyret and J. SantaLucia, Jr., Wayne
State University; (b) J. SantaLucia, Jr., Proc. Natl. Acad. Sci. U. S. A.,
1998, 95, 1460; (c) N. Peyret, P. A. Seneviratne, H. T. Allawi and J.
SantaLucia, Jr., Biochemistry, 1999, 38, 3468.
11 (a) E. Biala, A. S. Jones and R. T. Walker, Tetrahedron, 1980, 36,
155; (b) B. C. Froehler, S. Wadwani, T. J. Terhorst and S. R. Gerrard,
1848 | Org. Biomol. Chem., 2007, 5, 1845–1848
This journal is
The Royal Society of Chemistry 2007
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