Oligonucleotides containing a nucleotide analog with an ethynylfluorobenzene as nucleobase surrogate

Article abstract of DOI:10.1016/S0040-4039(02)02153-6

The 5′-protected 3′-phosphoramidite of 1-(2′-deoxy-β-D-ribofuranosyl)-2-ethynyl-4-fluorobenzene was prepared and employed in the synthesis of oligonucleotides. The duplex of CTTTTCF^#TTCTT with AAGAAAGAAAAG, where F^# is the ethynylfluorobenzene-containing nucleotide, melts higher than the duplex containing a difluorotoluene moiety.

Full text of DOI:10.1016/S0040-4039(02)02153-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 8755–8758
Oligonucleotides containing a nucleotide analog with an
ethynylfluorobenzene as nucleobase surrogate
Niels Griesang and Clemens Richert*
Department of Chemistry, University of Constance, Universita¨tsstraße 10, D-78457 Konstanz, Germany
Received 1 August 2002; accepted 23 September 2002
Abstract—The 5%-protected 3%-phosphoramidite of 1-(2%-deoxy-b-D-ribofuranosyl)-2-ethynyl-4-fluorobenzene was prepared and
employed in the synthesis of oligonucleotides. The duplex of CTTTTCFcTTCTT with AAGAAAGAAAAG, where Fc is the
ethynylfluorobenzene-containing nucleotide, melts higher than the duplex containing a difluorotoluene moiety. © 2002 Elsevier
Science Ltd. All rights reserved.
1. Introduction
found in natural A:T and G:C base pairs. Modified
nucleobases may engage in duplex stabilizing interac-
tions by forming additional hydrogen bonds, by binding
via the major or the minor groove, or by offering
additional surface sites for stacking.3,4 For example,
clamp-like binding in the major groove has been
reported to stabilize C:G base pairs.5 Propinyl sub-
stituents at position 5 of pyrimidines have been shown
to increase duplex stability via stacking.6
Nucleotide analogs with apolar aromatic rings as
replacements for natural nucleobases have been shown
to have interesting properties, both in terms of Watson–
Crick duplexes containing these analogs and in terms of
polymerase-catalyzed reactions.1 One analog of
thymidine in particular has attracted attention: the
C-nucleoside containing a difluorotoluene moiety as
nucleobase surrogate (‘F’, Fig. 1).2 This analog is incor-
porated opposite to deoxyadenosine residues in poly-
merase-catalyzed primer extension reactions and forms
base pairs that are isosteric to thymine:adenine base
pairs. Therefore, this C-nucleotide may be used as a lead
structure for developing new nucleotide analogs that
can engage in base pairing to deoxyadenosine residues.
We have an interest in tuning the stability of T:A base
pairs containing unmodified deoxyadenosine residues by
modifying thymidine residues. In this case, the lack of a
hydrogen bonding site at the 2-position of the adenine
ring makes the formation of a third hydrogen bond
difficult. Therefore, surrogates of thymine carrying sub-
stituents capable of engaging in additional van der
Waals or stacking interactions, such as a fluorobenzene
ring with a 2-ethynyl group (Fc, Fig. 1), were of
interest.
We have become interested in interactions between
modified nucleobases and natural nucleobases that go
beyond the hydrogen bonding and stacking interactions
Figure 1.
Keywords: C-nucleosides; base pairing; oligonucleotides.
* Corresponding author. Present address: Institut fu¨r Organische Chemie, Universita¨t Karlsruhe (TH), Fritz-Haber-Weg 6, D-76131 Karlsruhe,
Germany. Tel. +49 (0)721 608 2091; fax: +49 (0)721 608 4825; e-mail: cr@rrg.uka.de
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02153-6

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N. Griesang, C. Richert / Tetrahedron Letters 43 (2002) 8755–8758
2. Results and discussion
But 8 and 5%-AAGAAAGAAAAG-3% gave a duplex
stable enough to obtain a full sigmoidal UV melting
curve. Compared to unmodified duplex
Synthesis of phosphoramidite 1 (Scheme 1) started
from commercially available 2,7 which in the present
case was synthesized from less expensive 2-bromo-5-
fluoroaniline in 84% yield in analogy to a protocol of
Heaney and Miller for a similar substrate.8 Sonogashira
coupling9,10 of 2 with 1.2 equivalents of trimethylsilyl-
acetylene furnished 3 in 95% yield after distillation.11
Bromide 3 was converted to its Grignard derivative
under continuous ‘starting’ of the reaction with 1,2-
dibromoethane, transmetallated with CdCl2,12 and
reacted with chloroglycosyl donor 413 to yield 53% of a
mixture of epimers 5a and 5b.14 The 1H NMR spectrum
suggested that the undesired a-anomer 5a predomi-
nated in this mixture by a factor of 8–10. The epimers
proved difficult to separate, but desilylation to 6a/b in
71% yield allowed chromatographic separation, fol-
lowed by epimerization of 6a to a mixture of 6a and
6b.15 The combined yield of isolated nucleosides after
epimerization in toluene was 69%. Other solvents and a
one pot deprotection/epimerization with BF3·OEt2 gave
lower yields. Desired b-epimer 6b16 was deprotected to
7 in 99% yield, 5%-protected with a dimethoxytrityl
group in 79% yield, and phosphitylated under DIPAT
activation17 to obtain 1 in 76% after chromatography.
CTTTTCTTTCTT: AAGAAAGAAAAG, the melting
point of 8: AAGAAAGAAAAG is significantly
depressed (Table 1). When compared to the duplex
CTTTTCFTTCTT: AAGAAAGAAAAG, however,
where a difluorotoluene moiety replaces the thymine,
the duplex of 8 shows a higher melting point,20
suggest-
ing that the ethynyl group does provide a stabilizing
effect compared to a fluoro substituent.
Given that the methyl group of difluorotoluene is
absent in our base analog, the stabilizing effect of the
ethynyl group may be stronger than the melting point
increase suggests, since the stacking interactions pro-
Table 1. UV-melting points of duplexes at 1.6 mM strand
concentration, 10 mM PIPES buffer, pH 7.0, 10 mM
MgCl2, and 100 mM NaCl, determined at 260 nm
Duplexa
Tm (°C)b
42.290.5
29.590.7
39.4d
DTm (°C)c
CTTTTCTTTCTT:
AAGAAAGAAAAG
CTTTTCFcTTCTT:
AAGAAAGAAAAG
CTTTTCTTTCTT:
AAGAAAGAAAAG
CTTTTCFTTCTT:
AAGAAAGAAAAG
–
−12.7
–
With phosphoramidite 1 in hand, DNA syntheses pro-
21.4d
−18.0
ceeded via
a
standard protocol.18 Two modified
oligonucleotides, 5%-CTTTTCFcTTCTT-3% (8), where
Fc denotes the nucleotide with the fluoroethynyl ring
as nucleobase surrogate (Scheme 1), and 5%-
TTTTAAFcAAT-3% (9), were prepared19 together with
unmodified control and target strands. Duplexes of 9
with its complementary strand melted too low to allow
determination of an accurate UV-melting point (Tm).
a Sequences are given 5%- to 3%-terminus. The residue letter at the site
of modification is in boldface.
b UV melting point. For the first two entries, this is the mean of
melting points from four curves one standard deviation.
c Melting point difference to unmodified control duplex.
d From Ref. 1b, see also Ref. 20.
Scheme 1.

N. Griesang, C. Richert / Tetrahedron Letters 43 (2002) 8755–8758
8757
vided by this methyl group are missing. Therefore it is
promising to pursue ethynyl substituents at position 2
of pyrimidine nucleobase analogs further. They may
also be useful for probing substrate–polymerase
complexes.21
(d) Seela, F.; Becher, G. Nucleic Acids Res. 2001, 29,
2069–2078; (e) Okamoto, A.; Tanaka, K.; Saito, I.
Bioorg. Med. Chem. Lett. 2002, 12, 97–99.
5. Lin, K.-Y.; Matteucci, M. D. J. Am. Chem. Soc. 1998,
120, 8531–8532.
6. (a) Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang,
T.; Froehler, B. C. Nature Biotechn. 1996, 14, 840–844;
(b) Barnes, T. W.; Turner, D. H. J. Am. Chem. Soc. 2001,
123, 4107–4118 and references cited therein.
Acknowledgements
7. Available from ABCR GmbH, D-76151 Karlsruhe,
Germany.
8. Heaney, H.; Millar, I. T. Org. Syn. Collective Vol. V,
1120–1123.
9. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 50, 4467–4470.
10. Model protocol for a related substrate: Van Meurs, P. J.;
Janssen, R. A. J. J. Org. Chem. 2000, 65, 5712–5719.
11. To a mixture of 1-bromo-4-fluoro-2-iodobenzene (2, 42.4
g, 0.141 mol), Pd(PPh3)2Cl2 (990 mg, 1.41 mmol), and
CuI (376 mg, 1.97 mmol) in NEt3 (300 mL) under Ar was
added trimethylsilylethyne (16.6 g, 23.9 mL, 0.169 mol)
within 1 h. After 2 h at r.t., half conc. aqueous NH4Cl
solution (400 mL) was added, the organic phase sepa-
rated and the aqueous phase extracted with CH2Cl2
(3×100 mL). Distillation from the combined organic
phases (0.1 Torr, 65°C) yielded 3 (36.45 g, 0.134 mol,
95%). 1H NMR (CDCl3, 250 MHz) l (ppm)=7.50 (dd,
J=5.19, 8.8 Hz, 1H), 7.19 (dd, J=3.0, 8.8 Hz, 1H), 6.89
(ddd, J=3.0, 7.9, 8.8 Hz, 1H), 0.28 (s, 9H); Anal. calc: C
48.72; H 4.46, found C 48.48; H 4.42.
This work was supported by Deutsche Forschungsge-
meinschaft, grants No. RI 1063/2-1 and FOR 434, and
Fonds der Chemischen Industrie. The authors wish to
thank Jan Rojas, Sukunath Narayanan, and Frank
Brotzel for help with DNA synthesis, UV melting curve
experiments, and the acquisition of MALDI-TOF mass
spectra.
References
1. Selected references: (a) Schweitzer, B. A.; Kool, E. T. J.
Org. Chem. 1994, 59, 7238–7242; (b) Schweitzer, B. A.;
Kool, E. T. J. Am. Chem. Soc. 1995, 117, 1863–1872; (c)
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X.-F.; Rumney, S.; Kool, E. T. J. Am. Chem. Soc. 1997,
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Struc. Biol. 1998, 5, 950–954; (g) Matray, T. J.; Kool, E.
T. J. Am. Chem. Soc. 1998, 120, 6191–6192; (h) Morales,
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2001, 30, 1–22; (j) McMinn, D. L.; Ogawa, A. K.; Wu,
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Tae, E. J.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg,
F. E. J. Am. Chem. Soc. 2001, 123, 7439–7440; (m)
Parsch, J.; Engels, J. W. Helv. Chim. Acta 2000, 83,
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Wenzel, T. Helv. Chim. Acta 1996, 79, 488–498.
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(b) Guckian, K. M.; Krugh, T. R.; Kool, E. T. J. Am.
Chem. Soc. 2000, 122, 6841–6847; (c) Cubero, E.; Sherer,
E. C.; Luque, F. J.; Orozco, M.; Laughton, C. A. J. Am.
Chem. Soc. 1999, 121, 8653–8654.
3. For recent reviews, see e.g.: (a) Herdewijn, P. Antisense
Nucleic Acid Drug Dev. 2000, 10, 297–310; (b) Kool, E.
T.; Morales, J. C.; Guckian, K. M. Angew. Chem. Int.
Ed. Engl. 2000, 39, 990–1012, Angew. Chem. 2000, 112,
1046–1068.
12. Chaudhuri, N. C.; Kool, E. T. Tetrahedron Lett. 1995,
36, 1795–1798.
13. (a) Fox, J. J.; Yung, N. C.; Wempen, I.; Hoffer, M. J.
Am. Chem. Soc. 1961, 83, 4066–4068; (b) Hoffer, M.
Chem. Ber. 1969, 23, 2777–2779; (c) Wang, Z. X.; Duan,
W.; Wiebe, L. I.; Balzarini, J.; De Clerq, E.; Knaus, E. E.
Nucleosides Nucleotides Nucl. Acids 2001, 20, 11–40.
14. Bromide 3 (16.27 g, 60 mmol) and Mg (7.3 g, 300 mmol)
in THF (80 mL) were treated with 1,2-dibromoethane
(11.3 g, 60 mmol) in THF (20 mL) slowly enough to
maintain a mild reflux (1 h) (attention: ethene gas is
liberated). The solution was heated to reflux until GC-
MS showed disappearance of 3 (approx. 2 h). Solids were
filtered off under Ar after cooling, and the solution
treated with CdCl2 (5.5 g, 30 mmol) (attention: CdCl2 is
very toxic and carcinogenic), followed by heating to
reflux for 4 h. The mixture was cooled to 0°C and treated
with 4 (11.8 g, 27.6 mmol), followed by stirring for 16 h
while warming to approx. 10°C. The mixture was treated
with NH4Cl solution (100 mL) and water (100 mL). The
aqueous phases were extracted with CH2Cl2 (3×50 mL),
the combined organic phases dried over Na2SO4, and
evaporated. Flash chromatography (silica, toluene)
yielded a mixture of 5a and 5b (8.44 g, 14.4 mmol; 53%).
Rf (silica, CH2Cl2) 0.65; EI-MS (70 eV) 584, 428, 139,
111.
4. Selected references: (a) Groebke, K.; Hunziker, J.; Fraser,
W.; Peng, L.; Diederichsen, U.; Zimmermann, K.;
Holzner, A. Leumann, C.; Eschenmoser, A. Helv. Chim.
Acta 1998, 81, 375–474; (b) Fidanza, J. A.; McGall, G.
H. Nucleosides Nucleotides 1999, 18, 1293–1295; (c)
Nguyen, H.-K.; Fournier, O.; Asseline, U.; Dupret, D.;
Thuong, N. T. Nucleic Acids Res. 1999, 27, 1492–1498;
15. The epimers (5a/b) (560 mg, 0.956 mmol) in THF (5 mL)
were treated with TBAF solution (1 M in THF, 1.15 mL)
for 1 h. Sat. NH4Cl solution (15 mL) was added, fol-
lowed by extraction with CH2Cl2 (3×10 mL), washing
with brine, drying of the combined organic phases over
Na2SO4, evaporation, and flash chromatography (silica,
petroleum ether/ethyl acetate, 10:1) to yield 348 mg

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N. Griesang, C. Richert / Tetrahedron Letters 43 (2002) 8755–8758
18. Oligonucleotides were synthesized on an ABI 381 DNA
(0,679 mmol, 71 %) of 6a/b. Rf (silica, hexanes/ethyl
acetate 4:1) 0.42 (6a) and 0.5 (6b). Epimerization of 6a
(1.1 g, 2.14 mmol) in toluene (20 mL) with BF3·OEt2 (1.2
g, 1.08 mL, 8.56 mmol, added at rt) was induced by
refluxing for 6 h. The solution was cooled, treated with
CH2Cl2 (10 mL) and sat. NaHCO3 solution (40 mL). The
organic phase was separated, the aqueous phase was
extracted with CH2Cl2 (3×20 mL), the combined organic
phases concentrated and chromatographed as given
above. Combined yield of 6a and 6b, obtained in equal
amounts, 760 mg (1.48 mmol, 69%). Spectroscopic data
for 6b: 1H NMR (CDCl3, 250 MHz) l 8.01, 7.97 (2 d,
J=8.6 Hz, 2× 2H), 7.54 (dd, J=8.6, 5.8 Hz, 1H), 7.47,
7.41 (2 d, J=8.6 Hz, 2× 2H), 7.18 (dd, J=8.8, 2.4 Hz,
1H), 7.03 (td, J=8.5, 2.8 Hz, 1H), 5.62 (dd, J=10.5, 5.3
Hz, 1H), 5.58 (d, J=6.7 Hz, 1H), 4.70 (dd, J=11.7, 4.2
Hz, 1H), 4.66 (dd, J=11.7, 4.3 Hz, 1H), 4.52 (dd, J=6.9,
4.2 Hz, 1H), 3.36 (s, 1H), 2.77 (dd, J=14.3, 4.9 Hz, 1H),
2.10–1.96 (m, 1H); 19F NMR (CDCl3) l 114.6; EI-MS
(70 eV) 512, 139, 111.
synthesizer on a 1.0 mmolar scale, system software version
1.5. Total yields for modified oligonucleotides were
within a factor of three of unmodified control strands
prepared under the same conditions.
19. For 8, HPLC: CH3CN gradient (C18 column, 0.1 M
TEAA buffer) 0% B for 5 min, to 20% B in 45 min,
elution at 47 min; MALDI-TOF MS for C120H152FN25-
O77P11 ([M–H]−) calc: 3537.4, found 3534.0. For 9,
HPLC: same gradient as for 8, elution at 46 min;
MALDI-TOF MS for
C103H125FN30O58P9 ([M–H]−)
calc: 3010.1, found 3007.5.
20. Different values have been published. In Ref. 1b, the
melting point is given as 21.4°C. In the erratum (J. Am.
Chem. Soc. 1996, 118, 931) the authors state that a-and
b-anomers give little change in results. In 1997 (Ref. 2a),
the melting point is given as 27.4°C, whereas in 2000
(Ref. 3b), the authors again cite the earlier results (Ref.
1b), i.e. a melting point of 21.4°C and a DTm of 18.0°C.
The melting point of control duplex CTTTTCTT-
TCTT:AAGAAAGAAAAG at 5 mM strand concentra-
tion and identical buffer conditions is given as 39.8°C in
Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org.
Chem. 1998, 63, 9652–9656 and as 43.2°C in Ref. 1g. In
any case, the melting point for 8 with its complement is
higher than either of the melting points reported for the
duplex containing difluorotoluene.
16. Anomers were assigned based on the results of ROESY
experiments, where a H1% to H4% cross peak was observed
for the b-anomer, but was absent for the a-anomer, and
by comparison of coupling constants and chemical shifts
of non-exchangeable deoxyribose resonances with those
of reference C-nucleosides known from the literature: (a)
Chaudhuri, N. C.; Ren, R. X.-F.; Kool, E. T. Synlett
1997, 341–347; (b) Ref. 13c.
21. See e.g.: (a) Dzantiev, L.; Alekseyev, Y. O.; Morales, J.
C.; Kool, E. T.; Romano, L. J. Biochemistry 2001, 40,
3215–3221; (b) Kool, E. T. Curr. Opin. Chem. Biol. 2000,
4, 602–608 and Refs 1j and 1k.
17. Kierzek, R.; Kopp, D. W.; Edmonds, M.; Caruthers, M.
H. Nucleic Acids Res. 1986, 14, 4751–4764.