A nonafluoro nucleoside as a sensitive 19F NMR probe of nucleic acid conformation

Article abstract of DOI:10.1021/ol800872a

(Chemical Equation Presented) A nucleoside carrying a perfluorinated tert-butyl group (4) was prepared by a Sonogashira coupling of 5-iodo-2′-deoxyuridine with 4,4,4-trifluoro-3,3-bis(trifluoromethyl)-1- butyne in nearly quantitative yield and subsequently incorporated into DNA oligomers. Thermal denaturation studies showed that 4 had a negligible effect on duplex stabilty when compared to thymidine. Transition from single strand to duplex was monitored by ¹⁹F NMR spectroscopy at micromolar concentrations of oligomers, demonstrating the sensitivity of 4 as an NMR reporter nucleoside.

Full text of DOI:10.1021/ol800872a

ORGANIC
LETTERS
2008
Vol. 10, No. 13
2745-2747
A Nonafluoro Nucleoside as a Sensitive
19F NMR Probe of Nucleic Acid
Conformation
Nivrutti B. Barhate,† Rekha N. Barhate,† Pavol Cekan,‡ Gary Drobny,† and
Snorri Th. Sigurdsson*,‡
Department of Chemistry, UniVersity of Washington, Seattle, Washington 98195, and
UniVersity of Iceland, Science Institute, Dunhaga 3, 107 ReykjaVik, Iceland
snorrisi@hi.is
Received April 16, 2008
ABSTRACT
A nucleoside carrying a perfluorinated tert-butyl group (4) was prepared by a Sonogashira coupling of 5-iodo-2′-deoxyuridine with 4,4,4-
trifluoro-3,3-bis(trifluoromethyl)-1-butyne in nearly quantitative yield and subsequently incorporated into DNA oligomers. Thermal denaturation
studies showed that 4 had a negligible effect on duplex stabilty when compared to thymidine. Transition from single strand to duplex was
monitored by 19F NMR spectroscopy at micromolar concentrations of oligomers, demonstrating the sensitivity of 4 as an NMR reporter nucleoside.
Fluorine has many advantages as a probe for NMR spec-
troscopy of nucleic acids and other biopolymers.1–3 The
fluorine atom has a single naturally occurring isotope (19F),
which has a spin of one-half. High gyromagnetic ratio
contributes to high sensitivity of 19F (approximately 83% of
enables 19F NMR studies without background signal interfer-
ence. Furthermore, the chemical shift of 19F has been shown
to be very sensitive to its environment. For example,
conformational equilibria of bistable RNAs,6 Mg2+-induced
folding of the hammerhead ribozyme,7 and metal ion binding
to the TAR RNA8 have been studied by 19F NMR by
incorporation of fluorinated nucleosides (1 and/or 2, Scheme
1) and monitoring changes in their 19F chemical shift.
Heteronuclear 1H-19F and homonuclear 19F-19F NOE
experiments have also been performed on RNA9 and long-
range 1H-19F scalar couplings correlated with torsion angles
of glycosidic bonds.10
1
the sensitivity of H) and facilitates long-range distance
measurements through dipolar-dipolar coupling. For ex-
ample, 19F-31P rotational-echo double-resonance (REDOR)
SSNMR has been used to monitor conformational changes
in DNA and RNA upon ligand binding.4,5 Moreover, the
near-nonexistence of fluorine atoms in biological systems
† University of Washington.
‡ University of Iceland.
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Acids Res. 2003, 31, 5084
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Drobny, G. P. Nucleic Acids Res. 2005, 33, 3447
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U.S.A. 2001, 98, 5503.
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10.1021/ol800872a CCC: $40.75
Published on Web 06/06/2008
2008 American Chemical Society

The DNA sequence 5′-d(G4GCGCAC) was prepared by
automated solid-phase synthesis using phosphoramidite 7.
After deprotection and purification of the DNA by denaturing
polyacrylamide gel electrophoresis, mass spectrometric- and
HPLC-analysis of the enzymatic digest verified incorporation
of 4 into the oligomer (Supporting Information). We have
previously shown that the CF3-group in 3 is converted to
5-cyano-2′-deoxyuridine in aqueous ammonia at 55 °C,
commonly used for deprotection of DNA prepared by
chemical synthesis.13 On the other hand, 4 is stable under
those conditions for extended periods of time (>15 h, data
not shown). Furthermore, we have not noticed any decom-
position of 4, or oligonucleotides containing 4, that have been
stored at 25 °C in buffered aqueous solutions at neutral pH
7 for up to a week. The effects of nucleoside 4 on duplex
stability were determined by thermal denaturation experi-
ments on DNA duplexes containing 4 (Table 1). Incorpora-
Scheme 1. Fluorinated Nucleosides 1-4
Incorporation of multiple magnetically equivalent fluorine
atoms into nucleic acids enables 19F NMR experiments to
be performed at lower concentrations, thereby reducing the
material requirements and minimizing potential aggregation
problems. More equivalent fluorine atoms would also enable
longer distance measurements by 19F-31P REDOR SS-
NMR.11,12 We have previously incorporated nucleoside 3
into DNA but found that the CF3 group was not stable.13 In
this paper, we describe the synthesis of nonafluoro nucleoside
4, which contains nine magnetically equivalent fluorine
atoms, incorporation into DNA, and its use for studying
nucleic acid conformation at micromolar concentrations by
19F NMR spectroscopy.
Table 1. Melting Temperatures of DNA Duplexes I-VIa
b
duplex
sequence
TM(°C)
∆TM (°C)
5′-d(GTGCGCAC)
3′-d(CACGCGTG)
I
49.5 (0.5
Nucleoside 4 was conveniently synthesized in one step
by palladium(0)-catalyzed Sonogashira coupling of 5-iodo-
2′-deoxyuridine with 4,4,4-trifluoro-3,3-bis(trifluoromethyl)-
butyne (Scheme 2). No N3-alkylation was observed, unlike
5′-d(G4GCGCAC)
3′-d(CACGCG4G)
II
50.0 (0.5
60.6 (0.3
61.2 (0.5
61.5 (0.4
61.8 (0.4
+ 0.5
5′-d(GACCTCGCATCGTG)
3′-d(CTGGAGCGTAGCAC)
III
IV
V
5′-d(GACC4CGCATCGTG)
3′-d(CTGGAGCGTAGCAC)
+ 0.6
+ 0.9
+ 1.2
Scheme 2. Synthesis of Nonafluorophosphoramidite 7
5′-d(GACCTCGCATCGTG)
3′-d(CTGGAGCG4AGCAC)
5′-d(GACC4CGCATCGTG)
3′-d(CTGGAGCG4AGCAC)
VI
a 2 µM duplex in 10 mM sodium phosphate, 100 mM NaCl, 0.1 mM
Na2EDTA, pH 7.0. b ∆TM is the difference in the TM value between the
duplex having a modified base and that having a natural base.
tion of 4 had a very minor effect on duplex stability when
compared to oligomers containing T in the same positon(s).
For one or two modifications per duplex, the differences in
TM were e1.2 °C. All the modified duplexes had a slightly
higher TM, as observed for 5-alkyno-modified pyrimidines
in DNA.15
To evaluate the usefulness of nucleoside 4 for the study
of nucleic acid conformation,
a
solution of 5′-
the reaction of 3,3,3-trifluoropropyne with 5-iodo-2′-deox-
yuridine, which yielded exclusively the N-alkylated product
under similar conditions.14 Tritylation and phosphitylation
furnished phosphoramidite 7 in 72% overall yield (three
steps).
d(CACGA4GCGAGGTC) was treated with aliquots of the
complementary oligonucleotide 5′-d(GACCTCGCATCGTG)
and the 19F NMR spectra recorded. The single strand (Figure
1, top spectrum) has a chemical shift of -67.36 ppm, relative
to CCl3F. Addition of 0.4 equiv of the complementary strand
yielded a new peak at -66.91 ppm, which integrates for ca.
40% of the fluorine. Upon further addition, the signal
observed for the single strand decreased, along with the
(11) Merritt, M. E.; Sigurdsson, S. T.; Drobny, G. P. J. Am. Chem. Soc.
1999, 121, 6070
(12) Louie, E. A.; Chirakul, P.; Raghunathan, V.; Sigurdsson, S. T.;
Drobny, G. P. J. Magn. Reson. 2006, 178, 11
.
.
(13) Markley, J. C.; Chirakul, P.; Sologub, D.; Sigurdsson, S. T. Bioorg.
Med. Chem. Lett. 2001, 11, 2453.
(15) Ahmadian, M.; Zhang, P. M.; Bergstrom, D. E. Nucleic Acids Res.
1998, 26, 3127.
(14) Chirakul, P.; Sigurdsson, S. T. Tetrahedron Lett. 2003, 44, 6899.
2746
Org. Lett., Vol. 10, No. 13, 2008

Figure 2. Molecular model of a duplex DNA containing nucleoside
4 (stereoview), showing the position of the perfluorinated tert-butyl
group (dark gray) in the major grove of the DNA duplex.
In summary, we have described a short and efficient
synthesis of a stable nonafluorinated nucleoside that does
not perturb the stability of DNA duplexes. To our knowledge,
this is the first example of conjugation of a perfluorinated
tert-butyl group to biopolymers. We have demonstrated that
nucleoside 4 is a sensitive NMR probe of nucleic acid
conformation, both in terms of changes in the 19F chemical
shift and signal intensity arising from nine magnetically
equivalent fluorine atoms. Modeling studies indicate that the
perfluorinated tert-butyl group is accommodated in the major
groove of RNA (data not shown) and if verified experimen-
tally, this probe should be applicable for RNA studies, for
example to investigate conformational equilibria of bistable
RNAs.6 This reporter group might also be useful for studying
conformational changes in nucleic acids due to tertiary
folding or ligand binding.16 Fluorine has been shown to alter
physiochemical and pharmacokinetic properties of organic
compounds.17 Incorporation of nucleoside 4 into nucleic acid
oligomers, perhaps at multiple sites, could enhance the
efficacy of cellular delivery and activity of therapeutic nucleic
acids.
Figure 1.
DNA duplex formation monitored by 19F NMR spec-
troscopy. 19F NMR spectra of single stranded 5′-
d(CACGA4GCGAGGTC) (A) and after addition of 0.4 equiv (B),
0.6 equiv (C), 0.8 equiv (D), and 1.0 equiv (E) of the complemen-
tary strand 5′-d(GACCTCGCATCGTG). Spectra were recorded in
deuterated 10 mM phosphate buffer, pH 7.0, 100 mM NaCl, 0.1
mM EDTA. The concentration of the fluorinated oligomer was 3.3
µM. Recorded on a Bruker NMR DRX 500 at 298 K.
concomitant increase in the signal at -66.91 ppm, and
disappeared after addition of 1 equiv of the complementary
strand. These data are consistent with formation of a duplex
(Table 1) that has a different 19F chemical shift than the single
strand.
The NMR titration experiment illustrates two aspects of
the sensitivity of nucleoside 4 as a probe of nucleic acid
conformation. First, the NMR experiments can be carried
out at micromolar concentrations of the fluorinated oligomer
using a 500 MHz spectrometer. Second, the chemical shift
of the perfluorinated tert-butyl group is sensitive to its
environment. The 19F chemical shift of the duplex differed
from that of the single strand by ca. 0.5 ppm. The reason
for this difference might be placement of the fluorines close
to a phosphodiester in the duplex (Figure 2). The fact that
the fluorine atoms yield a single signal in the duplex indicates
that there is free rotation around both single bonds flanking
the triple bond. Restriction of this rotation due to nucleic
acid conformational change or intermolecular complex
formation would likely give multiple fluorine signals, which
is another potentially useful feature of this probe.
Acknowledgment. We thank the NIH (RO1 EB03152)
for financial support.
Supporting Information Available: Synthetic protocols
and spectral data for compounds 4, 6, and 7; preparation
and characterization of fluorinated DNA. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL800872A
(16) Kreutz, C.; Kahlig, H.; Konrat, R.; Micura, R. Angew. Chem., Int.
Ed. 2006, 45, 3450.
(17) Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
Org. Lett., Vol. 10, No. 13, 2008
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