Synthesis and DNA incorporation of an ethynyl-bridged cytosine C-nucleoside as guanosine surrogate

Article abstract of DOI:10.1021/ol7025334

(Chemical Equation Presented) As a guanosine mimic that lacks the preference for syn or anti conformation a cytosine C-nucleoside was synthesized connecting the nucleobase at the anomeric center by an ethynyl linker. The key step was a Sonogashira cross coupling of 5-iodocytosine with 1′-ethynyl-2′-deoxyribose. The new C-nucleoside incorporated into G/C-alternating oligonucleotides emerged as guanosine substitute, however, with reduced duplex stability. B-Form DNA was strongly stabilized by the new surrogate even in typically Z-DNA forming sequences and in Z-form inducing environment.

Full text of DOI:10.1021/ol7025334

ORGANIC
LETTERS
2007
Vol. 9, No. 25
5311-5314
Synthesis and DNA Incorporation of an
Ethynyl-Bridged Cytosine C-Nucleoside
as Guanosine Surrogate
Daniel Heinrich, Thomas Wagner, and Ulf Diederichsen*
Institut fu¨r Organische und Biomolekulare Chemie, UniVersita¨t Go¨ttingen,
Tammannstrasse 2, D-37077 Go¨ttingen, Germany
udieder@gwdg.de
Received October 17, 2007
ABSTRACT
As a guanosine mimic that lacks the preference for syn or anti conformation a cytosine C-nucleoside was synthesized connecting the nucleobase
at the anomeric center by an ethynyl linker. The key step was a Sonogashira cross coupling of 5-iodocytosine with 1 -ethynyl-2 -deoxyribose.
′
′
The new C-nucleoside incorporated into G/C-alternating oligonucleotides emerged as guanosine substitute, however, with reduced duplex
stability. B-Form DNA was strongly stabilized by the new surrogate even in typically Z-DNA forming sequences and in Z-form inducing
environment.
Properties and recognition potential of large biomolecules
like oligonucleotides are not only determined by the primary
structure but also strongly depend on parameters like solvent,
temperature, and salt concentration.1 Conversion between
DNA helix topologies like the right-handed A- and B-DNA
or the left-handed Z-DNA double helix can be induced by
changing the environment. The various DNA-forms encode
information since they are specifically recognized by small
molecules2 or proteins3 on the basis of hydrogen bonding or
shape recognition in the duplex grooves. Therefore, it is of
interest to specifically enforce one kind of DNA topology
or to stabilize dsDNA by ribosyl or nucleobase modifications
to preferentially obtain a defined DNA-form. Initially, it was
our intention to stabilize Z-DNA providing a guanosine
surrogate that resembles the required but energetically less
favored syn conformation. Nucleoside modifications known
to favor Z-DNA either address the required 2′-endo ribosyl
conformation4 or introduce substituents at C8 of purines5 or
at C5 of pyrimidines6 to sterically enhance the likelihood
(1) (a) Saenger, W. Principles of Nucleic Acid Structure; Springer: New
York, 1984. (b) Rich, A.; Nordheim, A.; Wang, A. H.-J. Ann. ReV. Biochem.
1984, 53, 791.
(2) (a) Spingler, B.; Antoni, P. M. Chem. Eur. J. 2007, 13, 6617. (b)
Qu, X.; Trent, J. O.; Fokt, I.; Priebe, W.; Chaires, J. B. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 12032. (c) Kim, Y.-G.; Park, H.-J.; Kim, K. K.;
Lowenhaupt, K.; Rich, A. Nucleic Acids Res. 2006, 34, 4937.
(3) (a) Travers, A. A. DNA-Protein Interactions; Chapman & Hall:
London, 1993. (b) Lilley, D. M. J. DNA-Protein: Structural Interactions;
IRL Press: Oxford, U.K., 1995.
(4) (a) Buff, R.; Hunziker, J. Synlett 1999, 905. (b) Buff, R.; Hunziker,
J. HelV. Chim. Acta 2002, 85, 224. (c) Lafer, E. M.; Mo¨ller, A.; Nordheim,
A.; Stollar, B. D.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3546.
(d) Xu, Y.; Ikeda, R.; Sugiyama, H. J. Am. Chem. Soc. 2003, 125, 13519.
(5) (a) Sugiyama, H.; Kawai, K.; Matsunaga, A.; Fujimoto, K.; Saito,
I.; Robinson, H.; Wang, A. H.-J. Nucleic Acids Res. 1996, 24, 1272. (b)
Uesugi, S.; Shida, T.; Ikehara, M. Biochemistry 1982, 21, 3400.
(6) Behe, M.; Felsenfeld, G. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 1619.
(7) (a) Kool, E. T. Acc. Chem. Res. 2002, 35, 936. (b) Kool, E. T. Annu.
ReV. Biophys. Biomol. Struct. 2001, 30, 1.
(8) (a) Diederichsen, U. Angew. Chem., Int. Ed. 1998, 37, 1655. (b)
Diederichsen, U.; Biro, C. Bioorg. Med. Chem. Lett. 2000, 10, 1417.
(9) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975,
16, 4467.
(10) Franc¸ois, P.; Perilleux, D.; Kempener, Y.; Sonveaux, E. Tetrahedron
Lett. 1990, 31, 6347.
(11) Srivastava, P. C.; Robbins, R. K.; Takusagawa, F.; Berman, H. M.
J. Heterocyclic Chem. 1981, 18, 1659.
(12) Gait, M. J. Oligonucleotide Synthesis, A Practical Approach, IRL
Press, Oxford 1984.
10.1021/ol7025334 CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/14/2007

for a syn nucleotide with respect to the thermodynamically
favored anti conformation (Figure 1).
anomeric center in â-configuration by an ethynyl spacer
fulfills the requirements of a guanosine surrogate with proper
hydrogen-bonding pattern. For incorporation in oligonucle-
otides by solid-phase synthesis, the preparation of phos-
phoramidite 2 was required that is amidine protected at the
nucleobase and equipped with the temporary dimethoxytrityl
protection at 5′-OH.
The key step of the synthesis of phosphoramidite 2 was
the Sonogashira cross coupling9 to connect the 5-iodo
cytosine with a 1′-ethynyl-2′-deoxyribosyl derivative (Scheme
1). The ethynyl group was introduced in analogy to other
alkyl groups10 at the anomeric center by Grignard reaction
of ethynyl magnesium bromide with 1-chloro-3,5-di-O-(p-
chlorobenzoyl)-2-deoxy-D-ribofuranose (3). Since the chlo-
robenzoyl protecting groups were cleaved in the substitution
step, 12 equiv of Grignard reagent were required to obtain
the resulting C-nucleoside 4. Silyl protection of the ethynyl
deoxyribose leads to ethynyl deoxyribose 5 ready for
Sonogashira cross coupling. 5-Iodocytidine was persilylated
with hexamethyldisilazane (HMDS) before it was used as
reagent for the coupling with ethynyl deoxyribose 5 catalyzed
by tetrakis(triphenylphosphine)palladium(0) and copper io-
dide to yield C-nucleoside 6. Deprotection of the hydroxy
groups was accomplished with 80% acetic acid in water at
40 °C since the standard reagent TBAF lead to separation
problems. Therefore, protection of the exocyclic amino group
of cytosine had to be done after desilysation owing to
hydrolysis sensitivity of the dimethyl amino methylene group
under the acetic acid cleavage conditions used. Previous to
nucleobase protection the tritylation needed to be done since
the dimethyl amino methylene group was completely cleaved
under tritylation conditions. Therefore, the fully deprotected
nucleoside 7 was treated with dimethoxytrityl-chloride in
pyridine and a catalytic amount of DMAP to yield the DMT-
protected nucleoside 8. In a first attempt to prepare the ribosyl
phosphoramidite with ethynyl-bridged cytosine, benzoyl
protection of the nucleobase was applied. A mixture obtained
of up to three times protected C-nucleosides turned out to
be disadvantageous. Therefore, protection of the exocyclic
amino group was done by treatment of nucleoside 8 with
dimethoxy amino methylene in DMF at room-temperature
providing nucleoside 9 in 76% yield as a pure â-anomer.
Remarkably, the high yield was obtained, although separation
from the R-anomer by column chromatography was ac-
complished at this stage.
Figure 1. Guanosine in anti and syn conformation: The sterically
disfavored syn conformation also suffers from stronger phosphodi-
ester repulsion.
Our approach for a new syn guanosine surrogate is based
on a linkage of the recognition unit at the anomeric center
without any conformational preference for the respective
torsion angle given by an ethynyl spacer combined with a
hydrogen-bonding pattern comparable to guanosine (Figure
2). Rotation around the ethynyl linker axis in cytosinyl
Figure 2. â-Ethynyl cytosine C-nucleoside 1 as surrogate for
guanosine in anti and syn conformation.
nucleoside 1 should facilitate the conversion between the
right- and left-handed DNA-forms. In addition, the ethynyl
linked cytosine nucleoside 1 might be of interest within the
field of artificial nucleosides and nucleobases in dsDNA.7,8
It has the potential to function as a versatile nucleobase with
respect to order and orientation of hydrogen-bonding rec-
ognition.
The design of the artificial nucleoside 1 is based on the
resemblance of the hydrogen-bonding pattern with the
Watson-Crick site of guanine (Figure 2) in combination with
the purine distance and the orientation of donor/acceptor
functionalities. Free rotation around the ethynyl linker should
allow equally well imitation of syn and anti conformation.
Therefore, nucleoside 1 with a cytosine linked at C5 to the
The configuration of the C-nucleosides at the anomeric
center was determined by 1H NMR spectroscopy. A pseudot-
riplet is typically found for the H-C1′ of â-nucleosides,
whereas a doublet of doublets is representing the R-isomer.
Furthermore, the chemical shift and the multiplicity of the
H-C2′ protons are characteristic for the anomers.11 Finally,
treatment of nucleoside 9 with 2-cyanoethyl-N,N-diisopro-
pylidene (10) and DIPEA in anhydrous DCM at room
temperature lead to the corresponding phosphoramidite 2 in
44% yield recovering 30% of nucleoside 9.
Phosphoramidite 2 was used in solid-phase synthesis of
DNAoligomersd(CGCG*CG)(11)andd(CGCG*CGCGCG*CG)
(12) with G* indicating the cytosinyl nucleoside 1 using
5312
Org. Lett., Vol. 9, No. 25, 2007

Scheme 1
standard protocols.12 The constitutional integrity of olig-
strong stabilizing effect in Z-DNA functioning as a syn
guanosine surrogate or to prohibit the conformational
reorganization from B- to Z-form of the double helix.
omers 11 and 12 was indicated by high-resolution
mass spectrometry. The sequences for incorporation of the
newly synthesized nucleotide 10 were oriented on the
assumption that the ethynyl linked cytosine 1 might be
suitable as a syn guanosine mimic for Z-DNA induction.
The respective natural DNA oligomers with alternating
C/G-sequence d(CG)3 (13) and d(CG)6 (14) are easily
converted into Z-form DNA with increasing NaCl concentra-
tion.13
The influence of guanosine surrogate 1 on the double helix
stability was investigated by temperature-dependent UV
spectroscopy. At 150 mM NaCl concentration both modified
oligomers 11 (Tm < 5 °C, >4% hyperchromicity (H), 3.3
µM) and 12 (Tm ) 50 °C, 12% H, 3.3 µM) containing two
or four modifications in the double strand, respectively,
provided significantly lower duplex stabilities compared to
the C/G-counterparts 13 (Tm ) 42 °C, 3% H, 3.3 µM) and
14 (Tm > 80 °C, >8% H, 3.3 µM) (Figure 3). The distinct
loss of stability in double strands containing the modified
nucleotide 1 compared to the C/G-sequences indicates that
the surrogates are not perfectly placed as guanine mimetic.
Induction of Z-form DNA with 4 M NaCl leads to a loss of
stability for the C/G oligomers 13 (Tm ) 27 °C, 8% H, 3.3
µM) and 14 (Tm ) 71 °C, 17% H, 3.3 µM), whereas the
modified oligomer 12 (Tm ) 56 °C, 14% H, 3.3 µM) was
even stabilized by high salt concentration (Supporting
Informatioin). The C-nucleoside 1 seems to have either a
Figure 3. UV melting curves of the modified oligomers
d(CGCG*CG) 11 (purple) and d(CGCG*CGCGCG*CG) 12 (blue)
in comparison to the hexamer d(CG)3 13 (black) and the dodecamer
d(CG)6 14 (green), each 3.3 µM, in 0.1 M NaH2PO4 buffer, pH 7,
150 mM NaCl.
CD spectroscopy at various temperatures and NaCl
concentrations were used to address the DNA topo-
logical preference and the conformational change be-
tween the helical forms. The modified oligomer 12 was
used in comparison to C/G-dodecamer 14 to exemplary
show the B- to Z-form transition induced by increasing
(13) Pohl, F. M.; Jovin, T. M. J. Mol. Biol. 1972, 67, 375.
Org. Lett., Vol. 9, No. 25, 2007
5313

the NaCl concentration (Figures 4 and 5). As known from
literature the C/G-alternating dodecamer 14 turns from
Figure 5. CD spectra of dodecamer d(CG)6 14 (20 µM, in 0.1 M
NaH2PO4 buffer, pH 7 at 20 °C) at 0 M NaCl (black), 1 M NaCl
(green), and 4 M (blue) NaCl concentration.
Figure 4. CD spectra of dodecamer d(CGCG*CGCGCG*CG) 12
(20 µM, in 0.1 M NaH2PO4 buffer, pH 7) at 20 °C at 0 M NaCl
(black), 1 M NaCl (green), and 4 M NaCl (blue).
recognition unit at the anomeric center. This provides free
rotation of the artificial nucleobase without preference to
orientation in syn- or anti-like conformation. The hydrogen
donor/acceptor pattern of the recognition unit resembles the
guanine Watson-Crick site. The new surrogate turned out
to stabilize the DNA duplex in its B-form when incorporated
in DNA that is destined to form Z-DNA under high salt
conditions. Studies toward the potential of the new C-
nucleoside to differentiate between A-DNA and B-form DNA
are currently under investigation.
B-DNA at 0 and 1 M NaCl concentration to Z-DNA at 4 M
NaCl.1 In contrast, it was not possible to induce Z-DNA in
the double strand of oligomer 12 that contained four
C-nucleotide 1 modifications. The B-form DNA was pre-
dominant at all salt concentrations. It is quite remarkable
that the guanosine surrogate 1 obviously fits well within the
B-DNA duplex and additionally seems to prohibit the
topological change to the otherwise favored Z-DNA. The
influence of the temperature on the DNA helix topology was
also investigated providing only a marginal loss in helix
propensity with increasing temperature. No deviation from
the B-form DNA was indicated (Supporting Information).
The results obtained with oligomer 12 were essentially
confirmed by respective CD spectra of the self-pairing duplex
of hexamer 11 that only contains two nucleoside surrogates
1. Nevertheless, the overall lower stability of the duplex was
recognized in lower helix propensity and the requirement to
investigate the conformational analysis at lower temperatures
(Supporting Information).
Acknowledgment. Financial support of the Deutsche
Forschungsgemeinschaft (Di 542/5) is gratefully acknowl-
edged.
Supporting Information Available: Complete experi-
mental details for the preparation and characterization of
compounds 2 - 9; analytical data of oligomers 11 and 12;
temperature-dependent UV and CD spectra at various NaCl
concentrations and temperatures of oligomers 11-14. This
material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, the synthesis of a guanosine surrogate was
presented that has the unique feature of a â-ethynyl linked
OL7025334
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Org. Lett., Vol. 9, No. 25, 2007