Triazole-linked analogue of deoxyribonucleic acid (TLDNA): Design, synthesis, and double-strand formation with natural DNA

Article abstract of DOI:10.1021/ol801230k

(Chemical Equation Presented) A new triazole-linked analogue of DNA ( ^TLDNA) has been designed and synthesized using click chemistry. The chain elongation reaction using copper-catalyzed Huisgen cycloaddition was successful and gave the artificial oligonucleotide that formed a stable double strand with the complementary strand of natural DNA.

Full text of DOI:10.1021/ol801230k

ORGANIC
LETTERS
2008
Vol. 10, No. 17
3729-3732
Triazole-Linked Analogue of
Deoxyribonucleic Acid (TLDNA): Design,
Synthesis, and Double-Strand Formation
with Natural DNA
Hiroyuki Isobe,*,† Tomoko Fujino,† Naomi Yamazaki,† Marine Guillot-Nieckowski,‡
and Eiichi Nakamura‡
Department of Chemistry, Tohoku UniVersity, Aoba-ku, Sendai 980-8578, Japan, and
Department of Chemistry, The UniVersity of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
isobe@mail.tains.tohoku.ac.jp
Received June 16, 2008
ABSTRACT
A new triazole-linked analogue of DNA (TLDNA) has been designed and synthesized using click chemistry. The chain elongation reaction using
copper-catalyzed Huisgen cycloaddition was successful and gave the artificial oligonucleotide that formed a stable double strand with the
complementary strand of natural DNA.
Analogues of DNA that have nucleobases displayed on
artificial backbones are attracting much interest. Analogues
that can be polymerized by bond-forming reactions other than
P-O bond formation are especially important,1 and peptide
nucleic acid (PNA) has emerged as one of the most
successful examples among such analogues that form stable
multiple strands with complementary DNA.2 The success
of PNA is backed up by the strong synthetic background of
carbonyl/peptide chemistry, which highlights the stringent
requirement needed for the bond-forming reaction of the
elongation process. Thus, an ideal reaction for elongation
should give high yield, generate inoffensive byproducts, and
proceed under simple reaction conditions. We conjectured
that a copper-catalyzed version of the Huisgen [3 + 2]
cycloaddition would satisfy such conditions3-6 and thus
designed a triazole-linked analogue of DNA (TLDNA), as
shown in Figure 1. Herein, we report on the synthesis of
(3) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
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† Tohoku University.
Nakamura, E. Org. Lett. 2007, 9, 4611–4614
(7) Eschenmoser, A. Chimia 2005, 59, 836–850
(8) Horwitz, J. P.; Chua, J.; Urbanski, J. A.; Noel, M. J. Org. Chem.
1963, 28, 942–944
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4261–4266
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‡ The University of Tokyo.
.
(1) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543–584.
(2) Peptide Nucleic Acids: Protocols and Applications, 2nd ed.;
Nielsen, P. E., Eds.; Horizon Bioscience: Norfolk, 2004; and references
cited therein.
.
.
10.1021/ol801230k CCC: $40.75
Published on Web 07/26/2008
2008 American Chemical Society

mediated nucleophilic ring opening reaction with lithium
acetylide (Scheme 1, see the Supporting Information for the
experimental details).9 After the mesylation of 5, the desired
azide 7 bearing a trimethylsilyl-protected acetylene moiety
was obtained by nucleophilic substitution of the mesylate 6
using sodium azide.10 Another monomer 9 for the terminus
was prepared from the common intermediate 5 through
desilylation and Mitsunobu substitution reactions. All the
reactions proceeded to give moderate to good yield and were
scalable to multigram-scale synthesis.
With monomers 7 and 9 in hand, we began examining
the coupling reaction in solution. After screening several
parameters, such as the solvent and copper reagents, we
found that the dimethyl sulfide complex of copper bromide
was suited for the Huisgen [3 + 2] cycloaddition, and
obtained 2-mer TLDNA 10 in 82% yield after a reaction time
of 15 h in THF (Scheme 2). The efficacy of the reaction for
Figure 1. Structure of natural DNA (1) and a triazole-linked
analogue (TLDNA, 2) with a retrosynthetic analysis to the monomer
(3). The natural phosphate linkage, O-P-O, was replaced by a
triazole linkage, N-C-C, shown in red.
oligothymine TLDNA and present the initial results on double-
strand formation with a complementary DNA strand.
The molecular design of TLDNA commenced with the
replacement of the phosphodiester linker of natural DNA
by a triazole ring (Figure 1). We kept the methylene bridge
at pseudo-5′-position to maintain the six-bond periodicity
of the oligonucleotides7 as well as the flexibility of the
oligonucleotide chains. We expect that the presence of
the sugar-like ring may also be advantageous in retaining
any puckering flexibility similar to that of natural DNA.
Therefore, the monomer for the elongation reaction should
feature an acetylene group at the pseudo-5′-position and
an azide function at the pseudo-3′-position (i.e., compound
3 in Figure 1).
Scheme 2.
Solution-Phase Synthesis of the 3-mer TLDNA
Monomers for TLDNA were readily accessed from natu-
rally occurring thymidine. Thus, the oxetane compound 4,
derived from thymidine using a reported method,8 was
converted to the acetylene derivative 5 by a Lewis acid
Scheme 1.
Synthesis of Monomers of TLDNA
the elongation process was demonstrated by the fact that the
reaction did not require an excess of the substrate, in contrast
to the need for an excess of the elongating phosphoramidite
monomer in a standard DNA synthesis.11,12 A popular
catalyst, a combination of CuSO4 and ascorbate,5 was also
effective for the elongation process, but the reaction did not
proceed to completion even after a reaction period of a few
days. The ensuing desilylation of 10 with TBAF gave the
3730
Org. Lett., Vol. 10, No. 17, 2008

2-mer TLDNA that was used in the next coupling reaction.
Due to the low solubility of the 2-mer TLDNA in THF, the
second elongation reaction with the azide 7 was carried out
in a polar solvent and gave 3-mer TLDNA 11 in 76% yield
when the reaction was carried out in an aqueous mixture
(THF/t-BuOH/H2O).
With the aim of producing longer oligomers, we then
examined a solid-phase synthesis and succeeded in synthe-
sizing the 10-mer TLDNA. We reoptimized the reaction
conditions for elongation and deprotection on a solid support
for the synthesis of 2-mer TLDNA. The reaction was carried
out with the terminal monomer 12 loaded on a poly(ethylene
glycol)-polystyrene-based resin (NovaSyn TG amino resin,
0.27 mmol/g; Scheme 3), and the yield was estimated after
successful using TBAF because the TLDNA cleaved from
the resin under the nucleophilic conditions. Then we found
that a copper-mediated desilylation proceeded smoothly to
give the unprotected acetylene derivative 13 without a
cleavage reaction taking place.13 Although the elongation
reaction with 13 on a solid support proceeded in an aqueous
solvent (THF/t-BuOH/H2O), we found that microwave
irradiation dramatically accelerated the reaction in THF.6
Thus, the 3-mer TLDNA was obtained in 84% yield from
the reaction carried out in THF under microwave irradiation
for a period of 1.5 h. Having finalized the reaction conditions
on a solid support, we repeated the deprotection/elongation
reactions and obtained the 10-mer TLDNA 15 after HPLC
purification on a triazole-capped hydrophilic interaction
chromatography (HILIC) column (isolated yield ) 0.61%,
19 steps). Improvement of the reaction and purification
conditions would increase the yield of the final product (see
the Supporting Information for details). The polythymine
TLDNA 15 was soluble in water up to ca. 7 µM in water,
and the solubility increases in the presence of polar organic
solvents such as acetonitrile. Implementation of hydrophilic
residue such as polyethylene glycol through click chemistry
may increase the solubility in water and thus expand the
scope of TLDNA.
Scheme 3.
Solid-Phase Synthesis of 10-mer TLDNA
Finally, we found that TLDNA formed a stable double
strand with a natural DNA strand. The formation of the
double strand was first confirmed by monitoring the hypo-
chromic effect from the base stacking using UV-visible
spectroscopy.14,15 The hypochromic effect at 260 nm was
observed when TLDNA 15 was hybridized with the natural
target d(T)2(A)10(T)2 in SSPE buffer (100 mM sodium
chloride, 10 mM sodium phosphate, and 0.10 mM ethylene-
diamine tetraacetic acid (EDTA), pH 7). The stoichiometric
ratio of each strand in the hybridized complex was deter-
mined to be 1:1 using the Job plot of the hypochromic change
(Figure S1, Supporting Information).16 The melting temper-
ature (Tm) was much higher (61.1 °C) than that of a control
natural DNA d(T)10 (20.0 °C), which shows that the analogue
bound to the target strand more tightly than the natural DNA.
This stability may be due to the neutral backbone that does
not have any repulsive interactions with the anionic phos-
phate backbone of the natural target.2 Considering the fact
that a similar triazole linker with a longer methylene bridge
destabilizes the double strand,17,18 we think that the six-bond
periodicity of TLDNA is a crucial design for the double strand
formation. The structural complementarity of TLDNA for
natural DNA is visible in the molecular model (Figure 2b).
(12) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311.
(13) Ito, H.; Arimoto, K.; Sensui, H.; Hosomi, A. Tetrahedron Lett. 1997,
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4175.
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cleavage from the resin. Thus, the first coupling reaction
proceeded smoothly under conditions similar to those of the
solution-phase reaction, and the 2-mer TLDNA was obtained
quantitatively. However, the subsequent desilylation was not
.
(17) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429–
4443
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(18) Lucas, R.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y.
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Org. Lett., Vol. 10, No. 17, 2008
3731

oligonucleotides. The cycloaddition way, examined for the
first time for chain elongation, has proven useful for the
synthesis of oligomers. As click chemistry tolerates a wide
range of functionalities,5 this method provides a powerful
strategy for synthesizing artificial oligonucleotides, including
various types of conjugates, and will contribute to the
growing scope of oligonucleotide chemistry.20-26 The toler-
ance of the reaction for other nucleobases has already been
demonstrated previously,20 and the variation of the nucleo-
base in future will expand the scope of TLDNA. Structural
features of the new analogue, such as the rigid π-rich
backbone or metal-coordination of the triazole rings may also
flourish the structural/supramolecular chemistry of oligo-
nucleotides.
Acknowledgment. We thank Prof. T. Wada (The Uni-
versity of Tokyo) for helpful discussions, Prof. M. Hirama
(Tohoku University) for MALDI-MS measurements, Prof.
N. Teramae (Tohoku University) for CD measurements, and
Nacalai Tesque for a sample column. This work was partly
supported by MEXT (KAKENHI, Houga 20655026), JST
(PRESTO) and the Kurata Memorial Foundation. M.G.-N.
thanks JSPS for a postdoctoral fellowship.
Supporting Information Available: Experimental pro-
cedures and spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 2.
Double-strand formation of TLDNA with a natural
complementary strand. (a) Melting curves of the 10-mer double
strands. Filled circle: TLDNA 15 with natural DNA (d(T)2(A)10(T)2).
Open square: natural DNA d(T)10 with d(T)2(A)10(T)2. (b) Molecular
models of the double strand between 15 and d(A)10 showing the
structural complementarity of two strands (left: side view, right:
top view). The triazole linkers are shown in CPK models (Gray:
C, red: O, white: H, blue: N), and d(A)10 strand are shown in light
blue. Top view shows the periodicity of the artificial triazole linkers
matches well with that of natural phosphate linkers. Details of the
molecular simulation are described in the Supporting Information.
OL801230K
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The helical structure of the double strand was experimentally
confirmed by the CD spectra (Figure S2, Supporting Infor-
mation).15,19 Analysis of the temperature-dependent change
of the CD spectra also confirmed the high Tm value (Figure
S3, Supporting Information). Investigations on the structural
details of the new double helix, for instance, by thermody-
namic analysis,15 are now underway and will be reported in
the near future.
.
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.
In summary, we have reported on the synthesis of a new
analogue of DNA using a highly efficient and selective route
that should be amenable to large-scale preparation of longer
.
.
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