Synthesis and base-pairing properties of C-nucleotides having 1-substituted 1H-1,2,3-triazoles

Article abstract of DOI:10.1016/j.bmcl.2009.04.063

Oligonucleotides including C-nucleotides having 1-substitued 1H-1,2,3-triazoles as artificial nucleobases were conveniently synthesized by the post-elongation modification method using the copper(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC)

Full text of DOI:10.1016/j.bmcl.2009.04.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3316–3319
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and base-pairing properties of C-nucleotides having 1-substituted
1H-1,2,3-triazoles
*
*
Motoi Nakahara, Takeshi Kuboyama, Akihiro Izawa, Yoshiyuki Hari , Takeshi Imanishi, Satoshi Obika
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oligonucleotides including C-nucleotides having 1-substitued 1H-1,2,3-triazoles as artificial nucleobases
were conveniently synthesized by the post-elongation modification method using the copper(I)-cata-
lyzed alkyne–azide 1,3-dipolar cycloaddition (CuAAC) reaction. The base-pairing properties of the tria-
zole nucleobase analogs in forming duplexes with oligonucleotides were investigated by the Tm
experiments.
Received 19 March 2009
Revised 14 April 2009
Accepted 16 April 2009
Available online 20 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Click chemistry
Nucleobase analogues
Oligonucleotides
Post-elongation modification method
Triazoles
Chemically modified oligonucleotides are currently attracting
much attention because of their applications as potent tools for
molecular biology, as diagnostic probes and/or as potential materi-
als for oligonucleotide-based therapy.1 In particular, modification
of a nucleobase moiety is widely used to increase base-discrimina-
tion ability and to enhance the stability of duplex or triplex nucleic
acids.1–5 These properties of base-modified oligonucleotides are
very important and useful for their application to many oligonu-
cleotide-based technologies.
The copper(I)-catalyzed alkyne–azide 1,3-dipolar cycloaddition
(CuAAC),6–9 giving a 1,2,3-triazole derivative, has been extensively
studied in both organic chemistry and chemical biology. For exam-
ple, syntheses of biomolecule-functional molecule conjugates,10–13
novel bioactive compounds,14,15 and circular oligonucleotides16–18
have been achieved by CuAAC. Thus, to develop a new class of
base-modified oligonucleotides, we considered that employing a
post-elongation modification method with CuAAC would produce
1-substituted 1H-1,2,3-triazole nucleobase analogs (Scheme 1).
Here, we demonstrate the synthesis of an oligonucleotide con-
Synthesis of the phosphoramidite derivative 1b was achieved as
shown in Scheme 2. As previously reported,14 2 was prepared as an
anomeric mixture (a
:b = ca. 3:1),19 which was then treated with
sodium methoxide in MeOH to give 3.20 After protection of the pri-
mary hydroxyl group of 3 with dimethoxytrityl (DMTr),21 each
anomer was readily separated by silica gel chromatography. Phos-
phitylation reactions on 4
a and 4b provided the desired phospho-
ramidites
1a
and 1b in 76% and 69% yields, respectively.22
Phosphoramidite 1b was then incorporated into a 15-mer homo-
pyrimidine oligonucleotide on an automated DNA synthesizer
using a standard phosphoramidite protocol.23 Using a trityl moni-
tor, the coupling efficiency of 1b was estimated to be >99%. The ob-
tained oligonucleotide ON-1 was purified by RP-HPLC and its
composition was confirmed by MALDI-TOF-MS.
Next, ON-1 was reacted with benzylazide to determine the opti-
mal conditions for CuAAC (Scheme 3). The conversion efficiency
was evaluated by RP-HPLC analysis (Fig. 1). Under condition A
[copper(II) sulfate (2 equiv), sodium ascorbate (2 equiv), benzylaz-
ide (2.2 equiv) in 10% THF (aq) buffer], the reaction proceeded
moderately at room temperature, and ca. 50% conversion to ON-2
was observed after 15 h (Fig. 1a). The reaction almost went to com-
pletion during the same period of time under condition B [cop-
per(II) sulfate (4 equiv), sodium ascorbate (4 equiv), benzylazide
(5 equiv) in 10% THF (aq) buffer] (Fig. 1b). As previously reported,9
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) effec-
tively promoted CuAAC in our experiments. After several attempts,
we found that the reaction went to completion within 90 min un-
der condition C [copper(II) sulfate (2 equiv), sodium ascorbate
(4 equiv), TBTA (4 equiv), benzylazide (10 equiv) in 30% DMSO
(aq) buffer] (Fig. 1c and Table 1, entry 1).
taining 1-ethynyl-2-deoxy-b-D-ribofuranose, and the efficient con-
version of the ethynyl group into several 1,2,3-triazoles, thereby
producing novel nucleobase analogs. Moreover, the duplex-form-
ing ability of the obtained oligonucleotides containing triazole C-
nucleotides with ssDNA is evaluated by Tm experiments.
* Corresponding authors. Tel./fax: +81 6 6879 8201 (Y.H.); tel.: +81 6 6879 8200;
fax: +81 6 6879 8204 (S.O.).
E-mail addresses: hari@phs.osaka-u.ac.jp (Y. Hari), obika@phs.osaka-u.ac.jp (S.
Obika).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.063

M. Nakahara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3316–3319
3317
N
N
N
N
R1
N
N
R1
R2
N3
N3
R2
Rn
HO
N3
O
incorporation
into
"CuAAC"
OH
oligonucleotides
N
N
Rn
N
Scheme 1. Schematic representation of the synthesis of oligonucleotides bearing 1-substituted 1H-1,2,3-triazole nucleobase analogs by post-elongation modification
methods using CuAAC.
m
m
m
m
X
ON-2
conditions
5'-TTTTT CT T CT CT CT-3'
TolO
HO
ON-1
(i)
O
O
N
benzylazide
CuSO4
sodium ascorbate
with or without TBTA
N
N
Ph
O
OTol
OH
O
2
3
X
=
O
P
DMTrO
DMTrO
O
O
O
O
(ii)
+
OH
OH
4β
Scheme 3. Conversion of ON-1 to ON-2 by CuAAC with benzylazide.
4α
DMTrO
O
(iii)
4
α
O
P
CN
iPr2N
O
1
α
DMTrO
(iii)
(iv)
O
4
β
O
P
CN
iPr2N
O
1
β
O
O
5'-TTTTTmCTXTmCTmCTmCT-3'
X
=
ON-1
O
P
O
O
Scheme 2. Synthesis of phosphoramidite 1 and incorporation into an oligonucleo-
tide. Reagents and conditions: (i) NaOMe, MeOH, rt, 3 h, 92%; (ii) dimethoxytrityl
chloride, pyridine, rt, 2 h, 56% for 4
a and 18% for 4b; (iii) (iPr2N)2PO(CH2)2CN,
Figure 1. HPLC analysis of CuAAC between ON-1 and benzylazide. The reaction was
carried out at room temperature under (a) condition A [copper(II) sulfate (2 equiv),
sodium ascorbate (2 equiv), benzylazide (2.2 equiv) in 10% THF (aq) buffer] for 15 h;
(b) condition B [copper(II) sulfate (4 equiv), sodium ascorbate (4 equiv), benzylaz-
ide (5 equiv) in 10% THF (aq) buffer] for 15 h and (c) condition C [copper(II) sulfate
(2 equiv), sodium ascorbate (4 equiv), TBTA (4 equiv), benzylazide (10 equiv) in 30%
DMSO (aq) buffer] for 90 min. The peaks at 8.9 and 12.0 min correspond to ON-1
(reactant) and ON-2 (product), respectively. The samples for the corresponding
peaks were collected and characterized by MALDI-TOF-MS. HPLC conditions:
reversed phase HPLC (Waters Xterra RP column) with acetonitrile/water containing
100 mM triethylamine-acetic acid (TEAA) buffer (pH 7.0) as mobile phase, linear
gradient 8–20% acetonitrile/water (30 min, 1.0 mL/min).
diisopropylammonium tetrazolide, MeCN/THF = 3:1, rt, 4 h, 76% for 1a and 69% for
1b; (iv) automated DNA synthesizer. In the ON-1 sequence, mC stands for 20-deoxy-
5-methylcytidine.
Following optimization of the reaction conditions for CuAAC be-
tween ON-1 and benzylazide, we evaluated the reaction of ON-1
with several other azide compounds (Table 1 and Scheme 4). In
addition to benzylazide (entry 1), primary azides (entries 2 and
3), a secondary azide (entry 4), a tertiary azide (entry 5) and aro-

3318
M. Nakahara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3316–3319
Table 1
MALDI-TOF-MS data and yields of the oligonucleotides obtained via Scheme 4a
Entry
R
Obtained oligonucleotides
MALDI-TOF-MS data
Conversion efficiencyb (%)
Calcd. [MꢀH]ꢀ
Found [MꢀH]ꢀ
1
2
3
4
5
6
4529.06
4528.47
4596.19
4562.37
4522.96
4573.22
4531.03
4557.79
98
100
97
91
38
79
81
(ON-2)
HO
10
4595.20
4561.12
4521.08
4573.15
4529.06
4559.04
(ON-3)
S
(ON-4)
(ON-5)
(ON-6)
Me
(ON-7)
7
HO2C
(ON-8)
a
The reaction was conducted for 90 min at room temperature under condition C [copper(II) sulfate (2 equiv), sodium ascorbate (4 equiv), TBTA (4 equiv), azide compound
(10 equiv) in 30% DMSO (aq) buffer].
b
The conversion efficiency was evaluated by RP-HPLC analysis from the peak areas of ON-1 and the obtained oligonucleotide.
N
R-N3 (10 eq.)
Cu SO4 (2 eq.)
sodium ascorbate
(4 eq.)
ON-2–8 stabilized the duplex better than ON-1, presumably due to
the stacking effect of the triazole nucleobases, while duplexes of
ON-2–8 with ssDNA targets (Y = A, G, T and C) were less stable than
the full-match one comprising ON-9 and ssDNA (Y = A). ON-2–8
were also found to form the most stable duplex with ssDNA
(Y = G) among all ssDNA targets, indicating that a nitrogen atom
in the triazole structure can make a hydrogen bond with the 2-
NH2 or 1-NH group of G. In comparison with benzyl-substituted
ON-2, ON-4 bearing a (phenylthio)methyl group was favorable
for duplex formation, and it would be because the hydrophobicity
of a nucleobase moiety increased by an additional sulfur atom.
Interestingly, ON-4 had almost the same stability against all ssDNA
targets (the Tm values ranged from 37 °C to 40 °C). This suggests
that 1-(phenylthio)methyl-1H-1,2,3-triazole is a new candidate
as a non-discriminatory nucleobase, namely a universal base,24
though more minute examination is naturally required. Results
of ON-5 and ON-7 demonstrated that an aromatic ring at the 1-po-
sition of a triazole moiety increased the duplex stability, probably
due to the stacking interaction with the neighboring base-pairs.
ON-8 with a carboxyl group destabilized the duplex compared to
ON-7 with a methyl group at the same position, indicating that
the hydrophilic group in that position was unsuitable for duplex
formation. In addition, it should be noted that ON-6 showed du-
DNA
O
R
N
N
O
ON-1
TBTA (4 eq.)
30%DMSO-aqueousbuffer
O
P
O
O
ON-2-8
(conditionC)
DNA
Scheme 4. CuAAC reaction of ON-1 with several azide reagents.
matic azides (entries 6 and 7) were treated with ON-1 using condi-
tion C at room temperature for 90 min. Although the reaction with
a tertiary azide (entry 5) afforded ON-6 in only moderate yield,
probably due to steric hindrance of the tertiary azide, most reac-
tions proceeded smoothly, and the desired oligonucleotides ON-
2–5, ON-7 and ON-8 bearing the corresponding 1-substituted
1H-1,2,3-triazoles as an artificial nucleobase were successfully ob-
tained (entries 1–4, 6 and 7).
Finally, the duplex-forming ability of the oligonucleotides ON-
2–8 containing triazole nucleobase analogs with ssDNA, 50-AGA-
GAGAYAGAAAAA-30 (Y = A, G, T or C), was examined by Tm exper-
iments and compared with those of ethynyl derivative ON-1 and
natural ON-9. The Tm values are summarized in Table 2. In general,

M. Nakahara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3316–3319
3319
Table 2
Tm values (°C) for duplex between ON-1–9 and ssDNA targetsa
References and notes
1. Methods in Molecular Biology; Herdewijn, P., Ed.Oligonucleotide Synthesis:
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2. For reviews, see: (a) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol. 2003, 7,
717; (b) Luyten, I.; Herdewijn, P. Eur. J. Med. Chem. 1998, 33, 515.
3. Triple Helix Forming Oligonucleotides; Malvy, C., Harel-Bellan, A., Pritchard, L. L.,
Eds.; Kluwer Academic: Boston, 1999.
Oligonucleotides
Target ssDNA (50-AGAGAGAYAGAAAAA-30)
Y = A
Y = G
Y = T
Y = C
ON-1
ON-2
ON-3
ON-4
ON-5
ON-6
ON-7
ON-8
ON-9b
32
32
35
37
34
30
39
33
52
32
38
39
40
37
37
42
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31
33
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29
34
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38
34
34
36
33
39
4. Guianvarc’h, D.; Fourrey, J. L.; Maurisse, R.; Sun, J. S.; Benhida, R. Bioorg. Med.
Chem. 2003, 11, 2751.
5. (a) Obika, S.; Hari, Y.; Morio, K.; Imanishi, T. Tetrahedron Lett. 2000, 41, 221; (b)
Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T. Angew. Chem., Int. Ed. 2001, 40,
2079. Angew. Chem. 2001, 113, 2149; (c) Obika, S.; Hari, Y.; Sekiguchi, M.;
Imanishi, T. Chem. Eur. J. 2002, 8, 4796; (d) Hari, Y.; Obika, S.; Sakaki, M.; Morio,
K.; Yamagata, Y.; Imanishi, T. Tetrahedron 2002, 58, 3051; (e) Hari, Y.; Obika, S.;
Sekiguchi, M.; Imanishi, T. Tetrahedron 2003, 59, 5123; (f) Hari, Y.; Obika, S.;
Inohara, H.; Ikejiri, M.; Une, D.; Imanishi, T. . Chem. Pharm. Bull. 2005, 53, 843;
(g) Obika, S.; Inohara, H.; Hari, Y.; Imanishi, T. Bioorg. Med. Chem. 2008, 16,
2945.
a
The measurement was carried out under 10 mM phosphate buffer (pH 7.0),
100 mM NaCl and 3 lM of each oligonucleotide.
b
The sequence is 50-TTTTTmCTTTmCTmCTmCT-30.
6. For recent reviews, see: (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2001, 40, 2004; (b) Kolb, H. C.; Sharpless, K. B. Drug Discovery
Today 2003, 8, 1128.
7. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
8. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
plex formation even though it has a bulky adamantyl group on the
nucleobase moiety.
9. Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853.
10. Wolfbeis, O. S. Angew. Chem., Int. Ed. 2007, 46, 2980.
11. (a) Geci, I.; Filichev, V. V.; Pedersen, E. B. Chem. Eur. J. 2007, 13, 6379; (b)
Kumar, R.; El-Sagheer, A.; Tumpane, J.; Lincoln, P.; Wilhelmsson, L. M.; Brown,
T. J. Am. Chem. Soc. 2007, 129, 6859.
12. Manetsch, R.; Krasinski, A.; Radic, Z.; Raushel, J.; Taylor, P.; Sharpless, K. B.;
Kolb, H. C. J. Am. Chem. Soc. 2004, 126, 12809.
13. Zhang, Y. H.; Gao, Z. X.; Zhong, C. L.; Zhou, H. B.; Chen, L.; Wu, W. M.; Peng, X. J.;
Yao, Z. J. Tetrahedron 2007, 63, 6813.
14. Wamhoff, H.; Warnecke, H. Arkivoc 2001, 95.
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2008, 18, 3321.
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17. Lietard, J.; Meyer, A.; Vasseur, J. J.; Morvan, F. J. Org. Chem. 2008, 73, 191.
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In conclusion, we achieved the synthesis of oligonucleotides
including C-nucleotides having 1-substituted 1H-1,2,3-triazoles
as artificial nucleobases by the post-elongation modification meth-
od using the CuAAC reaction between a 1-ethynyl-2-deoxy-b-D-
ribofuranose moiety in an oligonucleotide and several azide com-
pounds. In light of its simplicity and versatility, this method would
be quite useful for finding new 1,2,3-triazole-based nucleobase
having distinguished functions. Moreover, the Tm experiments of
the obtained oligonucleotide derivatives showed that 1-(phenyl-
thio)methyl-1H-1,2,3-triazole could act as a universal base. Thus,
this nucleobase analog may be used as an ambiguous site in prim-
ers for PCR and sequencing.24 Currently, further investigation on
this potential universal nucleobase is in progress.
19. In Ref. 14, only the a-anomer of 2 was isolated and employed for alkyne-azide
1,3-dipolar cycloaddition.
20. Recently, a similar synthetic procedure for compound 3 (anomeric mixture)
was reported, see: Heinrich, D.; Wagner, T.; Diederichsen, U. Org. Lett. 2007, 9,
5311.
Acknowledgments
21. Compound 4b was also synthesized by an alternative route, see: Chiba, J.;
Takeshima, S.; Mishima, K.; Maeda, H.; Nanai, Y.; Mizuno, K.; Inouye, M. Chem.
Eur. J. 2007, 13, 8124.
This work was supported in part by a Grant-in-Aid for Science
Research (KAKENHI) from the Japan Society for the Promotion of
Science (JSPS), the Molecular Imaging Research Program from the
Ministry of Education, Culture, Sports, Science and Technology, Ja-
pan (MEXT) and the Osaka University Life Science Young Indepen-
dent Researcher Support Program from the Japan Science and
Technology Agency (JST).
22. Selected data of 1a and 1b. 1a:
31P NMR (CDCl3) d 147.9, 148.3; HRMS (FAB) m/z
calcd for C37H45N2NaO6P (M+Na+): 667.2913; found 667.2909. 1b: 31P NMR
(CDCl3) d 148.5, 149.1; HRMS (FAB) m/z calcd for C37H45N2NaO6P (M+Na+):
667.2913; found 667.2915.
23. The phosphoramidite
1a was also successfully incorporated into an
oligonucleotide on an automated DNA synthesizer.
24. For a review, see: Loakes, D. Nucleic Acids Res. 2001, 29, 2437.