Triazole-linked oligonucleotides with mixed-base sequences: Synthesis and hybridization properties

Article abstract of DOI:10.1002/ejoc.201101700

A new class of backbone-modified oligonucleotide analogs has emerged since the discovery of the Cu^I-catalyzed [3+2] azide/alkyne cycloaddition reaction. These are oligonucleotide analogs with 1,4-disubstituted 1,2,3-triazoles as the internucleo

Full text of DOI:10.1002/ejoc.201101700

FULL PAPER
DOI: 10.1002/ejoc.201101700
Triazole-Linked Oligonucleotides with Mixed-Base Sequences: Synthesis and
Hybridization Properties
Anna Varizhuk,[a,b] Alexandr Chizhov,[c] Igor Smirnov,[b,d] Dmitry Kaluzhny,[a] and
Vladimir Florentiev*[a]
Keywords: Cylcoaddition / Nitrogen heterocycles / Oligonucleotides / Antisense agents / Hybridization
A new class of backbone-modified oligonucleotide analogs
has emerged since the discovery of the CuI-catalyzed [3+2]
pendence of the effect of this modification on duplex stability,
we have designed partially modified mixed-base oligonu-
azide/alkyne cycloaddition reaction. These are oligonucleo- cleotides, which can be prepared by using a modified dinu-
tide analogs with 1,4-disubstituted 1,2,3-triazoles as the in-
ternucleotide linkages. Of all such analogs known, only the
triazole-linked deoxythymidine decamer [(dT)10] has been
reported to show enhanced binding affinity to complemen-
tary DNA. Importantly, it is a fully modified (dT)10 analog. To
date, sequentially heterogeneous oligonucleotides bearing
the same backbone modification have not been described.
With the goal of investigating sequence and regularity de-
cleoside block. In this paper we report the synthesis of a di-
thymidine phosphoramidite analog with a triazole linker, its
use in oligonucleotide synthesis and hybridization data of the
resulting oligonucleotide analogs. The effect of single and
multiple modifications on stability of mixed-base duplexes is
assessed and compared with published data for the oligo(T)/
oligo(A) duplex. We also compared the effect of the linker
concerned with that of a shorter triazole linker.
et al. exhibits enhanced binding affinity to complementary
DNA.[6] This (dT)10 analog is a fully modified oligonucleo-
tide, which can be prepared from a thymidine derivative by
repeating functionalization and CuAAC ligation steps. The
melting temperature (Tm) of the corresponding duplex is
about 40 °C higher than that of the isosequential unmodi-
fied duplex. The authors conjecture that this is due to the
structural complementarity and the absence of the repul-
sion between neutral triazole and anionic natural phospho-
diester linkages. We believe it would be of interest to clarify
whether this remarkable enhancement in duplex stability is
sequence-dependent. Because the synthesis of fully modi-
fied mixed-base triazole-linked oligonucleotides could be
complex – requiring preparation of four different mono-
mers and optimization of oligomerization conditions – we
employed a different strategy, based on the use of a modi-
fied dinucleoside block.
In this paper, we report the synthesis of a dithymidine
phosphoramidite block with a triazole linker, its incorpora-
tion in oligonucleotides and hybridization data of modified
oligonucleotides. The effect of single and multiple modifica-
tions on stability of mixed-base duplexes is assessed and
compared with published data for the oligo(T)/oligo(A) du-
plex. We also compare the effect of the linker concerned
with that of a shorter triazole linker, reported in our pre-
vious work.[7]
Introduction
The discovery of the copper(I) catalysis of the Huisgen
1,3-dipolar cycloaddition reaction by Sharpless and Meldal
in 2002 had a marked impact on the field of artificial nu-
cleosides and oligonucleotides. A number of 1,2,3-triazole-
containing nucleoside analogs exhibiting antibacterial or
antiviral activity have been obtained recently by using a
CuI-catalyzed [3+2] azide/alkyne cycloaddition (CuAAC)
reaction as the key step.[1] Furthermore, a new class of
backbone-modified oligonucleotides, with 1,4-disubstituted
1,2,3-triazoles as the internucleotide linkages, has emerged
in the last decade.[2] These oligonucleotides are regarded as
potential posttranscriptional gene-silencing agents and may
become an alternative to well-known backbone-modified
oligonucleotides, such as locked nucleic acids, phos-
phorothioates, oligonucleotides with amide internucleotide
linkages, and so on.[3–5]
Of all triazole-linked oligonucleotides reported to date,
only the deoxythymidine decamer analog reported by Isobe
[a] Engelhardt Institute of Molecular Biology,
Vavilov str. 32, 119991 Moscow, Russian Federation
Fax: +7-499-135-140
E-mail: flor@imb.ac.ru
[b] Institute for Physical-Chemical Medicine of Ministry of Public
Health,
117312 Moscow, Russia
[c] N. D. Zelinsky Institute of Organic Chemistry,
Leninsky prospect 47, 119991 Moscow, Russia
[d] Belozerskii Institute of Physico-Chemical Biology,
119899 Moscow, Russia
Results and Discussion
We started the synthesis of the triazole-linked dinucleo-
tide with the preparation of acetylenic nucleoside compo-
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101700.
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Varizhuk, A. Chizhov, I. Smirnov, D. Kaluzhny, V. Florentiev
FULL PAPER
nent 6 from aldehyde 1 (Scheme 1). The latter was readily
Aldehyde 4 was treated with (dibromomethyl)triphenyl-
accessed from 3Ј-O-(tert-butyldiphenylsilyl)thymidine[8] by phosphonium bromide (prepared by Wolkoff’s method[12]
)
oxidation with o-iodoxybenzoic acid.[7] Wittig-type conden- in the presence of Zn in dioxane at reflux temperature to
sation of aldehyde 1 with the ylide derived from methyltri- give dibromomethylene derivative 5 in 80% yield. Treat-
phenylphosphonium bromide in the presence of BuLi in ment of compound 5 with BuLi in THF afforded acetylenic
THF afforded terminal olefin 2 in 84% yield. A methyltri- nucleoside 6 in 77% yield.
phenylphosphonium bromide/BuLi ratio of 1:1 is crucial to
Alkyne 6 was coupled with 3Ј-azido-3Ј-deoxy-5Ј-O-di-
obtain a high yield of nucleoside 2. A lack of BuLi leads to methoxytritylthymidine (Scheme 2) in the presence of
the formation of a polar by-product, presumably similar to CuSO4·5H2O and sodium ascorbate. The CuAAC reaction
the phosphonium salt reported by Matsuda et al.[9], and in was carried out in a two-phase solvent system (H2O/
the presence of an excess of BuLi β-elimination of the tert- CH2Cl2),[13] which has proven to be favorable for the click
butyldimethylsilyloxy group occurs.
coupling of poorly water-soluble compounds, to give dinu-
cleoside 7 in 80% yield. The silyl protecting group was re-
moved by using 0.5 m TBAF in THF. Treatment of di-
nucleoside 8, bearing a free hydroxy group, with 2-cyano-
ethyl-N,N,NЈ,NЈ-tetraisopropylphosphoramidite in the pres-
ence of 1H-tetrazole and pyridine in CH2Cl2 afforded target
phosphoramidite 9 in 72% yield.
Scheme 1. Synthesis of the acetylenic component of the triazole-
linked dinucleotide. Reagents and conditions: (a) PPh3CH3+Br–,
BuLi, THF; (b) (i) 9-BBN-H, THF; (ii) NaBO3·4H2O, THF/H2O/
MeOH
(5:2:3);
(c) Dess–Martin
periodinane,
CH2Cl2;
(d) PPh3CHBr2+Br–, Zn, dioxane, reflux; (e) nBuLi, THF, –70 °C.
Compound 2 was subjected to hydroboration with
9-borabicyclo[3.3.1]nonane (9-BBN-H) in THF and subse-
quent oxidative treatment with NaBO3·4H2O to give com-
pound 3 in 86% yield. Sodium perborate was chosen as the
oxidizing agent, because the conventional reagent, H2O2 in
alkaline solution, has been reported to cause side reactions
with a similar nucleoside.[10] Oxidation of alcohol 3 with o-
iodoxybenzoic acid gave the corresponding carboxylic acid;
Scheme 2. Synthesis of the phosphoramidite dinucleoside block
with a triazole internucleoside linkage. Reagents and conditions:
(a) CuSO4, sodium ascorbate, CH2Cl2/H2O; (b) TBAF, THF;
(d) NCCH2CH2OP(NiPr2)2, 1H-tetrazole, pyridine, CH2Cl2.
The structures of nucleosides 2–6 and dinucleosides 7–9
however, when Dess–Martin periodinane was used, alde- were ascertained by high-resolution mass spectrometry, and
hyde 4 was obtained in 89% yield. the structures of 7 and 8 were confirmed by COSY NMR
All attempts to convert aldehyde 4 into terminal alkyne spectroscopy. The COSY NMR experiments enabled as-
6 by using an Ohira–Bestmann reaction were unsuccessful. signment of signals to the protons of the 5Ј-deoxy-5Ј-ethen-
Neither a one-pot procedure with in situ generation of di- ylthymidine and 3Ј-amino-3Ј-deoxythymidine moieties in
1
methyl (1-diazo-2-oxopropyl)phosphonate (Ohira–Best- the H NMR spectra of the dinucleosides. C–H correlation
mann reagent) from tosyl azide and dimethyl (2-oxopropyl)- NMR experiments enabled assignment of 13C NMR spec-
phosphonate,[11] nor a more complex strategy involving iso- tra of the dinucleosides (for COSY and C–H correlation
lation of the intermediate Ohira–Bestmann reagent afforded NMR spectra, see Supporting Information).
alkyne 6. Therefore, formyl Ǟ ethynyl group transforma-
tion was realized in two steps, through the intermediate di- synthesis of modified oligonucleotides by using standard
bromoalkene. phosphoramidite protocols. The coupling time was in-
Dinucleoside block 9 was used directly for solid-phase
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Triazole-Linked Oligonucleotides
Table 1. MALDI-TOF mass spectra of oligonucleotides and melting temperatures of their duplexes with an unmodified DNA complement
(duplex concentration 2.5ϫ10–6 m).
Code
Sequence of modified strand (5ЈǞ3Ј)[a]
m/z [M + H]+ (calcd.)
Tm [°CϮ0.5] (ΔTm [°C])[b]
1
2
3
4
5
TTAACTTCTTCACATTC
XAACTTCTTCACATTC
TTAACTTCTTCACAXC
TTAACTTCXCACATTC
XAACTTCXCACAXC
5072.2 (5071.32)
5041.8 (5042.44)
5042.0 (5042.44)
5042.5 (5042.44)
4984.1 (4984.58)
50.3
50.4 (+0.1)
47.2 (–3.1)
41.9 (–8.4)
37.7 (–12.6)
[a] X: triazole-linked dithymidine fragment. Sequence of a non-modified strand 5Ј-GAATGTGAAGAAGTTAA-3Ј. [b] ΔTm: difference
between Tm of modified and natural duplexes.
creased to 15 min for the modified phosphoramidite. No two main minima. The most stable conformer is displayed
decrease in coupling efficiency was observed (98–99% step- in Figure 2. As evident from Figure 2, duplex distortion
wise coupling yields were achieved for both the modified may arise from the lesser twist of the triazole linker than
and unmodified amidites). Along with the modified oligo- the phosphordiester one. The results imply that the intro-
nucleotides, an unmodified complement and unmodified duction of a triazole linker concerned results in a longer
isosequential oligonucleotides were synthesized. All the oli- helix pitch. This agrees with the conclusion Isobe et al.
gonucleotides were purified by reverse-phase HPLC and made based on their molecular-modeling studies.[6]
characterized by using MALDI-TOF mass spectrometry
(Table 1).
Modified oligonucleotides and their unmodified counter-
part were hybridized to ssDNA. Thermal stability of the
duplexes was examined by monitoring the change in hyper-
chromicity at 260 nm (Figure 1). Sequences and melting
temperatures of the duplexes are shown in Table 1.
Figure 2. Superimposition of optimized conformations of a natural
duplex fragment (green strands) and a modified one (red strands).
The arrow points to the triazole internucleotide linker. Modified
Figure 1. Melting curves of the modified and unmodified duplexes.
Legends represent duplex code numbers (see Table 1). Temperature
gradient: 0.2 °C/min.
strand sequence: 5Ј-CATGTtriazoleTCATG-3Ј.
As evident from Table 1 the impact of the 5Ј-terminal
Overall duplex conformation is rather close to a classical
modification on oligonucleotide hybridization is insignifi- β-helix in our case. CD spectra of modified oligonucleotides
cant. The 3Ј-terminal modification destabilizes the duplex hybridized to complementary ssDNA confirm that the for-
to a certain extent (ΔTm = –3.1 °C), and the middle-strand mer adopt β-form geometry upon formation of the duplex
modification leads to a drastic loss in duplex stability (ΔTm (Figure 3).
= –8.4 °C). Average ΔTm per modification is –4.0 °C (calcu-
lated as a sum of all ΔTm values divided by the total those obtained earlier for mixed-sequence oligonucleotide
number of modifications).
analogs with shorter (four-atom) triazole linkers.[7] Both the
Our hybridization results are generally consistent with
To understand what changes in oligonucleotide geometry four-atom (C3Ј to C5Ј) linker described in our previous
underlie the destabilization, we performed molecular-mod- work and the five-atom linker discussed herein tend to de-
eling studies on a modified backbone fragment in a duplex. stabilize duplexes. The above triazole linkers have a length
We designed 12 initial structures differing in the position of closest to that of natural phosphodiester linkages. Further
the triazole ring relative to the duplex axis. Computational linker elongation – introduction of an additional methylene
minimization of the energy of these structures resulted in group on one side of a triazole or imidazole ring – is known
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1:49 (B), 1:25 (C), 1:14 (D). 1H and 13C NMR spectra were re-
corded with a Bruker AMXIII-400 NMR spectrometer (Germany).
Abbreviations used: ethT, 5Ј-deoxy-5Ј-ethenylthymidine moiety;
aT, 3Ј-amino-3Ј-deoxythymidine moiety; DMTr, 4,4Ј-dimethoxytri-
tyl. The signals were assigned to protons by using COSY experi-
ments. The NMR spectroscopic data were processed and are pre-
sented by using MestReNova version 7.0.3 (Mestrelab Research
SL, Spain). MALDI-TOF mass spectra were acquired with a
Bruker Microflex mass spectrometer (Bruker, Germany) in a posi-
tive ion linear mode (+20 kV). Oligonucleotides were desalted with
mZipTipC18 pipette tips (Millipore, USA) before MALDI-TOF
mass spectra analysis. Each spectrum was acquired with 200 laser
shots (N2 gas laser, 337 nm) with a solution of 35 g/mL of 3-hy-
droxypicolinic acid with dibasic ammonium citrate as the matrix.
HRMS (ESI) data were acquired with a Bruker microTOF II spec-
trometer (Germany) in a positive ion mode (capillary voltage
–4500 V, end plate offset –500 V, interface capillary temperature
180 °C, nitrogen dry gas, 4.0 L/min, scanning range m/z = 50–
3000 Da, scanning frequency 1 scan/s). Internal calibration was
done by using Electrospray Calibration Solution (Fluka). Oligonu-
cleotides were synthesized with an Applied Biosystems 3400 DNA
synthesizer (USA) by using standard phosphoramidite protocols
and purified by using preparative-scale reverse-phase HPLC on a
250 mmϫ4.0 mm Hypersil C18 column with detection at 260 nm.
Chromatography of dimethoxytrytil-protected oligonucleotides was
performed with a 10–50% gradient of CH3CN in 0.05 m triethylam-
monium acetate. Detritylated oligonucleotides were further purified
in a 0–25% gradient of CH3CN in triethylammonium acetate
buffer. Melting curves of the duplexes were recorded with a Shim-
adzu UV 160-A spectrophotometer (Japan) and with a thermo-
stated cell in 20 mm sodium phosphate buffer, 100 mm NaCl,
0.1 mm EDTA, pH = 7.0, concentration of each duplex being
2.5ϫ10–6 m. Samples were denatured at 95 °C for 5 min and slowly
cooled to 20 °C prior to analysis. Duplex absorbance at 260 nm
was measured as a function of temperature. Data was recorded
Figure 3. CD spectra of the modified and unmodified duplexes.
Legends represent duplex code numbers (see Table 1).
to decrease duplex stability.[14] In view of hybridization data
reported by Isobe et al.,[6] five-atom linkers were regarded
as the most promising.
Unfortunately, the results of the present work do not
confirm this and are not in accord with the hybridization
data obtained for the fully modified (dT)10 analog. We sug-
gest that distinct geometry of oligo(T)/oligo(A) DNA frag-
ments[15] may be somewhat responsible for the outstanding
stability of the regular triazole-modified decamer duplex.
Conclusions
We have reported the synthesis of the dinucleoside phos-
phoramidite block with a triazole internucleoside linkage. every 0.5 °C from 20 to 70 °C. Thermodynamic parameters of du-
plex formation were obtained by performing nonlinear regression
analysis by using DataFit version 9.0.059 (Oakdale Engineering,
USA). A calculation method taking into account the temperature
dependence of the UV absorbance of duplexes and single strands
was applied. Circular dichroism (CD) spectra were obtained with
a Jasco J-715 spectropolarimeter at 20 °C. Samples were annealed
in the same buffer and under the same conditions as for the thermal
denaturation studies. The CD values (Δε) are given per mol of nu-
cleotides. Conformational analysis of the modified duplex fragment
was performed by molecular mechanics calculations by using the
HyperChem. 8.0.9 Amber force field (HyperCube, Inc., USA). The
double-stranded B-DNA (5Ј-CATGTTCATG-3Ј) was generated by
using the nucleic acid feature in the database of HyperChem. The
phosphate linker in the TT fragment was cut off, and a triazole
linker, constructed by using a model building option of Hyper-
Chem, was inserted manually. Partial charges on triazole linker
atoms were calculated by using the ab initio method (6-31G**). We
obtained 12 initial structures by performing geometry optimizing
with the restrained C4Ј–C5Ј–C4(triazole)–C5(triazole) with a dihe-
dral angle ranging from 0 to 270° through 30° increments. A dis-
tance-dependent dielectric constant was used. After the restraint
was removed, the geometry of 12 initial structures was further opti-
mized.
Surprisingly, incorporation of this block in sequentially
heterogeneous oligonucleotides was disadvantageous for
their hybridization ability, whereas the fully modified oligo-
(dT) analog is known to bind tightly to a complementary
DNA fragment. Thus, we believe that the effect of this
modification is likely to be highly dependent on the regular-
ity of the oligonucleotide structure. The phosphoramidite
block we have described is a useful intermediate for further
synthesis and investigation of triazole-functionalized oligo-
nucleotides.
Experimental Section
General: All reagents were commercially available unless otherwise
mentioned and used without further purification. 3Ј-Azido-3Ј-de-
oxythymidine was a kind gift from the Association AZT (Russia).
Dess–Martin reagent was prepared according to a published pro-
cedure.[16] All solvents were purchased from Khimmid (Russia). Di-
oxane was dried with sodium hydroxide and distilled, CH2Cl2 was
distilled from phosphorus pentoxide, pyridine was distilled from
calcium hydride, and THF was distilled from lithium aluminium
hydride prior to use. Flash column chromatography was performed
on silica gel Kieselgel 60 (0.040–0.063 mm, Merck, Germany). TLC 3Ј-O-tert-Butyldiphenylsilyl-5Ј-deoxy-5Ј-methylenethymidine (2): To
was performed on silica gel Kieselgel 60 F254 precoated plates
(Merck) with detection by UV light with the following solvent sys-
tems (compositions expressed as v/v): ethanol/CH2Cl2, 1:95 (A),
a suspension of methyltriphenylphosphonium bromide (0.97 g,
2.72 mmol) in THF (10 mL), a solution of butyllithium (1.8 mL,
1.6 m in hexane) was added. The mixture was stirred at room tem-
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Triazole-Linked Oligonucleotides
perature for 2 h and then cooled to 0 °C. A solution of 1-(3-O-tert-
butyldiphenylsilyl-2-deoxy-β-d-erithro-pentadialdo-1,4-furanosy1)-
5-methyluracil (1) (650 mg, 1.36 mmol) in THF (10 mL) was added
dropwise. The mixture was stirred at room temp. for 18 h before
being poured into a saturated solution of NH4Cl (30 mL) and ex-
tracted with ethyl acetate (3ϫ30 mL). The combined organic layers
were washed with brine (30 mL), dried with Na2SO4 and concen-
trated. The residue was purified by flash column chromatography
with ethanol and CH2Cl2 (1:100, v/v) as eluents to give compound
2 as a colorless foam (0.54 g, 84% yield). Rf = 0.56 (solvent system
NMR). Aldehyde 4 was used without further purification in the
next reaction step. Rf = 0.59 (solvent system B). 1H NMR
3
(400 MHz, DMSO): δ = 11.26 (s, 1 H, 3-H), 9.49 (dd, J6Ј,5Јa
=
3
2.6 Hz, J6Ј,5Јb = 1.3 Hz, 1 H, 6Ј-H), 7.66–7.40 (m, 10 H, Ph2Si),
3
7.33 (d, 4J = 1.1 Hz, 1 H, H6), 6.21 (dd, 3J1Ј,2Јa = 7.9 Hz, J1Ј,2Јb
=
6.4 Hz, 1 H, 1Ј-H), 4.35–4.27 (m, 2 H, 3Ј-H, 4Ј-H), 2.58 (ddd,
3
2
3J5Јa,4Ј = 8.3 Hz, J5Јa,6Ј = 2.6 Hz, J5Јa,5Јb = 16.5 Hz, 1 H, 5Јa-H),
3
3
2
2.42 (ddd, J5Јb,4Ј = 4.3 Hz, J5Јb,6Ј = 1.3 Hz, J5Јa,5Јb = 16.5 Hz, 1
3
3
2
H, 5Јb-H), 2.16 (ddd, J1Ј,2Јa = 7.9 Hz, J2Јa,3Ј = 5.8 Hz, J2Јa,2Јb
=
13.9 Hz, 2 H, 2Јa-H), 2.09 (ddd, 3J1Ј,2Јb = 6.4 Hz, J2Јb,3Ј = 2.7 Hz,
3
1
4
A). H NMR (400 MHz, DMSO): δ = 11.27 (s, 1 H, 3-H), 7.63–
2J2Јa,2Јb = 13.9 Hz, 2 H, 2Јb-H), 1.74 (d, J = 1.1 Hz, 3 H, 5-CH3),
4
3
7.43 (m, 10 H, Ph2Si), 7.34 (d, J = 1.1 Hz, 1 H, 6-H), 6.23 (t, J 1.05 (s, 9 H, CH3, tBuSi) ppm. 13C NMR (101 MHz, DMSO): δ =
3
3
= 7.0 Hz, 1 H, 1Ј-H), 5.74 (ddd, J5Ј,6Јa(trans) = 17.1 Hz, J5Ј,6Јb(cis) 200.8 (6Ј-CH), 163.5 (4-C), 150.4 (2-C), 135.2 (6-CH), 135.12
3
= 10.6 Hz, J5Ј,4Ј = 7.1 Hz, 1 H, 5Ј-H), 5.12–5.05 (m, 2 H, 6Јa-H
and 6Јb-H), 4.29–4.22 (m, 2 H, 3Ј-H and 4Ј-H), 2.16–2.11 (m, 2 H,
(4ϫo-CH, Ph), 132.7 (2ϫC, Ph), 130.1 and 130.0 (4ϫp-CH, Ph),
127.9 (4ϫm-CH, Ph), 109.8 (5-C), 84.2 (1Ј-CH), 80.8 (4Ј-CH), 75.6
2Јa-H, 2Јb-H), 1.74 (d, 4J = 1.1 Hz, 3 H, 5-CH3), 1.05 (s, 9 H, CH3 (3Ј-CH), 46.3 (5Ј-CH2), 37.9 (2Ј-CH2), 26.6 (3ϫCH3, tBu), 18.6
tBu) ppm. 13C NMR (101 MHz, DMSO): δ = 163.5 (4-C), 150.4 (C, tBu), 11.9 (5-CH3) ppm. HRMS: calcd. for C27H32N2O5Si [M
(2-C), 135.9 (5Ј-CH), 135.8 (6-CH), 135.3, 135.2 (4ϫo-CH, Ph),
131.9 (2ϫC, Ph), 130.0 (4ϫp-CH, Ph), 127.88, 1127.85 (4ϫm-
CH, Ph), 117.6 (6Ј-CH2), 109.67 (5-C), 87.1 (1Ј-CH), 83.9 (4Ј-CH),
76.4 (3Ј-CH), 38.2 (2Ј-CH2), 26.6 (3ϫCH3, tBu), 18.6 (C, tBu),
12.0 (5-CH3) ppm. HRMS: calcd. for C27H32N2O4Si [M + Na]+
499.2024; found 499.2010.
+ Na]+ 515.1973; found 515.1972.
3Ј-O-tert-Butyldiphenylsilyl-5Ј-deoxy-5Ј-(2,2-dibromoethenyl)-
thymidine (5): To a stirred solution of nucleoside 4 (0.89 g,
1.8 mmol) and (dibromomethyl)triphenylphosphonium bromide
(1.85 g, 3.59 mmol) in dioxane (4 mL), Zn (260 mg, 3.95 mmol) was
added. The reaction mixture was stirred at reflux temperature for
3 h, then cooled to room temperature and filtered. The filtrate was
concentrated, the residue dissolved in CH2Cl2 (50 mL) and washed
with brine (30 mL). The organic layer was separated, dried with
Na2SO4 and concentrated. Purification by flash column
chromatography with ethanol/CH2Cl2 (1–3:100, v/v) as eluent af-
forded compound 5 as a colorless foam (0.93 g, 80% yield). Rf =
3Ј-O-tert-Butyldiphenylsilyl-5Ј-deoxy-5Ј-(hydroxymethyl)thymidine
(3): A solution of compound 2 (0.47 mg, 0.99 mmol) in THF
(4 mL) was cooled to 0 °C, and 9-BBN-H (1.46 g, 5.97 mmol,
6 equiv.) was added. The reaction mixture was allowed to warm
slowly and stirred at 20 °C for 20 h. The reaction mixture was then
cooled to 0 °C, and methanol (5.1 mL) was added dropwise. When
gas evolution had ceased, water (7.7 mL) was added, followed by
1
0.46 (solvent system A). H NMR (400 MHz, DMSO): δ = 11.28
4
NaBO3·4H2O (5.04 g, 24 mmol, 24 equiv.). The resulting mixture (s, 1 H, 3-H), 7.72–7.41 (m, 10 H, Ph2Si), 7.32 (d, J = 1.0 Hz, 1
3
3
was stirred vigorously at room temperature for 30 h and filtered to
remove the precipitate. The filtrate was diluted with ethyl acetate
(50 mL) and washed with brine (2ϫ25 mL). The organic layer was
dried with Na2SO4 and concentrated. The residue was purified by
flash column chromatography with ethanol and CH2Cl2 (2:100,
v/v) as eluents to give compound 3 as a colorless foam (0.42 g, 86%
yield). Rf = 0.43 (solvent system B). 1H NMR (400 MHz, DMSO):
H, 6-H), 6.43 (t, J5Ј,6Ј = 7.1 Hz, 1 H, 6Ј-H), 6.19 (dd, J1Ј,2Јa =
7.8 Hz, 3J1Ј,2Јb = 6.5 Hz, 1 H, 1Ј-H), 4.32–4.22 (m, 1 H, 3Ј-H), 3.99–
3.88 (m, 1 H, 4Ј-H), 2.21–2.13 (m, 3 H, 2Јa-H and 5Ј-CH2), 2.10
(ddd, J1Ј,2Јb = 6.5 Hz, J3Ј,2Јb = 3.2 Hz, J2Јa,2Јb = 13.5 Hz, 1 H,
3
3
2
4
2Јb-H), 1.76 (d, J = 1.0 Hz, 3 H, 5-CH3), 1.05 (s, 9 H, CH3, tBu)
ppm. 13C NMR (101 MHz, DMSO): δ = 163.5 (4-C), 150.3 (2-C),
136.0 (6-CH), 135.1 (4ϫo-CH, Ph), 134.8 (6Ј-CH), 132.7 (2ϫC,
Ph), 130.1 (4 ϫ p-CH, Ph), 127.94 and 127.92 (4 ϫ m-CH, Ph),
4
δ = 11.25 (s, 1 H, 3-H), 7.66–7.40 (m, 10 H, Ph2Si), 7.32 (d, J =
1.1 Hz, 1 H, 6-H), 6.20 (dd, 3J1Ј,2Јa = 8.0 Hz, 3J1Ј,2Јb = 6.2 Hz, 1 H, 109.7 (5-C), 90.1 (7Ј-C), 84.1 (1Ј-CH), 83.1 (4Ј-CH), 75.2 (3Ј-CH),
3
1Ј-H), 4.41 (t, J = 5.0 Hz, 1 H, 6Ј-OH), 4.26–4.21 (m, 1 H, 3Ј-H), 38.1 (2Ј-CH2), 36.2 (5Ј-CH2), 26.6 (3ϫCH3, tBu), 18.5 (C, tBu),
3
3
3
3.97 (ddd, J3Ј,4Ј = 2.7 Hz, J4Ј,5Јa = 6.0 Hz, J4Ј 5Јb = 7.3 Hz, 1 H, 12.0 (5-CH3) ppm. HRMS: calcd. for C28 H3 2Br2 N2 O4 Si
4Ј-H), 3.40–3.29 (m, 2 H, 6Ј-CH2), 2.13–2.06 (m, 1 H, 2Јa-H), 2.03
[M + Na]+ 669.0390; found 669.0390.
3
3
2
(ddd, J1Ј,2Јb = 6.2 Hz, J2Јb,3Ј = 2.8 Hz, J2Јa,2Јb = 13.3 Hz, 1 H,
3Ј-O-tert-Butyldiphenylsilyl-5Ј-deoxy-5Ј-ethynylthymidine (6): Nu-
cleoside 5 (0.73 g, 1.13 mmol) was dissolved in THF (15 mL), and
butyllithium (4 mL, 1.6 m in hexane) was added dropwise at –70 °C.
The reaction mixture was stirred at –70 °C for 30 min and allowed
to warm to room temperature. Saturated NH4Cl (30 mL) was
added and the resulting mixture was extracted with CH2Cl2
(3ϫ30 mL). The combined organic layers were washed with water
(50 mL), dried with Na2SO4 and concentrated. The residue was
purified by flash column chromatography with ethanol/CH2Cl2
(1:100, v/v) as eluent to give compound 6 as a colorless foam
(0.73 g, 77 % yield). Rf = 0.39 (solvent system A). 1H NMR
(400 MHz, DMSO): δ = 11.28 (s, 1 H, 3-H), 7.65–7.44 (m, 10 H,
4
2Јb-H), 1.75 (d, J = 1.1 Hz, 3 H, 5-CH3), 1.57–1.50 (m, 2 H, 5Ј-
CH2), 1.04 (s, 9 H, CH3 tBu) ppm. 13C NMR (101 MHz, DMSO):
δ = 163.5 (4-C), 150.4 (2-C), 135.8 (6-CH), 135.2 (4ϫo-CH, Ph),
132.9 and 132.8 (2ϫC, Ph), 129.9 (4ϫp-CH, Ph), 127.9 (4ϫm-
CH, Ph), 109.6 (5-C), 83.6 (1Ј-CH), 83.5 (4Ј-CH), 76.1 (3Ј-CH),
57.3 (6Ј-CH2), 38.2 (2Ј-CH2), 36.1 (5Ј-CH2), 26.6 (3ϫCH3, tBu),
18.6 (C, tBu), 12.0 (5-CH3) ppm. HRMS: calcd. for C27H34N2O5Si
[M + Na]+ 517.2129; found 517.2122.
3Ј-O-tert-Butyldiphenylsilyl-5Ј-deoxy-5Ј-formylthymidine (4): Nu-
cleoside 3 (1.3 g, 2.6 mmol) was dissolved in CH2Cl2 (10 mL), and
Dess–Martin periodinane (2.2 g, 5.2 mmol) was added. The re-
sulting suspension was stirred at room temperature. After 3 h, the
reaction mixture was diluted with ethyl acetate (60 mL), poured
into a cold solution of Na2S2O3·5H2O (8.5 g, 34 mmol) in saturated
NaHCO3 (60 mL), and shaken for 5 min. The reaction mixture was
allowed to cool to room temperature and filtered. The organic layer
was separated, washed with NaHCO3 (30 mL), water (30 mL) and
brine (30 mL), dried with Na2SO4 and concentrated to provide al-
dehyde 4 as a white foam (1.14 g, 89% yield, Ͼ95% pure by 1H
4
Ph2Si), 7.43 (d, J = 1.1 Hz, 1 H, 6-H), 6.26 (dd, J1Ј,2Јa = 8.1 Hz,
J1Ј,2Јb = 6.2 Hz, 1 H, 1Ј-H), 4.36–4.32 (m, 1 H, 3Ј-H), 4.00–3.96
4
(m, 1 H, 4Ј-H), 2.84 (t, J7Ј,5Јa(5Јb) = 2.6 Hz, 1 H, 7Ј-H), 2.38 (ddd,
4
2
3J5Јa,4Ј = 6.4 Hz, J5Јa,7Ј = 2.6 Hz, J5Јa,5Јb = 17.1 Hz, 1 H, 5Јa-H),
3
4
2
2.25 (ddd, J5Јb,4Ј = 5.5 Hz, J5Јb,7Ј = 2.6 Hz, J5Јa,5Јb = 17.1 Hz, 1
4
H, 5Јb-H), 2.14–2.08 (m, 2 H, 2Јa-H, 2Јb-H), 1.74 (d, J = 1.1 Hz,
3 H, 5-CH3), 1.05 (s, 9 H, CH3, tBuSi) ppm. 13C NMR (101 MHz,
DMSO): δ = 163.5 (4-C), 150.3 (2-C), 135.5 (6-CH), 135.24 and
Eur. J. Org. Chem. 2012, 2173–2179
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2177

A. Varizhuk, A. Chizhov, I. Smirnov, D. Kaluzhny, V. Florentiev
FULL PAPER
135.21 (4ϫo-CH, Ph), 132.7 and 135.6 (2ϫC, Ph), 130.1 (4ϫp-
CH, Ph), 127.9 (4ϫm-CH, Ph), 109.7 (5-C), 84.0 (1Ј-CH), 83.8 (4Ј-
CH), 80.3 (6Ј-C), 75.1 (3Ј-CH), 73.1 (7Ј-CH), 38.1 (2Ј-CH2), 26.7
3.73 (s, 6 H, CH3O, DMTr), 3.30–3.28 (m, 2 H, 5Ј-CH2, aT), 3.08
(dd, J4Ј,5Јa = 5.3 Hz, J5Јa,5Јb = 15.1 Hz, 1 H, 5Јa-H, ethT), 2.95
3
2
3
2
(dd, J4Ј,5Јb = 7.6 Hz, J5Јa,5Јb = 15.1 Hz, 1 H, 5Јb-H, ethT), 2.80–
3
2
(3ϫCH3, tBu), 22.2 (5Ј-CH2), 18.5 (C, tBu), 12.4 (5-CH3) ppm. 2.72 (m, 2 H, 2Ј-CH2, aT), 2.18 (dt, J1Ј,2Јa(3Ј) = 6.7 Hz, J2Јa,2Јb
=
=
HRMS: calcd. for C28H32N2O4Si [M + Na]+ 511.2024; found
511.2017.
13.6 Hz, 1 H, 2Јa-H, ethT), 2.08 (ddd, J1Ј,2Јb = 6.4 Hz, J2Јb,3Ј
3
3
3.8 Hz, 2J2Јa,2Јb = 13.6 Hz, 1 H, 2Јb-H, ethT), 1.77 (d, 4J = 1.2 Hz,
3 H, 5-CH3, aT), 1.60 (d, 4J = 1.1 Hz, 3 H, 5-CH3, ethT) ppm. 13
C
4-[3Ј-O-(tert-Butyldiphenylsilyl)-5Ј-deoxythymidin-5Ј-yl]-1-[3Ј-
deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)thymidin-3Ј-yl]-1H-1,2,3-triazole
(7): To a stirred solution of compound 6 (0.37 g, 0.76 mmol) and
3Ј-amino-3Ј-deoxy-5Ј-O-dimethoxytritylthymidine (0.47 g,
0.83 mmol) in CH2Cl2 (2 mL) and water (2 mL), CuSO4·5H2O
(19 mg, 0.08 mmol) and sodium ascorbate (49 mg, 0.23 mmol) were
added. The resulting solution was stirred at room temp. for 2 h.
The reaction mixture was diluted with CH2Cl2 (10 mL) and brine
(10 mL). The organic layer was separated, dried with Na2SO4 and
concentrated. The residue was purified by flash column chromatog-
raphy with ethanol/CH2Cl2 (1:100, v/v) as eluent to give dinucleo-
side 7 as a hard foam (0.64 g, 80% yield). Rf = 0.63 (solvent system
C). 1H NMR (400 MHz, DMSO): δ = 11.37 (s, 1 H, 3-H, ethT),
11.26 (s, 1 H, 3-H, aT), 7.91 (s, 1 H, 7Ј-H, ethT), 7.64 (d, 4J =
1.2 Hz, 1 H, 6-H, ethT), 7.54 (d, 4J = 1.1 Hz, 1 H, 6-H, aT), 7.61–
NMR (101 MHz, DMSO): δ = 163.6 (4-C, ethT, aT), 158.1 (2ϫp-
C, PhOMe, DMTr), 150.4 (2-C, ethT) 150.3 (2-C, aT), 144.5 (6Ј-C,
ethT), 143.6 (C, Ph, DMTr), 136.2 (6-CH, ethT), 136.0 (6-CH, aT),
135.2 (2ϫC, PhOMe, DMTr), 129.6 (4ϫo-CH, PhOMe, DMTr),
127.8 (2ϫo-CH, Ph, DMTr), 127.6 (2ϫm-CH, Ph, DMTr), 126.7
(p-CH, Ph, DMTr), 122.3 (7Ј-CH, ethT), 113.2 (4 ϫ m-CH,
PhOMe, DMTr), 109.8 (5-C, ethT), 109.6 (5-C, aT), 86.0 (4Ј-CH,
ethT), 85.9 (C, DMTr), 83.9 (1Ј-CH, ethT), 83.6 (1Ј-CH, aT), 82.2
(4Ј-CH, aT), 75.4 (3Ј-CH, ethT), 63.0 (5Ј-CH2, aT), 59.2 (7Ј-CH,
ethT), 55.0 (2ϫCH3, DMTr), 38.2 (2Ј-CH2, ethT), 37.0 (2Ј-CH2,
aT), 29.3 (5Ј-CH2, ethT), 12.0 (5-CH3, aT), 11.8 (5-CH3, ethT)
ppm. HRMS: calcd. for C43H45N7O10 [M + Na]+ 842.3120; found
842.3126.
4-{3Ј-Deoxy-3Ј-[(2-cyanoethoxy)(diisopropylamino)phosphanyl-
oxy]-5Ј-deoxythymidin-5Ј-yl}-1-[3Ј-deoxy-5Ј-O-(4,4Ј-dimethoxy-
trityl)thymidin-3Ј-yl]-1H-1,2,3-triazole (9): Dinucleoside 8 (0.35 g,
0.43 mmol) and tetrazole (45 mg, 0.64 mmol) were dissolved in pyr-
idine (5 mL), and the solvent was removed in vacuo. This operation
was repeated twice. The residue was dissolved in of CH2Cl2 (5 mL).
The mixture was stirred in the presence of molecular sieves at room
temp. for 30 min, and 2-cyanoethyl N,N,NЈ,NЈ-tetraisopropylphos-
phoramidite (148 μL, 0.64 mmol) was added. The resulting mixture
was stirred at room temp. for a further 30 min, then poured into
cold saturated NaHCO3 (50 mL) and extracted with CH2Cl2
(3ϫ40 mL). The combined organic layers were washed with water,
dried with Na2SO4, filtered and concentrated. The residue was
purified by flash column chromatography with ethanol/CH2Cl2/
TEA (2–3:100:0.1, v/v/v) as eluent to give amidite 9 as a hard foam
(0.31 g, 72% yield). Rf = 0.26 (solvent system C). 31P NMR
(162 MHz, DMSO): δ = 150.29, 150.02 ppm. HRMS: calcd. for
C52H62N9O11P [M + Na]+ 1042.4199; found 1042.4188.
3
6.81 (m, 23 H, ArH, DMTr, TBDPS), 6.39 (t, J1Ј,2Јa(2Јb) = 6.5 Hz,
3
3
1 H, 1Ј-H, aT), 6.24 (dd, J1Ј,2Јa = 6.3 Hz, J1Ј,2Јb = 7.9 Hz, 1 H,
1Ј-H, ethT), 5.49–5.38 (m, 1 H, 3Ј-H, aT), 4.39–4.33 (m, 1 H, 3Ј-
3
H, ethT), 4.27–4.21 (m, 1 H, 4Ј-H, aT), 4.15 (td, J3Ј,4Ј = 2.4 Hz,
3J4Ј,5Јa(5Јb) = 6.7 Hz, 1 H, 4Ј-H, ethT), 3.72 (s, 6 H, CH3O, DMTr),
3.29–3.24 (m, 2 H, 5Ј-CH2, aT), 2.82–2.73 (m, 2 H, 5Ј-CH2, ethT),
2.75–2.63 (m, 2 H, 2Ј-CH2, aT), 2.13–2.03 (m, 2 H, 2Ј-CH2, ethT),
4
4
1.73 (d, J = 1.2 Hz, 3 H, 5-CH3, aT), 1.60 (d, J = 1.1 Hz, 3 H,
5-CH3, ethT), 1.00 (s, 9 H, CH3, tBu) ppm. 13C NMR (101 MHz,
DMSO): δ = 163.6 (4-C, ethT), 163.5 (4-C, aT), 158.1 (2ϫp-C,
PhOMe, DMTr), 150.3 (2-C, ethT, aT), 144.5 (6Ј-C, ethT), 143.1
(C, Ph, DMTr), 136.2 (6-CH, ethT), 135. 9 (6-CH, aT), 135.1
(4ϫo-CH, Ph, TBDPS), 135.1 (2ϫC, PhOMe, DMTr), 132.8 and
132.7 (2ϫC, Ph, TBDPS), 129.9 (2ϫp-CH, Ph, TBDPS), 129.6
(4ϫo-CH, PhOMe, DMTr), 127.8 (4ϫm-CH, Ph, TBDPS), 127.74
(2ϫo-CH, Ph, DMTr), 127.68 (2ϫm-CH, Ph, DMTr), 126.7 (p-
CH, Ph, DMTr), 122.1 (7Ј-CH, ethT), 113.1 (4ϫm-CH, PhOMe,
DMTr), 109.8 (5-C, ethT), 109.6 (5-C, aT), 85.9 (C, DMTr), 85.5
(4Ј-CH, ethT), 84.0 (1Ј-CH, ethT), 83.9 (1Ј-CH, aT), 82.2 (4Ј-CH,
aT), 75.3 (3Ј-CH, ethT), 63.0 (5Ј-CH2, aT), 59.1 (7Ј-CH, ethT),
54.9 (2ϫCH3, DMTr), 38.1 (2Ј-CH2, ethT), 37.0 (2Ј-CH2, aT), 29.1
(5Ј-CH2, ethT), 26.6 (3 ϫ CH3, tBu, TBDPS), 18.5 (C, tBu,
TBDPS), 12.0 (5-CH3, aT), 11.8 (5-CH3, ethT) ppm. HRMS: calcd.
for C59H63N7O10Si [M + Na]+ 1080.4298; found 1080.4276.
Supporting Information (see footnote on the first page of this arti-
cle): COSYDQF and C-H correlation NMR spectra, MALDI-
TOF mass spectra and conformational analysis details.
Acknowledgments
We thank A. K. Shchyolkina and O. F. Borisova (Engelhardt Insti-
tute of Molecular Biology) for helpful discussions. This work was
supported by the Russian Foundation for Basic Research (11-04-
00131-a and 11-04-12155-ofi-m-2011).
1-[3Ј-Deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)thymidin-3Ј-yl]-4-(5Ј-deoxy-
thymidin-5Ј-yl)-1H-1,2,3-triazole (8): To a stirred solution of dinu-
cleoside 7 (0.59 g, 0.56 mmol) in THF (1.7 mL), a solution of tetra-
butylammonium fluoride (1.7 mL, 1 m in THF) was added. The
mixture was stirred at room temperature for 2 h, diluted with satu-
rated NaHCO3 (50 mL) and extracted with CHCl3 (3ϫ40 mL).
The combined organic layers were washed with water, dried with
Na2SO4, filtered and concentrated. The residue was purified by
flash column chromatography with ethanol/CH2Cl2/TEA
(1:100:0.1, v/v/v) as eluent to give dinucleoside 8 as a hard foam
(0.45 g, 98 % yield). Rf = 0.47 (solvent system D). 1H NMR
(400 MHz, DMSO): δ = 11.35 (s, 1 H, 3-H, ethT), 11.25 (s, 1 H,
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3-H, aT), 8.06 (s, 1 H, 7Ј-H, ethT), 7.64 (d, J = 1.1 Hz, 1 H, 6-H,
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Received: November 28, 2011
Published Online: February 28, 2012
Eur. J. Org. Chem. 2012, 2173–2179
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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