Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems

Article abstract of DOI:10.1021/jo400651k

New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was

Full text of DOI:10.1021/jo400651k

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Article
Synthesis of Triazole-Linked Oligonucleotides with High Affinity to DNA
Complements and an Analysis of Their Compatibility with Biosystems
Anna M. Varizhuk, Dmitry N. Kaluzhny, Roman A. Novikov, Alexander O. Chizhov, Igor Pavlovich Smirnov,
Andrey N Chuvilin, Olga N. Tatarinova, Gleb Y. Fisunov, Galina E Pozmogova, and Vladimir L. Florentiev
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo400651k • Publication Date (Web): 03 Jun 2013
Downloaded from http://pubs.acs.org on June 7, 2013
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SYNTHESIS OF TRIAZOLEꢀLINKED
OLIGONUCLEOTIDES WITH HIGH AFFINITY
TO DNA COMPLEMENTS AND AN ANALYSIS
OF THEIR COMPATIBILITY WITH
BIOSYSTEMS
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Anna M. Varizhuk* §,‡ , Dmitry N. Kaluzhny‡, Roman A. Novikov‡, Alexandr O. Chizhov†, Igor P.
Smirnov§, Andrey N. Chuvilin§, Olga N. Tatarinova§, Gleb Y. Fisunov§, Galina E. Pozmogova§,
Vladimir L. Florentiev‡
§ Institute for PhysicalꢀChemical Medicine of Ministry of Public Health, Malaya Pirogovskaya
‡
str. 1a, 117312 Moscow, Russia; Engelhardt Institute of Molecular Biology, Vavilov str., 32,
119991 Moscow, Russia; † N.D. Zelinsky Institute of Organic Chemistry, Leninsky prospect, 47,
119991 Moscow, Russia.
*Address correspondence to: annavarizhuk@gmail.com
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Keywords: nucleosides, oligonucleotide analogs, click chemistry, hybridization
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ABSTRACT. New oligonucleotide analogs with triazole internucleotide linkages were
synthesized, and their hybridization properties were studied. The analogs demonstrated DNA
binding affinities similar to those of unmodified oligonucleotides. The modification was shown
to protect oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested
as PCR primers. Modifications remote from the 3’ꢀterminus were tolerated by polymerases. Our
results suggest that these new oligonucleotide analogs are among the most promising triazole
DNA mimics characterized to date.
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INTRODUCTION
Oligonucleotide (ON) analogs with triazole internucleotide linkages have shown great promise
in the field of antisense technology and as DNA hybridization probes.1ꢀ4 These analogs can be
obtained by the iterative click ligation of azide– and alkyneꢀbearing nucleoside derivatives5,6 or
by automated phosphoramidite synthesis with modified dinucleoside blocks.7 Although a
significant number of triazoleꢀlinked ONs have been described to date,5ꢀ9 few have demonstrated
hybridization properties that meet the criteria for duplex stability. Isobe et al. reported a triazole
modification that is particularly favorable for duplex stabilization9. Unfortunately, this
stabilization was later shown to be sequenceꢀdependent10. Nevertheless, triazoleꢀlinked ONs
remain an intensively studied class of DNA mimics, primarily due to their potential for
biocompatibility.11,12 Unlike most internucleotide modifications, some triazole modifications can
be tolerated by polymerases,13,14 which makes triazoleꢀlinked ONs attractive as candidate
diagnostic tools and therapeutic agents. Several promising types of triazole internucleotide
modifications are shown in Scheme1.
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This paper introduces a new triazole internucleotide modification. The triazole linkage
described herein is an isomer of a previously reported one (Scheme 1C).6,13 A series of ON
analogs bearing the triazole modification were obtained by automated solidꢀphase synthesis with
a modified dinucleoside phosphoramidite block. The properties of the triazoleꢀlinked ONs are
described.
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Scheme 1. Fragments of ON analogs with three different types of triazole internucleotide
modifications.6,9,19
RESULTS AND DISCUSSION
The triazoleꢀlinked dithymidine block used for incorporation into ONs was obtained by clickꢀ
ligation of two nucleoside components (Scheme 2). The azido component was synthesized from
5'ꢀOꢀbenzoylthymidine 1. An Oꢀazidomethyl group was introduced into the 3’ position of 1 in
two steps using a modified version of a method reported by Lonnberg and coworkers.15 The first
step was methylthiomethylation of the 3’ꢀhydroxide group, which could be performed either by a
modified Pummerer rearrangement,15 or by treatment with DMSO and benzoyl peroxide.16,17
Both approaches are rather efficient and afford 3’ꢀOꢀmethylthiomethyl deoxynucleoside
derivatives in comparable yields.15,16,18 We used the modified Pummerer rearrangement.
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Nucleoside 1 was treated with a mixture of dimethylsulfoxide and acetic anhydride in the
presence of acetic acid. The 3’ꢀOꢀmethylthiomethyl derivative (2) was obtained in 80% yield.
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Scheme 2. Synthesis of the phosphoramidite dinucleoside block with a triazole internucleoside
linkage. Reagents and conditions: (a) DMSO, Ac2O, AcOH; (b) Br2, LiN3, CH3CN; (c) K2CO3,
MeOH; (d) DMTrCl, pyridine; (e) CuSO4, sodium ascorbate, CH2Cl2/H2O; (f) TBAF, THF; (g)
NCCH2CH2OP(NiꢀPr2)2, 1Hꢀtetrazole, pyridine, CH2Cl2.
Nucleoside 2 was reacted with molecular bromine followed by lithium azide in dry CH3CN to
produce the 3’ꢀOꢀazidomethyl derivative (3) in 83% yield. The treatment of 3 with a K2CO3
suspension in dry methanol resulted in the removal of the benzoyl protecting group. A
dimethoxytrityl protecting group was introduced by treatment with dimethoxytrityl chloride in
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dry pyridine. The resulting azide (5) was coupled with alkyne 619 in the presence of CuSO4⋅5H2O
and sodium ascorbate to give dinucleoside 7 with a yield of 88%. The silyl protection was
removed with 0.5 M TBAF in THF. The treatment of dinucleoside 8, bearing a free hydroxyl,
with 2ꢀcyanoethylꢀN,N,N',N'ꢀtetraisopropylphosphoramidite in the presence of 1Hꢀtetrazole and
pyridine in dry CH2Cl2 afforded target phosphoramidite 9 in 74% yield.
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Dinucleoside block 9 was used directly for the solidꢀphase synthesis of modified ONs using
standard phosphoramidite protocols. The coupling time was increased to 15 minutes for the
modified phosphoramidite. No decrease in coupling efficiency was observed (98%ꢀ99% stepwise
coupling yields for both modified and unmodified amidites).
ONs bearing triazole fragments at the 5’ꢀterminus, near the 3’ꢀterminus and/or in the middle of
the chain were obtained and hybridized to complementary ssDNAs. The СD spectra of the
duplexes (Figure 1A) suggest that the modification did not cause any significant changes in
duplex geometry.
Figure 1. CD spectra (A) and melting curves (B) of the modified duplexes.
The impact of the modification on duplex stability was evaluated by UV melting experiments
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(Figure 1B). The results of the UVꢀmelting experiments are summarized in Table 1. As evident
from these data, the modification tends to destabilize duplexes slightly. However, the
destabilization is minor (average ꢁТm per linkage = ꢀ1.4°С) compared to those of previously
described isosequential duplexes bearing other triazole modifications (average ꢁТm = ꢀ5.3°С for
the triazole modification A (Scheme 1)19 and ꢀ4.0°С for the modification B (Scheme 1)10). Thus,
the new ON analogs are among the most promising triazoleꢀbearing DNA mimics in terms of
hybridization properties.
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Table 1. Sequences, MALDIꢀTOF MS data and melting temperatures of the duplexes formed by
modified ONs and their unmodified complement (duplex concentration 5ꢂ10ꢀ6 M).
m/z,
Sequence of a modified strand
Тm, °С ± 0.5, (ꢁТm, °С)
Code
[b]
found (calculated
(5'→3') [a]
for [M + H]+)
ON1
ON2
ON3
ON4
ON5
TTAACTTCTTCACATTC
XAACTTCTTCACAТТC
TTAACTTCTTCACAXC
TTAACTTCXCACAТТC
XAACTTCXCACAXC
5072.2 (5071.3)
5059.3 (5058.4)
5059.1 (5058.4)
5058.5 (5058.4)
5032.3 (5032.5)
50.3
50.0 (ꢀ0.3)
48.2 (ꢀ2.1)
49.1 (ꢀ1.2)
45.4 (ꢀ4.9)
[a] X –triazoleꢀlinked dithymidine fragment. [b] Tm difference between modified and natural duplexes
.
Because the new modification has a relatively insignificant impact on duplex geometry, we
hypothesized that it might be tolerated by ONꢀrecognizing enzymes. Oligonucleotides with a
triazole linkage isomeric to the one described here (‘isoꢀtriazole’; C in Scheme 1) are known to
be recognized by DNA and RNA polymerases.13,14 Plasmid constructs with such linkages have
proven functional in E. coli.11 Although the ‘isoꢀtriazole’ modification appeared to reduce duplex
stability (ꢁТm of approximately ꢀ8°С per modification) and caused local duplex distortion,12 it
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was tolerated in both primers and templates by Taq polymerase.13 To investigate the
compatibility of the new triazole linkage with the thermostable polymerases Taq and Pfu, we
synthesized a series of PCR primers with modifications at various positions. PCR with the Taq
polymerase was quite efficient with all but the 3’ꢀmodified primers, while the Pfu polymerase
only tolerated modifications near the 5’ terminus (Figure 2A). The sensitivity of Taq to primer
modification was evaluated more rigorously by qPCR (Figure 2B). Modifications at up to 8
nucleotides from the 3’ end resulted in a substantial decrease in the product accumulation rate..
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Figure 2. PCR with modified primers. A: Relative sensitivity of the Taq (left) and Pfu (right)
polymerases to primer modification. The first 4 lanes of the upper gels are the products of PCR
with unmodified primers. The numbers represent the positions of the modifications (number of
nucleotides from the 3’ꢀterminus) For primer sequences, see Supplementary data. B:
Accumulation kinetics curves obtained during qPCR with Taq polymerase.
Thus, the new triazoleꢀlinked ONs are generally usable as PCR primers, but only some
polymerases can tolerate the modifications, and the dependency of PCR efficiency on the
modification position should be taken into account. Modified PCR primers may be useful for
SNPꢀbased allele detection should they demonstrate an enhanced sensitivity to mismatches. To
assess the hybridization specificity of the new triazoleꢀlinked ONs, we synthesized a set of ON4
complements with mismatches opposite to the modified fragment of ON4. Melting temperatures
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of mismatched duplexes were obtained and compared with that of a fullyꢀmatched duplex.
Sequences of the mismatched duplexes and Tm data are given in Supplementary. Generally, the
triazole modification appears to impart slightly increased sensitivity to mismatches opposing the
nucleoside which precedes the triazole fragment.
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An important aspect of compatibility with biosystems is stability in biological liquids. Nonꢀ
natural internucleotide fragments are well known to confer increased stability to fully modified
ONs. We tested the effect of partial triazole modification on the rate of ON hydrolysis by DNAse
I. The triple modification of the 17ꢀmer caused an approximately 1.5ꢀ to 2ꢀfold increase in ON
halfꢀlife. The previously reported triazole modification10 (B in Scheme 1) had a similar effect on
the rate of enzymatic hydrolysis (see Supplementary data). It can be concluded that even the
partial triazole modification of internucleotide linkages may substantially improve ON
biostability.
CONCLUSION
A dinucleotide with a new triazole internucleotide modification was synthetized and
incorporated into ONs. The ONs bearing triazole fragments demonstrated high hybridization
affinity to DNA complements and increased resistance to nuclease digestion. ONs with 5’ꢀ
modified termini were functional as PCR primers. Our results suggest that these new ON analogs
are adequate mimics of native DNA with enhanced biostability and may be attractive tools for
diagnostics or the investigation of DNA structure.
EXPERIMENTAL
All reagents were commercially available unless otherwise mentioned and used without further
purification.1ꢀ(2,5,6ꢀTrideoxyꢀ3ꢀOꢀ(tertꢀbutyldiphenylsilyl)ꢀbetaꢀDꢀerythroꢀhexꢀ5ynofuranosyl)ꢀ
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5ꢀmethyluracil (6) was synthesized as previously described.19 All solvents were purchased from
Khimmid (Russia). Dichloromethane was distilled from phosphorus pentaoxide, pyridine was
distilled from calcium hydride, and THF was distilled from lithium alumohydride prior to use.
Column chromatography (CC) was performed on silica gel Kieselgel 60 (0.040ꢀ0.063 mm,
Merck, Germany). TLC was performed on silica gel Kieselgel 60 F254 precoated plates (Merck)
with detection by UV using the following solvent systems (compositions expressed as v/v):
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ethanol ꢀ methylene chloride 1:49 (A), 1:20 (B). H NMR and C NMR spectra were recorded
on a Bruker AMXIIIꢀ400 NMR spectrometer (Germany). Chemical shifts are given in parts per
million (ppm). The coupling constants (J) are given in Hz. Abbreviations used: dmT, 5'ꢀdeoxyꢀ
5'ꢀmethylenethymidine moiety; aT, 3'ꢀOꢀaminomethylthymidine moiety. Spectral assignments
were completed by COSY DQF and {1Hꢀ13C} HSQC experiments (see supporting information).
The NMR data were processed and presented using MestReNova version 7.0.3 (Mestrelab
Research SL, Spain).
Mass spectra
MALDI TOF mass spectra were acquired on a Bruker Microflex mass spectrometer (Bruker,
Germany) in a linear mode (+20 kV). Each spectrum was accumulated using 200 laser shots (N2
gas laser, 337 nm). The solution of 35g/ml of 3ꢀhydroxypicolinic acid with dibasic ammonium
citrate was used as a matrix. ESI HR mass spectra were acquired on a Bruker microTOF II
spectrometer (Germany) in a positive ion mode (capillary voltage – 4500 V, end plate offset –
500 V, interface capillary temperature 180ºC, nitrogen dry gas, 4.0 L/min, scanning range from
m/z 50 to m/z 3000 Da, scanning frequency 1 scan/s). Internal calibration was done using
Electrospray Calibration Solution (Fluka).
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Oligonucleotides
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ONs were synthesized on an Applied Biosystems 3400 DNA synthesizer (USA) using standard
phosphoramidite protocols and purified using preparative scale reverseꢀphase HPLC on a 250
mm x 4.0 mm2 Hypersil C18 column with detection at 260 nm. Chromatography of
dimethoxytrityl protected ONs was performed using 10ꢀ50% gradient of CH3CN in 0.05 M
TEAA. Detritylated oligonucleotides were further purified in 0ꢀ25% gradient of CH3CN in
TEAA buffer.
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UV-melting and CD
Melting curves of the duplexes were recorded on a Shimadzu UV 160ꢀA spectrophotometer
(Japan) using a thermostated cell in 20 mM sodium phosphate buffer, 100 mM NaCl, 01 mM
EDTA, pH 7.0, concentration of each duplex being 2.5ꢂ10ꢀ6M. Samples were denatured at 95°C
for 5 min and slowly cooled to 20°C prior to measurements. Duplex absorbance at 260 nm was
measured as a function of temperature. It was registered every 0.5°C from 20 to 70°C.
Thermodynamic parameters of duplex formation were obtained by performing nonlinear
regression analysis using DataFit version 9.0.059 (Oakdale Engineering, USA). The calculation
method taking into account temperature dependence of UV absorbance of duplexes and single
strands was applied. Circular dichroism (CD) spectra were obtained on a Jasco Jꢀ715
spectropolarimeter at 20°C using samples annealed in the same buffer and under the same
conditions as for the thermal denaturation studies. The CD values (ꢁε) are given per moles of
nucleotides.
PCR and qPCR
PCR products from 215ꢀmer and 222ꢀmer templates (fragments of a pGEM vector, Promega,
US) were generated using 2.5 ꢃL of 10× buffer in a total reaction volume of 25 ꢃL with 1 ng of
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the DNA template, 0.5 ꢃM of each primer, 0.2 mM dNTP and 0.5 units of Taq DNA polymerase
(Lytech, Russia) or Pfu DNA polymerase (Fermentas, US). 10×Taq buffer: 100mM TrisꢀHCl,
500mM KCl, 15mM MgCl2, pH 8.3; 10×Pfu buffer: 200mM TrisꢀHCl (pH 8.8 at 25°C), 100mM
KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1.0% Triton® Xꢀ100 and 1mg/ml nucleaseꢀfree BSA.
PCR cycling conditions: 95 °C (initial denaturation) for 2 min then 35 cycles of 95 °C
(denaturation) for 15 s, 55 °C (annealing) for 20 s and 72 °C (extension) for 30 s. The reaction
mixture was loaded onto a 2% agarose gel in 1 X TBE buffer. DNA bands were visualized by
staining with ethidium bromide after electrophoresis. Accumulation kinetic curves were obtained
by SYBR Green qPCR using the CFX C1000 realꢀtime PCR detection systems (BioꢀRad
Laboratories, Inc., USA). Data analysis and curve plotting was performed using the bundled
software.
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Nuclease hydrolysis
ON (0.35 nmole) was dissolved in 15 ꢃL of a reaction buffer (100 mM TrisꢀHCl (pH 7.5), 25 mM
MgCl2 and 1 mM CaCl2). A 5ꢀꢃL aliquot was taken as a control, and 1 u of DNaseI (Fermentas, US) in 1
ꢃL of storage buffer was added to the remaining solution. The mixture was incubated at 37°C for 15 min,
then an aliquot was removed and stored in a freezer at ꢀ20°C until the end of the reaction, and the mixture
was incubated at 37°C for another 15 min. Once all three samples were collected, 1 ꢃL of formamide was
added to each sample. The samples were heated to 95°C, then cooled on ice and loaded onto a denaturing
20% polyacrylamide gel for electrophoresis. The gel was run in TBE buffer (90 mM TrisꢀBorate (pH 8)
and 20 mM EDTA).
5'-O-Benzoyl-3'-O-(methylthiomethyl)thymidine (2).
5'ꢀOꢀBenzoylthymidine20 (2.1 g, 6 mmol) was dissolved in a mixture of DMSO (19 mL), acetic
acid (4 mL) and acetic anhydride (13 mL). The mixture was stirred for 3 days at room
temperature, then poured into cold saturated NaHCO3 (250 мл) and stirred for 2 more hours. The
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precipitate was filtered, washed on filter with water, airꢀdried and recrystallized from a mixture
of ethylacetate and hexane. Compound 2 was obtained as colorless needles (1.95 g, 80%), Rf
0.31 (Solvent system A).
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3
1H NMR (400 MHz, DMSO): δ = 11.31 (s, 1H, 3ꢀH), 8.00 (d, J = 7.6 Hz, 2H, oꢀH Bz), 7.68
(t, 3J = 7.4 Hz, 1H, pꢀH Bz), 7.55 (dd, 3Jo,m = 7.7 Hz, 3Jp,m = 7.4 Hz, 2H, mꢀH Bz), 7.41 (s, 1H, 6ꢀ
H), 6.17 (t, 3J1',2'a = J1',2'b = 7.1 Hz, 1H, 1'ꢀH), 4.75 (d, J = 11.5 Hz, 1H, 3'aꢀOCH2S), 4.73 (d,
3
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2J = 11.5 Hz, 1H, 3'bꢀ OCH2S), 4.59 – 4.52 (m, 2H, 3'ꢀH and 5'aꢀH), 4.48 (dd, J5'a,5'b = 11.8,
2
3J5'b,4' = 5.1 Hz, 1H, 5'bꢀH), 4.28ꢀ4.23 (m, 1H, 4'ꢀH), 2.36ꢀ2.31 (m, 2H, 2'aꢀH and 2'bꢀH), 2.10 (s,
3H, SCH3), 1.61 (s, 3H, 5ꢀCH3).
13C NMR (101 MHz, DMSO): δ 165.43 (C=O), 163.47 (4ꢀC), 150.27 (2ꢀC), 135.46 (6ꢀC),
133.50 (pꢀCH, Bz), 129.25 (C, Bz), 129.12 (oꢀCH, Bz), 128.78 (mꢀCH, Bz), 109.75 (5ꢀC), 84.14
(1'ꢀCH), 81.04 (4'ꢀCH), 75.98 (3'ꢀCH), 73.04 (OCH2S), 64.16 (5'ꢀCH2), 35.79 (2'ꢀCH2), 13.19
(CH3S), 11.78 (5ꢀCH3).
HR MS ESI: m/z 429.1083, calculated for [C19H22N2NaO6S]+ m/z 429.1091
3'-O-(Azidomethyl)-5'-O-benzoylthymidine (3).
To a stirred solution of nucleoside 2 (1.63 g, 4 mmol) in 60 mL of abs. CH3CN, Br2 (0.21 mL,
4 mmol) was added dropwise at 0°С. The mixture was stirred upon cooling for 30 min, then
lithium azide (0.98 g, 10 mmol) was added. The mixture foamed after the addition of lithium
azide. The resulting suspension was mixed for 10 min upon cooling and then for 2 hours at 60°С.
The solvent was evaporated under vacuum and the residue was dissolved in CH2Cl2 (50 mL) and
water (50 mL). The organic layer was separated, and the aqueous layer was extracted with
CH2Cl2 (2*25 mL). Combined organic layers were washed with brine, dried over Na2SO4,
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filtered and concentrated. The residue was purified by CC in 0—3% of ethanol in CH2Cl2 to give
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dinucleoside 3 as a hard foam (1.35 g, 82.5% yield). Rf = 0.33 (Solvent system A).
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1H NMR (400 MHz, DMSO): δ = 11.31 (s, 1H, 3ꢀH), 8.01 (d, J = 7.2 Hz, 2H, oꢀH Bz), 7.69
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(t, 3J = 7.2 Hz, 1H, pꢀH Bz), 7.53 (t, 3Jo,m = 3Jp,m = 7.2 Hz, 2H, mꢀH Bz), 7.40 (s, 1H, 6ꢀH), 6.18
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(t, 3J1',2'a = J1',2'b = 7.2 Hz, 1H, 1'ꢀH), 4.92 (d, J = 9 Hz, 1H, 3'aꢀOCH2N3), 4.89 (d, 2J = 9 Hz,
2
1H, 3'bꢀ OCH2N3), 4.59 – 4.50 (m, 2H, 3'ꢀH and 5'aꢀH), 4.48 (dd, J5'a,5'b = 12.0, 3J5'b,4' = 6.8 Hz,
1H, 5'bꢀH), 4.29ꢀ4.26 (m, 1H, 4'ꢀH), 2.41ꢀ2.37 (m, 2H, 2'aꢀH and 2'bꢀH), 1.61 (s, 3H, 5ꢀCH3).
13C NMR (101 MHz, DMSO): δ 165.45 (C=O), 163.49 (4ꢀC), 150.28 (2ꢀC), 135.54 (6ꢀCH),
133.52 (pꢀCH, Bz), 129.25 (C, Bz), 129.15 (oꢀCH, Bz), 128.79 (mꢀCH, Bz), 109.79 (5ꢀC), 84.00
(1'ꢀCH), 81.24 (4'ꢀCH), 81.18 (OCH2N), 78.01 (3'ꢀCH), 64.08 (5'ꢀCH2), 36.25 (2'ꢀCH2), 11.78
(5ꢀCH3).
HR MS ESI: m/z 424.1220, calculated for [C18H19N5NaO6]+ m/z 424.1228.
3'-O-(Azidomethyl)thymidine (4).
Finely powdered К2СО3 (0.22 g, 1.6 mole) was added to a solution of nucleoside 3 (1.3 g, 3.2
mmole) in 25 mL of abs. methanol and the suspension was stirred for 12 hours. Dry silica gel (20
mL) was added to the resulting transparent solution. The mixture was stirred for 10 min and the
solvent was removed in vacuum. The residue was onto CC in 10 mL of CH2Cl2 and eluted with
2ꢀ5% of ethanol in CH2Cl2 to give nucleoside 4 as a hard foam (0.9 g, 94.6% yield). Rf = 0.63
(Solvent system B).
3
1H NMR (400 MHz, DMSO): δ 11.28 (s, 1H, 3ꢀH), 7.68 (s, 1H, 6ꢀH), 6.14 (t, J1',2'a = 8.0 Hz,
3
3J1',2'b = 6.8 Hz, 1H, 1'ꢀH), 5.14 (dd, J5'OH,5'a = J5'OH 5'b = 5.2 Hz, 1H, 5'ꢀOH), 4.86 (s, 2H, 3'ꢀ
OCH2N), 4.39ꢀ4.35 (m, 1H, 3'ꢀH), 3.97ꢀ3.94 (m, 1H, 4'ꢀH), 3.63–3.58 (m, 2H, 5'aꢀH and 5'bꢀH),
2.30ꢀ2.22 (m, 2H, 2'aꢀH and 2'bꢀH),1.79 (s, 3H, 5ꢀCH3).
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13C NMR (101 MHz, DMSO): 13C NMR (101 MHz, DMSO) δ 163.59 (4ꢀC), 150.35 (2ꢀC),
135.85 (6ꢀCH), 109.46 (5ꢀC), 84.59 (1'ꢀCH), 83.67 (4'ꢀCH), 80.93 (OꢀCH2ꢀN), 78.31 (3'ꢀCH),
61.13 (5'ꢀCH2), 36.53 (2'ꢀCH2), 12.14 (5ꢀCH3).
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HR MS ESI: m/z 320.0962, calculated for [C11H15N5NaO5]+ m/z 320.0965.
3'-O-(Azidomethyl)-5'-O-(4,4'-dimethoxytrityl)thymidine (5).
Nucleoside 3 (0.85 g, 2.9 mmole) was evaporated with abs. pyridine (2x5mL) and dissolved in
5mL of abs. pyridine. Dimethoxytrityl chloride (1.19 g, 3.5 mmole) was added and the mixture
was stirred for 12 hours at room temperature. Then 0.2 mL of water was added, the mixture was
stirred for 10 more minutes and evaporated in vacuum. The residue was dissolved in 50 mL of
chloroform, washed with water (20 mL) and saturated NaHCO3 (20 mL), dried over Na2SO4, and
concentrated. The residue was purified by CC in 3% of ethanol in CH2Cl2 to give nucleoside 5
(1.6 g, 92 % yield) as a hard foam. Rf = 0.48 (Solvent system A).
1H NMR (400 MHz, DMSO): δ 11.31 (s, 1H, 3ꢀH), 7.50 (s, 1H, 6ꢀH), 7.45ꢀ6.88 (m, 13 H,
3
3
2
ArH DMTr), 6.16 (t, J1',2'a = J1',2'b = 7.2 Hz, 1H, 1'ꢀH), 4.85 (d, J = 9 Hz, 1H, 3'aꢀOCH2N),
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4.82 (d, J = 9 Hz, 1H, 3'bꢀ OCH2N), 4.49 (br.s, 1H, 3'ꢀH), 4.04 (d, J3',4' = 3.2 Hz, 1H, 4'ꢀH),
3.74 (s, 6H, CH3O DMTr), 3.27 (br.s, 2H, 5'CH2), 2.37 (br.s, 2H, 2'CH2), 1.50 (s, 3H, 5ꢀCH3).
13C NMR (101 MHz, DMSO) δ 163.52 (4ꢀC), 158.14 (pꢀC, PhOMe), 150.25 (2ꢀC), 144.55 (C,
Ph), 135.54 (6ꢀCH), 135.26 and 135.17 (C, PhOMe), 129.63 (oꢀCH, PhOMe), 127.82 (oꢀCH,
Ph), 127.58 (mꢀCH, Ph), 126.75 (pꢀCH, Ph), 113.19 (mꢀCH, PhOMe), 109.62 (5ꢀC), 85.95 (C,
DMTr), 83.71 (1'ꢀCH), 82.71 (4'ꢀCH), 81.03 (OCH2N), 78.00 (3'ꢀCH), 63.34 (5'ꢀCH2), 54.98 and
54.79 (CH3O DMTr), 36.59 (2'ꢀCH2), 11.64 (5ꢀCH3).
HR MS ESI: m/z 622.2271, calculated for [C32H33N5NaO7]+ m/z 622.2272.
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1-(5'-O-(4,4'-Dimethoxytrityl)thymidin-3'-O-methyl)-4-(3'-O-(tert-butyldiphenylsilyl)-5'-
deoxythymidin-5'-yl)-1H-1,2,3-triazole (7).
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To a stirred solution of compound 5 (0.54 g, 0.9 mmol) and 3'ꢀOꢀtertꢀbutyldiphenylsilylꢀ5'ꢀ
deoxyꢀ5'ꢀethynylthymidine19 (0.4 g, 0.8 mmol) in dichloromethane (5 mL) and water (5 mL),
CuSO4⋅5 H2O (30 mg, 0.13 mmol) and sodium ascorbate (130mg, 0.7 mmol) were added. The
resulting solution was stirred for 3 hours at room temperature. The reaction mixture was diluted
with dichloromethane (25 mL) and brine (25 mL). The organic layer was separated, washed with
water (20 mL) and saturated NaHCO3 (20 mL), dried over Na2SO4, and concentrated. The
residue was purified by CC in 1% of ethanol in CH2Cl2 to give dinucleoside 7 as a hard foam
(0.77 g, 88/% yield). Rf = 0.74 (Solvent system B).
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1H NMR (400 MHz, DMSO): δ = 11.37 (s, 1 H, 3ꢀH), 11.26 (s, 1 H, 3ꢀH), 8.16 (s, 1 H, 6'ꢀH
dmT), 7.57 (d, 4J = 1.3 Hz, 1 H, 6ꢀH dmT), 7.49 (1 H, d, 4J = 1.1 Hz, 6ꢀH aT), 7.50 – 6.83 (m, 23
H, ArH DMTr and TBDPS), 6.42 (t, 3J1',2'a(2'b) = 7.5 Hz, 1 H, 1'ꢀH dmT), 6.24 (dd, 3J1',2'a = 6.5 Hz,
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3J1',2'b = 7.9 Hz, 1 H, 1'ꢀH, aT), 5.84 (d, J = 11.0 Hz, 1H, 3'aꢀOCH2, aT), 5.78 (d, J = 11.0 Hz,
1H, 3'bꢀOCH2, aT), 5.07 (d, 3J4',3' = 2.7 Hz, 1 H, 4'ꢀH, dmT), 4.61 ꢀ 4.52 (m, 2 H, 3'ꢀH dmT and
3'ꢀH aT), 3.93 (td, 3J3',4' = 2.9 Hz, 3J4',5'a (5'b) = 3.3 Hz, 1 H, 4'ꢀH, aT), 3.72 (s, 6 H, CH3O DMTr),
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3.15 (dd, J4',5'a = 3.3 Hz, J5'a, 5'b = 10.9 Hz, 1H, 5'aꢀH, aT), 3.10 (dd, J4',5'b = 3.3 Hz, J5'a, 5'b
=
10.9 Hz, 1H, 5'bꢀH, aT), 2.40 – 2.22 (m, 3 H, 2'ꢀCH2 dmT and 2'aꢀH aT), 2.23ꢀ2.15 (m, 1 H, 2'bꢀ
H, aT), 1.64 (d, 4J = 1.3 Hz, 3 H, 5ꢀCH3, dmT), 1.42 (d, 4J = 1.1 Hz, 3 H, 5ꢀCH3, aT), 1.01 (s, 9
H, CH3 tꢀBu).
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13C NMR (101 MHz, DMSO): C NMR (101 MHz, DMSO) δ 163.5 (4ꢀC, dmT and aT),
158.1 (pꢀC PhOMe DMTr), 150.4 and 150.2 (2ꢀC, dmT and aT), 145.6 (5'ꢀC, dmT), 144.5 (C, Ph
DMTr), 135.8 (6ꢀCH, aT and dmT), 135.1 (C, PhOMe DMTr), 135.1 and 135.0 (oꢀCH, Ph2Si),
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132.6 and 132.4 (C, PhSi), 130.0 and 129.9 (pꢀCH, Ph2Si), 129.6 (oꢀCH, PhOMe DMTr), 127.84
(mꢀCH, Ph2Si), 127.75 (oꢀCH, Ph DMTr), 127.5 (mꢀCH, Ph DMTr), 126.7 (pꢀCH, Ph DMTr),
124.0 (6'ꢀCH, dmT), 113.2 (mꢀCH, PhOMe DMTr), 109.8 (5ꢀC, dmT), 109.7 (5ꢀC, aT), 86.1 (C,
DMTr), 84.3 (1'ꢀCH, dmT), 83.6 (1'ꢀCH, aT), 82.8 (4'ꢀCH, aT), 79.9 (4'ꢀCH, dmT), 78.7 (3'ꢀCH,
aT), 76.9 (3'ꢀCH, dmT), 76.6 (OCH2N), 63.5 (5'ꢀCH2, aT), 55.0 (CH3O DMTr), 38.9 (2'ꢀCH2,
dmT), 36.7 (2'ꢀCH2, aT), 26.6 (CH3, tꢀBu), 18.5 (C, tꢀBu), 12.0 (5ꢀCH3, dmT) , 11.5 (5ꢀCH3, aT).
HR MS ESI: m/z 1096.4252. Calculated for [C59H63N7NaO11Si]+ m/z 1096.4247.
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1-(5'-O-(4,4'-Dimethoxytrityl)thymidin-3'-O-methyl)-4-(5'-deoxythymidin-5'-yl)-1H-
1,2,3-triazole (8).
To a stirred solution of dinucleoside 7 (0.77 g, 0.7 mmol) in dry THF (1.4 mL), 1M solution of
tetrabutylammonium fluoride in dry THF (1.4 mL) was added. The mixture was stirred for 2
hours at room temperature, diluted with saturated NaHCO3 (50 mL) and extracted with CHCl3
(3×40 mL). Combined organic layers were washed with water, dried over Na2SO4, filtered and
concentrated. The residue was purified by CC in 6% of ethanol in CH2Cl2 + 0.1% TEA to give
dinucleoside 8 as a hard foam (0.58 g, 99% yield). Rf = 0.43 (Solvent system B).
1H NMR (400 MHz, DMSO): δ = 11.32 (s, 1 H, 3ꢀH), 11.28 (s, 1 H, 3ꢀH), 8.39 (s, 1 H, 6'ꢀH,
dmT), 7.66 (d, 4J = 1.0 Hz, 1 H, 6ꢀH, dmT), 7.46 (1 H, d, 4J = 0.8 Hz, 6ꢀH, aT), 7.38 – 6.84 (m,
13 H, ArH DMTr), 6.34 (t, 3J1',2'a(2'b) = 7.1 Hz, 1 H, 1'ꢀH dmT), 6.14 (t, 3J1',2'a(2'b) = 6.2 Hz, 1 H, 1'ꢀ
H, aT), 5.86 (d, 2J = 11.0 Hz, 1H, 3'aꢀOCH2, aT), 5.84 (d, 2J = 11.0 Hz, 1H, 3'bꢀOCH2, aT), 5.57
(d, 3J = 4.5 Hz, 1H, 3'ꢀOH, dmT), 4.93 (d, 3J4',3' = 2.9 Hz, 1 H, 4'ꢀH, dmT), 4.61 ꢀ 4.56 (m, 1 H,
3'ꢀH, aT), 4.47 ꢀ 4.41 (m, 1 H, 3'ꢀH dmT), 3.98 (td, 3J3',4' = 3.0 Hz, 3J4',5'a (5'b) = 3.9 Hz, 1 H, 4'ꢀH,
aT), 3.73 (s, 6 H, CH3O DMTr), 3.20 (dd, 3J4',5'a = 3.9 Hz, J5'a, 5'b = 10.9 Hz, 1H, 5'aꢀH, aT), 3.12
3
(dd, J4',5'b = 3.9 Hz, J5'a, 5'b = 10.9 Hz, 1H, 5'bꢀH, aT), 2.47 – 2.39 (m, 1 H, 2'aꢀH, dmT), 2.35ꢀ
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2.21 (m, 3 H, 2'ꢀCH2 aT and 2'bꢀH dmT), 1.67 (d, J = 1.3 Hz, 3 H, 5ꢀCH3, dmT), 1.58 (d, J =
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1.1 Hz, 3 H, 5ꢀCH3, aT).
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13C NMR (101 MHz, DMSO): δ 163.51 (4ꢀC, aT), 163.47 (4ꢀC, dmT), 158.1 (pꢀC PhOMe
DMTr), 150.4 and 150.2 (2ꢀC, dmT and aT), 146.4 (C, Ph DMTr), 144.5 (5'ꢀC, dmT), 135.9 (6ꢀ
CH, dmT), 135.3 (6ꢀCH, aT), 135.3 (C, PhOMe DMTr), 129.6 (oꢀCH, PhOMe DMTr), 127. 9
(oꢀCH, Ph DMTr), 127.5 (mꢀCH, Ph DMTr), 126.7 (pꢀCH, Ph DMTr), 123.9 (6'ꢀCH, dmT),
113.2 (mꢀCH, PhOMe DMTr), 109.7 (5ꢀC, dmT), 109.5 (5ꢀC, aT), 86.0 (C, DMTr), 84.3 (1'ꢀCH,
dmT), 83.7 (1'ꢀCH, aT), 82.7 (4'ꢀCH, aT), 79.9 (4'ꢀCH, dmT), 78.4 (3'ꢀCH, aT), 76.5 (OCH2N),
74.1 (3'ꢀCH, dmT), 63.5 (5'ꢀCH2, aT), 55.9 (CH3O DMTr), 38.7 (2'ꢀCH2, dmT), 36.5 (2'ꢀCH2,
aT), 12.1 (5ꢀCH3, dmT) , 11.5 (5ꢀCH3, aT).
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HR MS ESI: m/z 858.3076, calculated for [C43H45N7 Na O11]+ m/z 858.3069.
1-(5'-O-(4,4'-Dimethoxytrityl)thymidin-3'-O-methyl)-4-(3'-deoxy-3'-((2-
cyanoethoxy)(diisopropylamino)phosphinooxy)-5'-deoxythymidin-5'-yl)-1H-1,2,3-triazole
(9).
Dinucleoside 8 (0.59 g, 0.7 mmol) and tetrazole (98 mg, 1.4 mmol) were evaporated with abs.
pyridine (2×5 mL) and dissolved in abs. CHsCN, The mixture was stirred over molecular sieves
for 30 min at room temperature. 2ꢀCyanoethylꢀN,N,N’,N’ꢀtetraisopropylphosphoramidite
(324ꢃL, 1.4 mmol) was added. The mixture was stirred for 3 hours at room temperature, then
poured into 50 mL of cold saturated NaHCO3 (50 mL) and extracted with CH2Cl2 (3×40 mL).
Combined organic layers were washed with water, dried over Na2SO4, filtered and concentrated.
The residue was purified by CC in 2—3% of ethanol in CH2Cl2 + 0.1% TEA to give amidite 9 as
a hard foam (0.42 g, 74% yield). Rf = 0.45 (Solvent system B).
31P NMR (162 MHz, DMSO): δ = 150.84, 150.51 ppm.
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1H NMR (400 MHz, DMSO): δ = 11.26 (br.s, 2 H, 3ꢀH aT and dmT), 8.44; 8.421 (s, 1 H, 6'ꢀH,
dmT), 7.64; 7.63 (s, 1 H, 6ꢀH, dmT), 7.45 (1 H, s, 6ꢀH, aT), 7.38 – 6.85 (m, 13 H, ArH DMTr),
6.36; 6.33 (t, 3J1',2'a(2'b) = 8.0 Hz, 1 H, 1'ꢀH dmT), 6.14 (t, 3J1',2'a(2'b) = 6.5 Hz, 1 H, 1'ꢀH, aT), 5.95ꢀ
5.77 (m, 2H, 3'ꢀOCH2), 5.15; 5.04 (d, 3J4',3' = 2.8 Hz, 1 H, 4'ꢀH, dmT), 4.79 ꢀ 4.66 (m, 1 H, 3'ꢀH,
dmT), 4.62 ꢀ 4.55 (m, 1 H, 3'ꢀH, aT), 3.99 (td, 3J3',4' = 3.0 Hz, 3J4',5'a (5'b) = 3.9 Hz, 1 H, 4'ꢀH, aT),
3.78ꢀ3.62 (m, 2H, POCH2), 3.73 (s, 6 H, CH3O DMTr), 3.62ꢀ3.47 (m, 2H, CH iꢀPr), 3.22 (dd,
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3J4',5'a = 3.4 Hz, J5'a, 5'b = 10.4 Hz, 1H, 5'aꢀH, aT), 3.13 (dd, 3J4',5'a = 3.9 Hz, J5'a, 5'b = 10.5 Hz, 1H,
3
5'bꢀH, aT), 2.75; 2.70 (t, J = 4.6 Hz, 2 H, CH2CN), 2.64ꢀ2.56 (m, 1 H, 2'aꢀH, dmT ), 2.44ꢀ2.35
(m, 1 H, 2'bꢀH dmT), 2.35ꢀ2.15 (m, 2 H, 2'ꢀCH2, aT ), 1.70; 1.67 (s, 3 H, 5ꢀCH3, dmT), 1.41 (s, 3
H, 5ꢀCH3, aT), 1.13; 1.09 (d, 4J = 5.9 Hz, 3 H, CH3 iꢀPr).
13C NMR (101 MHz, DMSO): δ 163.5 (4ꢀC, dmT and aT, 158.2 (pꢀC PhOMe DMTr), 150.4
and 150.3 (2ꢀC, dmT and aT), 145.7 (C, Ph DMTr), 144.5 (5'ꢀC, dmT), 135.9 (6ꢀCH, dmT),
135.3 (6ꢀCH, aT), 134.9 (C, PhOMe DMTr), 129.6 (oꢀCH, PhOMe DMTr), 127.84 (mꢀCH,
Ph2Si), 127.79 (oꢀCH, Ph DMTr), 127.6 (mꢀCH, Ph DMTr), 126.7 (pꢀCH, Ph DMTr), 122.9 (6'ꢀ
CH, dmT), 118.8 (CN, CH2CN), 113.2 (mꢀCH, PhOMe DMTr), 109.7 (5ꢀC, dmT and aT), 86.1
(C, DMTr), 84.3 (1'ꢀCH, dmT), 83.5 (1'ꢀCH, aT), 82.7 (4'ꢀCH, aT), 76.6 (4'ꢀCH, dmT and 3'ꢀCH,
aT), 76.4 (OCH2N), 63.5 (5'ꢀCH2, aT), 58.4 (POCH2), 42.6 (CH, iꢀPr), 55.0 (CH3O DMTr), 37.9
(2'ꢀCH2, dmT), 36.6 (2'ꢀCH2, aT), 24.3 (CH3, iꢀPr), 19.6 (CH2, CH2CN), 12.1 (5ꢀCH3, dmT) ,
11.5 (5ꢀCH3, aT).
HR MS ESI: m/z 1058.4144, calculated for [C52H62N9NaO12P]+ m/z 1058.4148.
1 Here and further chemical shifts of the diastereomers are separated by semicolons
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Supporting Information. 1. Sequences of PCR primers and MALDIꢀTOF MS data; 2.
Electrophoretic analysis of DNAse I cleavage of native and triazoleꢀmodfied ONs; 3. Sequences,
MALDIꢀTOF MS data, Tm values and UVꢀmelting profiles of duplexes with mismatches. 4.
NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGMENT
We thank V.A. Karpov (Institute for PhysicalꢀChemical Medicine) for ON synthesis. This work
was supported by Russian Foundation for Basic Research [11ꢀ04ꢀ00131ꢀа].
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