Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents

Article abstract of DOI:10.1016/j.bioorg.2011.03.002

Triazolyl-functionalized oligonucleotide (ON) analogs have received much attention as potential antitumor and antiviral agents. The most promising of such analogs are those exhibiting high binding affinity toward native DNA/RNA, since they may prove to be

Full text of DOI:10.1016/j.bioorg.2011.03.002

Bioorganic Chemistry 39 (2011) 127–131
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and hybridization data of oligonucleotide analogs with triazole
internucleotide linkages, potential antiviral and antitumor agents
Anna Varizhuk a, Alexandr Chizhov b, Vladimir Florentiev a,
⇑
a Engelhardt Institute of Molecular Biology, Vavilov str., 32, 119991 Moscow, Russia
b N.D. Zelinsky Institute of Organic Chemistry, Leninsky prospect, 47, 119991 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 February 2011
Available online 15 March 2011
Triazolyl-functionalized oligonucleotide (ON) analogs have received much attention as potential antitu-
mor and antiviral agents. The most promising of such analogs are those exhibiting high binding affinity
toward native DNA/RNA, since they may prove to be efficient antisense or siRNA agents. To date, rela-
tively few ON analogs with triazole internucleotide linkages have been described. In this paper, we report
an improved synthesis of a modified dinucleoside phosphoramidite and hybridization data of ON analogs
with four-bond triazole internucleotide linkages. We believe these data are essential for comprehensive
analysis of the relation between the length of triazole internucleotide linkages and duplex stability.
Ó 2011 Elsevier Inc. All rights reserved.
Keywords:
Click chemistry
Oligonucleotide analogs
Hybridization
1. Introduction
are those exhibiting high binding affinity toward native DNA/
RNA, since they may prove to be efficient antisense or siRNA agents
Click chemistry is a relatively new synthetic approach to joining
organic molecules which mimics the approach used by nature. It
takes advantage of reactions that are stereospecific and proceed
in high yields under mild conditions in the presence of a diverse
range of functional groups. Huisgen CuI-catalyzed [3 + 2] azide-al-
kyne cycloaddition (CuAAC), one of the most popular and efficient
reactions within the concept of click chemistry [1], has recently
gained significant importance in nucleic acids field, notably oligo-
nucleotide labeling, immobilization, cross-linking and ligation
[2,3]. The reaction leads to formation of 1,4-disubstituted 1,2,3-tri-
azoles. A number of triazolyl-functionalized ON analogs have been
synthesized using 1,3-dipolar cycloaddition, including base modi-
fied ON analogs, sugar modified analogs and the analogs with tria-
zole rings in internucleotide linkages. Triazolyl-functionalized ON
analogs have received much attention because of their potential
antitumor and antiviral properties. In view of this therapeutic as-
pect, possibility of metal coordination, low toxicity of triazole rings
and their remarkable stability may prove to be of use. Incorpora-
tion of triazole rings in internucleotide linkages is a way to gener-
ate promising ON analogs with enhanced resistance to chemical
and enzymatic degradation. The most promising of such analogs
[4,5].
To date, relatively few ON analogs with triazole internucleotide
linkages have been described. One of the noteworthy triazoliyl-
linked ONs is oligo-dT analog synthesized by Isobe et al. [6], which
has been reported to form extremely stable duplex with its DNA
complement (DTm per modification = 4.6 °C). It has been sug-
gested that the six-bond backbone periodicity (from C30i to C3i0þ1),
characteristic of the abovementioned analog, is essential for duplex
stability since longer triazole internucleotide linkages have been
shown to destabilize duplexes [7,8]. The comparison between four-
and five-bond (from C40 to C30) triazole linkages (Scheme 1) has not
been made so far. Fully modified ON analogs with four-bond tria-
zole linkages have been reported by Nuzzi et al. [9]. Their study
has been continued by Chandrasekhar et al., who considered mod-
ification throughout the chain to be a limiting factor for further
exploration and synthesized dinucleoside phosphoramidite
8
(Scheme 2) to overcome this [10]. Single and multiple incorpora-
tion of a modified dinucleoside in ON chains at requisite sites al-
lows evaluating the influence of the number and the positions of
modifications on duplex stability. Modified dinucleoside block 8
has been successfully utilized in solid-phase ON synthesis. How-
ever, no data on DNA binding affinity of thus obtained ON analogs
has been reported. In this paper, we report on improved synthesis
of modified dinucleoside phosphoramidite 8 and hybridization
data of ON analogs with four-bond triazole internucleotide link-
ages. We believe these data are essential for comprehensive anal-
ysis of the relation between the length of triazole internucleotide
linkages and duplex stability.
Abbreviations: CuAAC, CuI-catalyzed [3 + 2] azide-alkyne cycloaddition; EDTA,
ethylenediamine tetra-acetic acid; IBX, 2-iodoxybenzoic acid; ON, oligonucleotide;
TEA, triethylamine; TEAA, triethylammonium acetate; TBAF, tetrabutylammonium
fluoride; THF, tetrahydrofuran.
⇑
Corresponding author. Fax: +7 499 135 1405.
E-mail address: flor@imb.ac.ru (V. Florentiev).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.03.002

128
A. Varizhuk et al. / Bioorganic Chemistry 39 (2011) 127–131
O
N
O
N
group transformation was Ohira–Bestmann reaction [16], and it
enabled us to avoid the intermediate step. Aldehyde 2 was reacted
with dimethyl-1-diazo-2-oxopropylphosphonate (Ohira–Best-
mann reagent), generated in situ from tosyl azide (prepared fol-
lowing the published procedure [17]) and dimethyl-2-
oxopropylphosphonate (prepared following the published proce-
dure [18]), in the presence of K2CO3 and methanol in abs. acetoni-
trile (24 h, 20 °C) to give 4 in a yield of 87%. Compound 4 was
subjected to cycloaddition with 30-azido-30-deoxy-50-O-dimeth-
oxytritylthymidine 5 in the presence of CuSO4ꢁ5H2O and sodium
ascorbate. Compounds 4 and 5 being poorly soluble in aqueous
medium and such typical solvent systems for CuAAC as H2O/tBuOH
or H2O/ethanol, we used a two-phase solvent system, CH2Cl2/H2O
[19]. Although CH2Cl2 is commonly used as a solvent for organic li-
gands in ligand-mediated CuAAC, in ligand-free CuAAC this solvent
system is quite new. The use of this system has been reported to
increase reaction rates. Indeed, the reaction of azide 5 and alkyne
4 in CH2Cl2/H2O afforded triazole dinucleoside 6 in 75% yield after
2 h (20 °C). The silyl protection was removed with 0.5 M TBAF in
THF (2 h, 20 °C). Treatment of thus obtained dinucleoside 7 with
2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphoramidite in the pres-
ence of 1H-tetrazole and pyridine in dichloromethane (2 h, 20 °C)
afforded target phosphoramidite 8 in 65% yield. Nucleoside 4 and
dinucleosides 6–8 were characterized by ESI HRMS and the struc-
tures of 6 and 7 were confirmed by COSY NMR spectroscopy (see
Fig. 1 for the spectrum of compound 7 and supplementary data
for the spectrum of compound 6). COSY NMR experiments enabled
assignment of signals to protons of 50-deoxy-50-methylenethymi-
dine and 30-amino-30-deoxythymidine moieties in 1H NMR spectra
of the dinucleosides, and CH correlation NMR experiments enabled
assignment of 13C NMR spectra of the dinucleosides (for 13C and C–
H correlation NMR spectra, see supplementary data).
NH
NH
O
O
O
O
O
N
O
NH
N
N
NH
N
N
N
N
O
N
O
O
O
A
B
Scheme 1. Fragments of modified ONs with four-bond and five-bond triazole
linkages.
2. Results and discussion
We started the synthesis of the target dinucleoside block with
the preparation of a key intermediate, acetylenic derivative 4
(Scheme 2). Synthesis of 4 from easily accessible 30-O-(tert-butyldi-
phenylsilyl)thymidine 1 [11] was realized in 2–3 steps. Alcohol 1
was oxidized with IBX in abs. ethyl acetate (4 h, 80 °C) [12].
Remarkably simple work-up procedure, including filtration and re-
moval of the solvent, afforded aldehyde 2 in near quantitative
yield. To prepare acetylenic derivative 4 from aldehyde 2, we em-
ployed two methods. The first method included two steps. Like
Chandrasekhar and et al., we converted aldehyde to terminal al-
kyne via intermediate dibromomethylene derivative. While Chan-
drasekhar et al. employed Corey–Fuchs [13] protocol to prepare
the dibromomethylene derivative, we employed a Wittig-type con-
densation of aldehyde 2 with the ylide derived from dibromometh-
yltriphenylphosphonium bromide (prepared by Wolkoff’s method
[14]) in the presence of Zn in abs. dioxane under reflux (3 h), which
gave compound 3 in 80% yield. (Condensation of aldehydes with
the abovementioned ylide has been reported as the first stage of
a one-pot procedure for synthesis of alkynes from aldehydes
[15]. In that procedure, t-BuOK was used to generate ylide in the
first stage and to convert dibromo derivatives to target alkynes
in the second stage. However, the yield of compound 7 obtained
by this general procedure was disappointingly low). Compound 3
was treated with BuLi in abs. THF (0.5 h, ꢀ70 °C) to give compound
4 in 83% yield. The second method we used for formyl-to-ethynil
Dinucleoside block 8 was utilized directly for solid-phase syn-
thesis of modified ONs using standard phosphoramidite protocols.
Coupling time was increased to 15 min for the modified phospho-
ramidite. No decrease in coupling efficiency was observed (98–99%
step-wise coupling yields for both modified and unmodified ami-
dites). Along with the modified ONs, a wild-type complement
and isosequential ONs were synthesized. Purification of the ONs
was carried out by reverse-phase HPLC. All the ONs were charac-
terized by MALDI mass spectrometry (Table 1). Thermal dissocia-
tion of the modified duplexes and their wild-type counterpart
O
N
O
N
O
O
NH
NH
NH
NH
Br
HO
O
O
O
N
O
N
O
Br
O
O
O
O
a
b
c
OTBDPS
OTBDPS
OTBDPS
OTBDPS
1
2
3
4
d
O
N
O
N
O
N
NH
NH
NH
DMTrO
O
N
O
O
DMTrO
DMTrO
f
O
O
O
N
NH
O
O
O
N
O
N
DMTrO
NH
N
O
N
N
O
NH
NH
N
N
e
g
4 +
N
N
N
O
O
O
O
O
N
N3
O
O
5
O
P
OTBDPS
6
OH
CN
(i-Pr)2N
7
8
Scheme 2. Synthesis of the phosphoramidite dinucleosideblockwith
a triazole internucleoside linkage. Reagents and conditions: (a) IBX, ethylacetate, 80 °C; (b)
PPh3CHBr2+Brꢀ, Zn, dioxane, reflux; (c) n-BuLi, THF, ꢀ70 °C;(d) Ohira–Bestmann reagent, K2CO3, MeOH, acetonitrile; (e) CuSO4, ascorbate Na, CH2Cl2/H20; (e) and (f) TBAF,
THF; (g) NCCH2CH2OP(NPri )2, 1H-tetrazole, pyridine, CH2Cl2.
2

A. Varizhuk et al. / Bioorganic Chemistry 39 (2011) 127–131
129
ArH (DMTr)
H6 (aT) H6 (dmT)
H6' (dmT)
2
3
4
5
6
7
8
8.0
7.0
6.0
5.0
4.0
3.0
2.0
f2 (ppm)
Fig. 1. COSY NMR spectrum of compound 6. dmT = 50-deoxy-50-methylenethymidine moiety; aT = 30-amino-30-deoxythymidine moiety.
in extra homologation step). However, short linkages distort the
double helix. Our results add to the growing body of evidence that
the six-bond (from C30i to C3i0þ1) backbone periodicity is crucial for
the double-strand formation.
Table 1
MALDI-TOF mass spectra of ONs and melting temperatures of the corresponding
duplexes (duplex concentration 2.5 ꢂ 10ꢀ6 M).
Sequence
m/z, found (calculated for
Tm, °C 0.5, (
DTm,
(50 ? 30)*
[M + H]+)
°C**
)
TTAACTTCACATTC
TTAACTTCACAXC
XAACTTCACATTC
TTAACXCACATTC
XAACXCACAXC
50.3
4. Experimental
5030 (5028.37)-
5029 (5028.37)
5030 (5028.37)
4944 (4942.45)
47.3 (ꢀ3.0)
49.6 (ꢀ0.7)
39.9 (ꢀ10.4)
33.0 (ꢀ17.3)
All reagents were commercially available unless otherwise
mentioned and used without further purification. 30-Deoxy-30-azi-
dothymidine was provided by Association AZT (Russia). All sol-
vents were purchased from Khimmed (Russia). Dioxane was
dried over sodium hydroxide and distilled, dichloromethane was
distilled from phosphorus pentaoxide, pyridine was distilled from
calcium hydride, and THF was distilled from lithium alumohydride
prior to use. Flash column chromatography (CC) was performed on
silica gel Kieselgel 60 (0.040–0.063 mm, Merck, Germany). TLC was
performed on silica gel Kieselgel 60 F254 precoated plates (Merck)
with detection by UV using the following solvent systems (compo-
Notes:
*
X – modified dinucleoside fragment with a triazole internucleotide linkage.
Tm difference between modified and natural duplexes.
**
was measured. The resulting curves enabled evaluation of the
melting temperatures of the duplexes (Table 1).
As evident from Table 1, the modification causes destabiliza-
tion, the extent of which depends on the position of the modified
fragment. Modification in the middle of the strand dramatically de-
creases duplex stability, which is evident of serious distortion of
duplex structure. 30-Terminal modification also causes significant
destabilization. Average
lated as a sum of all Tm values divided by the total number of
modifications). The overall effect of multiple modifications roughly
equals the sum of the effects of single modifications.
sitions expressed as v/v): ethanol–methylene chloride 1:65 (A),
1:49 (B), 1: 25 (C), 1:9 (D). 1H NMR and 13C NMR spectra were re-
corded on a Bruker AMXIII-400 NMR spectrometer (Germany).
Chemical shifts are given in parts per million (ppm). The coupling
constants (J) are given in Hz. Abbreviations used: dmT, 50-deoxy-
50-methylenethymidine moiety; aT, 30-amino-30-deoxythymidine
moiety. The signals were assigned to protons using COSY experi-
ments. The NMR data were processed and presented using Mest-
ReNova version 6.1.1 (Mestrelab Research SL, Spain). MALDI TOF
mass spectra were acquired on a Bruker Ultraflex II mass spec-
trometer (Germany) in a positive ion mode. ESI HR mass spectra
were acquired on a Bruker maXis spectrometer (Germany) in a po-
sitive 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 from m/z 50 to m/z 3000 Da, scanning
frequency 1scan/s). Internal calibration was done using Electro-
spray Calibration Solution (Fluka).
D
Tm per modification is ꢀ5.3 °C (calcu-
D
3. Conclusion
In summary, we have reported on improved synthesis of the
dinucleoside phosphoramidite block, utilization of the modified
block in ON synthesis and hybridization data of thus obtained
ON analogs. The data reported provide the missing link in compar-
ative analysis of various triazolyl modifications. ON analogs with
four-bond (from C40 to C30) internucleotide linkages seem an
attractive alternative to the analogs with five-bond linkages since
preparation of the former requires less synthetic effort (no need

130
A. Varizhuk et al. / Bioorganic Chemistry 39 (2011) 127–131
ONs were synthesized on an Applied Biosystems 3400 DNA syn-
(30 mL) was added. The mixture was extracted with dichlorometh-
ane (3 ꢂ 30 mL). Combined organic layers were washed with water
(50 mL), dried over Na2SO4 and concentrated. The residue was
purified by CC in 1% of ethanol in CH2Cl2 to give compound 4 as
a colorless foam (665 mg, 83% yield). Rf = 0.48 (Solvent system A).
thesizer (USA) using standard phosphoramidite protocols and puri-
fied using preparative scale reverse-phase HPLC on a 250 mm ꢂ
4.0 mm2 Hypersil C18 column with detection at 260 nm. Chroma-
tography of dimethoxytrytil-protected ONs was performed using
10–50% gradient of CH3CN in 0.05 M TEAA. Detritylated oligonucle-
otides were further purified in 0–25% gradient of CH3CN in TEAA
buffer. Melting curves of the duplexes were recorded on a Shima-
dzu UV 160-A spectrophotometer (Japan), equipped with a ther-
mostatic system, in 20 mM sodium phosphate buffer, 100 mM
NaCl, 01 mM EDTA, pH 7.0, concentration of each duplex being
2.5 ꢂ 10ꢀ6 M. Samples were denatured at 95 °C for 5 min and
slowly cooled to 20 °C prior to measurements. A260 (duplex absor-
bance) was measured as a function of temperature registered every
0.5 °C from 20 to 70 °C. Thermodynamic parameters of duplex for-
mation were obtained by performing nonlinear regression analysis
using DataFit version 9.0.059 (Oakdale Engineering, USA). The cal-
culation method taking into account temperature dependence of
UV absorbance of duplexes and single strands was applied.
4.3.2. From nucleoside 2
To a suspension of K2CO3 (829 mg, 6 mmol) and tosyl azide
(473 mg, 2.4 mmol) in abs. acetonitrile (20 mL), dimethyl-2-
oxopropylphosphonate (399 mg, 2.4 mmol) in abs. acetonitrile
(5 mL) was added. The reaction mixture was stirred for 2 h at r.t.,
then a solution of compound 2 (957 mg, 2 mmol) in abs. methanol
(6 mL) was added, and the mixture was left overnight at r.t. The
solvent was removed, and the residue was partitioned between
water (30 mL) and dichloromethane (50 mL). The aqueous layer
was extracted with dichloromethane (30 mL). Combined organic
layers were washed with brine (30 mL), dried over Na2SO4 and
concentrated. The residue was purified by CC in 1% of ethanol in
CH2Cl2 to give compound 4 as a colorless foam (823 mg, 87% yield).
Rf = 0.48 (Solvent system A). 1H NMR (400 MHz, DMSO-d6):
4.1. 1-(2-Deoxy-3-O-tert-butyldiphenylsilyl-b-
D
-erythro-pentadialdo-
d = 11.32 (1 H, s, H3), 7.67–7.42 (10 H, m, ArH6), 7.40 (1 H, d, 4J
4
1.1, H6), 6.34 (1 H, t, J 7.1, H10), 4.57 (1 H, t, J4 ,3 2.2, J4 ,6 2.2,
0
0
0
0
1,4-furanosyl)-5-methyluracil (2)
4
H40), 4.53–4.49 (1 H, m, H30), 3.75 (1H, d, J4 ,6 2.2, H60), 2. 26–
2.21 (2 H, m, H20a and H20b), 1.75 (3 H, d, 4J 1.1, 5-CH3), 1.04 (9
H, s, t-BuSi). MS: m/z Calcd for C27H30N2O4Si, [M + Na]+ 497.1867.
Found: 497.1871.
0
0
30-O-(tert-Butyldiphenylsilyl)thymidine
1 (0.9 g, 1.87 mmol)
was dissolved in abs. ethyl acetate (13 mL), and IBX (1.57 g,
5.62 mmol) was added. The resulting suspension was immersed
in an oil bath set to 80 °C and stirred vigorously open to the atmo-
sphere. After 3.5 h, the reaction mixture was cooled to room tem-
perature and filtered. The filter cake was washed with 2 ꢂ 10 mL of
ethyl acetate, and the combined filtrates were concentrated to pro-
vide 877 mg (97% yield, >95% pure by 1H NMR) of the desired alde-
hyde 2 as a white foam. The aldehyde 2 was used without
purification for the next step. Rf = 0.42 (Solvent system C). 1H
NMR (400 MHz, DMSO-d6): d = 11.31 (1 H, s, H3), 9.35 (1 H, d,
4.4. 1-(30-Deoxy-50-O-(4,40-dimethoxytrityl)thymidine-30-yl)-4-(30-O-
(tert-butyldiphenylsilyl)-40-de(hydroxymethyl)thymidine-40-yl)-1H-
1,2,3-triazole (6)
To a stirred solution of compound 4 (300 mg, 0.63 mmol) and
30-azido-30-deoxy-50-O-dimethoxytritylthymidine
5
(396 mg,
0.7 mmol) in dichloromethane (3 mL) and water (3 mL), Cu-
SO4ꢁ5H2O (10 mg, 0.063 mmol) and sodium ascorbate (39 mg,
0.21 mmol) were added. The resulting solution was stirred for
2 h at r.t. The reaction mixture was diluted with dichloromethane
(5 mL) and brine (5 mL). The organic layer was separated, dried
over Na2SO4 and concentrated. The residue was purified by CC in
1% of ethanol in CH2Cl2 to give dinucleoside 6 as a hard foam
(497 mg, 75% yield). Rf = 0.37 (Solvent system B). 1H NMR
(400 MHz, DMSO-d6): d = 11.38 (1 H, s, H3), 11.30 (1 H, s, H3),
8.13 (1 H, s, H60 dmT), 7.66 (1 H, d, 4J 0.9, H6 aT), 7.54 (1 H, d, 4J
1.0, H6 dmT), 7.62–6.81 (23 H, m, ArH DMTr and Ph2Si), 6.46 (1
H, t, J 7.0, H10 dmT), 6. 34 (1 H, t, J 6.0, H10 aT), 5.55–5.47 (1 H,
J5 ,4 0.4, H50), 7.69–7.38 (11 H, m, ArH and H6), 6.33 (1 H, t, J 7.1,
0
0
H10), 4.79–4.72 (1 H, m, H30), 4.48 (1 H, d, J3 ,4 1.6, H40), 2.12 (2
H, m, H20a and H20b), 1.76 (3 H, d, 4J 0.8, 5-CH3), 1.06 (9 H, s, t-
BuSi).
0
0
4.2. 1-(2,5,6-Trideoxy-6,6-dibromo-3-O-(tert-butyldiphenylsilyl)-b-D-
erythro-hex-5-enofuranosyl)-5-methyluracil (3)
To a stirred solution of nucleoside 2 (0.86 g, 1.8 mmol) and dibro-
momethyltriphenylphosphonium bromide (1.85 g, 3.59 mmol) in
abs. dioxane (4 mL), 260 mg (3.95 mmol) of Zn was added. The reac-
tion mixture was stirred at reflux for 3 h, then cooled to r.t. and fil-
tered. The filtrate was evaporated, dissolved in dichloromethane
(50 mL) and washed with brine (30 mL). The organic layer was sep-
arated, dried over Na2SO4 and concentrated. Silica gel column chro-
matography (CC) in 1–3% of ethanol in CH2Cl2 afforded compound 3
as a colorless foam (912 mg, 80% yield). Rf = 0.55 (Solvent system A).
1H NMR (400 MHz, DMSO-d6): d = 11.27 (1 H, s, H3), 7.65–7.42 (10
m, H30 aT), 5.04 (1 H, d, J4 ,3 2.6, H40dmT), 4.64–4.60 (1 H, m, H30
0
0
dmT), 4.36–4.30 (1 H, m, H40 aT), 3.71 (6 H, s, CH3O DMTr), 3.28
(2 H, d, J4 ,5 -CH2 3.9, 50-CH2 aT), 2.80 (1 H, ddd, J1 ,2 a 6.0, J3 ,2 a 8.6,
0
0
0
0
0
0
2
J2 a,2 b 14.1, H20a aT), 2.75–2.66 (1 H, m, H20b aT), 2.42–2.31 (2
H, m, 20-CH2 dmT), 1.64 (3 H, d, 4J 1.0, 5-CH3 dmT), 1.62 (3 H, d,
4J 0.9, 5-CH3 aT), 1.02 (9 H, s, t-BuSi). MS: m/z Calcd for
0
0
C
58H61N7O10Si, [M + Na]+ 1066.4141. Found 1066.4133.
4
H, m, ArH), 7.37 (1 H, d, J 1.1, H6), 6.76 (1H, d, J 9.0, H50), 6.21 (1
H, t, J 6.9, H10), 4.50 (1H, dd, J5 ,4 9.0, J4 ,3 3.53, H40), 4.23–4.37 (1 H,
m, H30), 2.18–2.13 (2 H, m, H20a and H20b), 1.75 (3 H, d, 4J 1.1, 5-
CH3), 1.05 (9 H, s, t-BuSi).
4.5. 1-(30-Deoxy-50-O-(4,40-dimethoxytrityl)thymidine-30-yl)-4-(40-
de(hydroxymethyl)thymidine-40-yl)-1H-1,2,3-triazole (7)
0
0
0
0
To a stirred solution of dinucleoside 6 (842 mg, 0.81 mmol) in
dry THF (1.7 mL), 1 M solution of tetrabutylammonium fluoride
in dry THF (1.7 mL) was added. The mixture was stirred for 2 h
at r.t., diluted with saturated NaHCO3 (50 mL) and extracted with
CHCl3 (3 ꢂ 40 mL). Combined organic layers were washed with
water, dried over Na2SO4, filtered and concentrated. The residue
was purified by CC in 7% of ethanol in CH2Cl2 + 0.1% TEA to give
dinucleoside 7 as a hard foam (640 mg, 98% yield). Rf = 0.57 (sol-
vent system D). 1H NMR (400 MHz, DMSO-d6): d = 1H NMR
(DMSO): 11.36 (1 H, s, H3), 11.28 (1 H, s, H3), 8.41 (1 H, s, H60
4.3. 1-(2,5,6-Trideoxy-3-O-(tert-butyldiphenylsilyl)-b-D-erythro-hex-
5-ynofuranosyl)-5-methyluracil (4)
4.3.1. From nucleoside 3
To a stirred solution of nucleoside 3 (1.07 g, 1.688 mmol) in abs.
THF (17 mL), 6 mL of 1.6 M solution of butyllithium in abs. hexane
was added dropwise at ꢀ70 °C. The reaction mixture was stirred
for 30 min at ꢀ70 °C and allowed to heat to r.t. Saturated NH4Cl

A. Varizhuk et al. / Bioorganic Chemistry 39 (2011) 127–131
131
dmT), 7.65 (1 H, d, 4J 1.1, H6 aT), 7.60 (1 H, d, 4J 1.2, H6 dmT), 7.39–
6.82 (13 H, m, ArH DMTr), 6.40 (1 H, t, J 6.50, H10 aT), 6.36 (1 H, dd,
and terminal alkynes, Angew. Chem. 114 (2002) 2708–2711. Angew. Chem.
Int. Ed. 41 (2002) 2596–2599;
(b) C.W. Tornoe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase:
J1 ,2 a 7.6, J1 ,2 b 6.2, H10 dmT), 5.59 (1 H, d, J3 -OH ,3 4.3, 30-OH), 5.59–
0
0
0
0
0
0
0
[1.2.3]-triazoles
by
regiospecific
copper(I)-catalyzed
1.3-dipolar
0
5.53 (1 H, m, H30 aT), 4.94 (1 H, d, J4 ,3 2.9, H4 dmT), 4.55–4.49 (1 H,
0
0
cycloadditions of terminal alkynes to azides, J. Org. Chem. 67 (2002) 3057–
3062.
m, H30 dmT), 4.43–4.38 (1 H, dt, J3 ,4 6.6, J4 ,5 -CH2 4.0, H40 aT), 3.72
0
0
0
0
[2] F. Ambland, J.H. Cho, R.F. Schinazi, Cu(I)-catalyzed Huisgen azide–alkyne 1,3-
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chemistry, Chem. Rev. 109 (2009) 4207–4220.
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1388–1405.
(6 H, s, CH3O DMTr), 3.31 (2 H, d, J4 ,5 -CH2 4.0, 50-CH2 aT), 2.83–2.75
0
0
(2 H, m, J 8.54, 20-CH2 aT), 2.44 (1 H, ddd, J1 ,2 a 7.6, J3 ,2 a 6.1, 2J2 a,2 b
0
0
0
0
0
0
13.8, H20a dmT), 2.27 (1 H, ddd, J1 ,2 b 6.1, J3 ,2 a 3.3, 2J2 a,2 b 13.8, H20b
dmT), 1.67 (3 H, d, 4J 1.2, 5-CH3 dmT), 1.61 (3 H, d, 4J 1.1, 5-CH3 aT).
0
0
0
0
0
0
[4] N.M. Dean, C.F. Bennett, Antisense oligonucleotide-based therapeutics for
cancer, Oncogene 22 (2003) 9087–9096.
MS: m/z Calcd for
C42H43N7O10,
[M + Na]+ 828.2964. Found
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clinical experiences, Mol. Biotechnol. 33 (2006) 221–238.
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828.2963.
4.6. 1-(30-Deoxy-50-O-(4,40-dimethoxytrityl)thymidine-30-yl)-4-(30-
deoxy-30-((2-cyanoethoxy)(diisopropylamino)phosphinooxy)-40-
de(hydroxymethyl)thymidine-40-yl)-1H-1,2,3-triazole (8)
linked analogue of deoxyribonucleic acid (
TLDNA): design, synthesis, and
double-strand formation with natural DNA, Org. Lett. 10 (2008) 3729–3732.
[7] S.M. Frier, K.-H. Altmann, The ups and downs of nucleic acid duplex stability:
structure–stability studies on chemically-modified DNA:RNA duplexes,
Nucleic Acids Res. 25 (1997) 4429–4443.
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microwave-assisted synthesis of a 3,50-pentathymidine by copper (I)-catalyzed
[3 + 2] cycloaddition, Tetrahedron 64 (2008) 5467–5471.
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thymidine oligonucleotides with triazole internucleosidic linkages via
iterative Cu(I)-promoted azide–alkyne ligation chemistry, QSAR Comb. Sci.
26 (2007) 1191–1199.
Dinucleoside 7 (580 mg, 0.72 mmol) was dissolved in abs.
dichloromethane, pyridine (2 ꢂ 5 mL) and tetrazole (39 mg,
0.55 mmol) were added, and the mixture was stirred over molecu-
lar sieves for 30 min at r.t. 2-Cyanoethyl-N,N,N0,N0-tetra-
isopropylphosphoramidite (127.5 lL, 0.55 mmol) was added. The
mixture was stirred for 30 min at r.t., then poured into 50 mL of
cold saturated NaHCO3 (50 mL) and extracted with CH2Cl2
(3 ꢂ 40 mL). Combined organic layers were washed with water,
dried over Na2SO4, filtered and concentrated. The residue was puri-
fied by CC in 2–3% of ethanol in CH2Cl2 + 0.1% TEA to give amidite 8
[10] S. Chandrasekhar, P. Srihari, C. Nagesh, N. Kiranmai, N. Nagesh, M.M. Idris,
Synthesis of readily accessible triazole-linked dimer deoxynucleoside
phosphoramidite for solid-phase oligonucleotide synthesis, Synthesis 21
(2010) 3710–3714.
[11] S. Abbas, R.D. Bertram, C.J. Hayes, Commercially available 50-DMT
phosphoramidites as reagents for the synthesis of vinylphosphonate-linked
oligonucleic acids, Org. Lett. 3 (2001) 3365–3367.
as a hard foam (470 mg, 65% yield). Rf = 0.49 (Solvent system C). 31
P
[12] J.D. More, N.S. Finney, A simple and advantageous protocol for the oxidation of
alcohols with O-iodoxybenzoic acid (IBX), Org. Lett. 4 (2002) 3001–3003.
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(RCHO ? RC„CH or RC„CR0), Tetrahedron Lett. 13 (1972) 3769–3772;
(b) S. Takahashi, T. Nakata, Total synthesis of an antitumor agent, mucocin,
based on the ‘‘chiron approach’’, J. Org. Chem. 67 (2002) 5739–5752.
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(1975) 1333–1335.
NMR (133 MHz, DMSO-d6): d = 150.991, 150.575. MS: m/z Calcd
for C51H60N9O11P, [M + Na]+ 1028.4042. Found 1028.4033.
Acknowledgment
The work was supported by a grant from the Russian Founda-
tion for Basic Research.
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alkynes and bromoalkynes from aldehydes, Tetrahedron Lett. 40 (1999) 8575–
8578.
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Appendix A. Supplementary material
procedure for the synthesis of alkynes from aldehydes, Synlett
521–522;
6 (1996)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bioorg.2011.03.002.
(b) G. Roth, B. Liepold, S. Müller, H.J. Bestmann, Further improvements of the
synthesis of alkynes from aldehydes, Synthesis 1 (2004) 59–62.
[17] T.J. Curphey, Preparation of p-toluenesulfonyl azide, Org. Prep. Proced. Int. 13
(1981) 112–115.
[18] J. Pietruszka, A. Witt, Synthesis of the Bestmann–Ohira reagent, Synthesis 24
(2006) 4266–4268.
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