Synthesis and properties of oligonucleotides that contain a triazole-linked nucleic acid dimer

Article abstract of DOI:10.1002/jhet.532

New chemically modified oligonucleotides at the site of the backbone are needed to improve the properties of oligonucleotides. A practical synthesis for a triazole-linked nucleoside dimer based on a PNA-like structure has been developed. This involves synthesizing two uracil-based monomers that contain either an azide or an alkyne functionality, followed by copper-catalyzed 1,3-dipolar cycloaddition. This dimer was incorporated within an oligonucleotide via phosphoramidite chemistry and UV-monitored thermal denaturation data illustrates slight destabilization relative to its target complementary sequence. This chemically modified dimer will allow for a future investigation of its properties within DNA and RNA-based applications.

Full text of DOI:10.1002/jhet.532

May 2011
Synthesis and Properties of Oligonucleotides that Contain
a Triazole-Linked Nucleic Acid Dimer
533
Tim C. Efthymiou and Jean-Paul Desaulniers*
Faculty of Science, University of Ontario Institute of Technology, Oshawa, Ontario, L1H 7K4,
Canada
*E-mail: Jean-Paul.Desaulniers@uoit.ca
Received March 5, 2010
DOI 10.1002/jhet.532
Published online 8 February 2011 in Wiley Online Library (wileyonlinelibrary.com).
New chemically modified oligonucleotides at the site of the backbone are needed to improve the
properties of oligonucleotides. A practical synthesis for a triazole-linked nucleoside dimer based on a
PNA-like structure has been developed. This involves synthesizing two uracil-based monomers that con-
tain either an azide or an alkyne functionality, followed by copper-catalyzed 1,3-dipolar cycloaddition.
This dimer was incorporated within an oligonucleotide via phosphoramidite chemistry and UV-moni-
tored thermal denaturation data illustrates slight destabilization relative to its target complementary
sequence. This chemically modified dimer will allow for a future investigation of its properties within
DNA and RNA-based applications.
J. Heterocyclic Chem., 48, 533 (2011).
INTRODUCTION
involving chemical modification of the backbone within
the field of siRNA are less prevalent than sugar modifi-
cations. Some key examples include the negatively
charged backbone modification involving a phosphoro-
thioate [10] and boranophosphates [11]. Backbone-alter-
ing modifications such as a morpholino analogue [12]
and neutral amide bonds at 30 overhangs of siRNA
duplexes, have shown favorable results when utilized
for gene-knockdown studies [13,14]. Therefore, it is
possible that the RNAi pathway will adopt less conven-
tional backbone modifications at specific positions of the
oligonucleotide. Given that siRNA duplexes exhibit
thermodynamic asymmetry, being able to potentially
destabilize the 50 end of the guide strand with novel
backbone modifications may offer favorable properties.
Outside of the RNAi field, modified backbone constructs
could have many other potential applications such as in
antisense technology, artificial aptamer design or tem-
plate driven enzymatic reactions. To explore this pro-
spective, we are interested in pursuing alternative back-
bone modifications such as nonionic hydrophobic back-
bone mimics.
Oligonucleotides have found tremendous use as bio-
logical tools and as agents with therapeutic promise.
One promising example involves RNA interference
(RNAi), and exploiting this pathway has become a
major tool in elucidating gene function [1]. Furthermore,
utilization of the RNAi pathway by administering short
interfering RNAs (siRNAs) to cells [2] has offered ther-
apeutic hope in combating diseased cells with aberrant
gene expression profiles [3]. Despite awareness of this
potential, improvements in enzymatic stability, biodistri-
bution, cell-membrane permeability, and reducing off-
target effects are still needed [4,5]. One way to poten-
tially overcome these limitations involves altering the
stability and specificity profile of the siRNA through
chemical modification.
Within the areas of siRNA, the most common type of
chemical modification involves alterations within the
ribose moiety. Extensive studies involving 20-O-Me, flu-
orinated sugars and locked nucleic acids (LNA) have
shown favorable siRNA knockdown results depending
on its position and sequence context [6–9]. Studies
C
V
2011 HeteroCorporation

534
T. C. Efthymiou and J.-P. Desaulniers
Vol 48
Scheme 1. Reagents and conditions: (i) 1 equiv imidazole, 1 equiv
TBS-Cl, DCM, room temperature 96% yield of 1; (ii) 0.5 equiv
DIPEA, 0.5 equiv propargyl bromide, DCM, room temperature, 3 h,
71% yield of 2; (iii) 1.5 equiv uracil-1-yl acetic acid, 1.5 equiv
DIPEA, 1.5 equiv HBTU, DMF, 24 h, 44% yield of 3.
Figure 1. Chemical differences between peptide nucleic acid (left) and
triazole-linked nucleic acid (right).
Within the field of backbone modified oligonucleo-
tides, one of the most successful examples involves pep-
tide nucleic acid (PNA) [15] and this has shown remark-
able stability and specificity to its complementary tar-
gets [16,17]. PNA synthesis occurs by the successful
amide-bond formation between an acid and an amine
with high-yields. Therefore, we envisioned that a scaf-
fold based on a triazole functional group would serve as
a potentially good candidate (Fig. 1). To date, a number
of different triazole-based backbone modifications have
been reported [18–20] from the one presented herein.
uracil-based monomer with an azide. For the synthesis
of the uracil-based alkyne monomer, the first step
involved tert-butyldimethylsilyl (TBS) protection of eth-
anolamine and this was conducted using TBSCl under
standard basic conditions to afford 1 in 96% yield.
Alkylation of 1 with propargyl bromide generated the
alkyne 2 as a liquid in 71% yield. Utilizing peptide
bond coupling conditions involving HBTU, the alkyne 2
was amide-bond coupled to uracil-1-yl acetic acid [27]
to afford compound 3 as a white solid in 44% yield
(Scheme 1).
For the synthesis of the azide-based uracil monomer
6, 2-azidoethylamine 4 was prepared by the addition of
2-bromoethylamine to an aqueous solution of sodium
azide. Alkylation of 4 with a limiting amount of ethyl
2-bromoacetate afforded compound 5 as an off-colored
liquid in 83% yield. This liquid 5 was amide-bond
coupled with uracil-1-yl acetic acid using DCC/HOBt to
afford the azide-based uracil monomer, compound 6 in
62% yield (Scheme 2).
RESULTS AND DISCUSSION
The repetitive backbone unit in most analogues,
including PNA, contains six atoms. Although the six-
atom spacer unit dominates by choice for a large num-
ber of chemically modified nucleic acids, analogues
with shorter (five atom linker) [21–23] and longer back-
bones (seven atom linker) [24,25] have shown varying
degrees of specificity to its target, depending on its
sequence context and type of backbone. This suggests
that site-specific incorporation of unnatural nucleoside
backbones of different atom lengths can produce func-
tioning oligonucleotides, capable of binding to their
complement strand with varying specificity and stability.
As such, chemically modifying oligonucleotides could
offer an alternative way to fine-tune their functional
effects.
Scheme 2. Reagents and conditions: (i) 3 equiv NaN3, H2O, 75ꢀC, 24
h; (ii) 5.8 equiv NaOH, 0ꢀC, Et2O, 64% yield of 4; (iii) 1 equiv NEt3,
0.6 equiv ethyl 2-bromoacetate, DMF, 4 h, 83% yield of 5; (iv) 1.1
equiv uracil-1-yl acetic acid, 1.1 equiv DCC, 1.1 equiv HOBt, DMF,
24 h, 62% yield of 6.
We hypothesized that a copper (I)-catalyzed Huisgen
[3 þ 2] cycloaddition [26] based on a PNA-type struc-
ture would generate an alternative scaffold for bridging
adjacent nucleotides together. We report the synthesis of
a modified 1,4-triazole-linked uracil dimer through a
distance of seven atoms and report its compatibility with
hybridization to a complement sequence when appended
at the 50 or 30 end of a DNA oligonucleotide.
Our strategy involves the synthesis of a uracil-based
monomer possessing an alkyne functional group and a
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2011
Synthesis and Properties of Oligonucleotides that Contain a
Triazole-Linked Nucleic Acid Dimer
535
Scheme 3. Reagents and conditions: (i) 2 equiv sodium ascorbate, 0.5 equiv Cu(II)SO4, THF/t-BuOH/H2O (1:1:1), 24 h, 98% yield of 7; (ii) 2.5
equiv LiBH4, THF/MeOH (12:1), reflux 1.5 h, 76% yield of 8; (iii) 3 equiv DMT-Cl, pyridine, 24 h, 79% yield of 9; (iv) 3 eq TBAF, THF, 12 h,
85% yield of 10; (v) 2 equiv 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, 5.5 equiv DIPEA, 0.5 equiv DMAP, DCM, 8 h, 63% yield of
11.
With respect to the synthesis of the phosphoramidite,
the first task was to cyclize both monomers together.
Under copper (I) conditions, 1,4-triazole formation
occurred with copper sulfate and sodium ascorbate to
afford a white solid 7 in 98% yield. This newly formed
dimer 7 was selectively reduced with 2.5 equivalents of
LiBH4 in a methanol/THF mixture to afford the alcohol
8 in 76% yield. Alcohol 8 was treated with 4,40-dime-
thoxytrityl-chloride (DMT-Cl) to afford the compound 9
in 79% yield. The subsequent removal of the TBS group
using TBAF provided the alcohol 10 in 85% yield. This
was then followed by synthesis of phosphoramidite 11
by reacting alcohol 10 with 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite to afford a clear oil in 63%
yield (Scheme 3). With this phosphoramidite 11, its
incorporation within a DNA oligonucleotide was accom-
plished by employing solid-phase oligonucleotide syn-
thesis on a 394 ABI synthesizer.
cation at the 50 end and the 30 end, respectively. How-
ever, this suggests a certain degree of compatibility as
the Tm values for both modified oligonucleotides are
higher when compared to corresponding 12-mer sequen-
ces lacking the heterocyclic pyrimidine dimer (UU)
(Table 1). In comparison to other studies [28] and that
of Kraicsovits and coworkers, a similar Tm drop was
Table 1
UV-melting denaturation studies.
Entry
Sequence
Tm (ꢀC)
DTm (ꢀC)a
I
50 TTTTTCTCTCTCTT 30
50 TTTTTCTCTCTCUU 30
50 TTTTTCTCTCTC 30
50 UUTTTCTCTCTCTT 30
50 TTTCTCTCTCTT 30
40.9
38.1
37.1
36.4
35.3
0
II
ꢁ2.8
ꢁ3.8
ꢁ4.5
ꢁ5.6
III
IV
V
UV melting temperatures for duplexes to complement strand DNA (50-
AAG AGA GAG AAA AA-30). The total strand concentration ranged
from 2.4 to 3.0 lM in 90 mM NaCl, 10 mM sodium phosphate, and
1 mM EDTA at pH 7.0.
UV-melting denaturation studies indicate that moder-
ate destabilization occurs with the UU-dimer on both
the 50 and 30 position relative to the native strand (Table
1). A reduction in Tm of 4.5ꢀC and 2.8ꢀC is observed
when native TT is replaced with the UU-dimer modifi-
aFor the DTm values, the Tm of Entry I is the reference (Tm
¼
40.9 ꢀC).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

536
T. C. Efthymiou and J.-P. Desaulniers
Vol 48
(2.9 g, 71%); 1H NMR (500 MHz, CDCl3) d 0.07 (s, 6H),
0.90 (s, 9H), 1.67 (br s, 1H), 2.21 (s, 1H), 2.80 (t, 2H, J ¼ 5.5
Hz), 3.46 (s, 2H), 3.75 (t, 2H, J ¼ 5.1 Hz); 13C NMR (100
MHz, CDCl3) d ꢁ5.36, 25.89, 29.66, 38.18, 50.52, 62.32,
71.21, 82.15; ESI-HRMS (ESþ) m/z calcd for C11H23NOSi:
213.1549, found 214.1626 [M þ H]þ. Anal. Calcd. for
C11H23NOSi: C, 61.91; H, 10.86. Found: C, 61.56; H, 10.49.
N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-(prop-
2-yn-1-yl)acetamide (3). To a solution of uracil-1-yl acetic
acid (2.4 g, 14 mmol) in 240 mL of DMF was added diisopro-
pylethylamine (1.8 g, 14 mmol) and this solution was cooled
in an ice-water bath. The acid was activated with the addition
observed when compared to a PNA-DNA chimera that
contained a PNA element at the terminal end [29].
CONCLUSIONS
In conclusion, a facile and practical synthesis of a
new uracil-uracil dimer linked together through a tria-
zole functionality has been developed and is readily syn-
thesized from inexpensive starting material precursors.
Our alkyne-uracil monomer 3 was synthesized in 44%
yield and the azide-uracil monomer 6 was generated in
62% yield. Following heterocyclic cyclization, the phos-
phoramidite 11 was generated in four steps in 32%
yield. Incorporation of the heterocyclic dimer at both
the 50 and 30 ends of DNA oligonucleotides suggests a
certain degree of compatibility when analyzed by UV-
monitored thermal denaturation. The effects of this mod-
ification within different positions of other various oli-
gonucleotides will be investigated. Notwithstanding,
these oligonucleotides may have potential use when tar-
geted at messenger RNA in diseased cells, or for other
downstream applications.
of
O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-
phosphate (5.3 g, 14 mmol) to the stirring solution over the
course of 5 min, followed by the dropwise addition of 2 (2 g, 9.4
mmol) over 15 min. The reaction mixture was stirred for 24 h at
ambient temperature and then extracted with EtOAc and the or-
ganic layer was washed with H2O and brine (x3). The organic
layer was collected and concentrated in vacuo to afford the crude
product. This crude product was purified by flash chromatography
with a gradient of hexanes/EtOAc (7:3) to 100% EtOAc to elute
the title compound as a white solid (1.5 g, 44%). Compound 3 is
a pair of rotamers; the signals to the major (ma.) and minor (mi.)
1
rotamers are designated; H NMR (500 MHz, CDCl3) d 0.04 (s,
2H, mi.), 0.08 (s, 4H, ma.), 0.88 (s, 3H, mi.), 0.89 (s, 6H, ma.),
2.25 (br s, 0.66H, ma.), 2.41 (br s, 0.33H, mi.), 3.58 (t, 0.6H, J
¼ 5.3 Hz, mi.), 3.65 (t, 1.4H, J ¼ 5.2 Hz, ma.), 3.76 (t, 0.6H, J
¼ 5.3 Hz, mi.), 3.85 (t, 1.4H, J ¼ 5.0 Hz, ma.), 4.27 (2s, 2H),
4.68 (m, 2H), 5.71–5.74 (m, 1H), 7.13 (d, 0.66H, J ¼ 7.9 Hz,
ma.), 7.17 (d, 0.33H, J ¼ 7.9 Hz, mi.), 9.43 (br s, 0.66H, ma.),
9.48 (br s, 0.33H, mi.); 13C NMR (125 MHz, CDCl3) d ꢁ5.61,
ꢁ5.55, ꢁ5.41, ꢁ5.36, ꢁ3.72, ꢁ3.53, 17.94, 18.11, 18.28, 25.54,
25.69, 25.75, 25.81, 25.89, 25.96, 35.16, 38.67, 48.03, 48.06,
48.78, 49.55, 60.58, 61.67, 72.72, 73.59, 77.93, 78.13, 102.13,
102.24, 145.02, 145.09, 150.90, 150.93, 163.58, 166.30, 166.66;
ESI-HRMS (ESþ) m/z calcd for C17H27N3O4Si: 365.1771, found
366.1845 [M þ H]þ. Anal. Calcd. for C17H27N3O4Si: C, 55.86;
H, 7.45; N, 11.50. Found: C, 55.67; H, 7.80; N, 11.89.
2-Azidoethanamine (4). To a solution of NaN3 (23.8 g, 366
mmol) in 200 mL of water was added 2-bromoethylamine
hydrobromide (25 g, 122 mmol) and this solution was heated
to 75ꢀC. The reaction mixture was stirred for 24 h and was
then cooled in an ice-water bath. To the cooled solution was
added NaOH (28.5 g, 712.5 mmol) and the reaction mixture
was stirred until the NaOH was fully dissolved. To this aque-
ous solution was added Et2O (x3). The ether fractions were
collected, dried over Na2SO4, and evaporated in vacuo to
afford the title compound as a clear oil (6.7 g, 64%). The 1H
proton and 13C NMR shifts were confirmed with the report by
Mayer and Maier [32].
Ethyl 2-(2-azidoethylamino)acetate (5). To a solution of 4
(5 g, 58.1 mmol) in 100 mL of DMF was added triethylamine
(5.9 g, 58.1 mmol). Ethyl 2-bromoacetate (5.8 g, 34.9 mmol)
was then added dropwise over 15 min to the stirring solution
and the reaction was stirred for 4 h at room temperature. The
solution was then extracted with EtOAc, washed with H2O
(three times) and the EtOAc fractions were collected and dried
over Na2SO4. This solution was evaporated under reduced
pressure to yield a crude orange oil. The oil was loaded
directly onto a silica column for purification by gradient elut-
ing with hexanes/EtOAc (5:5) to 100% EtOAc to afford the
EXPERIMENTAL
Unless otherwise noted, all starting materials were obtained
from commercial sources and were used without any additional
purification. Anhydrous CH2Cl2, THF, and DMF were pur-
chased from Sigma-Aldrich and degassed by stirring under a
dry N2 atmosphere. Purification by flash chromatography was
carried out with Silicycle Siliaflash 60 (230–400 mesh) accord-
1
ing to the procedure of Still et al. [30] H NMR spectra were
recorded in DMSO-d6 or CDCl3 at 400 or 500 MHz and 13C
NMR spectra were recorded at 100 or 125 MHz. High-Resolu-
tion MS (HRMS) was recorded on a Micromass AutoSpec
Ultima Magnetic sector mass spectrometer.
2-(tert-Butyldimethylsilyloxy)ethanamine (1). To a solu-
tion of ethanolamine (9.6 mL, 159 mmol) in 100 mL of CH2Cl2
was dissolved imidazole (10.8 g, 159 mmol) and this solution
was cooled in an ice-water bath. To this solution was added
tert-butyldimethylsilyl chloride (24 g, 159 mmol) and the reac-
tion mixture was stirred overnight at room temperature. Sat.
NaHCO3 was added and the resulting mixture was partitioned.
The organic fractions were concentrated in vacuo to afford the
title compound as a clear light yellow oil (26.8 g, 96%) [31].
N-(2-(tert-Butyldimethylsilyloxy)ethyl)prop-2-yn-1-amine (2). To
a solution of 1 (10 g, 57 mmol) in 100 mL of CH2Cl2 was
added diisopropylethylamine (3.7 g, 28.5 mmol), and this solu-
tion was cooled in an ice-water bath. To this solution was
added 3-bromoprop-1-yne (3.4 g, 28.5 mmol) dropwise over
30 min. This reaction mixture was stirred at room temperature
until TLC analysis indicated the complete consumption of
starting material (3 h). Sat. NaHCO3 was added and the reac-
tion mixture was partitioned. The organic fraction was concen-
trated in vacuo, to afford an oil which was purified by silica
gel chromatography eluting with a gradient of hexanes/EtOAc
(7:3 to 3:7) to afford the title compound as a clear yellow oil
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2011
Synthesis and Properties of Oligonucleotides that Contain a
Triazole-Linked Nucleic Acid Dimer
537
title compound as a clear light yellow oil (5.0 g, 83%); 1H
NMR (400 MHz, CDCl3) d 1.25 (t, 3H, J ¼ 7.1 Hz), 1.98 (br
s, 1H), 2.80 (t, 2H, J ¼ 5.7 Hz), 3.39 (t, 2H, J ¼ 5.9 Hz),
3.40 (s, 2H), 4.17 (q, 2H, J ¼ 7.4 Hz); 13C NMR (100 MHz,
CDCl3) d 14.09, 48.01, 50.50, 51.33, 60.74, 172.07; ESI-
HRMS (ESþ) m/z calcd for C6H12N4O2: 172.0960, found
173.1034 [M þ H]þ. Anal. Calcd. for C6H12N4O2: C, 41.85;
H, 7.02; N, 32.54. Found: C, 41.65; H, 6.82; N, 32.12.
13.99, 14.04, 17.89, 18.04, 25.82, 25.90, 41.05, 47.31, 47.58,
47.91, 48.00, 60.18, 60.65, 60.83, 61.25, 100.57, 100.79,
123.70, 124.04, 124.12, 124.61, 143.18, 143.34, 143.41,
143.56, 146.12, 146.19, 146.29, 146.50, 146.54, 150.92,
150.98, 151.04, 151.07, 151.11, 163.76, 163.80, 163.81,
163.85, 163.88, 166.74, 166.82, 166.88, 166.90, 167.43,
167.97, 168.03, 168.85, 169.15; ESI-HRMS (ESþ) m/z calcd
for C29H43N9O9Si: 689.2953, found 690.3030 [M þ H]þ.
Anal. Calcd. for C29H43N9O9Si: C, 50.50; H, 6.28. Found: C,
50.47; H, 6.24.
Ethyl 2-(N-(2-azidoethyl)-uracil-1-yl-acetamido)acetate (6). To
a solution of uracil-1-yl acetic acid (1.1 g, 6.4 mmol) in 100 mL
of anhydrous DMF under N2 was added dicyclohexylcarbodii-
mide (1.3 g, 6.4 mmol) and 1N-hydroxybenzotriazole (1.0, 6.4
mmol). This solution was stirred for 15 min on an ice-water
bath, followed by 1 h of stirring at ambient temperature. To
this solution was added compound 5 (1 g, 5.8 mmol) dropwise
over 10 min, and the reaction mixture was stirred for 24 h af-
ter which the DCU precipitate was collected by filtration. The
filtrate was dried down in vacuo, dissolved in EtOAc, and
washed with water and brine (x3). The EtOAc solutions were
concentrated in vacuo. The resulting crude product was dis-
solved in a minimal amount of CH2Cl2 and purified by flash
chromatography with a gradient of hexanes/EtOAc (5:5) to
100% EtOAc to elute the title compound as a white solid (1.2
g, 62%). Compound 6 is comprised of a pair of rotamers, each
displaying signals of equal intensity; 1H NMR (500 MHz,
CDCl3) d 1.28 (t, 1.5H, J ¼ 7.1 Hz), 1.33 (t, 1.5H, J ¼ 7.1
Hz), 3.55 (s, 2H), 3.59 (t, 1H, J ¼ 5.6 Hz), 3.66 (t, 1H, J ¼
5.4 Hz), 4.12 (s, 1H), 4.21 (q, 1H, J ¼ 7.1 Hz), 4.26 (s, 1H),
4.28 (q, 1H, J ¼ 7.1 Hz), 4.49 (s, 1H), 4.72 (s, 1H), 5.74 (d,
0.5H, J ¼ 6.0 Hz), 5.75 (d, 0.5H, J ¼ 5.6 Hz), 7.20 (d, 0.5H,
J ¼ 2.0 Hz), 7.21 (d, 0.5H, J ¼ 2.0 Hz), 9.08 (br s, 1H); 13C
NMR (125 MHz, CDCl3) d 47.75, 47.82, 48.05, 48.31, 48.86,
49.84, 49.92, 50.95, 61.67, 62.31, 102.30, 102.38, 145.02,
145.14, 150.90, 150.92, 163.44, 167.16, 167.46, 168.64,
169.03; ESI-HRMS (ESþ) m/z calcd for C12H16N6O5:
324.1182, found 325.1262 [M þ H]þ. Anal. Calcd. for
C12H16N6O5: C, 44.44; H, 4.97; N, 25.91. Found: C, 44.40; H,
5.10; N, 25.41.
N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-((1-(2-
(uracil-1-yl-N-(2-hydroxyethyl)acetamido)ethyl)-1H-1,2,3-tri-
azol-4-yl)methyl)acetamide (8). To a solution of compound 7
(300 mg, 435 lmol) suspended in 12 mL of dry THF, drops of
MeOH were added until the compound was dissolved. To this
solution was then added LiBH4 (544 lL, 1.09 mmol) and the
reaction mixture was refluxed until TLC analysis indicated the
complete consumption of starting material (1.5 h). This reaction
mixture was quenched with MeOH and dried down in vacuo to
afford the crude product. The crude product was then redis-
solved in minimal CH2Cl2/MeOH and purified by flash column
chromatography eluting with a gradient of 10% NH4OH in
MeOH (5 to 15%) in CH2Cl2 to afford the title compound as a
white crystalline solid (213 mg, 76%). Compound 8 is a mixture
1
of rotamers with varying signal intensities; H NMR (500 MHz,
DMSO-d6) d 0.03 (s, 2H), 0.05 (s, 4H), 0.85, 0.87 (2s, 9H),
3.16–3.27 (m, 2H), 3.43–3.54 (m, 4H), 3.63–3.74 (m, 3H),
3.80–3.87 (m, 2H), 4.36–4.38 (m, 1H), 4.46 (t, 1.2H, J ¼ 6.3
Hz), 4.51 (t, 0.8H, J ¼ 6.3 Hz), 4.56–4.81 (m, 6H), 4.94–5.00
(m, 1H), 5.54–5.58 (m, 2H), 7.35–7.47 (m, 2H), 7.87, 8.04,
8.13, 8.23 (4s, 1H), 11.30 (br s, 2H); 13C NMR (125 MHz,
DMSO-d6) d ꢁ5.44, ꢁ5.37, 17.91, 18.05, 25.83, 25.92, 30.74,
41.04, 46.24, 46.55, 46.71, 46.92, 48.29, 48.32, 48.51, 49.07,
49.26, 58.49, 58.81, 58.85, 60.21, 60.85, 100.57, 100.63,
123.76, 124.11, 124.55, 143.23, 143.39, 143.43, 143.58, 146.48,
146.52, 146.58, 151.04, 151.08, 151.11, 163.84,þ163.87, 166.69,
166.74, 166.81, 167.43, 167.54; ESI-HRMS (ES ) m/z calcd for
C27H41N9O8Si: 647.2847, found 648.2918 [M þ H]þ. Anal.
Calcd. for C27H41N9O8Si: C, 50.06; H, 6.38; N, 19.46. Found:
C, 50.23; H, 6.84; N, 19.36.
Ethyl
2-(N-(2-(4-((N-(2-(tert-Butyldimethylsilyloxy)ethyl)-
N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(ura-
cil-1-yl)-N-(2-(4-((2-(uracil-1-yl)-N-(2-((2,3,3-trimethylbutan-2-
yl)oxy)ethyl)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)a-
cetamide (9). To a solution of compound 8 (1.06 g, 1.64
mmol) dissolved in 10 mL of dry pyridine under N2, was
added an excess of 4,40-dimethoxytrityl chloride (1.66 g, 4.91
mmol) until TLC analysis revealed the consumption of starting
material. This reaction mixture was stirred overnight at room
temperature. while under N2, after which the entire mixture
was extracted with CH2Cl2 and washed with H2O (two times).
The organic fractions were collected, dried over Na2SO4 and
subsequently condensed in vacuo to afford the crude product.
The crude was dissolved in minimal CH2Cl2/MeOH and puri-
fied by flash column chromatography eluting with a gradient
of MeOH (5 to 15%) in CH2Cl2 to afford the title compound
as a white solid (1.22 g, 79%). Compound 9 is a mixture of
rotamers with varying signal intensities; 1H NMR (400 MHz,
CDCl3) d 0.03 (s, 2H), 0.06 (s, 4H), 0.86, 0.88 (2s, 9H), 2.87–
2.94 (m, 1.5H), 3.03–3.08 (m, 0.5H), 3.20 (t, 1.5H, J ¼ 4.7
Hz), 3.26 (t, 0.5H, J ¼ 4.5 Hz), 3.29–3.31 (m, 0.2H), 3.36–
3.43 (m, 0.3H), 3.46–3.55 (m, 2.5H), 3.60–3.67 (m, 2H), 3.72–
3.75 (m, 0.75H), 3.76, 3.77 (2s, 7H), 3.80 (t, 1.25H, J ¼ 4.9
Hz), 4.51 (t, 1.5H, J ¼ 5.3 Hz), 4.55 (t, 0.5H, J ¼ 5.3 Hz),
uracil-1-yl-acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-ura-
cil-1-yl-acetamido)acetate (7). To a solution of compounds 3
(677 mg, 1.9 mmol) and 6 (600 mg, 1.9 mmol) dissolved in
18 mL of THF/t-BuOH/H2O (1:1:1) was added sodium ascor-
bate (733 mg, 3.7 mmol) and Cu(II)SO4 (231 mg, 925 lmol).
This reaction mixture was stirred for 24 h at room temperature
and then extracted with CH2Cl2. The organic layer was washed
with H2O (three times) to wash off the excess copper. The pre-
cipitate in the organic layer was collected by filtration and
washed with cold H2O to afford the crude product. This crude
product was redissolved in minimal CH2Cl2/MeOH for 1 h
and purified by flash column chromatography eluting with 5%
(10% NH4OH in MeOH) in CH2Cl2 to afford the title com-
pound as a white solid (1.3 g, 98%). Compound 7 is a mixture
of rotamers with varying signal intensities. 1H NMR (400
MHz, DMSO-d6) d 0.03 (s, 2H), 0.05 (m, 4H), 0.85, 0.86 (2s,
9H), 1.18 (t, 1.5H, J ¼ 7.2 Hz), 1.23 (t, 1.5H, J ¼ 7.0 Hz),
3.48–3.50 (m, 1H), 3.63–3.91 (m, 4H), 4.01–4.03 (m, 1H),
4.08 (q, 1.2H, J ¼ 7.3 Hz), 4.16 (q, 0.8H, J ¼ 7.3 Hz), 4.24,
4.29 (2s, 1H), 4.44–4.77 (m, 9H), 5.56 (d, 2H, J ¼ 7.8 Hz),
7.31–7.47 (m, 2H), 7.89, 8.05, 8.13, 8.24 (4s, 1H), 11.29 (br
m, 2H); 13C NMR (100 MHz, DMSO-d6) d ꢁ5.45, ꢁ5.39,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

538
T. C. Efthymiou and J.-P. Desaulniers
Vol 48
4.59 (s, 1H), 4.63–4.64 (m, 1H), 4.67 (s, 3H), 4.73 (d, 1H, J ¼
7.4 Hz), 5.51–5.55 (m, 1H), 5.62 (d, 0.1H, J ¼ 1.6 Hz), 5.64
(d, 0.1H, J ¼ 1.6 Hz), 5.64–5.68 (m, 0.8H), 6.57 (d, 0.1H, J ¼
7.8 Hz), 6.66 (d, 0.7H, J ¼ 7.8 Hz), 6.80–6.84 (m, 4.3H),
7.06–7.09 (m, 0.8H), 7.17–7.36 (m, 7.2H), 7.71, 7.81, 7.88,
7.94 (4s, 1H), 10.11 (br s, 2H); 13C NMR (100 MHz, CDCl3)
d ꢁ5.44, ꢁ5.16, 18.13, 18.29, 25.61, 25.83, 25.90, 41.33,
44.08, 47.64, 47.74, 48.27, 48.50, 48.62, 48.87, 48.93, 49.09,
55.23, 60.41, 60.66, 60.79, 60.98, 87.24, 87.39, 102.02,
102.21, 102.25, 113.15, 113.25, 124.37, 125.24, 127.18,
127.24, 127.86, 127.98, 128.15, 129.91, 130.08, 135.03,
135.09, 135.70, 143.49, 143.76, 144.01, 144.05, 144.65,
145.00, 145.10, 145.15, 145.21, 145.54, 151.26, 151.32,
151.38, 151.46, 158.42, 158.67, 158.71, 163.91, 164.00,
164.08, 166.48, 167.25, 167.67. ESI-HRMS (ESþ) m/z calcd
for C48H59N9O10Si 949.4154, found 972.4035 [M þ Na]þ.
Anal. Calcd. for C49H59N9O10Si: C, 60.68; H, 6.26; N, 13.27.
Found: C, 60.50; H, 6.28; N, 12.90.
Table 2
The oligonucleotides characterized by MALDI-TOF
mass spectrometry.
Calcd Observed
mass mass
(g/mol) (g/mol)
Observed
molecular
ion
Sequence
50 TTTTTCTCTCTCUU 30 4123
4122
3530
4164
3530
[M – H]ꢁ
[M þ H]ꢁ
50 TTTTTCTCTCTC 30
3529
50 UUTTTCTCTCTCTT 30 4123
50 TTTCTCTCTCTT 30
3529
[M – H þ K]ꢁ
[M þ H]ꢁ
Observed masses obtained from MALDI-TOF Mass Spectrometry.
Polyacrylamide gel-purified samples were desalted using Millipore
Zip-Tip C18-column micropipette tips to a final concentration of 5
lM. 5 lL of matrix solution (3-hydroxypicolinic acid at 25 mg/mL,
dissolved in 50/50 acetonitrile/water with 5 mg/mL ammonium citrate)
was added to each sample vial.
N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(uracil-
1-yl)-N-((1-(2-(2-(uracil-1-yl)-N-(2-hydroxyethyl)acetamido)ethyl)-
1H-1,2,3-triazol-4-yl)methyl)acetamide (10). To a solution of
compound 9 (1.12 g, 1.18 mmol) dissolved in 26 mL of dry
THF was added TBAF (926 mg, 3.54 mmol). This reaction
mixture was stirred for 12 h at room temperature and then
extracted with CH2Cl2. The organic layer was washed with
H2O and brine (ꢂ2), at which point the combined organic
fractions were dried over Na2SO4, and evaporated under
reduced pressure. The crude was dissolved in minimal
CH2Cl2/MeOH and purified by flash column chromatography,
first eluting with 100% CH2Cl2 followed by a gradient of
MeOH (5 to 15%) in CH2Cl2 to afford the title compound as a
yellow solid (840 mg, 85%). Compound 10 is a mixture of
rotamers with varying signal intensities; 1H NMR (400 MHz,
CDCl3) d 2.49 (br s, 1H), 2.98 (br s, 1H), 3.16–3.20 (br m,
2H), 3.27–3.31 (m, 3H), 3.45–3.57 (m, 4H), 3.68–3.81 (m,
8H), 4.45–4.80 (m, 8H), 5.50–5.53 (m, 0.8H), 5.56–5.60 (m,
1.2H), 6.63 (d, 0.15H, J ¼ 8.2 Hz), 6.66 (d, 0.35H, J ¼ 7.8
Hz), 6.78–6.83 (m, 4H), 7.17–7.31 (m, 8.5H), 7.69, 7.88, 7.90,
8.09 (4s, 1H), 10.20–10.42 (br m, 2H); 13C NMR (100 MHz,
CDCl3) d 22.13, 27.75, 29.14, 29.64, 42.18, 47.37, 48.29,
49.13, 50.35, 55.24, 59.15, 60.78, 68.46, 87.23, 87.32, 101.65,
101.91, 107.85, 113.16, 113.26, 124.97, 127.16, 127.99,
128.17, 129.92, 130.09, 135.15, 135.73, 143.56, 144.11,
144.28, 145.47, 146.36, 151.41, 151.47, 158.40, 158.64,
164.30, 164.39, 164.56, 167.63, 167.68; ESI-HRMS (ESþ) m/z
calcd for C42H45N9O10: 835.3289, found 858.3217 [M þ
Na]þ. Anal. Calcd. for C42H45N9O10: C, 60.35; H, 5.43.
Found: C, 60.72; H, 5.81.
2-(N-(2-(4-((N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-
2-(uracil-1-yl)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-
(uracil-1-yl)acetamido)ethyl (2-cyanoethyl) diisopropylphos-
phoramidite (11). To a solution of compound 10 (230 mg,
275 lmol) dissolved in 6 mL of dry CH2Cl2 under N2, was
added diisopropylethylamine (196 mg, 1.51 mmol) along with
4-dimethylaminopyridine (16.8 mg, 138 lmol). To this solu-
tion was added 2-cyanoethyl N,N-diisopropylchlorophosphora-
midite (195 mg, 825 lmol), until TLC analysis revealed the
maximum consumption of starting material. This mixture was
then stirred for 6 h at room temperature under N2 and dried in
vacuo to afford the crude product. The crude was dissolved in
minimal 2% triethylamine in hexanes/acetone and purified by
flash column chromatography eluting with a gradient of 2%
triethylamine in acetone/hexanes (1:1 to 4:1) to afford the title
compound as a white solid (180 mg, 63%). Compound 11 is a
mixture of rotamers with varying signal intensities; 1H NMR
(400 MHz, CDCl3) d 1.14–1.19 (m, 12H), 2.62–2.66 (m,
3.25H), 2.92 (t, 1H, J ¼ 3.9 Hz), 3.07 (t, 0.75H, J ¼ 4.3 Hz),
3.21 (t, 1.25H, J ¼ 4.7 Hz), 3.26 (t, 0.5H, J ¼ 4.3 Hz), 3.29–
3.37 (m, 0.25H), 3.54–3.61 (m, 5.5H), 3.70–3.77 (m, 2H), 3.78
(s, 6.25H), 3.81–3.90 (m, 2.25H), 4.52 (t, 1H, J ¼ 5.3 Hz),
4.55 (t, 0.5H, J ¼ 5.5 Hz), 4.61–4.65 (m, 2H), 4.70 (s, 2.5H),
4.74–4.77 (m, 1H), 5.52–5.55 (m, 1H), 5.66–5.70 (m, 1H),
6.56 (d, 0.33H, J ¼ 7.8 Hz), 6.63 (d, 0.66H, J ¼ 7.8 Hz),
6.81–6.84 (m, 4H), 7.17–7.31 (m, 12H), 7.70, 7.76, 7.89, 7.95
(4s, 1H); 13C NMR (100 MHz, CDCl3) d 14.10, 14.84, 20.45,
20.52, 22.66, 23.34, 24.58, 24.62, 24.65, 24.69, 24.77, 29.24,
29.34, 29.66, 31.72, 31.89, 33.80, 34.43, 41.51, 43.02, 43.14,
45.82, 47.67, 47.80, 48.29, 48.60, 48.71, 48.93, 53.76, 55.26,
58.08, 58.30, 60.53, 60.70, 60.80, 69.48, 87.29, 87.42, 102.08,
102.20, 102.25, 113.27, 117.90, 118.12, 125.13, 127.24,
128.01, 128.20, 130.12, 135.05, 135.09, 143.39, 143.82,
144.05, 145.07, 145.46, 151.24, 151.34, 151.45, 158.71,
158.75, 163.63, 163.87, 163.91, 166.67, 167.41, 167.74; ESI-
HRMS (ESþ) m/z calcd for C51H62N11O11P: 1035.4368, found
1058.4298 [M þ Na]þ.
Oligonucleotide synthesis. All standard b-cyanoethyl DNA
phosphoramidites, solid supports and reagents were purchased
from Glen Research. All oligonucleotides were synthesized on
an Applied Biosystems 394 DNA/RNA synthesizer using a 0.2
lM cycle with a 25 s coupling time for unmodified phosphora-
midites. All phosphoramidites were dissolved in anhydrous
acetonitrile to a 0.1M concentration immediately prior to syn-
thesis. Synthesis on solid phase was accomplished using 0.2
lM solid supports. Cleavage of the unmodified oligonucleo-
tides from their solid supports was performed through expo-
sure to 1.5 mL of 30% NH4OH for 2 h at room temperature,
followed by incubation in 30% NH4OH at 55ꢀC overnight. All
oligonucleotides were purified on a 20% denaturing polyacryl-
amide gel by utilizing the ‘‘crush and soak’’ method, followed
by ethanol precipitation and desalting through Millipore 3000
MW cellulose centrifugal filters. The oligonucleotides were
characterized by MALDI-TOF mass spectrometry (Waters Tof-
spec-2E) (Table 2).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2011
Synthesis and Properties of Oligonucleotides that Contain a
Triazole-Linked Nucleic Acid Dimer
539
Synthesis of 50 TTTTTCTCTCTCUU 30 and 50
UUTTTCTCTCTCTT 30. Phosphoramidite 11 was attached
to the 50 end of the growing oligonucleotide being synthesized
on the ABI 394 DNA/RNA synthesizer, with an increased cou-
pling time of 600 s. To perform the 30 modification, compound
11 was attached to a Universal III solid support (Glen
Research) via the ABI 394 synthesizer with a 600 s coupling
time. Cleavage from the Universal III solid support was per-
formed through exposure to 1.5 mL of 2M NH3 in MeOH for
30 min at room temperature, followed by incubation in 30%
NH4OH at 55ꢀC for 8 h.
Synthesized sequences. Oligonucleotides synthesized using
standard dT/dA supports: (i) 50-AAG AGA GAG AAA AA–30;
(ii) 50–TTT TTC TCT CTC TT–30; and (iii) 50–UUT TTC
TCT CTC TT–30. The UU–30 modified sequence synthesized
on the Universal III solid support: 50–TTT TTC TCT CTC
UU–30. The sequence 50-AAG AGA GAG AAA AA-30 was
purchased and purified from Integrated DNA Technologies.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet