Design and synthesis of dephosphono dna analogues containing 1,2,3-triazole linker and their uv-melting studies with DNA/RNA

Article abstract of DOI:10.1080/15257770.2011.644100

This article describes the synthesis of 3/5 linked 1,2,3-triazolyl dithymidine derivatives, their incorporation into oligonucleotides, and evaluation of their thermal stabilities toward complementary DNA/RNA. Copyright Taylor and Francis Group, LLC.

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Design and Synthesis of Dephosphono
DNA Analogues Containing 1,2,3-Triazole
Linker and Their UV-Melting Studies with
DNA/RNA
Vangala Madhuri a & Vaijayanti A. Kumar a
a Division of Organic Chemistry , National Chemical Laboratory ,
Pune , Maharashtra , India
Published online: 03 Feb 2012.
To cite this article: Vangala Madhuri & Vaijayanti A. Kumar (2012) Design and Synthesis of
Dephosphono DNA Analogues Containing 1,2,3-Triazole Linker and Their UV-Melting Studies with DNA/
RNA, Nucleosides, Nucleotides and Nucleic Acids, 31:2, 97-111, DOI: 10.1080/15257770.2011.644100
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Nucleosides, Nucleotides and Nucleic Acids, 31:97–111, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.644100
DESIGN AND SYNTHESIS OF DEPHOSPHONO DNA ANALOGUES
CONTAINING 1,2,3-TRIAZOLE LINKER AND THEIR UV-MELTING
STUDIES WITH DNA/RNA
Vangala Madhuri and Vaijayanti A. Kumar
Division of Organic Chemistry, National Chemical Laboratory, Pune, Maharashtra, India
This article describes the synthesis of 3ꢁ/5ꢁ linked 1,2,3-triazolyl dithymidine derivatives, their
2
incorporation into oligonucleotides, and evaluation of their thermal stabilities toward complemen-
tary DNA/RNA.
Keywords Oligonucleotide analogues; isostere; triazole; UV melting
INTRODUCTION
Chemical modification of oligonucleotides came to prominence in the
early 1990s with the advent of the antisense approach to control gene ex-
pression.[1] Among several nucleic acid analogues that have been studied
over the last two decades, only a very few are backbones with linkers that are
either shorter[2] or longer[3] than the four-atom linker O3ꢁ-P-O5ꢁ-C5ꢁ con-
necting sugar ring moieties in DNA and RNA. The four-atom amide substitu-
tion for four-atom phosphodiester linkage led to chimeric oligonucleotides
with amide-phosphodiester groups. Such chimeric oligonucleotides formed
complexes with complementary nucleic acids with a moderate RNA-binding
selectivity and with moderately diminished DNA affinity.[4] Amide linker is
known to be shorter than the phosphate linker, a five-atom amide linker, pro-
vided the requisite flexibility in the linker and thus stabilized the duplexes
with RNA.[5] The amide linkers in 2ꢁ-O-methyl substituted nucleosides also
led to the stabilization of RNA duplexes. The replacement of 3ꢁ-O with a
methylene group and the addition of a methylene group in either R or S
configuration also led to the stabilization of duplexes[6] (Figure 1). Some
amide-linked oligonucleotides were also reported by our group where a
Received 16 July 2010; accepted 18 November 2011.
Address correspondence to Vaijayanti A. Kumar, Division of Organic Chemistry, National Chemical
Laboratory, Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India. E-mail: va.kumar@ncl.res.in
97

98
V. Madhuri and V. A. Kumar
H-Bond acceptor
H-Bond acceptor
Electrophilic
carbon
Electrophilic
carbon
N
N
O
N R2
R2
R1
R1
N
H
H
H-Bond donor
H-Bond donor
R1 to R2 distance
= 5.0 Ao
R1 to R2 distance
= 3.9 Ao
FIGURE 1 Topological and electronic similarities of amides and 1,2,3-triazoles.
novel class of thioacetamido backbone[7] and an amino acid backbone ex-
tended DNA modification[8] were found to improve the binding affinity and
selectivity toward DNA/RNA recognition.
As triazole is known to be the nonclassical isostere of amide (Figure 1),
we envisaged the replacement of the amide linkage in the DNA dimer block
with a six-atom 1,2,3-triazole linkage (Figure 2). The planar aromatic cyclic
structure of triazole in the linker might require extended backbone flexibility
O
O
O
NH
NH
NH
HO
N
O
O
O
O
O
4
N
O
N
O
HO
O
3'
1
2
O
1
3'
2
O
N
N N
O
N
O
N
3
N
N
NH
NH
NH
3
NH
O
N
N
5
5'
O
5'
4
O
O
5
O
O
6
6
O
O
OMe
OH
(tt)
(C)
OH
(tt)
(B)
(A)
FIGURE 2 Chemical and structural representation of (A) amide-linked dimer, (B) 3ꢁ-O-alkynyl-5ꢁ-azide
triazolyl tt dimer, and (C) 5ꢁ-O-alkynyl-3ꢁ-azide triazolyl tt dimer.

DNA Analogues Containing 1,2,3-Triazole Linker
99
as compared with the amide linker. A recent article appeared where one-
atom extended 1,2,3-triazole linker was found to be suitable in acyclic amide-
linked PNA backbone.[9]
The Huisgen 1,3-dipolar cycloaddition between azides and alkynes, per-
formed by the recently discovered copper(I) catalysis[10], appears to be a
promising way to generate oligomers and particularly nonnatural triazole-
linked oligodeoxynucleotides. Previous studies, introducing a four-atom
triazole linkage in oligonucleosides backbone, have been realized using
thymidine as nucleoside (homothymidines) in both solution-phase and solid-
phase synthesis.[11] We propose an extended six-atom, 1,4-disubstituted-
1,2,3-triazole as the linker that would conserve the directional character
of DNA strands. While this work was under revision, a very interesting ap-
plication of the same six-atom triazolide linker group has appeared in the
literature.[12] Owing to the extended six-atom backbone, there is a possibil-
ity of cross pairing with both DNA and RNA. This modification may also
impart stability to the oligomers due to its neutral nature that will not have
any repulsive interactions with the anionic phosphate backbone of natural
DNA/RNA. The rigid planar, π-rich backbone and possibility of metal co-
ordination of the triazole rings may prove to be important in the structural
chemistry and biology of oligonucleotides. We report in this article, the two
triazole-linked dimers (Figure 2), when the triazole ring is formed at the
chiral C-3ꢁ carbon center and also when it is present at the achiral C-5ꢁ car-
bon of the sugar by synthesizing two different dimer units. Synthesis of the
two dimer blocks, triazolyl tt dimer from 3ꢁ-O-alkynyl and 5ꢁ-azido monomers
(Figure 2B) and triazolyl tt dimer from 5ꢁ-O-alkynyl and 3ꢁ-azido monomers
(Figure 2C), their incorporation into oligonucleotides, and UV-Tm
studies with complementary DNA and RNA are presented.
RESULTS AND DISCUSSIONS
Synthesis of Triazolyl Dithymidine tt Dimer Block (6) from
3ꢀ-O-Alkynyl Thymidine and 5ꢀ-Azido Thymidine
The free OH groups of thymidine were protected as trimethylsilyl (TMS)
ethers using TMS-Cl in pyridine followed by the addition of benzoyl chlo-
ride to get the N 3-benzoylated compound. The protection of the N3 atom
of thymine was necessary because if left unprotected, the latter alkylation re-
action with propargyl bromide gave exclusively N -alkylated product rather
than the required O-alkylated compound. The 5ꢁ-hydroxyl group of N 3-
benzoyl-thymidine was converted to its dimethoxttrityl (DMT) derivative by
treatment with DMTr-Cl in pyridine to get 2, which was alkylated using
propargyl bromide and NaH in dimethylformamide (DMF) to get the 3ꢁ-O-
alkyne derivative 3 in 55% yield (Scheme 1). Some amount of debenzoylation

100
V. Madhuri and V. A. Kumar
was observed under these reaction conditions as the thin layer chromatog-
raphy (TLC) of the reaction mixture showed some lower moving impurities
which could be a mixture of N - or O-alkylated products arising from the
debenzoylated compound. The yield of N-3 Bz-3ꢁ-O-alkylated product was
therefore only 55%.
O
N
O
N
O
N
NBz
O
NH
NBz
O
DMTO
HO
DMTO
O
O
O
O
iii
i, ii
OH
2
OH
1
O
3
O
iv
NBz
O
O
O
N
DMTO
N
NH
O
NH
O
N3
TsO
N
O
O
O
v
3, vi
O
O
OH
OH
N
NH
5
4
N
N
tt derivative
6
N
O
O
OH
Reagents: i. (a) TMS-Cl/pyridine (b) Bz-Cl (c) aq NH3 sol ii. DMTr-Cl/pyridine 85% iii. propargyl bromide/NaH,
iv. v. vi.
DMF 55%
Ts-Cl/pyridine 85% NaN3/DMF 87%
0.2% CuSO4, 0.5% Na-ascorbate, t-BuOH:H2O 80%
SCHEME 1 Synthesis of 3ꢁ-O-alkynyl-5ꢁ-azido triazolyl tt dimer unit (6).
Monomer 5 was synthesized from thymidine through monotosylation at
the 5ꢁ position using tosyl chloride in pyridine under dilute conditions to
get compound 4. Compound 4 was then treated with excess of sodium azide
in dry DMF to get compound 5, which was confirmed from the IR spectrum
showing characteristic absorption for azide group at 2100 cm−1. Derivatives
3 and 5 were then coupled using copper (I)-catalyzed cycloaddition in order
to obtain 1,4-dithymidine-substituted-1,2,3-triazole 6. This click reaction was
performed using CuSO4 and sodium ascorbate.[13]
Synthesis of Triazolyl Dithymidine tt Dimer Unit (12)
from 5ꢀ-O-Alkynyl Thymidine and 3ꢀ-Azido Thymidine
The two free hydroxyl groups of thymidine were protected as t-
butyldimethylsilyl (TBDMS) ethers using TBDMS-Cl in DMF, and the N3
position of thymine was protected as its benzoyl derivative using benzoyl
chloride in pyridine to get compound 7. In the next step, the primary 5ꢁ
hydroxyl group was deprotected selectively, keeping intact the secondary

DNA Analogues Containing 1,2,3-Triazole Linker
101
3ꢁ TBDMS ether using I2/MeOH[14] to get compound 8. This reaction was
carried out at room temperature for a period of 3–4 hours and was moni-
tored by TLC. It was observed that when the reaction was left for a longer
time, both primary and secondary TBDMS ethers were deprotected and N3-
benzoyl thymidine was obtained. The free 5ꢁ hydroxyl group in 8 was then
alkylated using propargyl bromide and NaH in DMF to get the alkylated
compound 9 in good yield.
The 3ꢁ-azido thymidine derivative was obtained from a known proce-
dure.[15] The alkyne monomer unit 9 and azide 10 were subjected to click
reaction using CuSO4 and sodium ascorbate. The reaction was carried out in
1:1 ratio of t-butanol and H2O to get the 1,4-dithymidine substituted 1,2,3-
triazole dimer 11 regioselectively in good yields. The TBDMS group was
deprotected using 1 M tetrabutylammonium fluoride (TBAF) in tetrahydro-
furan (THF) to get the desilylated product 12 (Scheme 2).
O
N
O
N
O
N
O
N
NBz
O
NBz
O
NH
NBz
O
HO
TBDMSO
O
HO
O
O
O
O
i
iii
ii
O
OTBDMS
OTBDMS
OH
1
OTBDMS
7
8
9
O
O
NH
O
NH
O
N
N
DMTO
N
O
DMTO
NH
O
O
O
O
DMTO
O
O
O
N
N
O
N
N
N3
10
N
N
NBz
, iv
v
NBz
N
O
N
O
O
O
tt derivative
OH
OTBDMS
12
11
Reagents: i. (a) TBDMS-Cl/DMF 95% (b) Bz-Cl/pyridine 90% ii. I2/MeOH 70% iii. propargyl bromide, NaH/DMF
iv. 10 v.
80%,
, 0.2eq CuSO4, 0.5eq Na-ascorbate t-BuOH:H2O 80% 1M TBAF/THF, 87%
SCHEME 2 Synthesis of 5ꢁ-O-alkynyl 3ꢁ-azide triazolyl dithymidine dimer unit (12).
Synthesis of tt and tt Phosphoramidite Building Blocks
The 3ꢁ hydroxyl group in the dimers 6 and 12 was phosphitylated by
treating with chloro(cyanoethoxy-N ,N ꢁ-diisopropylamino)phosphine to give

102
V. Madhuri and V. A. Kumar
O
N
O
N
NBz
O
NH
DMTO
DMTO
O
O
O
O
O
N N
O
N
i
6
O
N
NBz
O
N
i
NH
N
12
N
O
O
N
O
tt
tt
O
P
O
O
CN
N
O
N P
CN
14
13
DNA Synthesizer
Reagents: i. Chloro (cyanoethoxy-N, N'-diisopropylamino)phosphine, DIEA, DCM 87%
SCHEME 3 Synthesis of phosphoroamidites.
the phosphoramidites 13 and 14, ready for the use in an automated DNA
synthesizer for their incorporation into oligomers (Scheme 3).
Oligomers Synthesized by Incorporation of tt/tt Dimers,
Purification, and MALDI-TOF Analysis
A series of chimeric oligonucleotides (Table 1) containing one or two
dimer blocks incorporated at the predetermined positions were synthesized
by automated solid-phase synthesis using the phosphoramidite approach.
Control 18mer oligonucleotide (DNA-a) chosen was specific for the splice
correction of an aberrant β-globin intron (705 site).[16] The control 19mer
oligonucleotide (DNA-b) is the antisense sequence for bcr/abl b2a2 gene,
which is responsible for chronic myeloid leukemia.[17] The 10mer sequence
(DNA-c) is again the antisense sequence for bcr/abl b3a2 gene, which is
responsible for chronic myeloid leukemia.[17] The modified sequences were
synthesized by the incorporation of a single tt or tt blocks, or two modi-
fied tt or tt blocks were incorporated in the above-mentioned unmodified
DNA sequences. Oligonucleotides were synthesized on a 40 nmol scale in an
ABI 3900 High Throughput DNA synthesizer using standard β-cyanoethyl
phophoramidate chemistry on a controlled pore glass (CPG) resin. Final
deprotection was done by giving ammonia treatment at 55◦C for 5 hours.
The crude yield of the oligomers based on the scale of synthesis was found

DNA Analogues Containing 1,2,3-Triazole Linker
103
TABLE 1 Oligomers synthesized, their HPLC retention times, and MALDI-TOF mass
HPLC (rt)
(minutes)
Experimental
Observed
mass
Code
Sequences
mass
DNA-a
5ꢁ CCT CTT ACC TCA GTT ACA 3ꢁ
5ꢁ CCT CTT ACC TCA Gtt ACA 3ꢁ
5ꢁ CCT Ctt ACC TCA Gtt ACA 3ꢁ
5ꢁ CCT Ctt ACC TCA GTT ACA 3’
5ꢁ CCT CTT ACC TCA Gtt ACA 3ꢁ
5ꢁ CCT Ctt ACC TCA Gtt ACA 3ꢁ
5ꢁ CCT Ctt ACC TCA GTT ACA 3ꢁ
5ꢁ GAA GGG CTT TTG AAC TCT T 3ꢁ
5ꢁ GAA GGG Ctt TTG AAC TCT T 3ꢁ
5ꢁ GAA GGG Ctt ttG AAC TCT T 3ꢁ
5ꢁ GAA GGG CTT ttG AAC TCT T 3ꢁ
5ꢁ GAA GGG Ctt TTG AAC TCT T 3ꢁ
5ꢁ GAA GGG Ctt ttG AAC TCT T 3ꢁ
5ꢁ GAA GGG CTT ttG AAC TCT T 3ꢁ
5369.6
DNA-a-tt-1
DNA-a-tt-2
DNA-a-tt-3
DNA-a-tt-1
DNA-a-tt-2
DNA-a-tt-3
DNA-b
DNA-b-tt-1
DNA-b-tt-2
DNA-b-tt-3
DNA-b-tt-1
DNA-b-tt-2
DNA-b-tt-3
7.9
8.3
8.1
7.8
8.4
8.2
5371.35
5373.10
5371.35
5371.35
5373.10
5371.35
5833.90
5835.65
5837.40
5835.65
5835.65
5837.40
5835.65
5370.76
5370.91
5369.95
5375.66
5381.60
5381.30
7.3
8.4
7.4
7.9
8.1
7.8
5838.67
5847.82
5832.62
5837.73
5845.82
5835.69
to be 90% and the yield of the high-performance liquid chromatography
(HPLC) purified oligomers was found to be in the range of 60%–70%. The
sequences synthesized were deprotected and cleaved from the solid sup-
port. The purification was carried out by reversed-phase HPLC (RP-HPLC)
and the purified oligomers were characterized by MALDI-TOF (matrix-
assisted laser desorption/ionization [MALDI] time-of-flight [TOF]) mass
spectrometry.
UV-Tm of tt/tt Incorporated Oligomers Incorporated
into 18mer (DNA4)
The UV-Tm experiments of duplexes (1 µM) of each strand were
prepared in 10 mM sodium phosphate buffer, pH 7.2, containing NaCl
(100 mM) and ethylenediaminetetraacetic acid (EDTA; 0.1 mM) and were
annealed by keeping the samples at 90◦C for 5 minutes followed by slow
cooling to room temperature. Absorbance versus temperature profiles were
obtained by monitoring at 260 nm with CARY 300 BIO UV-Visible spec-
trophotometer scanning from 5◦C to 85◦C at a ramp rate of 0.2◦C per minute.
The data were processed using Microcal Origin 5.0 software and Tm values
derived from the derivative curves. Introduction of a single tt dimer unit
toward the 3ꢁ end in 18mer oligomer DNA-a-tt-1 showed the destabilization
of 13◦C and 12.3◦C with complementary DNA and RNA. The amount of
destabilization was same when the tt dimer was incorporated toward the 5ꢁ
end as in DNA-a-tt-3. A further destabilization was observed for the complex
DNA-a-tt-2:DNA1/RNA1 when two units of modified tt dimer were incorpo-
rated. The binding of triazolyl-substituted oligonucleotides containing single

104
V. Madhuri and V. A. Kumar
TABLE 2 UV-Tm (◦C) of DNA-a-tt /DNA-a-tt oligomers with complementary DNA/RNA
UV-Tm (◦C)
Code
DNA1
RNA1
DNA-a
53.8
58.1
DNA-a-tt-1/DNA-a-tt-1
DNA-a-tt-2/DNA-a-tt-2
DNA-a-tt-3/DNA-a-tt-3
40.8/41.8
36.3/30.8
43.0/37.5
45.8/36.0
35.4/32.8
44.6/39.3
mismatch at the middle position of the sequence was studied with comple-
mentary RNA1. For oligonucleotides DNA-a-tt-1, DNA-a-tt-3, and DNA-a-tt-2,
the decrease of binding affinity was 12.3◦C, 10.7◦C, and 9.2◦C, respectively,
in comparison with RNA1 when they were hybridized with one single-base
mismatch RNA4. The results are tabulated in Table 2. The destabilization of
tt dimer incorporated triazolyl oligomers DNA-a-tt-1, DNA-a-tt-2, and DNA-
a-tt-3 was found to be more when compared with tt triazolyl oligomers. DNA-
a-tt-1 and DNA-a-tt-3 triazolyl oligomers with a single tt dimer unit toward
the 3ꢁ and 5ꢁ end showed a binding affinity of 41.8◦C and 36.0◦C, respec-
tively. The ꢀTm between DNA-a-tt-1:RNA1 and DNA-a-tt-1:RNA1 and that of
DNA-a-tt-3:RNA1 and DNA-a-tt-3:RNA1 was found to be −9.8◦C and −5.3◦C,
respectively. The cumulative destabilization effect was observed when two
tt dimer units were incorporated. The UV-Tm derivative curves and their
results are shown in Figure 3.
UV-Tm of tt/tt Oligomers Incorporated into 19mer (DNA5)
The tt dimer building block when incorporated into the 19mer (DNA5)
in the middle of the sequence DNA-b-tt-1 decreased the Tm by 15.5◦C when
compared with the unmodified sequence. The binding of DNA-b-tt-1 with
RNA2 showed a decrease in Tm of 7.4◦C. The DNA-b-tt-2 triazolyl oligomer
with two tt dimer units incorporated together showed a Tm of 40.8◦C and
42.5◦C, respectively, with complementary DNA and RNA (Table 3). The
thermal stability of DNA-b-tt-3 sequence with DNA2/RNA2 was found to be
46.5◦C and 48.2◦C, respectively. The difference in Tm with single and double
TABLE 3 UV-Tm (◦C) of DNA-b-tt/DNA-b-tt oligomers with complementary DNA/RNA
UV-Tm (◦C)
Code
DNA2
RNA2
DNA-b
56.8
54.3
DNA-b-tt-1/DNA-b-tt-1
DNA-b-tt-2/DNA-b-tt-2
DNA-b-tt-3/DNA-b-tt-3
41.3/38.2
40.8/35.3
46.5/39.2
46.9/39.2
42.5/35.0
48.2/38.1

DNA Analogues Containing 1,2,3-Triazole Linker
105
FIGURE 3 UV derivative curves for DNA-a-tt oligomers with complementary DNA/RNA (10 mM sodium
phosphate buffer, pH 7.2, 100 mM NaCl, 0.1 mM EDTA). The values reported here are an average of
three independent experiments and are accurate to 0.5◦C.
modification is not very large, which means when the modified units are to-
gether, the degree of destabilization is not high as compared with oligomers
when the modified units are apart. The stabilization of tt dimer incorporated
DNA-b-tt-1 and DNA-b-tt-3 oligomers showed a Tm of 38.2◦C and 39.2◦C with
complementary DNA2 and 39.2◦C and 38.1◦C with complementary RNA2.
In comparison with DNA-b-tt-1 and DNA-b-tt-3 tt based triazolyl oligomers,
the decrease in Tm observed for DNA-b-tt-1, DNA-b-tt-3 with DNA2/RNA2
was 3.1◦C, 7.3◦C and 7.7◦C, 10.1◦C, respectively. The thermal stability of
DNA-b-tt-2 with two modifications showed a Tm of 35.3◦C and 35.0◦C with
DNA2 and RNA2, respectively. The UV-Tm derivative curves and their results
are shown in Figure 4.
FIGURE 4 UV derivative curves for DNA-b-tt oligomers with complementary DNA/RNA (10 mM sodium
phosphate buffer, pH 7.2, 100 mM NaCl, 0.1 mM EDTA). The values reported here are an average of
three independent experiments and are accurate to 0.5◦C.

106
V. Madhuri and V. A. Kumar
CONCLUSION
The 3ꢁ-O-alkynyl-5ꢁ-azido triazolyl dithymidine dimer unit (tt) and 5ꢁ-O-
alkynyl 3ꢁ-azide triazolyl dithymidine dimer unit (tt) have been synthesized
and incorporated into the oligomers by phosphoramidite method using an
automated DNA synthesizer. The tt oligomers showed 10◦C–13◦C of desta-
bilization when a single dimer unit was incorporated and destabilization
increased further when two dimer units are incorporated. The tt oligomers
also showed 10◦C–15◦C of destabilization when a single dimer unit was in-
corporated and destabilization increased further when two dimer units were
incorporated.
EXPERIMENTAL
General
All the reagents were purchased from Sigma-Aldrich and used without
˚
purification. DMF and pyridine were dried over KOH and 4 A molecular
sieves. THF was passed over basic alumina and dried by distillation over
sodium. TLCs were run on Merck 5554 silica 60 aluminum sheets. Col-
umn chromatography was performed for purification of compounds on
silica gel (100–200 mesh, LOBA Chemie). TLCs were performed using
dichloromethane–methanol or petroleum ether–ethyl acetate solvent sys-
tems for most compounds. Compounds were visualized with UV light and/or
by spraying with perchloric acid solution and heating. 1H (200 MHz) and 13C
(50 MHz) NMR spectra were recorded on a Bruker ACF 200 spectrometer
fitted with an Aspect 3000 computer, and all the chemical shifts (δ/ppm)
1
are referred to internal TMS for H NMR and chloroform-d and DMSO-d6
for 13C NMR. Mass spectra were recorded on a Finnigan-Matt mass spec-
trometer, while MALDI-TOF spectra were obtained from a KRATOS PCK-
ompact instrument. UV experiments were performed on a Perkin Elmer
λ35 UV-VIS spectrophotometer fitted with a Peltier temperature program-
mer and a Julabo water circulator. The DNA oligomers were synthesized on
CPG solid support using an ABI 3900 High Throughput DNA synthesizer by
β-cyanoethyl phosphoramidite chemistry followed by ammonia treatment
and their purities checked by HPLC prior to use. RNA was purchased from
Sigma-Aldrich.
CHARACTERIZATIONS: SPECTROSCOPIC DATA OF SELECTED
NEW COMPOUNDS
5ꢀ-O-Dimethoxytrityl 3ꢀ-Propynyl N3-Benzoyl Thymidine (3)
Compound 2 (1 g, 1.54 mmol) was taken in 10:1 dry DMF and ace-
tonitrile and cooled to 0◦C. NaH 60% in hexane (92 mg, 2.31 mmol) was

DNA Analogues Containing 1,2,3-Triazole Linker
107
added in portions followed by the addition of propargyl bromide (0.48 mL,
3.08 mmol) and the reaction was stirred for 2–3 hours with continuous mon-
itoring by TLC. Leaving the reaction for a long time led to the deprotection
of benzoyl group and the yield of the reaction also decreased. The reaction
mixture was quenched by adding few milliliters of MeOH to it. DMF was re-
moved under vacuum and the mixture was extracted into EtOAc (50 mL ×
2), washed with water, brine, and dried over Na2SO4. The crude mixture
was purified by column chromatography using 25% EtOAc in petroleum
ether and concentrated under vacuum to get compound 3 as a colorless
1
foam. Yield: 0.57 g (55%); IR (CHCl3) ν/cm−1 3307, 3020, 1652, 1215; H
NMR (200 MHz, CDCl3) δH 1.46 (s, 3H), 2.29–2.57 (m, 2H), 2.40 (t, 1H),
3.35–3.58 (m, 2H), 3.79 (s, 6H), 4.16 (m, 2H), 4.55 (m, 1H), 4.72 (m, 1H),
6.39 (dd, 1H, ꢀJ 1 = 6.0 Hz, ꢀJ 2 = 7.88 Hz), 6.81–6.87 (d, 4H, ꢀJ = 8.17
Hz), 7.28–7.75 (m, Ar), 7.91–7.95 (m, 2H); 13C NMR (50 MHz, CDCl3) δC
12.3, 36.5, 55.2, 60.4, 63.5, 70.6, 72.0, 76.5, 85.5, 86.3, 86.8, 110.3, 113.0,
127.1, 127.7, 127.9, 128.1, 129.1, 130.0, 134.3, 135.4, 139.5, 144.3, 150.2,
158.4, 158.6, 162.4; MS (EI) m/z 686.75 (calc.), found 709.73 (M+Na)+.
3ꢀ-O-Alkynyl 5ꢀ-Azide Dithymidine tt Dimer (6)
Individual monomer units 3 (1 g, 1.45 mmol) and 5 (0.31 g, 1.16 mmol)
were taken in 10 mL of 1:1 t-BuOH and water. CuSO4 (72.6 mg, 0.29 mmol)
and sodium ascorbate (144 mg, 0.72 mmol) were added to the reaction. The
reaction mixture was left overnight. t-BuOH was removed under reduced
pressure and the mixture was extracted into EtOAc (100 mL × 2), washed
with water, brine, and dried over Na2SO4. The crude mixture was purified by
column chromatography using 90% EtOAc/petroleum ether to 3% MeOH
in EtOAc and concentrated under vacuum to get compound 6 as a colorless
foam. Yield: 80%; IR (CHCl3) ν/cm−1 3017, 2929, 1702, 1508; 1H NMR (200
MHz, CDCl3) δH 1.36 (s, 3H), 1.76 (s, 3H), 2.17–2.36 (m, 5H), 3.32–3.52 (m,
2H), 3.81 (s, 6H), 4.03–4.18 (m, 2H), 4.26–4.56 (m, 4H), 5.22–5.25 (m, 1H),
6.0–6.24 (m, 2H), 6.72 (s, 1H), 6.84–6.89 (d, 4H, ꢀJ = 8.77 Hz), 7.26–8.04
(m, Ar), 9.8 (br, 1H); MS (EI) m/z 953.99 (calc.), found 976.33 (M+Na)+.
N3-Benzoyl-3ꢀ-O-tert-Butyldimethylsilyl Thymidine (8)
To compound 7 (2 g, 4.34 mmol) in 20 mL MeOH, I2 (∼300 mg) was
added and the reaction mixture was stirred for 3–4 hours at room temper-
ature with continuous monitoring by TLC. It was observed that if the reac-
tion was left for a long time, 3ꢁ-OTBDMS group also gets deprotected and
benzoyl thymidine was obtained. When the slightest formation of benzoyl
thymidine was observed, the reaction mixture was quenched immediately by
adding Na2S2O3 till the solution, which is dark brown, becomes colorless.
MeOH was removed under reduced pressure and the mixture was extracted

108
V. Madhuri and V. A. Kumar
into organic layer, washed with water, brine, and dried over Na2SO4. The
crude mixture was purified by column chromatography using 40% EtOAc
in petroleum ether and concentrated under vacuum to get compound 8 as
1
a colorless solid. Yield: 1.12 g (70%); H NMR (200 MHz, CDCl3) δH 0.07
(s, 6H), 0.087 (s, 9H), 1.88 (s, 3H), 2.09–2.26 (m, 3H), 3.65–3.87 (m, 3H),
4.37–4.44 (m, 1H), 6.14–6.21 (t, 1H, ꢀJ = 6.71 Hz), 7.44–7.64 (m, 3H),
7.90–7.93 (m, 2H); 13C NMR (50 MHz, CDCl3) δC −4.7, 12.6, 25.7, 40.9,
61.9, 71.8, 86.4, 87.8, 110.8, 129.1, 130.4, 131.5, 135.1, 136.7, 149.4, 163.0,
169.0; MS (EI) m/z 460.60 (calc.), found 462.03 (M+1)+, 484.09 (M+Na)+.
N3-Benzoyl-5ꢀ-O-Propynyl 3ꢀ-O-tert-Butyldimethylsilyl
Thymidine (9)
Compound 8 (1 g, 2.17 mmol) was taken in dry DMF (20 mL) and
acetonitrile (2 mL) and cooled to 0◦C. Propargyl bromide (0.28 mL, 3.26
mmol) was added and NaH (0.104 g, 2.60 mmol) was added in portions, and
the reaction mixture was stirred for 2–3 hours with continuous monitoring by
TLC. The reaction mixture was quenched by adding few milliliters of MeOH
to it. DMF was removed under vacuum and the mixture was extracted into
EtOAc (50 mL × 2), washed with water, brine, and dried over Na2SO4. The
crude mixture was purified by column chromatography using 30% EtOAc
in petroleum ether and concentrated under vacuum to get compound 9
as a colorless foam. Yield: 0.86 g (80%); IR (CHCl3) ν/cm−1 3308, 3019,
1
2955, 2930, 1707, 1671; H NMR (200 MHz, CDCl3) δH 0.00 (s, 6H), 0.80
(s, 9H), 1.60 (s, 3H), 2.00–2.39 (m, 3H), 3.55–3.76 (m, 1H), 4.13 (m, 1H),
4.34–4.71 (m, 4H), 6.23–6.29 (dd, 1H, ꢀJ 1 = 6.37 Hz, ꢀJ 2 = 7.01 Hz),
7.34–7.54 (m, 3H), 7.90–7.95 (m, 2H); 13C NMR (50 MHz, CDCl3) δC −4.6,
12.9, 25.68, 30.3, 41.2, 58.5, 63.7, 70.4, 70.6, 78.1, 85.8, 86.2, 110.4, 128.7,
129.5, 133.6, 150.0, 162.3; MS (EI) m/z 498.64 (calc.), found 499.57 (M+1)+,
521.58 (M+Na)+.
5ꢀ-O-Dimethoxytrityl 3ꢀ-O-tert-Butyldimethylsilyl
1,4-disubstituted 1,2,3-Triazolyl Dithymidine Dimer (11)
Individual monomer units 9 (1 g, 2.0 mmol) and 10 (0.91 g, 1.6 mmol)
were taken in 10 mL of 1:1 t-BuOH and water. CuSO4 (0.1 g, 0.401 mmol)
and sodium ascorbate (0.198 g, 1.0 mmol) were added to the reaction.
The reaction mixture was left overnight at room temperature. t-BuOH
was removed under reduced pressure and the mixture was extracted into
EtOAc (100 mL × 2), washed with water, brine, and dried over Na2SO4. The
crude mixture was purified by column chromatography using 85% EtOAc
in petroleum ether and concentrated under vacuum to get compound 11
as a colorless foam. Yield: 1.7 g (80%); IR (CHCl3) ν/cm−1 3017, 2929,
1
1702, 1508; H NMR (200 MHz, CDCl3) δH 0.00 (s, 6H), 0.87 (s, 9H), 1.24

DNA Analogues Containing 1,2,3-Triazole Linker
109
(s, 3H), 1.53 (s, 3H), 2.03–2.46 (m, 4H), 2.59–2.73 (m, 1H), 3.27–3.3 (m,
1H), 3.59–3.68 (m, 2H), 3.77 (s, 6H), 4.19–4.21 (m, 1H), 4.41–4.49 (m, 2H),
4.56–4.67 (m, 1H), 5.0–5.11 (m, 1H), 6.31–6.49 (m, 2H), 6.80–6.84 (d, 4H,
ꢀJ = 8.74 Hz), 7.25–8.02 (m, Ar); 13C NMR (50 MHz, CDCl3) δC −4.8,
12.0, 12.9, 17.9, 25.6, 29.6, 38.7, 41.4, 55.2, 62.2, 63.7, 71.9, 75.3, 78.9, 83.5,
85.7, 87.0, 110.3, 111.4, 113.3, 127.2, 128.0, 128.7, 129.4, 130.0, 133.6, 135.0,
144.1, 150.1, 150.5, 158.7, 166.1; MS (EI) m/z 1068.25 (calc.), found 1091.30
(M+Na)+.
5ꢀ-O-Alkynyl 3ꢀ-Azide Triazolyl Dithymidine tt Dimer Unit (12)
Compound 11 (1 g, 0.93 mmol) was taken in dry THF (10 mL) and
cooled to 0◦C. Then 1 M TBAF in THF (0.48 mL, 1.87 mmol) was added to
it at 0◦C. The reaction mixture was stirred for a period of 2 hours. THF was
removed and the reaction mixture was extracted into EtOAc (100 mL × 2),
washed with brine, and dried over Na2SO4. The crude mixture was purified
by column chromatography using EtOAc and concentrated under vacuum
to get compound 12 as a colorless foam. Yield: 0.77 g (87%): 1H NMR (200
MHz, CDCl3) δH 1.49 (s, 3H), 1.64 (s, 3H), 2.1–2.21 (m, 1H), 2.46–2.73 (m,
2H), 2.88–3.01 (m, 1H), 3.28–3.61 (m, 4H), 3.76 (s, 6H), 4.27–4.64 (m, 5H),
5.01 (s, 1H), 6.40–6.53 (m, 2H), 6.79–6.83 (d, 4H, ꢀJ = 8.98 Hz), 7.23–7.64
(m, Ar), 7.87 (s, 1H), 7.97–8.0 (d, 2H), 9.6 (hump, 1H) NH; 13C NMR (50
MHz, CDCl3) δC 11.9, 12.9, 14.2, 35.8, 40.7, 55.2, 60.4, 62.4, 64.3, 71.3, 83.5,
84.9, 87.1, 110.4, 111.1, 113.1, 123.5, 128.0, 128.6, 129.5, 130.0, 133.5, 135.0,
143.4, 144.1, 150.5, 150.6, 158.7, 162.9, 164.2, 166.3; MS (EI) m/z 953.99
(calc.), found 976.33 (M+Na)+.
Synthesis of 5ꢀ-O-(4,4ꢀ-Dimethoxy) Trityl-tt/tt-3ꢀ-O-
(2-Cyanoethyl-N,Nꢀ-Diisopropyl Phosphoramidite) Dimer (13, 14)
Compound 6/12 (0.35 g, 0.366 mmol) was dissolved in dry
dichloromethane (DCM; 10 mL) followed by the addition of diiso-
propyl ethyl amine (0.25 mL, 1.46 mmol) and chloro(2-cyanoethoxy-N,Nꢁ-
diisopropylamino)-phosphine (0.16 mL, 0.73 mmol), and the reaction mix-
ture was stirred at room temperature for 2 hours. The contents were then
diluted with dry DCM and washed with 5% NaHCO3 solution. The organic
phase was dried over anhydrous Na2SO4 and concentrated to foam. The
residue was dissolved in DCM and precipitated with hexane to obtain 13/14.
The phosphoramidite 13/14 were dried overnight over P2O5 and KOH in a
desiccator before applying on a DNA synthesizer. TLC shows two close mov-
ing spots for two diastereomers. Rf = 0.5, 2% methanol-dichloromethane;
Yield: 0.37 g (87%); 31P NMR (CDCl3) δ 149.0, 149.08.

110
V. Madhuri and V. A. Kumar
Synthesis of Complementary Oligonucleotides
The DNA oligonucleotides were synthesized on an Applied Biosystems
ABI 3900 High Throughput DNA synthesizer using standard β-cyanoethyl
phosphoramidate chemistry. The oligomers were synthesized in the 3ꢁ to 5ꢁ
direction on a polystyrene solid support, followed by ammonia treatment.
The oligonucleotides were desalted by gel filtration, their purity ascertained
by RP-HPLC on a C18 column was found to be more than 95%, and were
used without further purification. The RNA oligonucleotides were obtained
commercially.
HPLC Purification of Control/Triazolyl Oligomers
The purity of the oligomers was checked by RP-HPLC (C18 column,
0.1 N TEAA Buffer-ACN). The purity was again ascertained by analytical
RP-HPLC and integrity was confirmed by MALDI-TOF using THAP (2,4,6-
trihydroxyacetophenone) matrix with diammonium citrate as additive.
UV-Tm Studies of Triazolyl Oligomers
The binding affinity of triazolyl oligonucleotides with complementary
DNA/RNA was studied by a temperature-dependent UV absorbance ex-
periment. The Tm experiments of duplexes were carried out in 10 mM
sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM
EDTA. The triazolyl DNA:DNA/RNA chimeric oligonucleotides were indi-
vidually hybridized with the complementary DNA and RNA strands to obtain
duplexes.
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