Design and synthesis of LNA-based mercaptoacetamido-linked nucleoside dimers

Article abstract of DOI:10.1080/15257770.2013.783218

Three LNA-based mercaptoacetamido-linked nonionic nucleoside dimers T ^L-S-T, T-S-T^L, and T^L-S-T^L have been synthesized by HOBT and HBTU catalyzed condensation of silyl-protected 2-S-(thymidin-5 ′-yl)mercaptoacetic acid or 2-S-(2 ′-O,4 ′-C-methylenethymidin-5 ′-yl)mercaptoacetic acid with 3 ′-amino-3 ′-deoxy-5 ′-O-DMT-2 ′-O,4 ′-C- methylenethymidine or with 3 ′-amino-3 ′-deoxy-5 ′-O-DMT-β-thymidine followed by desilylation of the protected dimers. The 3 ′-O-phosphoramidite derivative of one of the nucleoside dimers was successfully prepared by condensation with [P(-Cl)(-OCH₂CH ₂CN)-N(iPr)₂}] in DCM in the presence of N,N-diisopropylethylamine (DIPEA), which is a building block for the preparation of mercaptoacetamido-linked oligonucleotides of therapeutic applications.

Full text of DOI:10.1080/15257770.2013.783218

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Design and Synthesis of LNA-Based
Mercaptoacetamido-Linked Nucleoside
Dimers
Vivek K. Sharma a , Sunil K. Singh a , Kapil Bohra a , Chandra Shekhar
Reddy L. a , Vinod Khatri a , Carl E. Olsen b & Ashok K. Prasad a
a Bioorganic Laboratory, Department of Chemistry, University of
Delhi, Delhi, India
b Faculty of Life Sciences, Department of Natural Sciences,
University of Copenhagen, Frederiksberg, Denmark
Published online: 14 Apr 2013.
To cite this article: Vivek K. Sharma , Sunil K. Singh , Kapil Bohra , Chandra Shekhar Reddy
L. , Vinod Khatri , Carl E. Olsen & Ashok K. Prasad (2013): Design and Synthesis of LNA-Based
Mercaptoacetamido-Linked Nucleoside Dimers, Nucleosides, Nucleotides and Nucleic Acids, 32:5,
256-272
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Nucleosides, Nucleotides and Nucleic Acids, 32:256–272, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.783218
DESIGN AND SYNTHESIS OF LNA-BASED
MERCAPTOACETAMIDO-LINKED NUCLEOSIDE DIMERS
Vivek K. Sharma,1 Sunil K. Singh,1 Kapil Bohra,1 Chandra Shekhar Reddy L.,1
Vinod Khatri,1 Carl E. Olsen,2 and Ashok K. Prasad1
1Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India
2Faculty of Life Sciences, Department of Natural Sciences, University of Copenhagen,
Frederiksberg, Denmark
Three LNA-based mercaptoacetamido-linked nonionic nucleoside dimers TL-S-T, T-S-TL, and
2
TL-S-TL have been synthesized by HOBT and HBTU catalyzed condensation of silyl-protected 2-S-
(thymidin-5 ꢁ-yl)mercaptoacetic acid or 2-S-(2 ꢁ-O,4 ꢁ-C-methylenethymidin-5 ꢁ-yl)mercaptoacetic acid
with 3 ꢁ-amino-3 ꢁ-deoxy-5 ꢁ-O-DMT-2 ꢁ-O,4 ꢁ-C-methylenethymidine or with 3 ꢁ-amino-3 ꢁ-deoxy-5 ꢁ-
O-DMT-β-thymidine followed by desilylation of the protected dimers. The 3 ꢁ-O-phosphoramidite
derivative of one of the nucleoside dimers was successfully prepared by condensation with [P(-Cl)(-
OCH2CH2CN){-N(iPr)2}] in DCM in the presence of N,N-diisopropylethylamine (DIPEA), which
is a building block for the preparation of mercaptoacetamido-linked oligonucleotides of therapeutic
applications.
[Supplementary materials are available for this article. Go to the publisher’s online
edition of Nucleosides, Nucleotides & Nucleic Acids for the following free supplemental
resource: supplementary information.doc.]
Keywords Locked nucleic acid; phosphate backbone modification; mercaptoace-
tamido-linkage; phosphoramidite-derivative
INTRODUCTION
The recent development in antisense, antigene, and RNA interference
technologies by using chemically modified oligonucleotides (ONs) has at-
tracted a great deal of both chemists and biologists.[1–5] The modified
Received 29 January 2013; accepted 4 March 2013.
We are thankful to the University of Delhi for providing financial support under DU-DST Purse
and R&D Grants. We express our sincere gratitude to Dr. Vaijayanti A. Kumar and Mrs. Anita D. Gunjal,
National Chemical Laboratory, Pune, for their help in the synthesis of phosphoramidite derivative of
one of the mercaptoacetamido-linked nonionic nucleoside dimers. We are also thankful to CIF-USIC
University of Delhi, Delhi, for NMR spectral data. V.K.S., S.K.S., K.B., and V.K. thank CSIR, New Delhi
and C.R.L. thanks DBT, New Delhi for the award of JRF/SRF.
Address correspondence to Ashok K. Prasad, Bioorganic Laboratory, Department of Chemistry,
University of Delhi, Delhi-110 007, India. E-mail: ashokenzyme@yahoo.com
256

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
257
O
O
T
C
O
O
O
O
B
B
HN
OC
HN
OC
O
O
CH2
S
CH2
S
O
O
P
O
P
H/OH
T
T
O
O
O-
O
O-
O
O
O
O
O
1b11
1c (T-S-T)20
1d (C-S-T)20
1a
FIGURE 1 Structure of DNA/RNA 1a; LNA 1b[11]; and Mercaptoacetamido-linked dimers 1c and 1d[20]
B = Nucleobases; T = Thymine; C = Cytosine.
.
oligonucleotides are also being increasingly utilized in nucleic acid nan-
otechnology, oligonucleotide-based diagnostics, gene-function determina-
tion, and drug target validation.[6–8] The novel utility of these agents resides
in their ability to selectively prevent the expression of a particular disease-
associated gene in a sequence specific manner. The ongoing synthetic stud-
ies have been focused on chemical modifications of backbone,[9] base,[10]
and sugar[11] functionalities of the natural DNA/RNA (1a, Figure 1) and
have resulted in significant progress toward establishing oligonucleotides as
viable therapeutic agents. One such modification in the sugar moiety has
resulted in locked nucleic acid (LNA 1b, Figure 1), where the furanose con-
formation is locked in an N -type (C3ꢁ-endo) form by the introduction of a
2ꢁ-O,4ꢁ-C methylene-linkage.[11–13] LNA has been found to be very useful for
antisense applications, since incorporation of one or more LNA monomer
unit(s) into an ON shows extraordinary thermal stability when hybridized
with either DNA, RNA, or LNA itself.[11,12,14]
Further, oligonucleotide analogues with differently modified back-
bone such as thioformacetal,[15] 5ꢁ-N -carbamate,[16] methylene(methy
limino),[17] amide-,[18] triazole,[19] etc. have been designed and synthe-
sized to circumvent the physical and biological limitations of natural phos-
phodiester linkage. Recently, Kumar et al.[20] have synthesized two 5-atom
mercaptoacetamido-linked dimers, T-S-T 1c and C-S-T 1d (Figure 1), as
compared to the 4-atom-linked natural DNA. The UV-Tm experiments for
binding affinity of these mixed backbone modified ONs with complemen-
tary DNA and RNA sequences revealed important results such as significantly
higher RNA-binding selectivity (ꢀTm/mod upto + 13◦C) as compared to
complementary DNA. Replacement of phosphate backbone with mercap-
toacetamido linkage in an ONs have several distinct advantages in terms of
its antisense properties, i.e., (i) it has strong binding affinity toward both
natural DNA and RNA, which may be due to the neutral backbone that does
not have any repulsive interactions with the anionic phosphate backbone;

258
V. K. Sharma et al.
FIGURE 2 Structures of natural dinucleotide 2; designed LNA-based mercaptoacetamido-linked non-
ionic nucleoside dimers 3, 4, and 5. The natural phosphate linkage O-P-O in 2 has been replaced by
mercaptoacetamido-linkage S-CH2-CO-NH in 3, 4, and 5, as shown in red (Color figure available online).
(ii) it provides a very high degree of nuclease resistance; and (iii) it en-
hances membrane permeability due to elimination of negative charge from
the backbone.
In this article we have designed and synthesized three LNA-based
mercaptoacetamido-linked nonionic nucleoside dimers i.e., 1-(3ꢁ-deoxy-
5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-β-thymidin-5ꢁꢁ-yl)
mercaptoacetamide (TL-S-T, 3); 1-(3ꢁ-deoxy-5ꢁ-O-DMT-β-thymidin-3ꢁ-yl)-4-
(5ꢁꢁ-deoxy-2ꢁꢁ-O,4ꢁꢁ-C-methylenethymidin-5ꢁꢁ-yl)mercaptoacetamide (T-S-TL,
4); and 1-(3ꢁ-deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-
deoxy-2ꢁꢁ-O,4ꢁꢁ-C-methylenethymidin-5ꢁꢁ-yl)mercaptoacetamide (TL-S-TL, 5)
containing the features of both, LNA- and mercaptoacetamido-linked
nucleoside dimers (Figure 2) and 3ꢁ-O-phosphoramidite derivative 27 of
one nucleoside dimer, which is a building block for the preparation of
mercaptoacetamido-linked oligonucleotides.
It was envisaged to synthesize the nucleoside dimer TL-S-T 3 by the
condensation of 3ꢁ-amino-3ꢁ-deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidine
(6) with 2-S-(β-thymidin-5ꢁ-yl)mercaptoacetic acid (9); nucleoside dimers
T-S-TL 4 and TL-S-TL 5 by condensing 3ꢁ-amino-3ꢁ-deoxy-5ꢁ-O-DMT-β-
thymidine (7); and nucleoside 6 with 2-S-(2ꢁ-O,4ꢁ-C-methylenethymidin-5ꢁ-
yl)mercaptoacetic acid (8). The aminonucleoside 6 has been synthesized
from 1,2-O-isopropylidene-5-O-methanesulfonyl-4-C-methanesulfonyloxy-
methyl-β-L-threofuranose 11, which was obtained from D-glucose through
trihydroxyfuranoside 10 following the procedure of Matin[21] in an overall
yields of 39%. The dimesylated compound 11 was converted into 3-azido-
3-deoxy-1,2-O-isopropylidene-5-O-methanesulfonyl-4-C-methanesulfonyloxy
methyl-α-D-ribofuranose 12 in two steps, i.e., triflation of the lone hydroxyl
group using triflic anhydride in DCM-pyridine followed by nucleophilic
displacement of the triflate with sodium azide in DMF in combined yields
of 50%. Acetolysis of compound 12 with acetic acid-acetic anhydride-conc.
H2SO4 yielded mixture of anomers 13a–13b in 84% yield, which on

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
259
Vorbru¨ggen coupling[22] with thymine resulted into the formation of
monoacetylated nucleoside 14 in 75% yield. The deacetylation followed by
cyclization was affected on monoacetylated nucleoside 14 using 2M NaOH
solution in water:dioxane to afford bicyclic nucleoside 15 in 90% yield.
The nucleoside 15 was demesylated to obtain 3ꢁ-azido-3ꢁ-deoxy-2ꢁ-O,4ꢁ-C-
methylenethymidine (17) in an overall yield of 72% through benzoylation
with sodium benzoate in dry DMF followed by basic hydrolysis of the resulted
benzoate 16 using 2M NaOH in THF:water (1:1) (Scheme 1). The azidobi-
cyclic nucleoside 17 was converted into DMT-protected aminonucleoside 6
by reaction with DMT-Cl in pyridine followed by reduction of the resulted
compound 18 with PPh3-pyridine and aq. ammonia solution in overall
yields of 68%. The synthesis of 3ꢁ-amino-3ꢁ-deoxy-5ꢁ-O-DMT-β-thymidine
(7) was accomplished from β-thymidine through DMT-protection, C2-O-
C3ꢁ-anhydro formation, azidation followed by reduction of the azide group
with PPh3-pyridine/aq. ammonia solution in overall yields of 49% following
literature procedure.[23]
SCHEME 1 Synthesis of 3ꢁ-amino-3ꢁ-deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidine (6). Reagents & con-
ditions: (i) (a) Trifluoromethanesulfonic anhydride, DCM:pyridine (10:1), –10◦C, (b) NaN3, DMF,
100◦C; (ii) AcOH, Ac2O, H2SO4 (100:10:0.1), 0◦C; (iii) T = Thymine, N ,O-bis-trimethylsilyl acetamide,
trimethylsilyltrifluoromethane sulfonate, acetonitrile, 80◦C; (iv) 2M NaOH, dioxane:water (1:1), 0◦C; (v)
Sodium benzoate, DMF, 100◦C; (vi) 2M NaOH, water:THF (1:1), 0◦C; (vii) 4,4ꢁ-Dimethoxytrityl chloride,
pyridine, 25◦C; (viii) PPh3, pyridine, 28% aqueous ammonia solution, 25◦C.
The synthesis of 2-S-(2ꢁ-O,4ꢁ-C-methylenethymidin-5ꢁ-yl)mercaptoacetic
acid (8) was accomplished from 2ꢁ-O,4ꢁ-C-methylenethymidine (LNA T, 20),
which was synthesized from diacetone-D-glucose 19 following multistep syn-
thetic procedure as reported in the literature (Scheme 2).[24] The primary
OH group in bicyclic nucleoside 20 was tosylated using p-toluenesulfonyl

260
V. K. Sharma et al.
SCHEME 2 Synthesis of 2-S-(5ꢁ-deoxy-3ꢁ-O-tert-butyldiphenylsilyl-2ꢁ-O,4ꢁ-C-methylenethymidine-5ꢁ-
yl)mercaptoacetic acid (8). Reagents & conditions: (i) p-Toluenesulfonyl chloride, pyridine, 0◦C;
(ii) tert-butyl diphenylsilyl chloride, imidazole, DMF, 25◦C; (iii) NaH, DMF, ethyl mercaptoacetate, 0◦C;
and (iv) 2M NaOH, methanol.
chloride in pyridine to afford nucleoside 21 in 65% yield, which on sily-
lation with tert-butyldiphenylsilyl chloride in DMF in the presence of im-
idazole afforded 5ꢁ-O-p-toluenesulfonyl-3ꢁ-O-tert-butyldiphenylsilyl-2ꢁ-O,4ꢁ-C-
methylenethymidine (22) in 90% yield. The targeted compound 8 was syn-
thesized in overall yields of 77% in two steps from compound 22, i.e., substitu-
tion of tosyl group with ethyl mercaptoacetate using sodium hydride in DMF
followed by de-esterification of the resulted compound 23 using 2M NaOH in
methanol (Scheme 2). The synthesis of 2-S-(β-thymidin-5ꢁ-yl)mercaptoacetic
acid (9) was accomplished from β-thymidine through tosylation, silylation,
ethoxycarbonylmethylthionation followed by de-esterification in an overall
yields of 51% using similar method applied for the synthesis of nucleoside 8.
The structure of compounds 6–23 was established on the basis of its spectral
data (IR, 1H-, 13C NMR, and HRMS) analysis. Further, the structure of known
compounds 6, 7, 10–13, and 17–20 was confirmed by comparing its physical
and spectral data with those reported in the literature.[23–28]
Finally, the synthesis of LNA-based mercaptoacetamido-linked nonionic
nucleoside dimer TL-S-T 3 was accomplished by HOBT/HBTU catalyzed
coupling of 3ꢁ-amino-nucleoside 6 and mercaptoacetic acid 9 in DMF to
afford 1-(3ꢁ-deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-
3ꢁꢁ-O-TBDPS-β-thymidin-5ꢁꢁ-yl)mercaptoacetamide 24 in 65% yield. The mer-
captoacetamide 24 was desilylated using tetrabutylammoniun fluoride to
obtain the desired mercaptoacatamido-linked nonionic nucleoside dimer 3
in 75% yield (Scheme 3).

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
261
SCHEME 3 Synthesis of mercaptoacetamido-linked nucleoside dimer TL-S-T
3
and its 3ꢁ-O-
phosphoramidite.
Further, nucleoside dimers T-S-TL 4 and TL-S-TL 5 were synthesized in
two steps, i.e. first by HOBT/HBTU catalyzed coupling of nucleoside 8 with
3ꢁ-aminonucleosides 7 and 6 to afford the silylated nucleoside dimers 25 and
26 in 62% yield in each cases. In the second step, desilylation of protected
mercaptoacetamides 25 and 26 using tetrabutylammoniun fluoride afforded
the desired mercaptoacatamido-linked nucleoside dimers 4 and 5 in 80%
and 70% yields, respectively (Scheme 4).
One of the LNA-based mercaptoacetamido-linked nonionic nucleoside
dimer TL-S-T 3 was condensed with [P(-Cl)(-OCH2CH2CN){-N(iPr)2}] in
DCM in the presence of N ,N -diisopropylethylamine (DIPEA) and converted
into its corresponding 3ꢁ-O-phosphoramidite 27, a building block for the syn-
thesis of mercaptoacetamido-linked oligonucleotide of therapeutic applica-
tion (Scheme 3). The structure of protected nucleoside dimers 24, 25, and
26; hydroxyl dimers TL-S-T 3, T-S-TL 4, and TL-S-TL 5; and phosphoramidite
SCHEME 4 Synthesis of mercaptoacetamido-linked nucleoside dimers T-S-TL 4 and TL-S-TL 5.

262
V. K. Sharma et al.
27 was unambiguously established on the basis of their spectral (IR, 1H-, 13C-,
31P NMR spectra, and HRMS) data analysis.
Synthesis of three LNA-based mercaptoacetamido-linked nonionic nu-
cleoside dimers TL-S-T 3, T-S-TL 4, and TL-S-TL 5 has been achieved us-
ing commonly available starting compound, i.e., D-glucose or thymidine.
Phosphoramidite derivative of one of the nucleoside dimers, TL-S-T 3
has been prepared, which can be used for the synthesis of LNA-based
mercaptoacetamido-linked nonionic oligonucleotides of potential antisense
application.
EXPERIMENTAL SECTION
Melting points (m.p.) were determined on Buchi M-560 instrument and
are uncorrected. The IR spectra were recorded on a Perkin–Elmer model
2000 FTIR spectrometer by making KBr disc for solid samples and thin film
1
for oils. The H- and 13C NMR spectra were recorded on a Bruker Avance
spectrometer at 300 MHz and 75.5 MHz or at Jeol alpha-400 spectrometer at
400 MHz and 100.5 MHz, using TMS as internal standard. HR-ESI-TOF-MS
analyses were carried out on a microTOF-Q instrument from Bruker Dal-
tonics, Bremen. The optical rotations were measured with Rudolph autopol
II automatic polarimeter using light of 546 nm wavelength. Analytical TLCs
were performed on precoated Merck silica-gel 60F254 plates; the spots were
detected either under UV light or by charring with 4% alcoholic H2SO4.
Silica gel (100–200 mesh) was used for column chromatography.
3ꢁ-Amino-3ꢁ-deoxy-5ꢁ-O-DMT-β-thymidine (7).23 It was synthesized from
β-thymidine according to the literature procedure as light yellow foam solid
24
in 49% yield. m.p.: 110–112◦C; [α]D = +1.9 (c = 0.1, CHCl3); HR-ESI-TOF-
MS: m/z 566.2241 ([M+Na]+), calcd. for [C31H33N3O6+Na]+ 566.2262.
4-C-Hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pento-furanose
(10).21 It was synthesized according to the literature procedure as white
24
solid in 65% yield. m.p.: 99–100◦C (lit21a mp: 98–99◦C); [α]D = −5.69 (c
= 0.34, MeOH); {lit21a [α]D = −5.73 (c 0.34, MeOH)}; HR-ESI-TOF-MS m/z
243.0840 ([M+Na]+), calcd. for [C9H16O6+Na]+ 243.0839.
1,2-O-Isopropylidene-5-O-methanesulfonyl-4-C-methanesulfonyloxymet
hyl-β-L-threo-pentofuranose (11).21a It was synthesized from 10 according
to the literature procedure as white solid in 60% yield. m.p.: 131–132◦C
25
25
(Lit.21a m.p.: 132–133◦C), [α]D = −8.4 (c 0.25, CHCl3) {Lit.21a [α]D
=
−8.0 (c 0.25, CHCl3)}; HR-ESI-TOF-MS m/z 399.0387 ([M+Na]+), calcd.
for [C11H20O10S2+Na]+ 399.0390.
3-Azido-3-deoxy-1,2-O-isopropylidene-5-O-methanesulfonyl-4-C-methan
esulfonyloxymethyl-β-D-ribofuranose (12).25b To a stirred solution of
compound 11 (5.0 g, 13.30 mmol) in dichloromethane:anhydrous pyridine
(10:1, 45 mL) was added dropwise the solution of trifluoromethanesulfonyl

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
263
chloride (3.35 mL, 19.95 mmol). After stirring the mixture for 1.5 hours
at −10◦C, 50 mL ice-cold water was added and the mixture was extracted
with chloroform (3 × 100 mL). The chloroform layer was washed with
saturated bicarbonate solution (3 × 50 mL), and dried over anhydrous
sodium sulfate. Excess of solvent was evaporated at reduced pressure and
the residue thus obtained was heated with NaN3 (0.95 g, 14.62 mmol) in
DMF (70 mL) at 100◦C to afford compound 12 (2.70 g) in an overall yield
of 50% as sticky colorless solid. Rf = 0.5 (5% methanol in chloroform).
35
[α]D = + 78.7 (c 0.1, CHCl3). HR-ESI-TOF-MS m/z 424.0449 ([M+Na]+),
calcd. for [C11H19N3O9S2+Na]+ 424.0455.
1,2-Di-O-acetyl-3-azido-3-deoxy-5-O-methanesulfonyl-4-C-methanesul
fonyloxymethyl-α,β-D-ribofuranose (13a–13b).27 It was synthesized from 12
according to the literature procedure as colorless viscous oil 13a–13b (α:β
= ca. 2:8, 4.22 g, 84% yield). HR-ESI-TOF-MS m/z 468.0352 ([M+Na]+),
calcd. for [C12H19N3O11S2+Na]+ 468.0353.
2ꢁ-O-Acetyl-3ꢁ-azido-3ꢁ-deoxy-5ꢁ-O-methanesulfonyl-4ꢁ-C-methanesulfony
loxymethyl-thymidine (14). N ,O-bis(Trimethysilyl)acetamide (BSA) (12.5
mL, 50.29 mmol) was added to an anomeric mixture of compound 13a–13b
(5.60 g, 12.57 mmol) and thymine (2.38 g, 18.86 mmol) in dry acetonitrile
(120 mL), under nitrogen atmosphere. The reaction mixture was refluxed
for 1 hour to get a clear solution. Reaction mixture was brought to
room temperature (r.t.) and then trimethylsilyl triflate (TMSOTf) (4.0 mL,
21.37 mmol) was added, and refluxing of the reaction was continued further
for 14 hours. The solution was cooled to r.t., extracted with chloroform
(3 × 100 ml), and washed with saturated aqueous bicarbonate solution.
The organic layer was further washed with water and brine, dried over
sodium sulfate, concentrated under reduced pressure and the residue thus
obtained was purified on silica gel column chromatography using methanol
in chloroform as a gradient solvent system to give 14 (4.82 g, 75%) as
light yellow foam solid. Rf = 0.5 (10% methanol in chloroform). m.p.:
35
70–72◦C. [α]D = –9.9 (c 0.1, CHCl3). IR (thin film) νmax: 3029, 2942,
2123, 1753, 1695, 1358, 1216, 1176, 1085, 1003, 966, 823, and 756 cm−1.
1H NMR (CDCl3, 300 MHz): δ 9.52 (1H, brs), 7.08 (1H, s), 5.73–5.65 (2H,
m), 4.94 (1H, J = 6.6 Hz, d), 4.49–4.33 (4H, m), 3.14 (3H, s), 3.11 (3H, s),
2.20 (3H, s), 1.92 (3H, s); 13C NMR (CDCl3, 75.5 MHz): δ 169.97, 163.64,
150.31, 137.99, 112.05, 92.55, 83.52, 74.35, 68.13, 67.15, 62.86, 37.86,
37.76, 20.49, 12.23; HR-ESI-TOF-MS m/z 534.0563 ([M+Na]+), calcd. for
[C15H21N5O11S2+Na]+ 534.0571.
3ꢁ-Azido-3ꢁ-deoxy-5ꢁ-O-methanesulfonyl-2ꢁ-O,4ꢁ-C-methylenethymidine
(15). To a stirred solution of compound 14 (4.70 g, 9.19 mmol) in
dioxane:water (22 mL, 1:1) was added 2M NaOH (21.4 mL) and reaction
mixture was stirred at r.t. for 1 hour. The reaction mixture was extracted
with ethyl acetate (3 × 100 ml), washed with bicarbonate solution and brine,

264
V. K. Sharma et al.
dried over sodium sulfate, and concentrated under reduced pressure. The
residue thus obtained was purified by silica gel column chromatography
using methanol in chloroform as gradient solvent system to give 15 (2.88 g,
90%) as off white solid. Rf = 0.5 (10% methanol in chloroform). m.p.:
35
77–79◦C. [α]D = + 64.4 (c 0.1, CHCl3); IR (thin film) νmax: 3179, 3026,
2121, 1691, 1464, 1355, 1276, 1175, 1111, 1059, 1024, 998, 966, 812, and
1
757 cm−1; H NMR (DMSO-d6, 300 MHz): δ 11.39 (1H, brs), 7.45 (1H, s),
5.55 (1H, s), 4.79 (1H, J = 12.0 Hz, d), 4.64 (1H, J = 12.0 Hz, d), 4.60 (1H,
s), 4.20 (1H, s), 3.88–3.80 (2H, m), 3.32 (3H, s), 1.79 (3H, s); 13C NMR
(DMSO-d6, 75.5 MHz): δ 163.85, 149.93, 134.35, 108.67, 86.59, 85.84, 78.52,
70.89, 65.09, 60.60, 36.96,12.27; HR-ESI-TOF-MS m/z 396.0579 ([M+Na]+),
calcd. for [C12H15N5O7S+Na]+ 396.0584.
3ꢁ-Azido-5ꢁ-O-benzoyl-3ꢁ-deoxy-2ꢁ-O-4ꢁ-C-methylenethymidine (16). A mix
ture of compound 15 (3.5 g, 10.02 mmol) and sodium benzoate (2.89 g,
20.05 mmol) was suspended in anhydrous DMF (170 mL) and was stirred for
4 hours at 100◦C. The mixture was cooled to r.t., filtered, and concentrated
under reduced pressure. The residue thus obtained was redissolved in ethyl
acetate (200 mL) and washed with saturated NaHCO3 (2 × 200 mL) and
H2O (4 × 200 mL). The organic phase was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue thus obtained was
purified by silica gel column chromatography using methanol in chloroform
as gradient solvent system to afford the nucleoside 16 (3.19 g, 80%) as white
25
solid. Rf = 0.5 (8% methanol in chloroform). m.p.: 168–170◦C; [α]D = +
89.2 (c 0.1, CHCl3); IR (thin film) νmax: 3178, 3041, 2959, 2827, 2130, 1709,
1
1686, 1467, 1270, 1111, 1060, 1026, 940, 907, 871, and 719 cm−1; H NMR
(DMSO-d6, 300 MHz): δ 11.40 (1H, brs), 8.05 (2H, J = 8.1 Hz, d), 7.74–7.57
(3H, m), 7.34 (1H, s), 5.56 (1H, s), 4.79 (2H, s), 4.61 (1H, s), 4.37 (1H,
s), 3.94 (2H, J = 16.8 and 8.7 Hz, dd), 1.59 (3H, s); 13C NMR (DMSO-d6,
75.5 MHz): δ 165.16, 163.80, 149.90, 134.11, 133.81, 129.33, 128.92, 108.53,
86.59, 86.45, 78.51, 71.14, 66.32, 60.65, 12.04; HR-ESI-TOF-MS m/z 422.1062
([M+Na]+), calcd. for [C18H17N5O6+Na]+ 422.1071.
3ꢁ-Azido-3ꢁ-deoxy-2ꢁ-O,4ꢁ-C-methylenethymidine (17).28 To a stirred solu-
tion of compound 16 (3.11 g, 7.78 mmol) in THF:water (1:1, 50 mL) was
added 2M NaOH (11.8 mL), and the mixture was stirred at 0◦C for 1 hour.
After completion of reaction acetic acid (3.0 mL) was added, and the solu-
tion was concentrated under reduced pressure. Purification was performed
on silica gel column using methanol in chloroform as gradient solvent sys-
tem to yield azido-LNA 17 (2.07 g, 90%) as a white powder. m.p.: 94–95◦C
35
(Lit.28 m.p.: 94–96◦C); [α]D = + 87.1 (c 0.1, MeOH); HR-ESI-TOF-MS m/z
318.0816 ([M+Na]+), calcd. for [C11H13N5O5+Na]+ 318.0809.
3ꢁ-Azido-3ꢁ-deoxy-5ꢁ-O-DMT-2ꢁ-O-4ꢁ-C-methylenethymidine (18).25a It was
synthesized from 17 according to the literature procedure to afford
18 (0.45 g, 75%) as white foam solid. m.p.: 125–126◦C, (Lit.25a m.p.:

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
265
22
125–128◦C); [α]D = +19.1 (c 0.75, acetone); HR-ESI-TOF-MS: m/z
620.2107 ([M+Na]+), calcd. for [C32H31N5O7+Na]+ 620.2116.
3ꢁ-Amino-3ꢁ-deoxy-5ꢁ-O-DMT-2ꢁ-O-4ꢁ-C-methylenethymidine (6).25a It was
synthesized from 18 according to the literature procedure to afford 6
(0.10 g, 90% yield) as light yellow foam solid. m.p.: 130–132◦C, (Lit.25a m.p.:
22
131–134◦C). [α]D = + 22.9 (c 1, acetone). HR-ESI-TOF-MS: m/z 594.2214
([M+Na]+), calcd. for [C32H33N3O7+Na]+ 594.2211.
2ꢁ-O,4ꢁ-C-methylenethymidine (20).24 Compound 20 was synthesized
from D-glucose according to the literature procedure as a white solid
23
material. m.p.: 196–197◦C (Lit.24 m.p.: 196–198◦C); [α]D = + 57.9
(c = 1, EtOH); HR-ESI-TOF-MS: m/z 293.0752 ([M+Na]+), calcd. for
[C11H14N2O6+Na]+ 293.0744.
5ꢁ-O-p-Toluenesulfonyl-2ꢁ-O,4ꢁ-C-methylenethymidine (21). p-Toluene
sulfonyl chloride (12 mmol) dissolved in 10 mL of dry pyridine was added
drop wise during 4 hours to the solution of 20 (10 mmol) in 30 mL of dry
pyridine at 0◦C. The reaction mixture was stirred for additional 4 hours at
r.t.. Pyridine was removed under reduced pressure and residue thus obtained
was dissolved in 100 mL ethyl acetate and the organic layer was washed with
(2 × 50 mL) 10% aq. NaHCO3 solution followed by cold water (2 × 50 mL).
The organic layer was dried over anhydrous Na2SO4 and was evaporated at
reduced pressure. The crude product so obtained was purified over silica gel
column using methanol in chloroform as gradient solvent system to afford
21 as white solid (2.75 g, 65% yield). Rf = 0.4 (10% methanol in chloro-
22
form); m.p.: 100–102◦C; [α]D = + 71.7 (c = 0.1, MeOH); IR (KBr) νmax
:
3401, 3198, 3070, 2952, 1690, 1597, 1466, 1363, 1275, 1176, 1054, 983, and
810 cm−1; 1H NMR (CDCl3, 300 MHz): δ 9.72 (1H, brs), 7.82 (2H, J = 8.2 Hz,
d), 7.46 (1H, s), 7.39 (2H, J = 8.2 Hz, d), 5.52 (1H, s), 4.60 (1H, s), 4.41
(2H, s), 4.23 (1H, s), 4.00 (1H, brs), 3.99 (1H, J = 8.0 Hz, d), 3.79 (1H, J =
7.9 Hz, d), 2.46 (3H, s), 1.85 (3H, s); 13C NMR (CDCl3, 75.5 MHz): δ 164.35,
150.20, 149.55, 145.64, 134.36, 132.30, 130.21, 110.56, 87.18, 86.22, 79.15,
77.01, 69.94, 64.27, 21.70, 12.45; HR-ESI-TOF-MS: m/z 447.0838 ([M+Na]+),
calcd. for [C18H20N2O8S+Na]+ 447.0833.
5ꢁ-O-p-Toluenesulfonyl-3ꢁ-O-tert-butyldiphenylsilyl-2ꢁ-O,4ꢁ-C-methylene
thymidine (22). A solution of compound 21 (6.0 mmol), tert-butyldiphe
nylsilyl chloride (7.2 mmol), imidazole (14.8 mmol) in 20 mL of anhydrous
DMF was stirred for 6 hours under nitrogen atmosphere at r.t.. Excess of
DMF was removed in vacuo and the residue was dissolved in 200 mL of ethyl
acetate, washed with water (3 × 50 mL) followed by brine (2 × 30 mL).
The organic layer was dried over anhydrous Na2SO4 and solvent was then
removed in vacuo. The residue thus obtained was purified by silica gel
column chromatography using ethyl acetate in petroleum ether as gradient
solvent system to afford 22 as light yellow crystalline solid (3.51 g, 90%
yield). Rf = 0.5 (50% ethyl acetate in petroleum ether); m.p.: 95–96◦C;

266
V. K. Sharma et al.
23
[α]D = + 6.2 (c 0.1, MeOH); IR (KBr) νmax: 3181, 3048, 2955, 2859,
1689, 1597, 1466, 1370, 1274, 1178, 1104, 1054, 823, 744, and 705 cm−1;
1H NMR (CDCl3, 300 MHz): δ 8.71 (1H, brs), 7.81–7.34 (15H, m), 5.47
(1H, s), 4.84–4.35 (2H, m), 4.14 (1H, J = 7.2 Hz, d), 4.02–3.79 (3H, m),
2.48 (3H, s), 1.74 (3H, s), 1.03 (9H, s); 13C NMR (CDCl3, 75.5 MHz):
δ 163.48, 149.13, 145.60, 135.61, 135.33, 133.71, 132.35, 131.98, 131.39,
130.45, 130.37, 130.15, 128.06, 127.95, 110.20, 86.86, 85.81, 78.47, 71.50,
63.70, 26.90, 21.72, 19.04, 12.37; HR-ESI-TOF-MS: m/z 663.2162 ([M+H]+),
calcd. for [C34H38N2O8SSi+H]+ 663.2191.
Ethyl 2-S-(3ꢁ-O-tert-butyldiphenylsilyl-5ꢁ-deoxy-2ꢁ-O,4ꢁ-C-methylenethymi
din-5ꢁ-yl)mercaptoacetate (23). A solution of NaH (0.29 g, 60% in hex-
ane, 7.25 mmol.) and ethyl mercaptoacetate (5.5 mmol) in anhydrous DMF
(5 mL) was stirred for 30 minutes. Compound 22 (4.50 mmol) dissolved in
DMF (5 mL) was then added slowly at 0◦C. The reaction mixture was stirred
for 1 hour at r.t.. Excess of DMF was removed under reduced pressure, and
the residue was dissolved in 200 mL ethyl acetate. The organic layer was
washed with water (3 × 50 mL) and with brine (2 × 30 mL). The organic
layer was dried over anhydrous Na2SO4 and solvent was removed in vacuo.
The crude thus obtained was purified on silica gel column using methanol in
chloroform as gradient solvent system to afford 23 as viscous oil (2.26 g, 82%
23
yield). Rf = 0.5 (50% ethyl acetate in petroleum ether); [α]D = + 41.2 (c
0.1, MeOH); IR (KBr) νmax: 3183, 3069, 2931, 2858, 1690, 1459, 1275, 1155,
1113, 1051, 943, 856 and 744 cm−1; 1H NMR (CDCl3, 300 MHz): δ 9.20 (1H,
brs), 7.63–7.31 (11H, m), 5.51 (1H, s), 4.22–4.14 (3H, m), 3.93 (2H, s), 3.91
(1H, s), 3.70 (1H, s), 3.33–3.11 (3H, m), 1.78 (3H, J = 1.0 Hz, d), 1.28 (3H,
J = 7.2 Hz, t), 1.09 (9H, s); 13C NMR (CDCl3, 75.5 MHz): δ 169.80, 163.62,
149.25, 145.90, 135.66, 135.57, 135.43, 134.24, 132.16, 131.80, 130.39, 130.34,
130.14, 127.98, 127.93, 109.85, 88.39, 86.96, 78.75, 73.61, 72.87, 61.60, 34.79,
28.88, 26.75, 21.07, 19.10, 12.18; HR-ESI-TOF-MS: m/z 633.2028 ([M+Na]+),
calcd. for [C31H38N2O7SSi+Na]+ 633.2061.
2-S-(5ꢁ-Deoxy-3ꢁ-O-tert-butyldiphenylsilyl-2ꢁ-O,4ꢁ-C-methylenethymidin-
5ꢁ-yl)mercaptoacetic acid (8). Sodium hydroxide (2M, 5 mL) solution was
added to a solution of 23 (5.30 mmol) in 10 mL methanol. The reaction
mixture was stirred for 30 minutes. After completion of reaction on
analytical TLC examination, the sodium salt of the acid and excess NaOH
present in the solution was neutralized by DOWEX-50H+ resin. The resin
was filtered and washed with 2:1 mixture of methanol:water. The filtrate
was concentrated and dried under vacuum to afford 8 as white solid (2.32 g,
94% yield). Rf = 0.5 (50% methanol in chloroform); m.p.: 208–210◦C;
23
[α]D = +34.5 (c 0.1, MeOH); IR (KBr) νmax: 3449, 3064, 2931, 2858,
1689, 1580, 1469, 1427, 1389, 1273, 1156, 1110, 1052, 855 and 704 cm−1;
1H NMR (DMSO-d6, 300 MHz): δ 11.26 (1H, brs), 7.58–7.32 (11H, m), 5.30
(1H, s), 4.04 (1H, J = 8.1 Hz, d), 3.82 (1H, J = 8.1 Hz, d), 3.74 (1H, s),

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
267
3.61 (1H, s), 3.28 (2H, s), 3.12 (2H, s), 1.64 (3H, s), 1.02 (9H, s); 13C NMR
(DMSO-d6, 75.5 MHz): δ 172.60, 163.58, 149.39, 135.13, 134.95, 133.72,
132.17, 131.44, 130.17, 127.87, 127.77, 108.69, 88.34, 86.03, 78.10, 73.14,
72.46, 38.58, 27.69, 26.47, 18.63, 12.22; HR-ESI-TOF-MS: m/z 605.1731
([M+Na]+), calcd. for [C29H34N2O7SSi+Na]+ 605.1748.
2-S-(5ꢁ-Deoxy-3ꢁ-O-tert-butyldiphenylsilyl-β-thymidin-5ꢁ-yl)mercaptoacetic
acid (9). It was synthesized from β-thymidine following the same procedure
used for the synthesis of mercaptoacetic acid 8 from 20 as white solid (2.70 g,
27
92% yield). m.p.: 69–70◦C; [α]D = + 29.13 (c 0.1, MeOH); IR (thin film)
νmax: 3180, 3071, 2932, 2859, 1702, 1474, 1428, 1364, 1276, 1197, 1112,
1
998, 924, 823, 756, 703, and 669 cm−1; H NMR (CDCl3, 400 MHz): δ 9.48
(1H, brs), 7.65–7.61 (4H, m), 7.45–7.40 (6H, m), 7.21 (1H, J = 1.2 Hz,
d), 6.27 (1H, J = 6.8 Hz, t), 4.24–4.20 (1H, m), 4.12–4.08 (1H, m), 3.12
(2H, J = 33.2 Hz and J = 14.8 Hz, dd), 2.63–2.62 (1H, m), 2.58–2.53
(1H, m), 2.37–2.31 (1H, m), 1.95–1.88 (1H, m), 1.83 (3H, s), 1.07 (9H,
s); 13C NMR (CDCl3, 100 MHz): δ 174.46, 164.60, 150.34, 136.02, 135.70,
132.86, 130.20, 127.97, 111.22, 86.15, 84.58, 74.52, 40.17, 34.29, 34.20,
26.81, 18.95, 12.29; HR-ESI-TOF-MS m/z 577.1788 ([M+Na]+), calcd. for
[C28H34N2O6SSi+Na]+ 577.1799.
General procedure for the synthesis of silylated dimers 24–26. To a so-
lution of mercaptoacetic acid 8/9 (1.72 mmol) in dry DMF (5 mL), HBTU
(2.06 mmol), DIPEA (5.20 mmol), and HOBt (0.1 mmol) were added and
stirred for 15 minutes at 25◦C. Amine 6/7 (1.70 mmol) was dissolved in
5 mL DMF and was then added into the reaction mixture and the reac-
tion mixture was further stirred at 25◦C for 4 hours. The reaction mixture
was concentrated to dryness, dissolved in ethyl acetate (30 mL) and washed
with aqueous 5% NaHCO3 solution (2 × 20 mL). The organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The crude product so obtained was purified by silica gel column chromatog-
raphy using methanol in chloroform as gradient solvent system to afford
mercaptoacetamido-linked dimer 24 and 25/26 in 65% and 62% yields,
respectively.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-3ꢁꢁ-
O-tert-butyldiphenylsilyl-β-thymidin-5ꢁꢁ-yl)mercaptoacetamide (24). It was
obtained by the HOBT/HBTU catalyzed coupling reaction of acid 9 with
27
amine 6 as white solid (1.24 g, 65% yield). m.p.: 104–105◦C. [α]D
=
+26.71 (c 0.1, MeOH); IR (thin film) νmax: 3199, 3069, 2932, 2859, 1691,
1509, 1466, 1384, 1275, 1252, 1178, 1112, 1055, 1035, 978, 916, 830, 792,
756, 704, 665, and 613 cm−1; 1H NMR (CDCl3, 400 MHz): δ 8.90 (1H, brs),
8.58 (1H, brs), 8.01 (1H, brs), 7.62–7.60 (4H, m), 7.45–7.25 (18H, m), 6.82
(4H, J = 8.4 Hz, d), 6.17–5.82 (1H, m), 5.30 (1H, J = 5.2 Hz, d), 4.51 (1H,
J = 8.0 Hz, d), 4.47 (1H, J = 3.6 Hz, d), 4.23–4.21 (1H, m), 3.97–3.91 (1H,
m), 3.78–3.76 (6H, m), 3.43 (2H, m), 3.41 (1H, J = 7.2 Hz, d), 2.88–2.87

268
V. K. Sharma et al.
(2H, m), 2.46–2.41 (1H, m), 2.24–2.22 (3H, m), 1.84 (3H, s), 1.63 (3H, s),
1.06 (9H, s); 13C NMR (DMSO-d6, 100.6 MHz): δ 169.71, 163.77, 163.62,
158.13, 150.43, 149.91, 144.51, 135.75, 135.38, 134.97, 133.87, 132.82,
132.59, 130.07, 129.76, 128.00, 127.72, 126.75, 113.24, 109.81, 108.84, 87.63,
86.30, 85.88, 85.43, 84.05, 79.18, 78.54, 75.36, 71.15, 57.97, 55.00, 51.18,
38.42, 33.65, 26.66, 18.59, 12.41, 12.00; HR-ESI-TOF-MS m/z 1130.4031
([M+Na]+), calcd. for [C60H65N5O12SSi+Na]+ 1130.4012.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-β-thymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-3ꢁꢁ-O-tert-
butyldiphenylsilyl-2ꢁꢁ-O,4ꢁꢁ-C-methylenethymidin-5ꢁꢁ-yl)mercaptoacetamide
(25). It was obtained by the HOBT/HBTU catalyzed coupling reaction
of acid 8 with amine 7 as white solid (1.17 g, 62% yield). Rf = 0.7 (10%
24
methaol in chloroform); m.p.: 138–140◦C; [α]D = +18.3 (c 0.05, CHCl3);
IR (KBr) νmax: 3338, 3064, 2930, 2857, 1686, 1507, 1459, 1252, 1176, 1112,
1051, 829, and 703 cm−1; 1H NMR (CDCl3, 300 MHz): δ 9.47 (2H, 2 x brs),
7.63–7.56 (5H, m), 7.41–7.19 (17H, m), 6.83 (4H, J = 8.4 Hz, d), 6.34 (1H,
brs), 5.50 (1H, s), 4.66 (1H, s), 4.11 (1H, J = 7.8 Hz, d), 4.03 (1H, s),
3.87–3.82 (2H, m), 3.71 (7H, s), 3.46 (2H, s), 3.29 (2H, s), 3.06–2.88 (2H,
m), 2.40–2.30 (2H, m), 1.74 (3H, s), 1.37 (3H, s), 1.06 (9H, s); 13C NMR
(DMSO-d6, 100.6 MHz): δ 168.56, 163.70, 163.56, 158.14, 150.38, 149.43,
144.71, 135.71, 135.45, 135.19, 135.03, 133.58, 132.12, 131.47, 130.26,
129.77, 127.92, 127.85, 127.72, 126.79, 113.23, 109.67, 108.73, 87.85, 86.17,
85.88, 83.66, 83.15, 78.23, 73.03, 72.69, 63.66, 55.05, 49.61, 36.74, 35.84,
28.30, 26.49, 22.51, 18.65, 12.26, 11.72; HR-ESI-TOF-MS: m/z 1130.3980
([M+Na]+), calcd. for [C60H65N5O12SSi+Na]+ 1130.4012.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-3ꢁꢁ-
O-tert-butyldiphenylsilyl-2ꢁꢁ-O,4ꢁꢁ-C-methylene-thymidin-5ꢁꢁ-yl)mercaptoaceta
mide (26). It was obtained by the HOBT/HBTU catalyzed coupling reaction
of acid 8 with amine 6 as white solid (1.20 g, 62% yield). Rf = 0.7 (10%
25
methaol in chloroform); m.p.: 118–120◦C; [α]D = + 4.2 (c 0.05, CHCl3);
IR (KBr) νmax: 3199, 3069, 2931, 1691, 1608, 1509, 1465, 1252, 1177, 1112,
1
1053, 980, 831, and 703 cm−1; H NMR (CDCl3, 300 MHz): δ 7.62–7.22
(22H, m), 6.84 (4H, J = 8.7 Hz, d), 5.64 (1H, s), 5.48 (1H, s), 4.50 (1H, s),
4.45 (1H, J = 8.1 Hz, d), 4.09 (1H, J = 7.8 Hz, d), 3.92 (1H, s), 3.85 (1H, s),
3.78 (7H, s), 3.73 (2H, m), 3.48 (2H, s), 3.26 (2H, J = 14.4 Hz, q), 2.95 (1H, J
= 15.0 Hz, d), 2.81 (1H, J = 15.0 Hz, d), 1.69 (6H, s), 1.06 (9H, s); 13C NMR
(DMSO-d6, 100.6 MHz): δ 169.73, 163.75, 163.56, 158.12, 150.39, 149.89,
144.49, 135.79, 135.44, 135.28, 134.95, 133.11, 132.73, 110.01, 108.81,
87.59, 86.27, 85.87, 83.21, 80.68, 78.51, 72.88, 71.06, 57.88, 57.23, 54.99,
51.10, 45.67, 38.12, 33.58, 33.22, 26.68, 18.88, 12.39, 11.93;HR-ESI-TOF: m/z
1136.3997 ([M+H]+), calcd. for [C61H65N5O13SSi+H]+ 1136.4101.
General procedure for the synthesis of mercaptoacetamido-linked
dimers 3, 4, and 5. A solution of 24/25 or 26 (0.90 mmol) and TBAF
(1.20 mL, 1M in THF) in anhydrous tetrahydrofuran (15 mL) was stirred at

LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers
269
r.t. for 1 hour. Excess of tetrahydrofuran was evaporated in vacuo and residue
was redissolved in dichloromethane (50 mL). The solution was washed with
water (2 × 20 mL) followed by brine (2 × 20 mL). The organic layer was
dried over anhydrous Na2SO4 and evaporated to dryness. The crude thus ob-
tained was purified by silica gel column chromatography using methanol in
chloroform as gradient solvent system to afford mercaptoacetamido-linked
nucleosiode dimers 3/4/5 in 75%, 80%, and 70% yields, respectively.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-β-
thymidin-5ꢁꢁ-yl)mercaptoacetamide (3). It was obtained as white solid powder
30
(0.59 g, 75% yield). m.p.: 173–175◦C (decompose); [α]D = + 29.1 (c 0.05,
MeOH); IR (KBr) νmax: 3448, 3067, 2927, 1690, 1508, 1466, 1252, 1178,
1
1054, 977, 895, 823, 760, 727, and 705 cm−1; H NMR (CDCl3, 400 MHz):
δ 10.36 (1H, brs), 9.87 (1H, brs), 7.61 (1H, brs), 7.47 (2H, J = 7.2 Hz, d),
7.36–7.21(10H, m), 7.14 (1H, brs), 6.84 (4H, J = 9.2 Hz and J = 2.8 Hz,
dd), 5.98 (1H, s), 5.59 (1H, s), 5.04 (1H, J = 16.0 Hz, d), 4.59 (1H, J =
7.6 Hz, d), 4.25 (2H, J = 20.0 Hz, d), 3.96 (2H, J = 8.4 Hz, d), 3.78 (6H, s),
3.48 (2H, J = 23.2 Hz and J = 11.2 Hz, dd), 3.23 (2H, s), 2.79–2.71 (2H,
m), 2.28–2.25 (1H, J = 12.0 Hz, d), 1.84 (3H, s), 1.68 (3H, s); 13C NMR
(DMSO-d6, 100 MHz): δ 169.88, 163.80, 158.15, 150.45, 149.91, 144.60,
135.95, 135.25, 134.98, 133.92, 129.86, 127.96, 127.72, 126.79, 113.27,
109.77, 108.82, 87.65, 86.30, 85.89, 85.18, 83.81, 79.19, 78.53, 72.41, 71.16,
57.99, 55.05, 51.23, 39.18, 34.01, 12.40, 12.05; HR-ESI-TOF-MS m/z 892.2771
([M+Na]+), calcd. for [C44H47N5O12S+Na]+ 892.2834.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-β-thymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-2ꢁꢁ-O,4ꢁꢁ-C-methylene
thymidin-5ꢁꢁ-yl)mercaptoacetamide (4). It was obtained as white solid
powder (0.61 g, 80% yield). Rf = 0.5 (10% methaol in chloroform); m.p.:
24
158–160◦C; [α]D = + 102.2 (c 0.1, CHCl3); IR (KBr) νmax: 3353, 3064,
1
2930, 1694, 1509, 1466, 1252, 1178, 1105, 1042, 830, and 584 cm−1; H
NMR (DMSO-d6, 400 MHz): δ 11.35 (2H, brs), 8.54 (1H, J = 7.2 Hz, d),
7.53 (1H, s), 7.66 (1H, s), 7.37 (2H, J = 7.2 Hz, d), 7.30–7.20 (8H, m), 6.88
(4H, J = 8.8 Hz and J = 2.0 Hz, dd), 6.20 (1H, J = 6.4 Hz, t), 5.85 (1H, J
= 4.4 Hz, d), 5.43 (1H, s), 4.50–4.43 (1H, m), 4.13 (1H, s), 3.87 (2H, J =
4.4 Hz, d), 3.81 (1H, J = 8.0 Hz, d), 3.70–3.66 (7H, m), 3.32–3.25 (2H, m),
3.18–3.11 (2H, m), 3.00–2.90 (2H, m), 2.37–2.32 (1H, m), 2.18–2.12 (1H,
m), 1.75 (3H, s), 1.42 (3H, s); 13C NMR (DMSO-d6, 100 MHz): δ 168.87,
163.35, 158.14, 150.40, 149.95, 144.60, 135.80, 135.45, 135.27, 134.64,
129.80, 127.94, 127.74, 126.82, 113.25, 109.77, 108.65, 88.00, 86.51, 85.72,
83.67, 83.09, 79.09, 72.57, 70.68, 63.51, 55.07, 49.56, 45.74, 36.76, 35.98,
28.34, 12.40, 11.76; HR-ESI-TOF-MS: m/z 892.2851 ([M+Na]+), calcd. for
[C44H47N5O12S+Na]+ 892.2834.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(5ꢁꢁ-deoxy-2ꢁꢁ-
O,4ꢁꢁ-C-methylenethymidin-5ꢁꢁ-yl)mercaptoacetamide (5). It was obtained
as white solid powder (0.55 g, 70% yield). Rf = 0.5 (10% methaol in
24
chloroform); m.p.: 158–160◦C; [α]D = + 47.1 (c 0.05, CHCl3); IR (KBr)

270
V. K. Sharma et al.
νmax: 3195, 2924, 2853, 1694, 1609, 1509, 1463, 1251, 1178, 1053, 978, and
832 cm−1; 1H NMR (DMSO-d6, 400 MHz): δ 11.45 (2H, brs), 8.16 (1H, J =
8.4 Hz, d), 7.70 (1H, s), 7.58 (1H, s), 7.44 (2H, J = 7.6 Hz, d), 7.33–7.23
(8H, m), 6.89 (4H, J = 8.8 Hz and J = 2.0 Hz, dd), 4.85 (1H, s), 5.52 (2H, J
= 16.0 Hz, d), 4.44 (2H, J = 7.2 Hz, d), 4.32 (1H, s), 3.85–3.84 (2H, m), 3.73
(8H, s), 3.34–3.31 (2H, m), 3.26–3.19 (2H, m), 3.04 (2H, J = 14.8 Hz, d),
1.78 (3H, s), 1.54 (3H, s); 13C NMR (DMSO-d6, 100 MHz): δ 169.68, 163.69,
158.14, 149.90, 144.60, 134.97, 134.31, 133.86, 129.83, 129.78, 127.92,
127.66, 126.77, 113.25, 108.77, 87.56, 87.10, 86.47, 86.28, 85.84, 78.53,
77.48, 72.65, 72.00, 71.13, 57.93, 55.02, 50.88, 45.64, 34.59, 28.14, 12.21;
HR-ESI-TOF-MS: m/z 898.2925 ([M+H]+), calcd. for [C45H47N5O13S+H]+
898.2911.
1-(3ꢁ-Deoxy-5ꢁ-O-DMT-2ꢁ-O,4ꢁ-C-methylenethymidin-3ꢁ-yl)-4-(3ꢁꢁ-O-β-
cyanoethyl-N,N-diisopropylphosphoramidite-5ꢁꢁ-deoxy-β-thymidin-5ꢁꢁ-
yl)mercaptoacetamide (27). To a stirred solution of compound 3 (0.1g,
0.115 mmol) in dry DCM (2 mL), N ,N -Diisopropylethylamine(25 μL,
0.283 mmol) and 2-cyanoethyl N ,N -diisopropylamino chlorophospho-
ramidite (64 μL, 0.283 mmol) were added under argon atmosphere at 0◦C.
The reaction mixture was stirred at r.t. for 30 minutes. After completion of
reaction on analytical TLC examination, the reaction mixture was diluted
with dry DCM (20 mL) and washed with 5% NaHCO3 solution (2 × 10 mL).
The organic phase was dried and concentrated under reduced pressure.
Purification was done by flash column chromatography using hexane:
ethyl acetate (1:8) solvent gradient. The phosphoramidite 27 was dried
overnight over P2O5 and KOH in a desiccator before characterization. It
was obtained as white solid, Rf = 0.5 (ethyl acetate); 31P NMR (CDCl3): δ
148.96, 148.70; 1H NMR (CDCl3, 400 MHz): δ 7.59 (1H, s), 7.49 (1H, J =
4.0 Hz, d), 7.38–7.10 (11H, m), 6.86 (4H, J = 8.0 Hz, d), 6.01–5.99 (1H,
m), 5.84–5.83 (1H, m), 5.68 (1H, s), 5.04–4.93 (3H, m), 4.56–4.46 (3H, m),
3.95–3.60 (11H, m), 3.50–3.48 (2H, m), 3.24–3.21 (2H, m), 2.88–2.87 (3H,
m), 2.66–2.64 (2H, m), 1.72 (3H, s), 1.49 (3H, s), 1.30 (6H, J = 8.0 Hz, d),
1.20 (6H, J = 8.0 Hz, d); HR-ESI-TOF-MS: m/z 1092 ([M+Na]+), calcd. for
[C53H64N7O13PS+Na]+ 1092.
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