4′-Epi-DNA: A DNA Mimic Containing 4′-hydroxymethyl-α-l-Xylo-Thymidine with Compact Backbone like RNA

Article abstract of DOI:10.1080/15257770.2016.1202963

Synthesis of C4′-epi-DNA containing 3′→ 5″ linkages is reported for the first time. Crystal structure study of the monomer indicated that though the dihedral angle O3′-C3′-C4′-C5″ in this case would be like in RNA, the sugar conformation would remain like that in DNA. The study of the effect of this backbone configuration in DNA with respect to its binding to cDNA and RNA is reported in this note.

Full text of DOI:10.1080/15257770.2016.1202963

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
4′-Epi-DNA: A DNA Mimic Containing 4′-
hydroxymethyl-α-l-Xylo-Thymidine with Compact
Backbone like RNA
Seema Bagmare, Vedavati G. Puranik, Moneesha Fernandes & Vaijayanti A.
Kumar
To cite this article: Seema Bagmare, Vedavati G. Puranik, Moneesha Fernandes & Vaijayanti
A. Kumar (2016): 4′-Epi-DNA: A DNA Mimic Containing 4′-hydroxymethyl-α-l-Xylo-Thymidine
with Compact Backbone like RNA, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2016.1202963
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1202963
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Date: 16 August 2016, At: 06:50

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
ꢀ-Epi-DNA: A DNA Mimic Containing
ꢀ-hydroxymethyl-α-L-Xylo-Thymidine with
Compact Backbone like RNA
Seema Bagmarea, Vedavati G. Puranikb, Moneesha Fernandesa,
and Vaijayanti A. Kumara
aOrganic Chemistry Division; bCenter for Material Characterization, CSIR-National Chemical Laboratory,
Pune, India
ABSTRACT
ARTICLE HISTORY
Received  November 
Accepted  June 
Synthesis of C4ꢀ-epi-DNA containing 3ꢀ→ 5ꢀꢀ linkages is reported
forthefirsttime. Crystalstructurestudyofthemonomerindicated
that though the dihedral angle O3ꢀ-C3ꢀ-C4ꢀ-C5ꢀꢀ in this case would
be like in RNA, the sugar conformation would remain like that in
DNA. The study of the effect of this backbone configuration in
DNA with respect to its binding to cDNA and RNA is reported in
this note.
KEYWORDS
Antisense Oligonucleotide;
ꢀ-hydroxymethyl
nucleosides Cꢀ-epi-DNA
GRAPHICAL ABSTRACT
Introduction
Backbone-modified DNA oligomers as antisense oligonucleotides (ONs) are being
explored for therapeutic applications since the last two decades.[1–3] Numerous
examples of modified ONs and their antisense action are reported in the litera-
ture.[4–7] Different modifications of DNA, such as 2ꢀ-O-alkyl,[7] 2ꢀ-O-aminoalkyl,[8,9]
LNA,[10,11] as well as 2ꢀ-fluoro,[12,13] resulted in significant increase of the DNA:RNA
duplex stability. Most of these modifications drive the sugar into the C3ꢀ-endo
CONTACT Vaijayanti A. Kumar
ratory, Pune , India.
va.kumar@ncl.res.in
Organic Chemistry Division, CSIR-National Chemical Labo-
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
Supplemental data for this article can be accessed at publisher’s website.
©  Taylor & Francis Group, LLC

2
S. BAGMARE ET AL.
Figure . Cꢀ-endo and Cꢀ-endo sugar conformations and proposed Cꢀ-epi-DNA.
conformation (Figure 1, N-type) typical for the A-type RNA/RNA duplex,[14] which
results in complete loss of susceptibility to RNase H activity. However, arabinonu-
cleic acids and 2ꢀ-deoxy-2ꢀ-fluoro-arabinonucleic acids were shown[15–18] to be sub-
strates for RNase H-mediated cleavage of target RNA. In an NMR study, it was
demonstrated that the 2ꢀF-ANA adopted an eastern O-4ꢀ-endo-sugar conforma-
tion, closer to B-type DNA when hybridized to RNA.[19] The α-l-LNA[20,21] and
CeNA[22] were also reported to assume a DNA-like southern conformation. The
crystal structure of RNase H complexed with a DNA:RNA duplex also later estab-
lished that the enzyme specifically recognizes the A-form of the RNA strand and B-
form of the DNA strand in the RNA:DNA duplex.[23] It is apparent from the above
discussion that an advantageous strategy would be to improve the affinity to the tar-
get RNA while maintaining the C2ꢀ-endo sugar pucker in the modified nucleosides
(Figure 1, S-type).
ONs possessing a variously 4ꢀ-C-substituted 2ꢀ-deoxyribose moiety[24–27]
(Figure 2) have been reported. In these studies, the 5ꢀ-hydroxy→3ꢀ-hydroxy
backbone substituted at C4ꢀ- (Figure 2, I) or the 5ꢀ-hydroxy→4ꢀ-C-hydroxymethyl
backbone (Figure 2, II) were explored. In I, the geometry of the sugar remained S-
type and the phosphodiester linkages remain trans(pseudodiaxial), extended. Many
of these resulted in enhanced stability of the resulting duplexes with DNA and RNA
and also induced RNase H activity. The 4ꢀ-C-hydroxymethyl-α-l-RNA nucleic
acids were synthesized by Wengelꢀs group (Figure 2, III).[28] In this case, the C3ꢀ-
and C4ꢀ-centers taking part in the phosphodiester linkages are inverted compared
to natural linkages but still remain trans-oriented. The derived oligomers were
found to form less stable duplexes with DNA and RNA, the latter also being good
substrates for RNase H. The other isomer, 4ꢀ-C-hydroxymethyl-β-d-lyxofuranosyl

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Figure . ꢀ-C substituted DNA analogues and dXylo and Xylo nucleic acids.
nucleic acids (Figure 2, IV) where the C3ꢀcentre taking part in the phosphodiester
linkages is inverted, has a cis disposition of the substituents involved in the backbone
linkages.[28] Incorporation in a DNA strand led to RNA-selective binding, while
incorporation in an RNA strand led to stable duplex formation with both DNA and
RNA. In α-l-LNA, which is a locked analogue of 4ꢀ-C-hydroxymethyl-α-l-RNA[28]
also, the stereochemistry at both C4ꢀ- and C3ꢀ-centers is inverted and remains trans.
Similarly, 2ꢀ-deoxy-xylo-based nucleic acids[29] (Figure 2, V), in which the stereo-
chemistry is inverted only at the C3ꢀ-center compared to DNA, with cis-orientation
of the 5ꢀ- and 3ꢀ-backbone linkers, was investigated. Very recently, a xylo-nucleic
acid analogue with a 2ꢀ-hydroxy group was also studied.[30] The backbone sug-
gested for the present studies, containing modified 4ꢀ-hydroxymethyl-thymidine
(or 4ꢀ-hydroxymethyl-α-l-xylo-thymidine), i.e., α−4ꢀ-C-hydroxymethyl→cis-3ꢀ-
hydroxy backbone will be epimeric at the C4ꢀ position with respect to the DNA
backbone (hence termed 4ꢀ-epi-DNA), has not yet been studied and therefore is
of interest. We suppose that the cis orientation of C5ꢀ- and C3ꢀ-substitutions as
phosphodiester linkers (pseudoequatorial, pseudoaxial, Figure 1) would lead to
compact geometry as in the case of N-type sugar in RNA with pseudodiequatorial
orientation of phosphate groups. In this paper, we report the synthesis of this back-
bone as a DNA analogue containing 4ꢀ-substituted α-l-xylo-thymidine which is
envisaged to have the sugar in a 2ꢀ-endo (S-type) pucker that is normally observed in
DNA. The cis-geometry of the phosphodiester linker involving 4ꢀ-α-C5ꢀꢀH2OH and
3ꢀ-OH group may show internucleoside distance complementarity while binding to
compact A-type RNA geometry (Figure 1).
The synthesis of the monomer was accomplished from thymidine 1 (Scheme 1).
Compound 4 was obtained following the reaction sequence as reported in litera-
ture.[31] We applied a simple oxidation protocol for the synthesis of the 5ꢀ-aldehyde
using IBX[32] rather than Swern oxidation[33,34] (oxalyl chloride/DMSO/DIPEA) or
Moffatt’s oxidation (DMSO/DCC/TFA/pyridine) procedure.[35] The latter two oxi-
dation procedures require polar solvents and tedious work-up conditions whereas
IBX oxidation was simple and high yielding. Reaction of compound 4 with IBX

4
S. BAGMARE ET AL.
ꢀ
Scheme . Synthesis of C -epi-DNA monomer.
gave corresponding aldehyde 5 which was followed by aldol condensation with
formaldehyde in the presence of aqueous sodium hydroxide with concomitant
reduction using sodium borohydride employing known reaction conditions [36] to
give known compound 6. The α−4ꢀ-C-hydroxymethyl functionality (C5ꢀꢀ-OH) was
regioselectively protected by reaction with DMT-Cl to give nucleoside 7. Similar
selectivity for the reactivity of C5ꢀꢀ-OH was found earlier by Obika et al.[37] Protec-
tions of 5ꢀ-hydroxyl group as a silyl ether furnished compound 8. The DMT groups
in compound 8 were then removed using 2% dichloroacetic acid in DCM to give 5ꢀ-
O-(tert-butyldimethylsilyl)-4ꢀ-C-(hydroxymethyl)thymidine 9. The protection of
the α−4ꢀ-C-hydroxymethyl group by reaction with DMT-Cl furnished compound
10 which was then converted to its phosphoramidite derivative 11. Compound 11
was found to be unstable for characterization and easily decomposed, probably
due to the cis DMT-protected hydroxymethyl group and the reactive phospho-
ramidite. Its formation was confirmed by 31P NMR spectroscopy and was used in
the synthesis of oligomers immediately.
The sugar cꢀonformation of compound 9 was calculated from NMR H1ꢀ coupling
constants (JH1 -H2 ,2 = 8.84 and 5.43 Hz) using Sum rule[38] and was found to have
76% S-type sugar pucker. We further confirmed the sugar conformation using crys-
tal structure of compound 9 (Figure 3) which clearly indicated S-type sugar confor-
mation and the envisaged proximity of the O3ꢀ and O5ꢀꢀ centers to be engaged in
phosphodiester linkages.
ꢀ
ꢀ
The unmodified oligomers[39] were synthesized using an automated Bioau-
tomation MM4 DNA synthesizer and commercially available 5ꢀ-O-DMT-2ꢀ-deoxy-
3ꢀ-phosphoramidite building blocks using the β-cyanoethylphosphoramidite
method.[40,41] The chosen control 18mer oligonucleotide DNA1 was specific for the
Splice correction of an aberrant β-globin intron (705 site). The modified monomer
11 was incorporated at pre-determined positions in the unmodified sequence DNA1

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure . X-rayCrystalstructureofcompound9, thepercentageofprobabilityoftheellipsoidsis%.
using extended coupling time to yield the modified oligomers ON1 and ON2
(Table 1). The synthesized ONs were cleaved from the solid support with conc.
NH4OH and lyophilized. The 5ꢀ-O-silyl group was deprotected by treatment with
0.1 mL desilylation solution (1.5 mL NMP + 0.75 mL TEA + 1 mL 3HF: TEA)
at 65°C for two hours. The reaction mixture was then lyophilized and desalted to
Table . The synthetic oligomers, their characterization and Tm (°C) values of mer chimeric ONs:
DNA/RNA duplexes#.
UV-Tm°C (ꢀTm°C)
HPLC tR
(min)
MALDI-TOF Mass
Expected/Found
Sequence
DNA 
RNA
DNA
ON
ON
.
.
.
.
/.
/.
. (−.)
. (−.)
. (−.)
. (−.)
#DNA1:ꢀCCT CTT ACC TCA GTT ACAꢀ, ON1: ꢀCCT CTT ACC TCA GTT ACAꢀ ON2:ꢀCCT CTT ACC TCA GTTACAꢀꢀ
DNA2: ꢀTGT AAC TGA GGT AAG AGG ꢀ, RNA: ꢀUGU AAC UGA GGU AAG AGG ꢀ, T: represents modification. Tm
melting temperature (measured in the buffer  mM sodium phosphate,  mM NaCl, pH = .), of
Cꢀ-epi-DNA-modified ONs:DNA/RNA complexes. The values reported here are the average of  independent
=
experiments and are accurate to .°C. ꢀTm = Tm − Tm(control)
.

6
S. BAGMARE ET AL.
get the crude ONs. The purity of the oligomers listed in Table 1 was checked by
analytical RP-HPLC (C18 column, 0.1 N TEAA buffer-acetonitrile) which showed
more than 75–80% purity. These oligomers were subsequently purified by reverse
phase HPLC on a C18 column. The purity of the oligomers was again ascertained
by analytical RP-HPLC and found to be >95%. The modified oligomers were char-
acterized by MALDI-TOF mass spectrometric analysis (SI). The UV-melting pro-
files of the duplexes formed by oligonucleotides ON1 and ON2 with complemen-
tary DNA/RNA were studied (SI). The unmodified 18mer DNA1 sequence formed
complexes with complementary DNA2 and RNA with higher melting temperature
for DNA1:RNA complex over DNA1:DNA2 (ꢀTm= +3.4°C, Table 1). Introduc-
tion of a single monomeric unit towards the 3ꢀ-end in 18mer oligomer ON1, caused
destabilization of 2.9°C and 6.1°C with complementary DNA2 and RNA, respec-
tively. The effect of the second unit was less disturbing to the ON2:RNA duplex
stability and much less destabilization was observed for the ON2:RNA duplex
(ꢀTm= −1.8°C) compared to ON2:DNA2. Thus, intermittent change to 3ꢀ−5ꢀꢀ link-
age in continuous 3ꢀ−5ꢀ backbone at a single or more positions destabilized the
duplexes with both DNA and RNA.
In summary, NMR and crystal structure studies confirmed the S-type sugar
pucker for C4ꢀ-hydroxymethyl-thymidine derivative. Introduction of this 4ꢀ-
substituted thymidine monomer into oligomers containing 3ꢀ → 5ꢀꢀ linkages at
selected positions, destabilized complexes with both DNA and RNA. We found that
the experimental results did not corroborate with the proposed hypothesis that the
compact geometry due to RNA-like O3ꢀ-C3ꢀ-C4ꢀ-C5ꢀꢀ dihedral angle and cis axial-
equatorial orientation of phosphodiester groups at the modified site would be effec-
tive for stabilizing the DNA:RNA duplexes. The duplexes formed with both DNA
and RNA were largely destabilized similar to the instances of the other stereoiso-
mers studied before.
Experimental section
General: All the reagents were purchased from Sigma-Aldrich and used without
purification. DMF, CH3CN were dried over P2O5 and CaH2, respectively, and stored
˚
by adding 4 A molecular sieves. Pyridine, TEA were dried over KOH and stored
by adding KOH. THF was passed over basic alumina and dried by distillation
over sodium. Analytical TLCs were performed on Merck 5554 silica 60 aluminium
sheets. Column chromatography was performed for purification of compounds on
silica gel (60–120 mesh, Merck). For acid-sensitive (trityl-containing) compounds,
the column was packed and equilibrated with 0.5% triethylamine.TLCs were per-
formed using dichloromethane-methanol or petroleum ether–ethyl acetate solvent
systems. Visualization was accomplished with UV light and/or by spraying with per-
1
chloric acid reagent and heating. H and 13C NMR spectra were obtained using
Bruker ACF 200 (200 MHz) or 400 (400 MHz) spectrometers and all the chemi-
1
cal shifts (δ/ppm) are referred to internal TMS for H and chloroform-d for 13C
NMR. 1H NMR data are reported in the order of chemical shift, multiplicity (s, sin-
glet; d, doublet; t, triplet; br, broad; br s, broad singlet; m, multiplet and/ or multiple

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
resonance), number of protons. Mass spectra were recorded on Thermo Finni-
gan Surveyor MS-Q spectrometer, while HRMS was recorded by using a Thermo
Scientific Q-exactive mass spectrometer. RP-HPLC was carried out on a C18 col-
umn using Waters system (Waters Delta 600e quaternary solvent delivery system
and 2998 photodiode array detector and Empower2 chromatography software).
MALDI-TOF spectra were recorded on a Voyager-De-STR (Applied Biosystems)
MALDI-TOF instrument. UV experiments were performed on a Varian Cary 300
UV-VIS spectrophotometer fitted with a Peltier-controlled temperature program-
mer and a water circulator.
5ꢀ-O-(tert-butyldimethylsilyl)-thymidine (2)
Thymidine1 (10.0 g, 41.28 mmol) and imidazole (6.0 g, 90.75 mmol) were sus-
pended in 15 mL of dry DMF. TBS-Cl (6.5 g, 41.25 mmol) dissolved in 5 mL
of dry DMF was added dropwise at 0°C and the reaction was stirred at RT for
20 h. DMF was removed under vacuum and the residue was dissolved in ethyl
acetate, washed with water and then brine. The organic layer was dried over Na2SO4
and concentrated. Purification by column chromatography using 2% methanol in
dichloromethane gave the product 2 (12.1 g, Yield 80%).
1H NMR (200MHz, CDCl3) δ: 8.93 (s, 1H, exchanges with D2O), 7.53 (s, 1H),
6.35–6.42 (dd, J = 8.21, 5.81 Hz, 1H), 4.48 (m, 1H), 4.05 (m, 1H), 3.89 (m, 2H),
2.65 (br s, 1H, exchanges with D2O), 2.34–2.44 (m, 1H), 2.03–2.17 (m, 1H), 1.92 (s,
1H), 0.92 (s, 9H), 0.12 (s, 6H); 13C NMR (50 MHz, CDCl3) δ: 164.1, 150.6, 135.5,
110.9, 87.3, 85.0, 72.7, 63.6, 41.0, 25.8, 18.3, 12.5, −5.4, −5.5; MS (EI)m/z 356.1767,
found 379.11 (M+Na+); HRMS (ESI): calcd for C16H28N2O5SiNa: 379.1659, found
379.1648.
5ꢀ-O-(tert-butyldimethylsilyl)-3ꢀ-O-(4,4ꢀ-dimethoxytrityl)thymidine (3)
A mixture of 2 (10.0 g, 28.0 mmol) and DMT-Cl (11.4 g, 33.7 mmol) in 25 mL dry
pyridine was heated at 50°C for 12 h. Pyridine was removed under vacuum and
the crude product was purified by column chromatography on neutralized silica gel
using 50% ethyl acetate in petroleum ether to give product 3 (17.1g, Yield 92%).
1H NMR (200 MHz, CDCl3) δ: 8.32 (s, 1H, exchanges with D2O), 7.32–7.47
(m, 10H), 6.82–6.86 (d, J = 8.85 Hz, 4H), 6.37–6.45 (dd, J = 9.34, 5.31 Hz, 1H),
4.28 (d, J = 5.30 Hz, 1H), 4.03 (s, 1H), 3.80 (s, 6H), 3.66 (m, 1H), 3.27 (m, 1H),
1.86 (s, 3H), 1.70 (m, 2H), 0.81 (s, 9H), −0.04 (s, 3H), −0.09 (s, 3H); 13C NMR
(50 MHz, CDCl3) δ: 163.6, 158.6, 150.2, 145.0, 136.3, 136.2, 135.5, 130.2, 130.1,
128.2, 127.9, 127.0, 113.2, 110.8, 87.2, 86.6, 84.8, 74.9, 63.5, 55.2, 39.9, 25.7, 18.1,
12.3, −5.4, −5.7; MS (EI)m/z 658.3074, found 681.34 (M + Na+); HRMS (ESI):
calcd for C37H46N2O7SiNa: 681.2966, found 681.2956.
3ꢀ-O-(4,4ꢀ-dimethoxytrityl) thymidine (4)
Compound 3 (15.0 g, 22.7 mmol) dissolved in 40 mL anhydrous THF and 1N TBAF
in THF (34.1 mL, 34.1 mmol) was added. The reaction was stirred at RT for 1 h.

8
S. BAGMARE ET AL.
THF was removed under reduced pressure and the residue was dissolved in DCM,
washed with water and then brine. The organic layer was dried over Na2SO4 and
concentrated. The product was purified by column chromatography using 1.5% of
methanol in dichloromethane to yield product 4 as a white solid (9.8 g, Yield 79%).
1H NMR (200 MHz, CDCl3) δ: 8.59 (s, 1H, exchanges with D2O), 7.24–7.47 (m,
10H), 6.82 (d, J = 8.46 Hz,4H), 6.09–6.17 (dd, J = 8.72, 5.81 Hz, 1H), 4.36 (m, 1H),
3.98 (s, 1H), 3.79 (s, 6H), 3.63–3.69 (m, 1H), 3.35 (m, 1H), 2.50 (br s, 1H, exchanges
with D2O), 1.90–1.95 (m, 1H), 1.85 (s, 3H), 1.69 (s, 1H); 13C NMR (50 MHz,
CDCl3) δ: 163.7, 158.8, 150.3, 145.0, 137.1, 136.2, 136.1, 130.1, 128.2, 127.9, 127.0,
113.2, 110.8, 87.2, 87.1, 86.5, 74.2, 62.4, 55.2, 38.5, 12.3; MS (EI)m/z 544.2210,
found 567.21 (M+ Na+); HRMS (ESI): calcd for C31H32N2O7Na:567.2102, found
567.2091.
3ꢀ-O-(4, 4ꢀ-dimethoxytrityl)-5ꢀ-formyl Thymidine (5)
Compound 4 (3.00 g, 5.51 mmol) was dissolved in 20 mL acetonitrile in a flask
equipped with a reflux condenser. To it, IBX (4.63 g, 16.5 mmol) was added and the
reaction mixture was heated at 80°C for 3 h. After completion of reaction, the reac-
tion mixture was cooled to room temperature and filtered through a bed of celite.
The filtrate was concentrated and subjected to filtration column purification on neu-
tralized silica gel. The product eluted at 35–40% acetone in petroleum ether system
to get compound 5 (2.65 g, yield 89%) as a white foam. The product obtained was
immediately utilized for the next reaction.
3ꢀ-O-(4,4ꢀ-dimethoxytrityl)-4ꢀ-hydroxymethylthymidine (6)
To a stirred solution of 5 (2.62 g, 4.83 mmol) in dioxane, cooled in a waterbath,
35% HCHO (0.78 mL, 9.18 mmol) and 2N NaOH (4.6 mL, 9.18 mmol) were added.
Stirring was continued for 5 min. The reaction mixture was then cooled in an ice
bath and NaBH4 (0.349 g, 9.18 mmol) was added in portions. Stirring was contin-
ued in an icebath for 1 h and then allowed to attain room temperature, with stirring
continued for 3 h. To the reaction mixture, 1 mL water was added and the mix-
ture was neutralized using Dowex H+ resin. The resin was filtered off and the fil-
trate evaporated to dryness. The residue was purified by column chromatography
using 30% acetone in petroleum ether to give product 6 as a white solid (2 g, Yield
72%).
1H NMR (200 MHz, CDCl3) δ: 8.65 (s, 1H, exchanges with D2O), 7.28–7.46
(m, 10H), 6.98 (s, 1H), 6.83–6.88 (m, 4H), 6.12–6.18 (m, 1H), 4.57 (m, 1H), 4.02
(m, 1H), 3.75–3.80 (m, 8H), 3.39 (m, 1H), 3.08 (br s, 1H, exchanges with D2O),
2.62 (br s, 1H, exchanges with D2O), 2.0–2.15 (m, 1H), 1.81 (s, 3H), 1.41–1.55
(m, 1H); 13C NMR (50 MHz, CDCl3) δ: 163.6, 158.8, 150.5, 144.9, 136.6, 135.9,
135.8, 130.3, 128.2, 128.0, 127.2, 113.3, 111.1, 89.1, 87.3, 83.7, 72.6, 63.2, 62.1, 55.2,
38.8, 12.3; MS (EI)m/z 574.2315, found 597.17 (M+ Na+); HRMS (ESI): calcd for
C32H34N2O8Na:597.2207, found 597.2196.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
4ꢀ-C-[(4,4ꢀ-dimethoxytrityl)oxomethyl]−3ꢀ-O-(4,4ꢀ-dimethoxytrityl)thymidine (7)
To a solution of 6 (1.90 g, 3.31 mmol) in pyridine, DMT-Cl (1.34 g, 3.8 mmol) was
added and reaction mixture was allowed to stir for 12 h. Pyridine was removed under
vacuum and the crude product obtained was purified by column chromatography
on neutralized silica gel using 45% ethyl acetate in petroleum ether to give product
7 (2.25 g, Yield 78%).
1H NMR (200 MHz, CDCl3) δ: 8.60 (s, 1H, exchanges with D2O), 7.51–7.54 (m,
2H), 7.39–7.45 (m, 4H), 7.16–7.33 (m, 12H), 7.09 (m, 1H), 6.84–6.89 (m, 4H), 6.71–
6.77 (m, 4H), 6.26–6.32 (m, 1H), 4.33 (t, J = 8.09 Hz,1H), 3.83 (m, 1H), 3.77 (s, 12H),
3.55 (d, J = 10.36 Hz, 1H), 3.30 (d, J = 10.36 Hz, 1H), 2.96–3.09 (m, 1H), 2.32–2.47
(m, 1H), 2.08 (br s, 1H, exchanges with D2O), 1.84 (s, 3H), 1.49–1.62 (m, 1H); 13
C
NMR (100 MHz, CDCl3) δ: 163.6, 158.6, 158.6, 158.4, 158.4, 150.0, 145.1, 144.8,
136.5, 136.0, 135.9, 134.8, 135.7, 130.2, 130.1, 128.1, 127.9, 127.7, 127.0, 126.7, 113.2,
113.2, 113.0, 110.7, 88.5, 87.0, 86.7, 84.1, 71.6, 64.8, 62.9, 55.1, 38.8, 12.4; MS (EI)m/z
876.3622, found 875.50 (M+-1). HRMS (ESI): calcd for C53H52N2O10Na:899.3514,
found 899.3508.
5ꢀ-O-(tert-butyldimethylsilyl)-4ꢀ-C-[(4,4ꢀ-dimethoxytrityl)
oxomethyl]-3ꢀꢀ-O-(4,4ꢀ-dimethoxytrityl)thymidine(8)
Compound 7 (2.000 g, 2.28 mmol), imidazole (0.446 g, 6.84mmol) and TBS-Cl
(0.515 g, 3.42 mmol) were suspended in 4 mL of dry DMF. The reaction mixture
was stirred at RT for 3 h. DMF was removed under vacuum and the product was
extracted in ethyl acetate. The organic layer was dried over Na2SO4, concentrated
and the crude product was purified by column chromatography using 30% ethyl
acetate in petroleum ether to give pure product 8 (1.71 g, Yield 76%).
1H NMR (200 MHz, CDCl3) δ: 8.27 (s, 1H, exchanges with D2O), 7.50–7.53 (m,
2H), 7.34–7.43 (m, 4H), 7.22–7.31 (m, 3H), 7.07–7.18 (m, 9H), 7.09 (m, 1H), 6.80–
6.86 (m, 4H), 6.67–6.72 (m, 4H), 6.38 (t, J = 6.84 Hz, 1H), 4.21–4.27 (dd, J = 7.70,
4.55 Hz,1H), 3.92 (d, J = 10.7 Hz, 1H), 3.76 (s, 12H), 3.68 (d, merged with 3.76
peak, 1H), 3.24–2.31 (m, 2H), 1.87 (s, 3H), 1.63 (m, 2H), 0.77 (s, 9H), −0.07 (s, 3H),
−0.16 (s, 3H); 13C NMR (50 MHz, CDCl3) δ: 163.5, 158.5, 158.4, 150.0, 145.3, 144.9,
136.0, 135.8, 130.3, 130.2, 130.2, 130.1, 128.2, 128.1, 127.8, 127.7, 126.8, 126.7, 113.1,
113.1, 113.0, 110.7, 88.7, 86.9, 86.5, 83.0, 77.3, 65.1, 64.5, 55.1, 39.2, 25.8, 18.2, 12.5,
−5.4, −5.6; MS (EI)m/z 990.4487, found 1013.54 (M+ Na+); HRMS (ESI):calcd
for C59H66N2O10SiNa: 1013.4383, found 1013.4421.
5ꢀ-O-(tert-butyldimethylsilyl)-4ꢀ-hydroxymethylthymidine (9)
Compound 8 (1.5 g, 1.51 mmol) was dissolved in minimum quantity of DCM and
to it 2% dichloroacetic acid in DCM (total 18 mL + 0.3 mL of triisopropylsilane
as scavenger) was added in portions. The reaction was monitored by TLC. After
completion of the reaction, the mixture was diluted with DCM and the organic layer

10
S. BAGMARE ET AL.
was washed with saturated Na2CO3. The organic layer was dried over Na2SO4 and
concentrated. The crude product was purified by column chromatography using
60% ethyl acetate in petroleum ether to give 9 as a white solid (0.438 g, Yield 75%).
1H NMR (200 MHz, CDCl3) δ: 9.87 (s, 1H, exchanges with D2O), 7.55 (s, 1H),
6.45–6.52 (dd, J = 8.84, 5.43 Hz, 1H), 4.50 (d, J = 5.05 Hz, 1H), 4.32 (br s, exchanges
with D2O, 1H), 3.70–3.88 (m, 5H), 2.39–2.48 (m, 1H), 2.16–2.30 (m, 1H), 1.90 (s,
3H), 0.92 (s, 9H), 0.12 (s, 6H); 13C NMR (50 MHz, CDCl3) δ: 163.9, 150.7, 135.4,
111.3, 88.8, 84.7, 74.0, 66.1, 63.2, 41.4, 25.8, 18.3, 12.4. −5.4, −5.5; MS (EI)m/z
386.1873, found 409.16 (M+ Na+); HRMS (ESI): calcd for C17H30N2O6SiNa:
409.1771, found 409.1771.
5ꢀ-O-(tert-butyldimethylsilyl)-4ꢀ-C-[(4, 4ꢀ-dimethoxytrityl)oxomethyl]thymidine
(10)
To a solution of 9 (0.40 g, 1.03 mmol) in pyridine, DMT-Cl (0.42 g, 1.24 mmol)
was added and reaction mixture was allowed to stir for 12 h. Pyridine was removed
under vacuum and the crude product was purified by column chromatography on
neutralized silica gel using 40% ethyl acetate in petroleum ether to give product 10
(0.583 g, Yield 82%).
1H NMR (500 MHz, CDCl3) δ: 8.43 (s, 1H, exchanges with D2O), 7.55 (s, 1H),
7.88 (d, J = 7.33 Hz, 2H), 7.22–7.33 (m, 8H), 6.84 (m, 4H), 6.43–6.46 (m, 1H), 4.50
(s, 1H), 3.90 (d, J = 10.68 Hz, 1H), 3.80 (s, 6H), 3.62 (d, J = 10.68 Hz, 1H), 3.35 (d,
J = 9.77 Hz, 1H), 3.24 (d, J = 9.46 Hz, 1H), 2.88 (br s, exchanges with D2O, 1H),
2.34–2.38 (m, 1H), 2.20–2.26 (m, 1H), 1.91 (s, 3H), 0.92 (s, 9H), 0.12 (s, 3H), 0.11 (s,
3H); 13C NMR (125 MHz, CDCl3) δ: 163.5, 158.7, 150.1, 143.8, 135.5, 134.8, 134.7,
129.8, 129.8, 128.1, 127.7, 127.1, 113.4, 110.8, 88.8, 87.2, 84.6, 73.9, 66.5, 63.2, 55.2,
40.8, 25.9, 18.4, 12.4, −5.42, −5.45; MS (EI)m/z 688.3180, found 711.38 (M+ Na+);
HRMS (ESI): calcd for C38H48N2O8SiNa: 711.3078, found 711.3098.
5ꢀ-O-(tert-butyldimethylsilyl)-3ꢀ-O-[(2-cyanoethoxy)diisopropylaminop-
hosphino]−4ꢀ-C-[(4,4ꢀ-dimethoxytrityl)oxomethyl]thymidine (11)
Compound 10 (0.200 g, 0.29 mmol) was dissolved in dry dichloromethane (5 mL)
followed by the addition of diisopropylethylamine (0.126 mL, 0.726 mmol). Then
2-cyanoethyl-N, Nꢀ-diisopropyl-chloro phosphine (77 μL, 0.34 mmol) was added to
the solution at 0°C and the reaction mixture was stirred at room temperature for 3 h.
The contents were then diluted with DCM and washed with 5% NaHCO3 solution.
The product was purified by column chromatography using 1% triethylamine in
30% petroleum ether in DCM to give compound 11 (0.153 g, yield 60%).
31P NMR (400 MHz, CDCl3) δ: 149.3, 148.5.
Solid phase synthesis of DNA oligonucleotides by phosphoramidite method
Control 18mer oligonucleotide DNA1 chosen was specific for the Splice correc-
tion of an aberrant β-globin intron (705 site). The unmodified oligomers were

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
synthesized using an automated Bioautomation MM4 DNA synthesizer and com-
mercially available 5ꢀ-O-DMT-2ꢀ-deoxy-3ꢀ-phosphoramidite building blocks using
β-cyanoethylphosphoramidite method. The modified monomer unit 11 was incor-
porated at pre-determined positions using an extended coupling time of 6 min to
yield the modified oligomers efficiently. ON1 and ON2 were synthesized with one
and two modifications, respectively.
Purification and MALDI-TOF characterization of modified oligomers
The synthesized oligonucleotides were cleaved from the solid support with
conc. NH4OH, lyophilized. The silyl group was deprotected by adding 0.1 mL
desilylation solution (1.5 mL NMP + 0.75 mL TEA + 1 mL 3HF:TEA) and heated
at 65°C for two hours. The reaction mixture was then cooled and neutralized with
0.1 mL NH4HCO3 (0.5 M), lyophilized and desalted to get the crude oligonu-
cleotides. The purity of the oligomers listed in Table 1 was checked by analytical
RP-HPLC (C18 column, 0.1 N TEAA buffer-acetonitrile) which showed more than
75–80% purity. These oligomers were subsequently purified by reverse phase HPLC
on C18 column. The purity of the oligomers was again ascertained by analytical
RP-HPLC and found to be >95%. Their integrity was confirmed by MALDI-TOF
mass spectrometric analysis. THAP (2,4,6-trihydroxy acetophenone) matrix with
diammonium citrate as additive was used for MALDI-TOF (matrix assisted laser
desorption ionization time-of-flight) mass analysis of oligomers. HPLC retention
time and observed values of mass in MALDI-TOF spectrometry are listed in Table 1.
Biophysical studies of modified oligomers
UV-Tm studies were carried out to investigate the binding of the modified oligomers
to complementary DNA and RNA. 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. Chimeric DNA oligonucleotides were individually hybridized with the com-
plementary DNA and RNA strands, to obtain duplexes.
The binding affinity of 18mer chimeric ONs (ON1 and ON2) with complemen-
tary DNA2 and RNA was investigated by measuring the melting temperatures (Tm)
of the duplexes.
Supporting Information. X-ray data collection and refinement parameters, 1H,
13C NMR spectra of compounds 2–10, HRMS of compounds 2–10, 31P NMR spec-
trum of compound 11, HPLC profiles and MALDI-TOF spectra of ON1 and ON2,
UV melting graphs.
Acknowledgment
VAK and MF thank CSIR, New Delhi for research funding (BSC0123). SB thanks UGC, New
Delhi for research fellowship.

12
S. BAGMARE ET AL.
References
1. Bennet, C. F.; Swayze, E. E. RNA targeting therapeutics: molecular mechanisms of antisense
oligonucleotides as a therapeutic platform. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259–293.
2. Kole, R.; Krainer, A. R.; Altman, S. RNA therapeutics: beyond RNA interference and anti-
sense oligonucleotides. Nat. Rev. Drug Discov. 2012, 11, 125–140.
3. Levin, A. A. A review of the issues in the pharmacokinetics and toxicology of phosphoroth-
ioate antisense oligonucleotides. Biochim. Biophys. Acta 1999, 1489, 69–84.
4. Kurreck, J. Antisense technologies. Improvement through novel chemical modifications. Eur.
J. Biochem. 2003, 270, 1628–1644.
5. Shukla, S.; Sumaria, C. S.; Pradeepkumar, P. I. Exploring chemical modifications for
siRNA therapeutics: A structural and functional outlook. Chem. Med. Chem. 2010, 5, 328–
349.
6. Micklefield, J. Backbone modification of nucleic acids: Synthesis, structure and therapeutic
applications. Curr. Med. Chem. 2001, 8, 1157–1179.
7. Lesnik, E. A.; Guinosso, C. J.; Kawasaki, A. M.; Sasmor, H.; Zounes, M.; Cummins, L. L.;
Ecker, D. J.; Cook, P. D.; Freier, S. M. Oligodeoxynucleotides containing 2ꢀ-O-modified
adenosine: Synthesis and effects on stability of DNA:RNA duplexes. Biochemistry 1993, 32,
7832–7838.
8. Manoharan, M. 2ꢀ-carbohydrate modifications in antisense oligonucleotide ther-
apy:Importance of conformation, configuration and conjugation. Biochim. Biophys.
Acta 1999, 1489, 117–130.
9. Martin, P. EinneuerZugangzu 2-O-Alkylribonucleosiden und Eigenchaftenderen Oligonu-
cleotide. Helv. Chim. Acta 1995, 78, 486–504.
10. Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K., Kumar, R.; Meldgaard, M.; Olsen,
C. E.; Wengel, J. LNA(Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine,
5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and
unprecedented nucleic acid recognition. Tetrahedron 1998, 54, 3607–3630.
11. Kurreck, J.; Wyszko, E.; Gillen, C.; ErdmannV. A. Design of antisense oligonucleotides stabi-
lized by locked nucleic acids. Nucleic Acids Res. 2002, 30, 1911–1918.
12. Deleavey, G. F.; Watts, J. K.; Alain, T.; Robert, F.; Kalota, A.; Aishwarya, V.; Pelletier, J.;
Gewirtz, A. M.; Sonenberg, N.; DamhaM. J. Synergistic effects between analogs of DNA and
RNA improve the potency of siRNA-mediated gene silencing. Nucleic Acids Res. 2010, 38,
4547–4557.
13. Kawasaki, A. M.; Casper, M. D.; Freier, S. M.; Lesnik, E. A.; Zounes, M. C.; Cummins, L. L.;
Gonzalez, C.; Cook, P.D. Uniformly modified 2ꢀ-deoxy-2ꢀ-fluoro phosphorothioate oligonu-
cleotides as nuclease-resistant antisense compounds with high affinity and specificity for
RNA targets. J. Med. Chem. 1993, 36, 831–841.
14. Frieden, M.; Christensen, S. M.; Mikkelsen, N. D.; Rosenbohm, C.; Thrue,C. A.; Westergaard,
M.; Hansen, H. F.; Ørum, H.; Koch, T. Expanding the design horizon of antisense oligonu-
cleotides with alpha-L-LNA. Nucleic Acids Res. 2003, 31, 6365–6372.
15. Noronha, A. M.; Wilds, C. J.; Lok, C. N.; Viazovkina, K., Arion, D., Parniak, M.
A.; Damha, M. J. Synthesis and biophysical properties of arabinonucleic acids (ANA):
Circular dichroic spectra, melting temperatures, and ribonuclease H susceptibility of
ANA.RNA hybrid duplexes. Biochemistry 2000, 39, 7050–7062.
16. Lima, W. F.; Nichols, J. G.; Wu, H.; Prakash, T. P.; Migawa, M. T.; Wyrzykiewicz, T. K.; Bhat,
B.; Crooke, S. T. Structural requirements at the catalytic site of the heteroduplex substrate for
human RNase H1 catalysis. J. Biol. Chem. 2004, 279, 36317–36326.
17. Minasov, G.; Teplova, M.; Nielsen, P.; Wengel, J.; Egli,M. Structural basis of cleavage by RNase
H of hybrids of arabinonucleic acids and RNA. Biochemistry 2000, 39, 3525–3532.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
18. Damha, M. J.; Wilds, C. J.; Noronha, A.; Brukner, I.; Borkow, G.; Arion, D.; Parniak, M. A.
Hybrids of RNA and Arabinonucleic Acids (ANA and 2‘F-ANA) are substrates of ribonucle-
ase H. J. Am. Chem. Soc. 1998, 120, 12976–12977.
19. Denisov, A. Y.; Noronha, A. M.; Wilds, C. J.; Trempe, J.-F.; Pon, R. T.; Gehring, K.; Damha,
M. J. Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin:
comparison with 2ꢀ-fluoro-ANA/RNA and DNA/RNA hybrids. Nucleic Acids Res. 2001, 29,
4284–4293.
20. Nielsen, K. M.; Petersen, M.; Håkansson, A. E.; Wengel, J.; Jacobsen, J. P. Alpha-L-LNA
(alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic
study. Chemistry. 2002, 8, 3001–3009.
21. Nielsen, J. T.; Stein, P. C.; Petersen, M. NMR structure of an alpha-L-LNA:RNA hybrid: Struc-
tural implications for RNase H recognition. Nucleic Acids Res. 2003, 31, 5858–5867.
22. Wang, J.; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.; HendrixC.; Rosemeyer, H.;
Seela, F.; Van Aerschot, A.; Herdewijn, P. Cyclohexene Nucleic Acids (CeNA): Serum Stable
Oligonucleotides that Activate RNase H and Increase Duplex Stability with Complementary
RNA. J. Am. Chem. Soc. 2000, 122, 8595–8602.
23. Nowotny, M.; Gaidamakov, S. A.; Crouch, R. J.; Yang, W. Crystal structures of RNase H
bound to an RNA/DNA hybrid: Substrate specificity and metal-dependent catalysis. Cell
2005, 121, 1005–1016.
24. Wang, G.; Middleton, P. J.; Lin, C.; Pietrzkowski, Z. Biophysical and biochemical properties
of oligodeoxy-nucleotides containing 4ꢀ-C- and 5ꢀ-C-substituted thymidines. Bioorg. Med.
Chem. Lett. 1999, 9, 885–890.
25. Kanazaki, M.; Ueno, Y.; Shuto, S.; Matsuda, A. Highly Nuclease-resistant phosphodiester-
type oligodeoxynucleotides containing 4‘α-C-aminoalkylthymidines form thermally stable
duplexes with DNA and RNA. A candidate for potent antisense molecules. J. Am. Chem. Soc.
2000, 122, 2422–2432.
26. Fensholdt, J.; Thrane, H.; Wengel, J. Synthesis of oligodeoxynucleotides containing 4ꢀ-C-
(Hydroxymethyl)thymidine: Novel promising antisense molecules. Tetrahedron Lett. 1995,
36, 2535–2538.
27. Nielsen, K. D.; Kirpekar, F.; Roepstorff, P.; Wengel, J. Oligonucleotide analogues containing
4ꢀ-C-(Hydroxymethyl)uridine:Synthesis, evaluation and mass spectrometric analysis. Bio.
Med. Chem. 1995, 3, 1493–1502.
28. Gaubert, G.; Babu, B. R.; Vogel, S.; Bryld, T.; Vester, B.; Wengel, J. Synthesis and RNA-selective
hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides. Tetrahedron 2006, 62,
2278–2294.
29. Rosemeyer, H.; Seela, F. 1-(2ꢀ-Deoxy-β-D-xylofuranosyl)thymine building blocks for solid-
phase synthesis and properties of Oligo(2ꢀ-Deoxyxylonucleotides). Helv. Chim. Acta 1991,
74, 748–760.
30. Maiti, M.; Maiti, M.; Knies, C.; Dumbre, S.; Lescrinier, E.; Rosemeyer, H.; Ceulemans,
A.; Herdewijn, P. Xylonucleic acid: synthesis, structure, and orthogonal pairing properties.
Nucleic Acids Res. 2015, 43, 7189–7200.
31. Guangyu, W.; Seifert, W. E. Synthesis and evaluation of oligodeoxynucleotides containing
4ꢀ-C-substituted thymidines. Tetrahedron Lett. 1996, 37, 6515–6518.
32. More, J. D.; Finney, N. S. A simple and advantageous protocol for the oxidation of alcohols
with o-Iodoxybenzoic Acid (IBX). Org. Lett. 2002, 4, 3001–3003.
33. Omura, K.; Swern, D. Oxidation of alcohols by “activated” dimethyl sulfoxide. a preparative,
steric and mechanistic study. Tetrahedron 1978, 34, 1651–1660.
34. Mancuso, A. J.; Swern, D. Activated dimethyl sulfoxide: Useful reagents for synthesis. Syn-
thesis 1981, 165–185.
35. PfitznerK. E.; Moffatt, J. G. Sulfoxide-Carbodiimide Reactions. I. A Facile Oxidation of Alco-
hols. J. Am. Chem. Soc. 1965, 87, 5661–5670.

14
S. BAGMARE ET AL.
36. Jones, G. H.; Taniguchi, M.; Tegg, D.; Moffatt, J. G. 4ꢀ-Substituted nucleosides. 5. Hydrox-
ymethylation of nucleoside 5ꢀ-aldehydes. J. Org. Chem. 1979, 44, 1309–1317.
37. Obika, S.; Nanbu, D.; Hari, Y.; MorioK.-I.; In, Y.; Ishida, T.; Imanishi, T. Synthesis of 2ꢀ-O,4ꢀ-
C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3ꢀ-endo sugar
puckering. Tetrahedron Lett. 1997, 38, 8735–8738.
38. Altona, C.; Sundaralingam, M. Conformational analysis of the sugar ring in nucleosides and
nucleotides. New description using the concept of pseudorotation. J. Am. Chem. Soc. 1972,
94, 8205–8212.
39. Abes, S.; Turner, J. J.; Ivanova, G. D.; Owen, D.; Williams, D.; Arzumanov, A.; Clair, P.; Gait,
M. J.; Lebleu, B. Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery
peptide. Nucleic Acids Res. 2007, 13, 4495–4502.
40. Gait, M. J. Oligonucleotide Synthesis: A Practical Approach. IRL Press Oxford, 1984.
41. Agrawal, S. Methods in Molecular Biology. Vol.20, ed.N.J. Totowa, Humana Press, Inc.