Synthesis and properties of 2′-O-[R- and S-(2-amino-3-methoxy)propyl] (R-AMP and S-AMP) nucleic acids

Article abstract of DOI:10.1016/j.tet.2013.05.104

Substitution at 2′-position by either amino- or methoxy-pendant groups of the antisense oligonucleotides (AONs) is known to enhance their therapeutic value. A simple modification is described here in which we introduce both these groups in the form of ena

Full text of DOI:10.1016/j.tet.2013.05.104

Tetrahedron 69 (2013) 6404e6408
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Tetrahedron
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Synthesis and properties of 20-O-[R- and S-(2-amino-3-methoxy)
propyl] (R-AMP and S-AMP) nucleic acids
*
Venubabu Kotikam, Vaijayanti A. Kumar
Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India
a r t i c l e i n f o
a b s t r a c t
Substitution at 20-position by either amino- or methoxy-pendant groups of the antisense oligonucleo-
tides (AONs) is known to enhance their therapeutic value. A simple modification is described here in
which we introduce both these groups in the form of enantiospecific tethers at 20-position. Practical
Article history:
Received 7 March 2013
Received in revised form 10 May 2013
Accepted 22 May 2013
Available online 29 May 2013
synthesis of modified nucleosides using natural L-serine, en route to R-AMP- and S-AMP-AONs is pre-
sented. Such tethered ONs formed stable DNA:RNA duplexes and the stability was found to be marginally
better than the methoxyethyl/methoxypropyl-substituted MOE/MOP-AONs. The stereochemistry of the
tether effectively differentiated the hydrolytic cleavage of AONs and the R-AMP-AON was three times
more stable than the S-AMP-AONs after 4 h. In comparison, the MOE- or MOP-AONs were almost
completely digested by SVPD after 1 h.
Keywords:
MOE-AONs
Positively charged ON analogues
Antisense therapeutics
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
with complementary RNA compared to MOE-AONs.2 The thermal
stability of the duplexes containing the 20-O-aminoethyl/20-O-
Synthetic antisense or siRNA oligonucleotides (ONs) as gene
silencing agents inhibit viral replication and expression of disease-
causing genes based on the simple concept of nucleic acid sequence
recognition via WatsoneCrick hydrogen bonding by a comple-
mentary base sequence.1 Amongst the large number of 20-O-alkyl
derivatives studies so far,2 the 20-O-methoxyethyl substituted an-
tisense oligonucleotides (MOE-AONs) are being studied3 in several
ongoing in vivo studies and have shown excellent results.
The presence of amino functionality in the AONs could reduce
electrostatic charge-repulsions during duplex formation in some
backbone/nucleobase modifications of DNA that could reduce the
overall negative charges of the ONs.4 Conjugation or complexation
with positively charged peptides with oligonucleotides improves
cellular uptake of oligonucleotides and analogues.5 Introduction of
positively charged substitution as in 20-O-aminoethyl/20-O-amino-
propyl derived DNA oligomers shows increased stability towards
hydrolytic enzymes.6 The protonated amino group in the 20-O-
substituent of these modifications was found to displace a catalyt-
ically important metal cation of the hydrolytic enzyme.7 The
methoxy substitution as in OMe or MOE-AONs improves duplex
stability by improving hydration in the minor groove and by in-
creasing conformational pre-organization through a stable water
structure.8 The MOP-AONs marginally destabilize the duplexes
aminopropyl is also relatively less compared to the OMe or MOE-
AONs.2,6 We planned the synthesis of a novel modification by
combining the amino- and OMe-substitution patterns. The pres-
ence of a positively charged amino-functionality in conjunction
with the methoxy substituent on the propyl chain would ensure
that the resulting oligomers have advantages of both methoxy
and amino groups (Fig. 1). The aminomethoxypropyloxy (AMP)
substitution at 20-position, thus derived, might improve nuclease
stability of the AONs while maintaining duplex stability, compa-
rable to MOE-AONs. Although a large number of 20-O-chemical
* Corresponding author. Tel.: þ91 20 25902340; e-mail address: va.kumar@
ncl.res.in (V.A. Kumar).
Fig. 1. Design of R-AMP and S-AMP.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.05.104

V. Kotikam, V.A. Kumar / Tetrahedron 69 (2013) 6404e6408
6405
modifications are reported in the literature, this is perhaps the first
report where branched chain is tethered stereospecifically, to the
20-O-position of AONs. In addition to the presence of charged amino
group, the hydrolytic susceptibility of the functionalized AONs is
also known to be reduced with increasing size of the 20-O-sub-
stitution.2b For the present enantiomeric R/S-AMP tethers, due to
the conformational freedom, the steric bulk and electronic factors
may be expected to be similar for each of the isomers when in-
corporated in the in the ssDNA.9 We report here the synthesis of
two diastereomeric nucleoside analogues differing in chirality at
the 20-O-sustituent groups with a synergistic combination of
methoxy and amino pendant groups. Stereospecific synthesis of
protected [R/S-(2-amino-3-methoxy)propanol tethers (R-AMP and
amino group was then protected to get the trifluoroacetyl de-
rivatives S-11 and R-12, Scheme 2,16 suitable for the solid-phase
DNA synthesis protocols.
S-AMP) from L-serine, synthesis of appropriately protected R-AMP
and S-AMP phosphoramidite derivatives of uridine starting from
2,20-anhydrouridine are presented. The stereochemistry of the
tether effectively differentiated the hydrolytic cleavage of AONs and
the R-AMP-AON was three times more stable than the S-AMP-AONs
after 4 h. We also show that the thermal stability of S-AMP- and
R-AMP-derivatized AONs is marginally better than the MOE/MOP-
AONs. These oligomers exhibit stereochemistry-dependent pro-
tection against hydrolysis by snake venom phosphodiesterase
enzyme (SVPD) when the MOE/MOP ONs are completely digested.
Scheme 1. Synthesis of protected/activated [R/S-(2-amino-3-methoxy)propanol.
2. Results and discussion
There are previous literature reports2 aimed to achieve selective
alkylation of uridine at 20-position, such as treatment of uridine
with alkyl halides using DBTO10 or by employing 30,50-O- and
N-protected uridine in reactions with alkyl halides2a or cyanoeth-
ylation via a Michael addition of 30,50-O-protected uridine to the
acrylonitrile.11 Alternatively, regiospecific 2,20-anhydrouridine ring
opening is effected by reaction with borate esters generated from
12
Scheme 2. Synthesis of protected 20-O-[R/S(2-amino-3-methoxy)propyl uridine
phosphoramidite.
BH3 and large excess of appropriate alcohols to get the desired
products at high temperature and pressure or by treatment with
metal alkoxides.13 The yields were variable in each case and in the
case of N-2-hydroxyethylphthalimide, the reported yield was 21%.14
In the present design, we need to synthesize both the enantiomeric
amino alcohol tethers separately. In the above mentioned pro-
tocols, the alcohol components are used as reaction medium or the
alkylation is low yielding and we could not adopt them for the
present studies. Alternatively, we came across BF3$OEt2-promoted
2,20-anhydro ring opening strategy used by Saneyoshi,15 using ex-
cess trimethylsilylated alcohol as a nucleophile. We decided to
employ this methodology by some variance in the strategy. The
synthesis of protected [S/R-(2-amino-3-methoxy)propanol S-3 and
R-5 was thus undertaken. We achieved this starting from the same
The phosphoramidite derivatives S-13 and R-14 were then pre-
pared by phosphitylation using known method17 starting from S-11
and R-12, to be used in automated DNA synthesis. We chose a DNA
sequence of biological relevance that was used in the splice-
correction assay developed by Kang et al.18 The unmodified oligo-
mers were synthesized using Bioautomation MM4 DNA synthesizer
and commercially available phosphoramidite building blocks. The
phosphoramidites S-13 and R-14 were incorporated into sequences
using increased coupling time (6 min) to yield the modified
sequences. The site of the modified units in the sequences was
decided so that these units were in the middle of the sequence
(16e19, Table 1), were separated by 4e5 nucleosides (20e23, Table
1) or were at consecutive 30-end positions (24e28, Fig. 2). The AONs
were purified by HPLC subsequent to ammonia treatment and
characterized by MALDI-TOF mass spectrometric analysis. Their
purity was also re-checked by analytical HPLC and found to be
>95% prior to their use in experiments. The thermal stability of
DNA:RNA duplexes was evaluated using temperature dependent
UV measurements and the results were comparable to UMOE con-
taining oligomers (Table 1). The vicinal electronegative sub-
stituents, namely, O40, -20-O- and 2-amino- and 3-methoxy- on the
propyl chain at 20-O-position of uridine would be expected to
maintain the gauche orientations with respect to the O40-oxygen to
induce conformational change and hydration in the minor groove
as is known for 20-MOE derivatives.7,8 We examined the S-type to
N-type conformational equilibria for 20-MOE-uridine with S-7 and
R-8 and found that they are comparable (please see SI). This might
indeed be the case even in the oligomers as the stability of the
duplex structures was marginally higher with 20-O-R-AMP (19 and
chiral source, i.e., L-serine. Just by altering the sequence of reactions
we arrive at the enantiomeric tethers S-3 and R-5 having suitable
activation/protection, as shown in Scheme 1. Thus, N-Cbz-pro-
tected- -serine methyl ester 1 was O-methylated using silver oxide
L
and methyl iodide. After reduction, hydroxy compound R-2 was
obtained (83% overall yield) and was further activated as a silyl
ether S-3 (83%) using trimethylsilylchloride (TMS-Cl). Alternatively,
ester 1 was first protected as tert-butyldimethylsilyl ether (TBS) and
then the ester was reduced to get alcohol R-4 (87%). Methylation of
free hydroxyl group in R-4 gave TBS protected ether R-5 in 91%
yield. We slightly improved Saneyoshi15 strategy to get the desired
2,20-anhydro ring-opened products S-7 and R-8 by employing
2 equiv of S-3 and R-5, Scheme 2. We found that both the TMS- and
TBS-silyl ethers were equally reactive as nucleophiles in 2,20-
anhydrouridine ring-opening and the yields were comparable (60,
68%, respectively). Compounds S-7 and R-8 were then protected as
50-O-DMT to get S-9 and R-10, respectively, (Scheme 2). The N-Cbz
protecting group of the 20-alkoxy derivatives was removed under
hydrogenation conditions using Pd/C under pressure. The primary

6406
V. Kotikam, V.A. Kumar / Tetrahedron 69 (2013) 6404e6408
Table 1
digested by SVPD at the end of 1 h. At the same time, almost 90% of R-
AMP-AONs and 80% S-AMP-AONs were intact and were effectively
able to resist the hydrolytic cleavage. Furthermore, w42% of R-
tethered AON stereoisomer was still intact after 4 h and was almost
thrice more stable than the S-stereoisomer. Introduction of chirality
in the minor groove as in the case of R-AMP and S-AMP, did indeed
allow differential protection of one of the isomers while interacting
with the active site of the enzyme6 and thus this interaction may be
energetically different for two isomers, compared to the achiral,
acyclic, alkyl substitution, such as MOE or AE/AP.7,8 It could be pre-
sumed that the projection of the amino substituent in the minor
groove while interacting with chiral environment of the enzyme7
would play a crucial role for this result. The chirality of our new
compounds containing stereospecific 20-tethers thus allowed pref-
erential protection against nucleases synergistically while retaining
the duplex stability. The constrained chiral analogue of MOE syn-
thesized earlier also could effectively protect the AONs from enzy-
matic degradation but without chiral discrimination.21 The chirality
effect on protection of AONs against digestive enzymes, such as
SVPD was also observed when currently therapeutically used, fully
modified, stereorandom phosphorothioate oligonucleotides were
synthesized in chirally pure form.22
Modified DNA sequences, their MALDI-TOF mass analysis and biophysical evaluation
by UV-Tm (ꢀC) measurementsa
Sequences no.b
Mass
Calcd
Tm ꢀC
Obsd
RNAc
cctcttacctcagttaca 15
56.6
cctcttacctcagtUMOEaca 16
cctcttacctcagtUMOPaca 17
cctcttacctcagtUS-AMPaca 18
cctcttacctcagtUR-AMPaca 19
cctcttaccUMOEcagtUMOEaca 20
cctcttaccUMOPcagtUMOPaca 21
cctcttaccUS-AMPcagtUS-AMPaca 22
cctcttaccUR-AMPcagtUR-AMPaca 23
5429.9
5440.9
5459.6
5459.6
5490.9
5515.0
5549.0
5549.0
5430.0
5474.1
5460.2
5460.6
5492.7
5541.6
5549.8
5551.4
55.8(ꢁ0.8)d
55.3(ꢁ1.3)
56.2(ꢁ0.4)
56.2(ꢁ0.4)
57.4(þ0.8)
56.5(ꢁ0.1)
57.3(þ0.7)
57.5(þ0.9)
a
UV-Tm values were measured by annealing 1
m
M sequences with 1 mM cDNA/RNA
in sodium phosphate buffer (0.01 M, pH 7.2) containing 100 mM NaCl and is an
average of three independent experiments. (Accuracy is ꢂ0.5 ꢀC).
b
The lower case letters indicate unmodified DNA, UMOE denotes 20-O-methox-
yethyl uridine, UMOP denotes 20-O-methoxypropyl uridine, US-AMP and UR-AMP de-
note 20-O-[S-(2-amino-3-methoxy)propyl and 20-O-[R-(2-amino-3-methoxy)propyl
uridine derivatives, respectively.
c
50-uguaacugagguaagagg was the complementary RNA sequence.
Values in parenthesis denote D
Tm ꢀC compared to the unmodified duplex.
d
3. Conclusions
In this paper, efficient synthesis of novel, chiral R/S-AMP-AONs
is presented. The synergy of chirally placed methoxy and amino
groups substituents marginally improved the DNA:RNA duplex
stability. The oligomers were highly stable to nuclease digestion as
compared to MOE or POM where amino groups are absent. The
R-AMP-AONs were thrice as stable to enzymatic degradation
compared to the S-AMP-AONs.
4. Experimental section
4.1. (R)-Benzyl (1-hydroxy-3-methoxypropan-2-yl)carbamate,
R-2
Fig. 2. Preferential protection of AONs containing S-AMP and R-AMP substituents at
30-27e28 (tttttttttttttUS-AMPUS-AMP 27, tttttttttttttUR-AMPUR-AMP 28) compared to native
ttttttttttttttt 24, MOE-modified DNA tttttttttttttUMOEUMOE 25 and MOP-modified DNA
tttttttttttttUMOPUMOP 26.
N-Cbz protected- -serine-methyl ester 1 (39.5 mmol, 10 g) was
L
dissolved in dry acetonitrile (250 mL), followed by the addition of
Ag2O (98.8 mmol, 22.8 g) and MeI (197.5 mmol, 12.7 mL) and the
reaction mixture was vigorously stirred at room temperature for
12 h. Reaction mixture was filtered and the filtrate was concen-
trated under reduced pressure to give the O-methylated derivative
of 1. The crude colourless residue was dissolved in MeOH (500 mL)
and NaBH4 (150 mmol, 5.6 g) was added fraction wise at 0 ꢀC for
a period of 1 h and the mixture was kept for stirring at room
temperature for another 6e8 h. Excess NaBH4 was quenched with
saturated NH4Cl solution, followed by the removal of MeOH under
reduced pressure. The crude reaction mixture was extracted with
ethyl acetate. The organic extract was washed with brine solution
and dried over anhydrous sodium sulfate. Ethyl acetate was re-
moved under reduced pressure to give the crude product and was
purified through column chromatography (eluted in 20% ethyl ac-
etate in petroleum ether) to yield R-2 as a colourless liquid in 83%
23) and 20-O-S-AMP AONs (18 and 22) when compared with 20-O-
MOE/MOP (16,17/20,21) AONs.
We further explored the in vitro studies regarding the protection
of these newly synthesized oligomers tethered with US-AMP and UR-
AMP units against hydrolytic cleavage and compared the results with
the UMOE tether.1,2 MOE is being developed as antisense therapeutic
and is also finding newer applications in siRNA approach where only
two terminal units are modified with MOE.3 We therefore followed
literature precedence and synthesized
a homothyminyl se-
quence19,20 24 and modified it at the 30-terminus with two con-
secutive units of UMOE, UMOP, US-AMP or UR-AMP to get sequences 25,
26, 27 and 28, respectively, for comparison (Fig. 2). We digested
these sequences with SVPD under conditions reported earlier.19 The
products of the digestion were analyzed by RP-HPLC and percent
intact AON was plotted against time to understand the degradation
pattern for all the oligomers (Fig. 2). The results clearly indicate the
dependence of enzyme digestion on steric bulk and/or the presence
of protonable amino group as charged species, as both the oligomers
containing US-AMP 27 and UR-AMP 28 are much more stable than the
unmodified 24, MOE-modified 25 and MOP-modified 26 sequences.
The stereochemical influence of the AMP tethers in US-AMP (27) and
UR-AMP (28) AONs was clearly reflected in SVPD digestion experi-
ment, (Fig. 2). The MOE- and MOP-AONs were almost completely
(7.8 g) over two steps. 1H NMR (CDCl3, 200 MHz):
3.35 (s, 3H), 3.55e3.86 (m, 5H), 5.11 (s, 2H), 5.43 (br s, 1H),
7.36e7.37 (m, 5H). 13C NMR (CDCl3, 100 MHz):
51.8, 58.7, 62.1,
d 2.53 (br s, 1H),
d
66.4, 71.7, 127.7, 128.1, 136.0, 156.2. HRMS(EI) mass calcd for
C12H18O4N (MþH) 240.1230, found 240.1229.
4.2. (S)-Benzyl (1-methoxy-3-((trimethylsilyl)oxy)propan-2-
yl)carbamate, S-3
To a stirred solution of R-2 (16.7 mmol, 4 g) and NEt3
(83.5 mmol, 11.7 mL) in dry acetonitrile (70 mL), TMS-Cl

V. Kotikam, V.A. Kumar / Tetrahedron 69 (2013) 6404e6408
6407
(25.1 mmol, 3.1 mL) was added and stirring was continued for
another 1 h. The reaction mixture was diluted with ethyl acetate.
Water wash and brine wash were given to the organic layer. The
organic layer was dried over anhydrous sodium sulfate, solvents
removed in vacuo and the crude compound was purified through
column chromatography (eluted in 5% ethyl acetate in petroleum
ether) to give S-3 as a colourless liquid in 83% yield (4.3 g). 1H NMR
(eluted in 5% MeOH in DCM) to result S-7/R-8 as a white solid in
60e68% yield. S-7 1H NMR (DMSO-d6, 200 MHz):
3.21 (s, 3H),
d
3.49e3.63 (m, 5H), 3.82e3.9 (m, 3H), 4.03e4.10 (m, 1H), 5.01e5.05
(m, 3H), 5.14 (t, 1H, J¼4.69, 9.75 Hz), 5.60e5.65 (m, 1H), 5.79 (d, 1H,
J¼4.15 Hz), 7.35 (m, 5H), 7.92 (d, 1H, J¼7.76 Hz). 13C NMR (DMSO-d6,
200 MHz):
d 50.0, 58.1, 59.9, 65.3, 68.1, 69.1, 70.9, 81.5, 84.4, 86.1,
101.6, 127.7, 128.3, 136.9, 140.3, 150.3, 155.8, 163.1. HRMS(EI) mass
(CDCl3, 200 MHz):
d
0.10 (s, 9H), 3.32 (s, 3H), 3.35e3.39 (m, 1H),
calcd for C21H27O9N3Na (MþNa) 488.1640, found 488.1630. R-8 1H
3.47e3.50 (m, 1H), 3.56e3.60 (m, 1H), 3.68e3.73 (m, 1H), 3.84 (m,
NMR (DMSO-d6, 400 MHz): d 3.22 (s, 3H), 3.51e3.68 (m, 5H),
1H), 5.09 (s, 2H), 5.27 (br s, 1H), 7.29e7.34 (m, 5H). 13C NMR (CDCl3,
3.78e3.93 (m, 3H), 4.09e4.13 (m, 1H), 4.97e5.051 (m, 3H), 5.16 (t,
1H, J¼4.77, 9.54 Hz), 5.64e5.66 (m, 1H), 5.84 (d, 1H, J¼4.77 Hz), 7.36
(m, 5H), 7.92 (d, 1H, J¼8.03 Hz). 13C NMR (DMSO-d6, 100 MHz):
100 MHz): d 0.8, 51.3, 58.6, 60.6, 66.4, 70.3, 127.8, 128.2, 136.3, 155.7.
HRMS(EI) mass calcd for C15H26O4NSi (MþH) 312.1626, found
312.1622.
d 50.3, 58.4, 60.6, 65.5, 68.6, 69.3, 71.4, 81.8, 85.0, 86.3, 102.0, 128.0,
128.5, 137.2, 140.6, 150.7, 156.0, 163.4 HRMS(EI) mass calcd for
4.3. (R)-Benzyl (1-((tert-butyldimethylsilyl)oxy)-3-
hydroxypropan-2-yl)carbamate, R-4
C21H28O9N3 (MþH) 466.1820, found 466.1822.
4.6. 50-O-DMT-20-O-(N-benzyloxycarbonyl-20-O-S-AMP) and
20-O-(N-benzyloxycarbonyl-20-O-R-AMP) uridine: S-9/R-10
N-Cbz protected-L-serine-methyl ester 1 (39.5 mmol, 10 g) was
dissolved in dry DCM (200 mL), followed by the addition of imid-
azole (98.8 mmol, 6.7 g) and TBS-Cl (47.4 mmol, 7.1 g). The reaction
mixture was diluted with DCM and the DCM layer was washed with
water and brine solution. Organic layer was dried over anhydrous
sodium sulfate and solvent removed in vacuo to result the crude
TBS protected ester, which was directly subjected to NaBH4 re-
duction. The colourless residue was dissolved in methanol (500 mL)
and NaBH4 (150 mmol, 5.6 g) was added fraction wise at 0 ꢀC for
a period of 1 h and then continued stirring at room temperature for
another 6 h. Excess NaBH4 was quenched with saturated NH4Cl
solution, followed by the removal of MeOH under reduced pressure
and the crude compound was extracted with ethyl acetate. The
organic extract was washed with brine and dried over anhydrous
sodium sulfate. Ethyl acetate was removed under reduced pressure
to give the crude product and was purified through column chro-
matography (eluted in 15% ethyl acetate in petroleum ether) to
yield R-4 as a colourless liquid in 87% (11.6 g) over two steps. 1H
(3.44 mmol, 1.6 g) was dissolved in dry pyridine (10 mL) and
DMT-Cl (3.61 mmol, 1.22 g) and catalytic amount of DMAP
(w20 mg) were added. Reaction mixture was kept for stirring at
room temperature for 5e6 h. Pyridine was removed under reduced
pressure and the residue was diluted with ethyl acetate. 10%
aq NaHCO3, water and brine solution wash were given to the or-
ganic layer. The organic layer was dried over anhydrous Na2SO4 and
concentrated to dryness. Crude compound was column purified
(eluted in 70% ethyl acetate in petroleum ether) to result S-9/R-10
as a white foam in 89% yield. S-9 1H NMR (CDCl3, 200 MHz):
d 3.33
(s, 3H), 3.42e3.54 (m, 5H), 3.8 (s, 6H), 3.91e4.04 (m, 4H), 4.44 (br s,
1H), 5.09 (s, 2H), 5.23 (d,1H, J¼8.21 Hz), 5.32 (d,1H, J¼7.96 Hz), 5.89
(s, 1H), 6.83e6.87 (m, 4H), 7.31e7.40 (m, 14H), 8.02 (d, 1H,
J¼8.08 Hz), 8.26 (br s, 1H). 13C NMR (CDCl3, 125 MHz):
d 50.1, 55.0,
58.9, 61.0, 66.6, 68.2, 70.9, 71.4, 82.8, 82.9, 86.8, 87.5, 101.8, 113.1,
126.9, 127.8, 128.0, 128.3, 129.9, 130.0, 134.9, 135.2, 136.2, 139.9,
144.2, 150.2, 156.6, 158.4, 158.5, 163.8. HRMS(EI) mass calcd for
C42H45O11N3Na (MþNa) 790.2946, found 790.2931. R-10 1H NMR
NMR (CDCl3, 200 MHz):
3.66e3.84 (m, 5H), 5.11 (s, 2H), 5.38e5.41 (m, 1H), 7.35e7.38 (m,
5H). 13C NMR (CDCl3, 50 MHz):
d 0.05 (s, 6H), 0.88 (s, 9H), 2.64 (br s, 1H),
d
ꢁ5.6, 18.1, 25.7, 53.0, 63.0, 63.4,
(CDCl3, 400 MHz): d 3.31 (s, 3H), 3.50e3.52 (m, 5H), 3.76 (m, 6H),
66.7, 128.0, 128.4, 136.2, 158.2. HRMS(EI) mass calcd for
3.90e3.91 (m, 1H), 4.02e4.04 (m, 3H), 4.39e4.42 (m, 1H),
5.07e5.08 (m, 2H), 5.27 (d, 1H, J¼8.24 Hz), 5.64 (d, 1H, J¼8.24 Hz),
5.92 (d, 1H, J¼0.92 Hz), 6.82e6.84 (m, 4H), 7.27e7.39 (m, 14H), 7.96
C17H30O4NSi (MþH) 340.1939, found 340.1945.
4.4. (R)-Benzyl (1-((tert-butyldimethylsilyl)oxy)-3-
methoxypropan-2-yl)carbamate, R-5
(d, 1H, J¼8.24 Hz). 13C NMR (DMSO-d6, 100 MHz):
d 50.2, 55.0, 58.9,
61.2, 66.7, 68.3, 70.9, 71.5, 83.1, 83.2, 86.8, 87.2, 101.9, 113.1, 127.0,
127.8, 128.0, 128.3, 129.9, 130.0, 134.9, 135.1, 136.1, 139.8, 144.2,
150.3, 156.2, 158.50, 158.54, 163.7. HRMS(EI) mass calcd for
C42H45O11N3Na (MþNa) 790.2946, found 790.2946.
To a stirred solution of R-4 (29.4 mmol, 10 g) and MeI
(147.4 mmol, 9.5 mL), Ag2O (73.5 mmol, 16.9 g) was added and the
reaction mixture was vigorously stirred at room temperature for
12 h. Reaction mixture was filtered and the filtrate was concen-
trated under reduced pressure. Crude compound was purified
through column chromatography (eluted in 5% EtOAc in petroleum
ether) to result R-5 as a colourless liquid in 91% (9.4 g) yield. 1H
4.7. 50-O-DMT-20-O-(N-trifluoroacetyl-20-O-S-AMP) and 20-O-
(N-trifluoroacetyl-20-O-R-AMP) uridine: S-11/R-12
The 50-DMT protected 20-O-functionalized uridine derivative
S-7/R-8 (2.3 mmol, 1.8 g) was dissolved in MeOH (10 mL) followed
by the addition of 10% PdeC (10% w/w, 0.18 g). Then reaction
mixture was subjected to catalytic hydrogenation at 65 psi of hy-
drogen pressure for 6 h. After the TLC analysis, reaction mixture
was filtered over Celite and the removal of methanol in vacuo gave
the free amine. Without further purification, the amine was sub-
jected to trifluoroacetyl protection. To the crude amine (2.2 mmol,
1.4 g) dissolved in MeOH (15 mL), NEt3 (3.3 mmol, 0.46 mL) was
added. Ethyltrifluoroacetate was added to reaction mixture and the
mixture was kept for stirring at room temperature for 8e10 h.
MeOH was removed on rota evaporator and the reaction mixture
was diluted with ethyl acetate. The organic layer was washed with
water and 5% aq NaHCO3 and the organic layer was dried over
anhydrous Na2SO4, concentrated in vacuo. Crude compound was
column purified to furnish S-11/R-12 as a white foam in 86e88%
NMR (CDCl3, 200 MHz):
3.37e3.77 (m, 4H), 3.78e3.86 (m, 1H), 5.10 (s, 2H), 7.35e7.38 (m,
5H). 13C NMR (CDCl3, 100 MHz):
d 0.05 (s, 6H), 0.88 (s, 9H), 3.33 (s, 3H),
d
ꢁ5.6, 18.1, 25.7, 51.5, 58.8, 61.3,
66.6, 70.4, 128.0, 128.4, 136.4, 155.9. HRMS(EI) mass calcd for
C18H32O4NSi (MþH) 354.2095, found 354.2089.
4.5. A typical procedure for 20-O-functionalization of 2,20-
anhydrouridine
Desiccated 2,20-anhydrouridine 6 (6.6 mmol,1.5 g) was dissolved
in dry DMA (10 mL) followed by the addition of BF3$OEt2 (10 mmol,
1.2 mL) under argon atmosphere. After 2 min, the activated/pro-
tected silyl ethers 3/5 (13.2 mmol) were added and stirred at 130 ꢀC
for 8e12 h. DMA was removed partially in vacuo, followed by the
dilution with MeOH and the reaction mixture was column purified

6408
V. Kotikam, V.A. Kumar / Tetrahedron 69 (2013) 6404e6408
yield (eluted in 55% ethyl acetate in pet-ether). S-11 1H NMR (CDCl3,
500 MHz): 3.40 (s, 3H), 3.52e3.63 (m, 5H), 3.81 (s, 6H), 3.82e3.85
Supplementary data
d
(m, 1H), 3.93 (dd, 1H, J¼1.8 and 4.9 Hz), 4.03e4.06 (m, 1H), 4.09 (dd,
1H, J¼4.8 and 10.0 Hz), 4.33e4.37 (m, 1H), 4.42e4.46 (m, 1H), 5.30
(dd, 1H, J5,6¼2.1 and 8.24 Hz), 5.92 (d, 1H, J¼1.5 Hz), 6.85e6.86 (m,
4H), 7.25e7.39 (m, 9H), 8.00 (d, 1H, J5,6¼8.24 Hz). 13C NMR (CDCl3,
1H, 13C and HRMS mass spectra of selected compounds in
Scheme 1 and HPLC and MALDI-TOF-TOF mass spectra of oligomer
15e28, temperature dependent UV-melting profiles of oligomers
with complementary DNA/RNA sequences and overlay of HPLC
chromatograms of SVPD digestion experiments of oligomers
24e28. Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.tet.2013.05.104.
500 MHz):
d 49.43, 55.2, 59.3, 61.2, 68.5, 70.1, 70.6, 83.4, 83.6, 87.1,
87.5, 102.3, 113.2, 113.3, 127.2, 128.0, 128.1, 130.0, 130.1, 135.0, 135.2,
139.7, 144.2, 150.2, 158.7, 162.7. HRMS(EI) mass calcd for
C36H38O10N3F3Na (MþNa) 752.2402, found 752.2391. R-12 1H NMR
(DMSO-d6, 400 MHz):
d 3.24 (s, 3H), 3.43e3.45 (m, 3H), 3.64e3.68
References and notes
(m, 2H), 3.74 (s, 6H), 3.83 (m, 1H), 3.94e3.99 (m, 3H), 4.16e4.19 (m,
3H), 5.17 (d, 1H, J¼7.50 Hz), 5.27 (dd, 1H, J5,6¼2.0 and 8.17 Hz), 5.77
(d,1H, J¼2.75 Hz), 6.89e6.91 (m, 4H), 7.24e7.39 (m, 9H), 7.70 (d,1H,
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J5,6¼8.19 Hz). 13C NMR (CDCl3, 500 MHz):
d 49.44, 55.2, 59.2, 60.9,
68.4, 69.3, 70.4, 83.1, 83.4, 87.1, 87.8, 102.2, 113.1, 113.3, 127.0, 128.0,
128.1, 130.0, 131.0, 135.0, 135.2, 139.7, 144.3, 150.3, 158.6, 158.7.
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found 752.2405.
4.8. General procedure was followed for the synthesis of
phosphoramidite derivatives S-13 and R-14
To the compound S-11/R-12 (0.68 mmol, 500 mg) dissolved in
dry DCM (10 mL), DIPEA (1.7 mmol, 0.29 mL) was added. 2-Cya-
noethyl-N,N-diisopropyl-chloro phosphine (0.81 mmol, 0.18 mL)
was added to the reaction mixture at 0 ꢀC and continued stirring at
room temperature for 3 h. The contents were diluted with DCM and
washed with 5% NaHCO3 solution. The organic phase was dried
over anhydrous sodium sulfate and concentrated to white foam.
The residue was re-dissolved in DCM and the compound was pre-
cipitated with n-hexane to obtain corresponding phosphoramidite
derivatives in 88e92% yield.
Phosphoramidite S-13: 31P NMR (Acetonitrile, D2O as external
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d 149.22, 149.68. HRMS(EI) mass calcd for
1263e1273.
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C45H56O11N5F3P (MþH) 930.3661, found 930.3674.
d 149.52, 150.62. HRMS(EI) mass calcd for
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300
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were incubated at 37 ꢀC for 15 min. SVPD 5 ng/
L was added to the
oligonucleotide incubated at 37 ꢀC and aliquots of 20
L were re-
mM of oligonucleotide in
4539e4557.
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m
m
m
moved at time intervals of 0, 2, 5,10, 20, 30, 60, 90,120,150,180, 210
and 240 min. Aliquots were kept at 90 ꢀC for 2 min prior to their
analysis on RP-HPLC with an increasing gradient (A: 5% acetonitrile
and B: 30% acetonitrile in 0.1 N triethylammonium acetate, pH 7.0).
And the % of the intact oligonucleotides (based on the %area of the
peaks) was plotted against the time intervals.
Acknowledgements
V.B. thanks CSIR, New Delhi for research fellowship. V.A.K.
thanks CSIR, New Delhi for research grants.