Synthesis of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified oligonucleotides: Hybridization affinity, resistance to nuclease, and protein binding characteristics

Article abstract of DOI:10.1016/S0040-4020(03)01104-9

Synthesis of a series of 2′ -O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in h

Full text of DOI:10.1016/S0040-4020(03)01104-9

Tetrahedron 59 (2003) 7413–7422
Synthesis of 20-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified
oligonucleotides: hybridization affinity, resistance to nuclease,
and protein binding characteristics
Thazha P. Prakash, Andrew M. Kawasaki, Elena A. Lesnik, Namir Sioufi
*
and Muthiah Manoharan
Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc., 2280 Faraday Ave, Carlsbad, CA 92008, USA
Received 14 May 2003; revised 14 July 2003; accepted 14 July 2003
Abstract—Synthesis of a series of 20-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid
supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high
yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent
nuclease stability with t1/2.24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess
their transport and toxicity properties.
q 2003 Published by Elsevier Ltd.
1. Introduction
thesized and showed considerable promise.5 The stability
of a 20-O-DMAOE:RNA duplex was comparable to that of
the 20-O-MOE:RNA duplex of the same sequence. The
20-O-DMAOE modification also imparted excellent
resistance to nuclease digestion. Here we describe the
design and synthesis of several additional analogues of the
20-O-AOE in order to test two hypotheses. We postulated
that increasing the steric bulk at the 20-position would
improve the nuclease stability of oligonucleotides. In
addition, as these dialkyl modifications should 0 be more
lipophilic than either the 20-O-AOE or the 2 -O-MOE
modifications, the 20-O-AOE analogues may offer more
desirable protein binding and cellular permeation
properties.
Structural analogues of antisense oligonucleotides with
modified heterocycles, sugars, and/or backbones have been
synthesized in an attempt to improve binding affinity and
nuclease resistance, to modulate binding to selected
transport proteins, and to enhance cell permeation in
vivo.1 Some of the most successful analogues have resulted
from modification of the sugar at the 20-position.2 The 20-O-
modified oligonucleotides used with the ‘gapmer’ tech-
nology3 have emerged as the leading second generation
candidates for clinical 0 applications. Gapmer oligonucleo-
tides have one or two 2 -O-mo0dified regions and a 20-deoxy
phosphorothioate region; the 2 -deoxy region is required for
RNase H digestion of mRNA. We recently reported
synthesis and properties of 20-O-(2-aminooxyethyl)
modification (20-O-AOE), the pseudoisostere of 20-O-
methoxyethyl (20-O-MOE) modification.4 Unfortunately,
due to high chemical reactivity of 20-O-AOE, several
modified residues cannot be conveniently incorporated into
an oligonucleotide. Moreover, this modification did not
impart enhanced nuclease stability relative to the 20-O-MOE
modification, although du0plexes formed with RNA had
comparable affinity to 2 -O-MOE:RNA duplexes. The
dimethyl derivative of the 0 20-O-AOE modification, 20-O-
dimethylaminooxyethyl (2 -O-DMAOE), has been syn-
We envisaged that due to high reactivity of the aminooxy
group to aldehydes and ketones,5 it could be easily
converted into dialkyl derivatives by reaction with
aldehydes and ketones of interest followed by reduction of
the resulting methyleneaminooxy derivatives. The modified
nucleosides 20-O-[2-[(N,N-diethylamino)oxy]ethyl] (20-O-
DEAOE), 20-O-[2-[(N-isopropyl-N-methylamino)oxy]-
ethyl] (20-O-IPMAOE), and the cationic 20-O-[2-[[N-
methyl-N-[2-(amino)ethyl]amino]oxy]ethyl]
(20-O-
AEMAOE) were synthesized using this strategy. Each
novel nucleoside was incorporated into oligonucleotides
(Fig. 1). The thermal melting temperatures (Tm’s) of these
modified oligonucleotide:RNA duplexes were determined
and their stability to nuclease digestion and their albumin
binding properties were evaluated.
Keywords: hybridization affinity; nuclease resistance; protein binding.
*
Corresponding author. Tel.: þ1-617-252-0700x2319; fax: þ1-617-252-
0011; e-mail: mmanoharan@alnylam.com
0040–4020/$ - see front matter q 2003 Published by Elsevier Ltd.
doi:10.1016/S0040-4020(03)01104-9

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T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
Reduction of compound 2 in 1 M pyridinium p-toluene-
sulfonate (PPTS) in MeOH and NaBH3CN gave mono-
ethylaminooxy derivative. This was followed by treatment
with acetaldehyde under reductive conditions to give
diethylaminooxy derivative 4 in 75% yield. Compound 3
was reduced to the monomethylaminooxy derivative under
reductive conditions described for compound 2; this was
followed by treatment with acetone under reductive
conditions to give compound 5 in 67% yield. Reduction of
the compounds 2 and 3 proceeded smoothly at ambient
temperature. Desilylation of compounds 4 and 5 with
triethylamine trihydrofluoride and triethylamine in THF
gave 6 (74% yield) and 7 (71% yield0). These nucleosides
were then selectively protected at 5 -position with 4,40-
dimethoxytrityl chloride (DMTCl) and DMAP in anhydrous
pyridine to yield compounds 8 (86% yield) and 9 (92%
yield). The compounds 8 and 9 were converted into 30-
phosphoramidites using 2-cyanoethyl tetraisopropyl-
phosphorodiamidite and N,N-diisopropylammonium tetra-
zolide in acetonitrile at room temperature to yield 10 (70%
yield) and 11 (85% yield), respectively.
Figure 1. 20-modifications.
2. Results and discussion
2.1. Synthesis
phosphoramidites and solid supports
of
20-O-modified
nucleoside
We recently described the synthesis of 20-O-DMAOE
(Fig. 1) nucleosides and phosphoramidites.5 We used a
similar synthetic strategy to synthesize all novel nucleosides
described in this report. The 50-O-(tert-butyldiphenylsilyl)-
20-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]-
ethyl]-5-methyluridine 1 was a convenient intermediate for
the synthesis of all phosphoramidites described in this report
and was synthesized as reported.5 The phthalimido group in
compound 1 (Scheme 1) was deprotected with N-methyl-
hydrazine (NMH) at 08C to give the aminooxy derivative.
The aminooxy derivative was converted into the ethyl-
ideneaminooxy derivative 2 (55% yield) and methylene-
aminooxy derivative 3 (74% yield) using acetaldehyde in
MeOH and formaldehyde in MeOH, respectively.
The synthesis of phosphoramidite 15 is described in
Scheme 2. Compound 3 was reduced in 1 M PPTS in
MeOH with NaBH3CN to yield the methylaminooxy
derivative. The methylaminooxy derivative was treated
with 2-phthalimidoacetaldehyde under reductive conditions
to yield 12 in 60% yield. The 2-phthalimidoacetaldehyde
was generated from commercially available 2-phthalimido-
acetaldehyde diethyl acetal by stirring in CHCl3 with 50%
trifluoroacetic acid for 24 h followed by usual work-up. The
desilylation of compound 12 with triethylamine trihydro-
fluoride and triethylamine in THF gave 13 (52% yield).
Selective 4,40-dimethoxytritylation of compound 13 at 50-
position gave 14 (73% yield). Finally, phosphitylation of 14
at the 30-positon yielded 15 (85% yield). The phthalimido
group was used as the protecting group for the amino
Scheme 1. (i) (a) CH2Cl2, N-methylhydrazine (b) for 2: MeOH, CH3CHO,
rt; for 3: MeOH, HCHO, rt; (ii) (a) 1 M PPTS in MeOH, NaBH3CN, rt (b)
for 4: 1 M PPTS in MeOH, CH3CHO, NaBH3CN, rt, for 5: 1 M PPTS in
MeOH, CH3COCH3, NaBH3CN, rt; (iii) triethylamine trihydrofluoride,
THF, (C2H5)3N, rt; (iv) 4,40-dimethoxytrityl chloride, pyridine, DMAP, rt;
(v) (2-cyanoethyl)-N,N,N0,N0-tetraisopropylphosphorodiamidite, N,N-diiso-
propylammonium tetrazolide, CH3CN, rt.
Scheme 2. (i) (a) 1 M PPTS in MeOH, NaBH3CN, rt; (b) 1 M PPTS in
MeOH, 2-phthalimidoacetaldehyde, NaBH3CN, rt; (ii) triethylamine
trihydrofluoride, THF, (C2H5)3N, rt; (iii) 4,40-dimethoxytrityl chloride,
pyridine, DMAP, rt; (iv) (2-cyanoethyl)-N,N,N0,N0-tetraisopropylphos-
phorodiamidite, N,N-diisopropylammonium tetrazolide, CH3CN, rt.

T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
7415
tides containing each of the modified phosphoramidites was
more than 97%. The oligonucleotides were characterized by
ES MS (Table 2) and purity was assessed by HPLC and
capillary gel electrophoresis.
2.3. Evaluation of hybridization of the 20-O-DMAOE, 20-
O-DEAOE, 20-O-IPMAOE and 20-O-AEMAOE modified
oligonucleotides to complementary RNA and DNA by
thermal denaturation studies
Table 1 shows the Tm values of 0the 20-O-DMAOE, 20-O-
DEAOE, 20-O-IPMAOE and 2 -O-AEMAOE modified
oligonucleotides hybridized to complementary RNA. The
Tm’s of the modified oligonucleotides were compared to
those formed with unmodified DNA oligonucleotides. Free
energies of duplex formation DG 037 (Table 1) were obtained
from UV absorbance vs. temperature curves assuming a
two-state model with a line0ar sloping baseline.90 Substitution
with the 20-O-DMAOE, 2 -O-DEAOE and 2 -O-IPMAOE
nucleosides led to an increase in the melting temperatures
relative to unmodified oligonucleotides. A Tm enhancement
of 0.8–1.18C per substitution was observed when modified
bases were not contiguous, as in the oligonucleotides 23, 24,
and 25 (Table 1). In the oligonucleotides with ten adjacent
substitutions, 27, 28, and 29, the Tm enhancements were
1.4–1.58C per substitution. The DNA phosphorothioates
(PS) forms a duplex with complementary RNA destabilized
by approximately 20.88C per modification.10 Therefore,
stabilization due to the 20-modifications described in this
report corresponds to a net stabilization of more than 28C
per modification when compared to DNA PS, the first
generation antisense drug. Although the 20-O-DEAOE 0and
20-O-IPMAOE modifications are bulkier than the 2 -O-
DMAOE modification, there is no significant Tm differ-
ences. These data suggest that the RNA heteroduplex
tolerates significant bulk at 20-position without affecting
binding affinity. Charge is not as well tolerated, as shown by
melting of oligonu0 cleotide 31. This oligonucleotide has
adjacent cationic 2 -O-AEMAOE modifications and there is
only a moderate increase in Tm (0.58C per modification).
This observation is in agreement with the reported Tm for
cationic 20-O-(aminoalkyl)-modified oligonucleotides.11
Scheme 3. (i) Succinic anhydride, 1,2-dichloroethane, DMAP, (C2H5)3N,
608C; (ii) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetra-
fluoroborate (TBTU), 4-methyl morpholine, DMF, aminoalkyl controlled
pore glass (CPG), rt.
functionality during oligonucleotide6 synthesis. The
phthalimido group was deprotected, to provide amino
functionality, during the deprotection of protecting groups
at the exocyclic amino groups and cyanoethyl groups of the
backbone phosphate of oligonucleotides by the treatment
with aqueous ammonia containing 4% methylamine at 558C
for 6 h.
Compounds 8, 9, and 14 were also converted into the 30-
succinyl derivatives 16, 17, and 18 (Scheme 3) and loaded
on to the aminoalkyl controlled pore glass (CPG) according
to the literature procedure7 to yield the functionalized solid
supports 19, 20, and 21 (51–60 mmol g21).
2.2. Synthesis of 20-modified oligonucleotides
The modified oligonucleotides (Table 1) were synthesized
using phosphoramidites 10, 11, and 15 on a solid-phase
DNA synthesizer. Standard phosphoramidites were used for
incorporation of A, C, G, and T residues. Solid supports 19,
20, and 21 were used for the synthesis of oligonucleotides
34, 36, and 37. A 0.1 M solution of phosphoramidite in
anhydrous acetonitrile was used for the synthesis. Oxidation
of the internucleoside phosphite to the phosphate was
carried out using 1-S-(þ)-(10-camphorsulfonyl)oxaziridine
(CSO).8 The overall coupling efficiency for oligonucleo-
In contrast to the increase in the Tm observed with RNA, the
hybridization of oligonucleotides 27, 28, and 29 with
complementary DNA led to duplexes less stable than those
formed with unmodified DNA oligonucleotides
(20.9–21.18C per modification, Table 3). These data
Table 1. Effect of 20-O-DMAOE, 20-O-DEAOE, 20-O-IPMAOE and 20-O-AEMAOE modification on duplex stability against complementary RNA targets
Oligo No.
Sequence 50 –30
Modification
Tm (8C)
DTm (8C)
DTm/unit (8C)
2DG 037 (kcal mol21
)
22
23
24
25
26
27
28
29
30
31
d(TCC AGG TGT CCG CAT C)
DNA parent
62.3
66.8
65.5
65.4
48.3
62.9
63.3
62.6
61.8
63.8
14.9
17.8
19.8
19.5
13.0
17.8
17.0
15.8
17.6
19.1
d(TpCC AGG TpGTp CCG CATp C)
d(TOCC AGG TOGTO CCG CATO C)
d(T§CC AGG T§GT§ CCG CAT§ C)
d(GCG TTT TTT TTT TGC G)
20-O-DMAOE
20-O-DEAOE
20-O-IPMAOE
DNA parent
4.5
4.2
3.1
1.1
1.1
0.8
d(GCG TpTpTp TpTpTp TpTpTp TpGC G)
d(GCG TOTOTO TOTOTO TOTOTO TOGC G)
d(GCG T§T§T§ T§T§T§ T§T§T§ T§GC G)
D(CTC GTA CTT TTC CGG TCC)
d(CTC GTA CT†T† T†T†C CGG TCC)
20-O-DMAOE
20-O-DEAOE
20-O-IPMAOE
DNA parent
14.6
15.0
14.3
1.5
1.5
1.4
20-O-AEMAOE
2.0
0.5
Tp¼20-O-DMAOE-5-methyluridine, TO¼20-O-DEAOE-5-methyluridine, T§¼20-O-IPMAOE-5-methyluridine, T†¼20-O-AEMAOE-5-methyluridine. Each
Tm and DG 037 was the average of at least three experiments with standard deviations not exceeding ^0.58C for Tm and ^0.1 kcal mol21 for DG837 values.

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T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
Table 2. HPLC and mass spectral analysis of the 20-O-DMAOE, 20-O-
DEAOE, 20-O-IPMAOE and 20-AEMAOE oligonucleotides used for Tm
analysis
Oligo No.
Mass
HPLC tR (min)a
Calcd
Found
23
24
25
27
28
29
31
5246.4
5358.5
5358.5
5906.4
6187.6
6187.6
5919.2
5245.6
5358.9
5358.3
5905.6
6188.1
6188.1
5919.8
23.6
25.5
22.9
26.2
32.4
37.7
23.8
a
Water C-4, 3.9£300 mm, A¼50 mM triethyl ammonium acetate, pH 7,
B¼acetonitrile, 5–60% B in 55 min, flow 1.5 mL min21, l¼260 nm.
Table 3. Tm data for the oligonucleotides containing 20-O-DMAOE, 20-O-
DEAOE, 20-O-IPMAOE modification hybridized to complementary DNA
Oligo No.
Modifications
Tm (8C)
DTm (8C)
DTm (8C)/unit
26
27
28
29
DNA parent
54.2
45.4
42.9
43.5
20-O-DMAOE
20-O-DEAOE
20-O-IPMAOE
28.8
211.3
210.7
20.9
21.1
21.1
Figure 2. Time-dependent disappearance of oligonucleotides 32–37 in the
presence of snake venom phosphodiesterase. The oligonucleotides were
phosphodiester TTT TTT TTT TTT TTT TpTpTpTp, where Tp is 20-O-
DMAOE-5-methyluridine for oligonucleotide 32, 20-O-MOE-5-methyl-
uridine for 33, 20-O-DEAOE-5-methyluridine for 34, T for 35, 20-O-
IPMAOE for 36, and 20-O-AEMAOE for 37. Each symbol represents the
average of two separate measurements (n¼2).
Each Tm value was the average of at least three experiments with standard
deviations not exceeding ^0.58C.
demonstrated an RNA selective hybridization of 20-
modified oligonucleotides described in this report.
2.4. Resistance of 20-modified oligonucleotides to
degradation by snake venom phosphodiesterase
and their ultimate uptake into cells of target organs is due to
their avid binding to plasma proteins.13 Albumin and a2-
macroglobulin are the major protein species that bind to PS
antisense oligonucleotides with relatively high capacity.14
However, non-specific protein binding characteristics of PS
oligonucleotides are responsible for some of the undesired
hemodynamic effects.13 Ideally, modifications to antisense
oligonucleotides will either reduce the requirement for PS
linkages or moderate the non-specific binding of PS
oligonucleotides.
To evaluate the relative resistance of the 20-modified
oligonucleotides toward nucleases, the oligonucleotides
32–37 (Fig. 2) were digested with snake venom phospho-
diesterase (SVPD).12 The modifications were placed at the
30-end of the oligonucleotide and all internucleoside
linkages were phosphodiesters. The oligonucleotides were
50-32P labeled and the percentage of full-length oligonucleo-
tide was estimated after separation by gel electrophoresis.
Figure 2 shows the relative exonuclease stability of the
modified oligonucleotides compared to the unmodified
DNA. The half-lives of the disappearance of the full-length
20-O-AOE analogues were more than 24 h. All are more
resistant to degradation than 20-O-AOE4 and 20-O-MOE
modified oligonucleotide. This observation suggests that
modifications with significant steric bulk impart enhanced
nuclease stability to oligonucleotides. Both the 20-O-
AEMAOE and 20-O-IPMAOE modified oligonucleotides
were extremely0 stable to nuclease digestion. The amino
group in the 2 -O-AEMAOE modification is protonated
under physiological conditions (pKa¼9). The positive
charge probably contributes to enhanced nuclease resistance
of the 20-O-AEMAOE modified oligonucleotide. The
observed order of nuclease stability of cationic 20-O-
AEMAOE modified oligonucleotide is consistent with the
reported data for other cationic modifications.6,11
Figure 3. Fraction of oligonucleotides 28–29 and 38 bound to albumin
ploted as a function of albumin concentration. Each symbol represents the
average of three separate measurements (n¼3). Oligonucleotides 26 and 27
did not show 0 binding and could not fit into the no-linear regression
equation. 38: 5 d(GCG T10 GC G) 30, back bone phosphorothioates; 39: 50
d(GCG Tp10 GC G) 30, Tp is 20-O-MOE-5-methyluridine, back bone
phosphodiesters.
2.5. Binding of oligonucleotides to human serum
albumin
The distribution of oligonucleotide PS to peripheral tissues

T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
7417
Table 4. Effect of 20-modification on human serum albumin binding
constant
3. Experimental
3.1. General procedures
Oligoa No.
Chemistry
Kd (mM)
Back bone
Tp
Anhydrous MeCN (water content ,0.001%), standard
phosphoramidites and ancillary reagents for oligonucleotide
synthesis were purchased from PE Biosystems (Foster City,
CA). All other reagents and anhydrous solvents were
purchased from Aldrich and used without further purifi-
cation. Thin-layer chromatography was performed on pre-
coated plates (silica gel 60 F254 from EM Science) and
visualized with UV light and spraying with a solution of
p-anisaldehyde (6 mL), H2SO4 (8.3 mL), CH3COOH
26
27
28
29
38
39
PvO
PvO
PvO
PvO
PvS
PvO
Thymidine
200-O-DMAOE-5-methyluridine
No binding
No binding
153
2 -O-DEAOE-5-methyluridine
20-O-IPMAOE-5-methyluridine No binding
Thymidine
20-O-MOE-5-methyluridine
30
No binding
50 d(GCG Tp10 GCG) 30, PvO phosphodiesters, PvS phosphorothioates.
a
1
(2.5 mL) in C2H5OH (227 mL) followed by charring. H
NMR spectra were referenced using internal standard
(CH3)4Si and 31P NMR spectra using external standard
85% H3PO4. Mass spectra were recorded by Mass
Consortium, San Diego, CA and the College of Chemistry,
University of California, Berkeley, CA.
In order to evaluate the protein binding characteristics of
20-modified oligonucleotides with phosphodiester (PvO)
backbone described in this report, binding of the oligonu-
cleotides 26–29 and oligonucleotides 38–39 (Fig. 3) to
human serum albumin was measured by ultrafiltration
techniques.13 The equilibrium constant, Kd, (Table 4) was
determined from the data of fraction of oligonucleotide
bound to protein concentration. The constants were
determined from non-linear regression analysis of a fraction
of oligonucleotides bound as a function of free albumin
monomer concentration. An oligonucleotide with a lower
Kd value binds more tightly to the protein than one with
higher value of Kd. An oligonucleotide with Kd value more
than 300 mM is not considered to bind protein. The 20-O-
DEAOE-modified oligonucleotide with a phosphodiester
backbone exhibited moderate protein binding
(Kd¼153 mM) while the 20-deoxy, 20-O-MOE,15 20-O-
DMAOE and 20-O-IPMAOE/PvO showed no binding
(Table 4). The PS oligonucleotide had a Kd¼30 mM in
these experiments. The moderate protein binding properties
of 20-O-DEAOE modified oligonucleotides indicates that,
20-O-DEAOE modified PvO oligonucleotides are expected
to bind less tightly to protein compared to oligonucleotide
PS in vivo. Therefore, antisense drugs with 20-O-DEAOE
modified oligonucleotide PvO are expected to have fewer
side effects than oligonucleotide PS.
3.1.1. 50-O-(tert-Butyldiphenylsilyl)-20-O-[2-(ethylidene-
aminooxy)ethyl]-5-methyluridine (2). Compound
1
(10 g, 14.6 mmol) was dissolved in CH2Cl2 (146 mL) and
methylhydrazine (1.03 mL, 14.6 mmol) was added drop-
wise at 2108C. The reaction mixture was stirred at 210–
08C for 1 h. The white precipitate that formed was filtered
and washed thoroughly with CH2Cl2 (ice-cold). The organic
phase was evaporated to dryness. The residue obtained was
dissolved in MeOH (210 mL), and acetaldehyde (0.89 mL,
16 mmol) was added and stirred at room temperature for
12 h. The solvent was removed under reduced pressure and
the residue was purified by flash silica gel column
chromatography and eluted with ethyl acetate/hexane (6:4)
to yield compound 2 (4.64 g, 55%): Rf¼0.42, (ethyl acetate/
1
hexane, 7:3); H NMR (400 MHz, DMSO-d6) d 1.02 (s,
9H), 1.44 (s, 3H), 1.69 (dd, 3H, J¼5.6 Hz), 3.66 (m, 1H),
3.76 (m, 2H), 3.94 (m, 2H), 4.05 (s, 2H), 4.15 (m, 1H), 4.22
(m, 1H), 5.18 (d, 1H, J¼6 Hz), 5.9 (dd, 1H, J¼4.4 Hz), 7.36
(m, 1H), 7.40 (m, 7H), 7.63 (m, 5H), 11.38 (s, 1H); 13C
NMR (50 MHz, CDCl3) d 11.8, 15.1, 19.4, 27.0, 63.0, 68.6,
70.3, 72.0, 72.1, 82.7, 84.3, 87.0, 111.1, 127.9, 130.0, 132.3,
133.0, 135.0, 135.1, 135.4, 147.9, 150.5, 164.1; HRMS
(FAB) calcd for C30H39N3O7SiNaþ 604.2455, found
604.2471.
In conclusion, we have designed and synthesized 20-O-
DEAOE, 20-O-IPMAOE and the cationic 20-O-AEMAOE
modified oligonucleotides for antisense applications.
Efficient synthetic strategy for the synthesis of
pyrimidine nucleosides with these modifications and their
phosphoramidites and solid supports were developed. The
methods described herein provide general procedures for
functionalization of nucleosides at the 20-position. The use
of the 20-modified nucleoside building blocks in oligo-
nucleotide synthesis resulted in high coupling efficiency.
These modified oligonucleotides showed RNA-selective,
high binding affinity, and excellent nuclease stability. These
oligonucleotides showed reduced binding affinity to human
serum albumin relative to phosphorothioate oligo-
nucleotides. These favorable antisense properties make
them valid candidates for further in vivo evaluation0 for
antisense drug development. We have selected 2 -O-
DEAOE modification for further in vivo antisense
evaluation and such efforts are in progress in our
laboratory based on the target optimal binding, enhanced
nuclease resistance and protein binding compared to 20-O-
MOE.
3.1.2. 50-O-(tert-Butyldiphenylsilyl)-20-O-[2-(methylene-
aminooxy)ethyl]-5-methyluridine (3). From compound 1,
compound 3 was synthesized as described in the literature.5
3.1.3. 50-O-(tert-Butyldiphenylsilyl)-20-O-[2-[(N,N-
diethylamino)oxy]ethyl]-5-methyluridine (4). Compound
2 (4.5 g, 7.74 mmol) was dissolved in 1 M pyridinium
p-toluenesulfonate (PPTS) in MeOH (77.4 mL). To this,
NaBH3CN (0.97 g, 15.5 mmol) was added at 108C and the
mixture was stirred for 10 min. The reaction mixture was
allowed to come to room temperature and stirring continued
for an additional 4 h. The reaction was monitored by TLC
(ethyl acetate/hexane, 70:30). The solvent was removed
under reduced pressure and the oily residue obtained was
diluted with ethyl acetate (100 mL) and washed with water
(75 mL), aqueous NaHCO3 (5 wt%, 75 mL), and brine
(75 mL). The organic phase was dried over anhydrous
Na2SO4 and evaporated. The residue obtained was dissolved

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T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
in 1 M PPTS in MeOH (77.4 mL) and acetaldehyde
(0.48 mL, 8.52 mmol) was added and stirred at ambient
temperature for 10 min. To this NaBH3CN (0.97 g,
15.50 mmol) was added at 108C and stirred for 10 min.
The mixture was allowed to come to room temperature
and stirring continued for an additional 4 h. The solvent
was removed in vacuo and the residue obtained was
dissolved in ethyl acetate (100 mL), washed with water
(75 mL), 5% NaHCO3 (75 mL), and brine (75 mL). The
organic phase was dried over anhydrous Na2SO4 and
evaporated to dryness. The residue obtained was
purified by flash silica gel column chromatography and
eluted with CH2Cl2/MeOH/NEt3, 94:5:1 to afford 4 (3.55 g,
75.1%) as a white foam: Rf¼0.41 (ethyl acetate/hexane,
7:3); 1H NMR (400 MHz, DMSO-d6) d 0.95 (t, 6H,
J¼7.2 Hz), 1.03 (s, 9H), 1.43 (s, 3H), 2.58 (q, 4H,
J¼7.2 Hz), 3.59 (m, 1H), 3.73 (m, 3H), 3.81 (m, 1H),
3.88 (m, 1H), 3.96 (m, 2H), 4.23 (m, 1H), 5.21 (d, 1H,
J¼5.6 Hz), 5.95 (d, 1H, J¼6.4 Hz), 7.43 (m, 7H), 7.76 (m,
4H), 11.39 (s, 1H); 13C NMR (50 MHz, CDCl3) d 11.8,
19.4, 27.0, 52.3, 63.3, 68.8, 70.3, 72.3, 82.6, 84.5, 86.8,
111.0, 127.9, 130.0, 132.3, 133.0, 135.1, 135.4, 141.3,
150.5, 164.0; HRMS (FAB) calcd for C32H45N3O7SiCsþ
744.2081, found 744.2067.
3.1.5. 20-O-[2-[(N,N-Diethylamino)oxy]ethyl]-5-methyl-
uridine (6). A mixture of triethlyamine trihydrofluoride
(4.39 mL, 26.81 mmol) and triethylamine (1.87 mL,
13.41 mmol) in THF (53.6 mL) was added to compound 4
(3.28 g, 5.36 mmol). The reaction mixture was stirred at
room temperature for 18 h. The solvent was removed under
reduced pressure and the residue obtained was purified by
flash silica gel column chromatography and eluted with
CH2Cl2/MeOH/NEt3 (89:10:1) to afford 6 (1.49 g, 74.3%):
1
Rf¼0.38 (10% MeOH in CH2Cl2); H NMR (400 MHz,
DMSO-d6) d 0.97 (t, 6H, J¼7.2 Hz), 1.75 (s, 3H), 2.58 (q,
4H, J¼7.2 Hz), 3.55 (m, 4H), 3.66 (m, 2H), 3.83 (br s, 1H),
3.95 (t, 1H, J¼5.6 Hz), 4.11 (q, 1H, J¼4.8, 5.6 Hz), 5.05 (d,
1H, J¼5.6 Hz), 5.87 (d, 1H, J¼6 Hz), 7.75 (s, 1H), 11.31 (s,
1H); 13C NMR (50 MHz, CDCl3) d 11.8, 12.3, 52.2, 61.3,
68.9, 70.2, 72.3, 81.5, 85.1, 90.3, 110.6, 137.8, 150.6, 164.4;
HRMS (FAB) calcd for C16H28N3Oþ7 374.1927, found
374.1919.
3.1.6. 20-O-[2-[(N-Isopropyl-N-methylamino)oxy]ethyl]-
5-methyluridine (7). Compound 7 was obtained from
compound 5 (4.62 g, 7.55 mmol) and triethylamine tri-
hydrofluoride (6.15 mL, 37.76 mmol) and triethylamine
(2.63 mL, 18.88 mmol) in anhydrous THF (50.53 mL)
according to the procedure used for the synthesis of
compound 6 from 4 with the minor variations that follow.
After work up, the solvent was removed under reduced
pressure and the residue obtained was dissolved in ethyl
acetate (50 mL) and filtered. The filtrate was added
dropwise to hexane (1000 mL). The precipitate formed
was decanted, dissolved in CH2Cl2, and evaporated to
dryness to yield 7 (1.89 g, 71.31%): Rf¼0.36 (10% MeOH
in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) d 0.91 (s, 3H),
0.94 (s, 3H), 1.76 (s, 3H), 2.39 (s, 3H), 2.66 (m, 1H), 3.51–
3.66 (m, 6H), 3.84 (d, 1H, J¼3 Hz), 3.94 (t, 1H,
J¼5.28 Hz), 4.08–4.15 (m, 1H), 5.07 (d, 1H, J¼5.48 Hz),
5.87 (d, 1H, J¼5.52 Hz), 7.76 (s, 1H), 11.27 (br s, 1H); 13C
NMR (50 MHz, CDCl3) d 12.4, 18.7, 18.9, 41.6, 58.3, 61.6,
69.1, 70.7, 71.3, 81.6, 85.4, 91.0, 110.7, 138.1, 150.6, 164.3;
HRMS (FAB) calcd for C16H28O7Nþ3 374.1927, found
374.1933.
3.1.4. 50-O-(tert-Butyldiphenylsilyl)-20-O-[2-[(N-iso-
propyl-N-methylamino)oxy]ethyl]-5-methyluridine (5).
Compound 3 (12.12 g, 21.38 mmol) was dissolved in 1 M
PPTS in MeOH (213.8 mL). To this, NaBH3CN (2.69 g,
42.75 mmol) was added at 108C and the mixture was stirred
for 10 min. The reaction mixture was allowed to come to
room temperature and stirring continued for an additional
2 h. Solvent was removed under reduced pressure. The
residue obtained was dissolved in ethyl acetate (200 mL),
washed with water (150 mL), aqueous NaHCO3 (5 wt%,
150 mL), and brine (150 mL). The organic layer was
evaporated to dryness. The residue was dissolved in 1 M
PPTS in MeOH (213.8 mL). To this, acetone (1.88 mL,
25.66 mmol) was added and the reaction mixture was stirred
at room temperature for 15 min. NaBH3CN (2.69 g,
42.75 mmol) was added at 108C and the mixture was stirred
for 10 min. The reaction mixture was warmed up to room
temperature and stirring continued for 4 h. The solvent was
removed under reduced pressure and the residue obtained
was dissolved in ethyl acetate (300 mL), washed with water
(200 mL), aqueous NaHCO3 (5 wt%, 200 mL), and brine
(200 mL). The organic phase was dried over anhydrous
Na2SO4 and the solvent was removed under reduced
pressure. The residue obtained was purified by flash silica
gel column chromatography and eluted with ethyl acetate/
hexane (70:30) to afford 5 (8.73 g, 66.8%): Rf¼0.3 (ethyl
3.1.7. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N,N-diethyl-
amino)oxy]ethyl]-5-methyluridine (8). Compound
6
(1.25 g, 3.35 mmol) was dried over P2O5 in vacuo over-
night. It was then co-evaporated with pyridine (10 mL)
under reduced pressure and the residue was dissolved in
anhydrous pyridine (9.32 mL). To this, 4,40-dimethoxytrityl
chloride (1.45 g, 3.99 mmol) and DMAP (0.041 g,
0.34 mmol) was added and the reaction mixture was stirred
at ambient temperature for 12 h under argon atmosphere.
Pyridine was evaporated under reduced pressure and the
residue obtained was purified by flash silica gel column
chromatography and eluted with CH2Cl2/MeOH/NEt3
(89:10:1) to afford 8 (1.96 g, 86.64%): Rf¼0.47 (10%
MeOH in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) d 0.98,
(t, 6H, J¼7.12 Hz), 1.39 (s, 3H), 2.6 (q, 4H, J¼7.22 Hz),
3.22 (m, 2H), 3.73 (br s, 10H), 3.98 (br s, 1H), 4.07 (m, 1H),
4.23 (m, 1H), 5.18 (d, 1H, J¼5.76 Hz), 5.9 (d, 1H,
J¼5.38 Hz), 6.9 (d, 4H, J¼8.42 Hz), 7.24–7.41 (m, 9H),
7.49 (s, 1H), 11.39 (s, 1H); 13C NMR (50 MHz, CDCl3) d
11.7, 11.9, 52.3, 55.1, 62.2, 69.0, 70.4, 72.2, 82.8, 83.5,
86.7, 87.5, 110.8, 113.2, 127.0, 127.9, 128.1, 130.0, 135.3,
1
acetate/hexane, 70:30); H NMR (200 MHz, DMSO-d6) d
0.89 (d, 3H, J¼2.2 Hz), 0.92 (d, 3H, J¼1.96 Hz), 1.42 (s,
3H), 2.38 (s, 3H), 2.64 (m, 1H), 3.56–3.71 (m, 4H), 3.75–
3.8 (m, 2H), 3.87–4.02 (m, 2H), 4.2–4.27 (m, 1H), 5.22 (d,
1H, J¼5.42 Hz), 5.94 (d, 1H, J¼5.94 Hz), 7.37–7.48 (m,
6H), 7.51–7.7 (m, 4H), 11.39 (s, 1H); 13C NMR (50 MHz,
CDCl3, 30-O-acetyl derivative) d 11.9, 19.1, 19.4, 20.8, 27.0,
41.8, 58.3, 63.3, 69.6, 70.4, 71.5, 80.3, 82.3, 86.2, 111.5,
127.7, 128.0, 130.1, 130.2, 132.0, 132.8, 134.9, 135.2,
135.5, 150.5, 163.7, 170.2; MS (ES) m/z 610.3 (M2H)2;
HRMS (FAB) calcd for C32H45N3O7SiCsþ 744.2041, found
744.2017.

T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
7419
144.3, 150.5, 158.6, 164.2; HRMS (FAB) calcd for
C37H46N3Oþ9 676.3234, found 676.3214.
3.1.11. 50-O-(tert-Butyldiphenylsilyl)-20-O-[2-[[N-methyl-
N-(2-phthalimidoethyl)amino]oxy]ethyl]-5-methyluri-
dine (12). Compound 3 (2.3 g, 4.17 mmol) was dissolved in
1 M PPTS in MeOH (41.7 mL) and NaBH3CN (0.52 g,
8.35) was added at 108C and the mixture was stirred for
15 min. The reaction mixture was allowed to come to room
temperature and stirring continued for an additional 4 h. The
reaction was monitored by TLC (5% MeOH in CH2Cl2).
The solvent was removed under reduced pressure and the
residue obtained was diluted with ethyl acetate (50 mL) and
washed with water (30 mL), aqueous NaHCO3 (5 wt%,
30 mL), and brine (30 mL). The ethyl acetate layer was
dried over anhydrous Na2SO4 and evaporated to dryness to
yield a foam. This was then dissolved in 1 M PPTS in
MeOH (34 mL). a-phthalimidoacetaldehyde (0.72 g,
3.78 mmol) was added and the reaction mixture was stirred
at ambient temperature for 10 min. To this NaBH3CN
(0.43 g, 0.89 mmol) was added at 108C and stirred for
15 min. The reaction mixture was allowed to come to room
temperature and stirring continued for an additional 4 h. The
solvent was removed under reduced pressure and the oily
residue obtained was diluted with ethyl acetate (50 mL) and
washed with water (40 mL), aqueous NaHCO3 (5 wt%,
40 mL), and brine (25 mL). The organic phase was dried
over anhydrous Na2SO4 and evaporated to dryness. The
residue obtained was purified by flash silica gel column
chromatography and eluted with ethyl acetate/hexane
(60:40) to afford 12 (1.54 g, 60%): Rf¼0.68 (ethyl acetate);
1H NMR (200 MHz, DMSO-d6) d 1.04 (s, 9H), 1.41 (s, 3H),
2.46 (s, 3H), 2.79 (t, 2H, J¼6.34 Hz), 3.69–4.08 (m, 10H),
4.27 (m, 1H), 5.22 (d, 1H, J¼5.7 Hz), 5.95 (d, 1H,
J¼5.86 Hz), 7.39–7.7 (m, 11H), 7.84 (s, 4H), 11.38 (s,
1H); 13C NMR (50 MHz, CDCl3) d 11.8, 19.4, 27.0, 35.6,
45.8, 58.2, 63.2, 68.9, 70.1, 71.1, 82.8, 84.4, 86.9, 111.1,
123.1, 126.3, 127.9, 128.0, 128.4, 130.1, 131.9, 132.1,
132.1, 133.9, 135.2, 135.5, 147.2, 150.2, 163.7, 168.5;
HRMS (MALDI) calcd for C39H46O9N4SiNaþ 765.2932,
found 765.2922.
3.1.8. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N-isopropyl-
N-methylamino)oxy]ethyl]-5-methyluridine (9). Com-
pound 9 (2.33 g, 92% yield) was prepared from compound
7 (1.39 g, 3.93 mmol) in anhydrous pyridine (6.8 mL),
DMTCl (1.44 g, 4.2 mmol) and DMAP (0.072 g,
0.59 mmol) according to the procedure used for the
synthesis of compound 8 from compound 6: Rf¼0.42
(ethyl acetate/hexane, 80:20); 1H NMR (200 MHz, DMSO-
d6) d 0.91 (s, 3H), 0.94 (s, 3H), 1.37 (s, 3H), 2.4 (s, 3H), 2.66
(m, 1H), 3.2 (m, 2H), 3.69 (s, 4H), 3.72 (6H), 3.96–4.06 (m,
2H), 4.21 (m, 1H), 5.18 (d, 1H, J¼5.8 Hz), 5.88 (d, 1H,
J¼5.3 Hz), 6.89 (d, 4H, J¼8.62 Hz), 7.2–7.4 (m, 9H), 7.47
(s, 1H); 13C NMR (50 MHz, CDCl3) d 11.8, 18.9, 19.1, 41.7,
55.2, 58.3, 62.3, 69.2, 70.8, 71.3, 83.0, 83.6, 86.9, 87.8,
110.9, 113.3, 127.1, 128.0, 128.2, 130.2, 135.4, 144.4,
150.6, 158.7, 164.4; HRMS (FAB) calcd for
C37H45O9N3Naþ 698.3222, found 698.3221.
3.1.9. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N,N-diethyl-
amino)oxy]ethyl]-5-methyluridine-30-[(2-cyanoethyl)-
N,N-diisopropyl]phosphoramidite (10). Compound
8
(1.02 g, 1.51 mmol)) was co-evaporated with toluene
(10 mL) in vacuo. The residue was mixed with N,N-
diisopropylamine tetrazolide (0.13 g, 0.76 mmol) and dried
over P2O5 under reduced pressure overnight. To this,
anhydrous acetonitrile 0(7.6 mL) was added, followed by
2-cyanoethyl-N,N,N0,N -tetraisoporpylphosphorodiamidite
(1.92 mL, 6.04 mmol). The mixture was stirred at room
temperature under argon atmosphere until all of the starting
material disappeared (TLC, CH2Cl2/MeOH/Pyridine,
94:5:1). The solvent was removed under reduced pressure
and the residue was dissolved in ethyl acetate (100 mL),
washed with aqueous NaHCO3 (5 wt%, 75 mL), and brine
(75 mL). The organic layer was dried over anhydrous
Na2SO4 and the solvent was removed under reduced
pressure. The oily residue obtained was dissolved in
CH2Cl2 (5 mL) and added dropwise into vigorously stirring
hexane (20 mL). The white solid formed was collected by
decanting the hexane and dried under reduced pressure to
afford 10 as a white foam (0.92 g, 70%): Rf¼0.58 (CH2Cl2/
MeOH/pyridine, 94:5:1); 31P NMR (80 MHz, CDCl3) d
150.84, 150.54; HRMS (FAB) calcd for C46H62N5O10PNaþ
898.4132, found 898.4113.
3.1.12. 20-O-[2-[[N-Methyl-N-(2-phthalimidoethyl)ami-
no]oxy]ethyl]-5-methyluridine (13). Compound 13
(0.42 g, 52% yield) was prepared from compound 12
(1.2 g,
1.62 mmol),
triethylamine
trihydrofluoride
(2.64 mL, 16.2 mmol) and triethylamine (1.13 mL,
8.1 mol) and THF (16 mL) according to the procedure
used for the synthesis of compound 6 from compound 4:
Rf¼0.34, 10% MeOH in CH2Cl2; 1H NMR (200 MHz,
DMSO-d6) d 1.70 (s, 3H), 2.46 (s, 3H), 2.78 (t, 2H,
J¼6.35 Hz), 3.54–3.74 (m, 8H), 3.80 (d, 1H, J¼3.52 Hz),
3.97 (t, 1H, J¼5.26 Hz), 4.10 (q, 1H, J¼4.98 Hz), 5.05 (d,
1H, J¼5.58 Hz), 5.12 (t, 1H, J¼5.14 Hz), 5.86 (d, 1H,
J¼5.64 Hz), 7.75 (s, 1H), 7.84 (s, 4H), 11.29 (s, 1H); 13C
NMR (50 MHz, CDCl3) d 12.4, 35.6, 45.6, 58.1, 61.6, 69.1,
70.0, 71.0, 81.4, 85.2, 90.7, 110.7, 123.2, 132.0, 133.9,
138.1, 150.5, 164.2, 168.5; HRMS (FAB) calcd for
C23H28O9N4Naþ 527.1927, found 527.1917.
3.1.10. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N-iso-
propyl-N-methylamino)oxy]ethyl]-5-methyluridine-30-
[(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite (11).
Compound 11 (1.1 g, 85%) was prepared from compound
9 (1 g, 1.48 mmol), N,N-(diisopropyl)amine tetrazolide
(0.25 g, 1.408 mmol), anhydrous acetonitrile and 2-cyano-
ethyl-N,N,N ,N0-tetraisopropylphosphorodiamidite (1.88 mL,
5.92 mmol) according to the procedure used for the
synthesis of 10 from 8, except that the residue obtained
after work up was purified by flash silica gel column
chromatography and eluted with ethyl acetate/hexane/
pyridine (69:30:1): Rf¼0.62 (ethyl acetate/hexane/
3.1.13. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[[N-methyl-
N-(2-phthalimido)ethylamino]oxy]ethyl]-5-methyluri-
dine (14). Compound 14 (0.47 g, 73% yield) was prepared
from compound 13 (0.4 g, 0.79 mmol), DMAP (0.019 g,
0.16 mmol), anhydrous pyridine (1.9 mL), and DMTCl
(0.29 g, 0.87 mmol) according to the procedure described
pyridine, 69:30:1); 31P NMR (80 MHz, CDCl3)
d
150.86, 150.61; MS (FAB) m/z 898 (MþNa)þ; HRMS
(FAB) calcd for C46H62N5O10PNaþ 898.4162, found
898.4123.

7420
T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
for synthesis of compound 8 from compound 6: Rf¼0.35,
(5% MeOH in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) d
1.36 (s, 3H), 2.48 (s, 3H), 2.79 (t, 2H, J¼6.34 Hz), 3.21 (m,
2H), 3.73 (br s, 12H), 3.97 (m, 1H), 4.07 (m 1H), 4.22 (m,
1H), 5.16 (d, 1H, J¼6.12 Hz), 5.87 (d, 1H, J¼4.94 Hz), 6.89
(d, 3H, J¼4 Hz), 7.34–7.43 (m, 9H), 7.48 (s, 1H), 7.83 (s,
4H), 11.36 (s, 1H); 13C NMR (50 MHz, CDCl3) d 11.6,
35.4, 45.5, 54.8, 57.9, 62.0, 68.9, 69.9, 82.6, 83.2, 86.5,
87.3, 110.5, 113.0, 122.6, 125.8, 127.7, 128.0, 129.8, 131.8,
133.6, 135.3, 135.8, 144.2, 150.4, 158.3, 164.3, 168.2;
HRMS (FAB) calcd for C44H46O11N4Naþ 829.3061, found
829.3066.
1.10 (s, 3H), 1.13 (s, 3H), 1.39 (s, 3H) 2.61 (s, 3H), 2.68 (m,
4H), 2.93 (m, 1H), 3.41 (dd, 1H, J¼2.12, 8.9 Hz), 3.54–
4.02 (m, 5H), 3.82 (s, 6H), 5.37 (m, 1H), 6.07 (d, 1H,
J¼4.84 Hz), 6.86 (d, 4H, J¼8.74 Hz), 7.27–7.43 (m, 9H),
7.61 (s, 1H), 8.56 (br s, 1H); 13C NMR (50 MHz, CDCl3) d
11.8, 18.8, 29.2, 41.7, 55.3, 58.4, 62.4, 69.7, 71.0, 71.4,
80.6, 81.4, 87.0, 87.2, 111.4, 113.4, 127.3, 128.2, 130.2,
135.1, 135.6, 144.2, 150.8, 158.8, 164.6, 171.7, 175.5; MS
(FAB) m/zþ 776 (MþH)þ; HRMS (FAB) calcd for
C41H50N3O12 776.8423, found 776.8412.
3.1.17. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[[N-methyl-
N-(2-phthalimidoethyl)amino]oxy]ethyl]-30-O-succinyl-
5-methyluridine (18). Compound 18 (0.162 g, 90% yield)
was prepared from compound 14 (0.16 g, 0.2 mmol),
DMAP (0.013 g, 0.10 mmol), and succinic anhydride
(0.03 g, 0.3 mmol) according to the procedure used for the
synthesis of compound 16 from compound 8: Rf¼0.43 (10%
MeOH in CH2Cl2); 1H NMR (200 MHz, DMSO-d6) d 1.4 (s,
3H), 2.42 (s, 3H), 2.56 (m, 4H, overlapping with DMSO
peak), 2.75 (t, 2H, J¼6.29 Hz), 3.24 (m, 2H, overlapping
with H2O peak), 3.53–3.8 (m, 6H), 3.72 (s, 6H), 4.13 (br s,
1H), 4.37 (t, 1H, J¼5.86 Hz), 5.29 (t, 1H, J¼4.4 Hz), 5.87
(d, 1H, J¼6.36 Hz), 6.89 (d, 4H, J¼8.72 Hz), 7.21–7.39
(m, 9H), 7.49 (s, 1H), 7.82 (s, 4H), 11.42 (s, 1H), 12.24 (br s,
1H); 13C NMR (100 MHz, CDCl3) d 11.7, 28.9, 29.4, 35.7,
45.7, 55.3, 58.0, 62.3, 69.7, 71.0, 71.2, 80.5, 81.2, 87.1,
111.2, 113.3, 123.3, 127.2, 128.0, 128.2, 130.1, 132.1,
133.9, 135.1, 135.2, 135.4, 144.1, 150.2, 158.8, 163.7,
168.5, 171.4, 173.9; MS (FAB) m/z 929 [MþNa]þ, HRMS
(FAB) calcd for C48H50N4O14Naþ 929.9253, found
929.9242.
3.1.14. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[[N-methyl-
N-(2-phthalimidoethyl)amino]oxy]ethyl]-5-methyl-uri-
dine-30-[(2-cyanoethyl)-N,N-diisopropyl)phosphorami-
dite (15). Compound 15 (0.28 g, 85%) was prepared from
compound 14 (0.26 g, 0.32 mmol), N,N-diisopropylamine
tetrazolide (0.06 g, 0.32 mmol), anhydrous acetonitrile
(1.6 mL),
and
2-cyanoethyl-N,N,N0,N0-tetraisopropyl-
phosphorodiamidite (0.41 mL, 1.28 mmol) according to
the procedure used for the synthesis of compound 10 from
8, except that the residue obtained after work up was
purified by flash silica gel column chromatography and
eluted with ethyl acetate containing 0.5% of pyridine:
Rf¼0.28, ethyl acetate/hexane (60:40); 31P NMR (80 MHz,
CDCl3) d 150.82, 150.61; MS (FAB) m/z 1029 [MþNa]þ,
HRMS (FAB) calcd for C53H63O12N6PNaþ 1029.4132,
found 1029.4112.
3.1.15. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N,N-diethyl-
amino)oxy]ethyl]-30-O-succinyl-5-methyluridine
(16).
Compound 8 (0.2 g, 0.3 mmol) was mixed with succinic
anhydride (0.05 g, 0.45 mmol) and DMAP (0.02 g,
0.15 mmol) and dried over P2O5 in vacuo overnight. This
was then dissolved in anhydrous CH2Cl2 (0.8 mL) and
anhydrous triethylamine (0.08 mL, 0.6 mmol) was added
and the reaction mixture was stirred at r.t. for 8 h under inert
atmosphere. The reaction mixture was diluted with CH2Cl2
(20 mL) and washed with aqueous citric acid (ice cold,
15 mL, 10 wt%) and water (2£15 mL). The organic phase
was dried over anhydrous Na2SO4 and evaporated under
reduced pressure to afford 16 (0.23 g, 100%) as a foam:
3.1.18. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N,N-diethyl-
amino)oxy]ethyl]-5-methyluridine-30-O-succinyl-CPG
(19). Compound 16 (0.21 g, 0.27 mmol) was dried over
P2O5 in vacuo overnight. Anhydrous DMF (0.66 mL) was
added, followed by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluroniumtetrafluoroborate (TBTU, 0.09 g, 0.27 mmol)
and N-methylmorpholine (0.06 mL, 0.54 mmol) and vortexed
underargonatmospheretoyieldaclearsolution. Anadditional
anhydrous DMF (2.1 mL) and CPG (1.17 g, 115.2 mmol g21
,
˚
particle size 120/200, mean pore diameter 520 A) was added
and shaken for 18 h at room temperature. The solid support
was filtered, washed with DMF, acetonitrile and diethyl ether
dried in vacuo overnight and capped with a mixture of Ac2O/
pyridine/N-methylimidazole/THF (10:10:10:70) for 3 h at
room temperature. The solid support was filtered and washed
with acetonitrile and diethyl ether. The solid support (19) was
dried in vacuo and final loading (57^0.5 mmol g21) was
determined by the standard DMT assay.
1
Rf¼0.44 (10% MeOH in CH2Cl2); H NMR (200 MHz,
CDCl3) d 0.96 (t, 6H, J¼7.22 Hz), 1.44 (s, 3H), 2.55–2.7
(m, 8H), 3.26 (m, 2H), 3.42–3.7 (m, 4H), 3.74 (s, 6H), 4.14
(br s, 1H), 4.37 (m, 1H), 5.31 (br s, 1H), 5.90 (d, 1H,
J¼6.36 Hz), 6.91 (d, 4H, J¼8.3 Hz), 7.23–7.41 (m, 9H),
7.50 (s, 1H), 11.47 (s, 1H), 12.26 (br s, 1H); 13C NMR
(50 MHz, CDCl3) d 11.6, 28.9, 52.3, 55.1, 62.2, 69.6, 70.8,
72.5, 80.4, 81.1, 86.9, 87.1, 111.2, 113.2, 127.1, 128.0,
130.0, 135.0, 135.5, 144.0, 150.5, 158.7, 164.5, 171.5,
175.6; HRMS (FAB) calcd for C41H50N3Oþ12 776.3395,
found 776.3380.
3.1.19. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N-iso-
propyl-N-methylamino)oxy]ethyl]-5-methyluridine-30-
succinyl CPG (20). Compound 20 (51 mmol g21) was
synthesized from 17 by a procedure similar to that used for
the synthesis of compound 19 from compound 16.
3.1.16. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[(N-iso-
propyl-N-methylamino)oxy]ethyl]-30-O-succinyl-5-
methyluridine (17). Compound 17 (0.31 g, 98% yield) was
prepared from compound 9 (0.28 g, 0.41 mmol), succinic
anhydride (0.134 g, 1.34 mmol), and DMAP (0.071 g,
0.58 mmol) according to the procedure used for the
synthesis of compound 16 from compound 8: Rf¼0.38
3.1.20. 50-O-(4,40-Dimethoxytrityl)-20-O-[2-[[N-methyl-
N-(2-phthalimidoethyl)amino]oxy] ethyl]-5-methyluri-
dine-30-O-succinyl CPG (21). Compound 18 (0.15 g,
0.17 mmol) and DMAP (0.021 g, 0.17 mmol) was dissolved
in anhydrous acetonitrile. The reaction mixture was
1
(10% MeOH in CH2Cl2); H NMR (200 MHz, CDCl3) d

T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
7421
protected from moisture and 2,20-dithiobis(5-nitropyridine)
(0.07 g, 0.19 mmol) was added and stirred for 5 min at room
temperature. To this triphenyl phosphine (0.05 g,
0.17 mmol) in anhydrous acetonitrile (1.12 mL) was
added. The solution was stirred at ambient temperature for
10 min. Activated CPG (1.12 g, 115.2 mmol g21, particle
measured at 260 nm using a Gilford Response II spectro-
photometer. The buffer contained 100 mM Naþ, 10 mM
phosphate, 0.1 mM EDTA, pH 7. The 500 mL of buffer for
Tm analysis was prepared in a standard flask by adding
0.2669 g of NaH2PO4.H2O, 0.8218 g Na2HPO4.7H2O,
2.4482 g NaCl, 100 mL 0.5 M EDTA solution. The pH
was adjusted to 7 and the volume was adjusted to 500 mL.
Oligonucleotide and their complements were combined at
4 mM of each strand, heated for 5 min at 908C, and cooled
slowly to allow the formation of duplexes. Oligonucleotide
concentrations were calculated from the oligonucleotide
absorption at 260 nm at 858C using extinction coefficients
estimated according to the method of Puglisi and Tinoco.17
Oligonucleotide solutions were heated at a rate of
0.78C min21 in 1 cm path length cells and then cooled to
confirm reversibility and lack of evaporation. Tm values
were obtained from the absorbance versus temperature
curves. Standard deviations did not exceed ^0.58C. Each
Tm reported was an average of three experiments. The DTm
per modification was calculated by subtracting the Tm value
of a duplex containing modified oligonucleotide from the
Tm value of a duplex containing unmodified DNA (parent
duplex), and dividing by the number of modified residues in
the sequence.
˚
size 120/200, mean pore diameter 520 A) was added and
allowed to shake for 2 h. An aliquot was withdrawn and
loading capacity was determined (61.52 mmol g21). The
solid support was filtered and washed with CH3CN, CH2Cl2
and Et2O and dried in vacuo overnight and capped with a
mixture
of
Ac2O/pyridine/N-methylimidazole/THF
(10:10:10:70) for 3 h at room temperature. Finally, the
solid support was filtered and washed with CH3CN, CH2Cl2
and Et2O. The loading of 21 (60^0.5 mmol g21) was
determined by standard DMT assay.
3.2. Oligonucleotide synthesis
A 0.1 M solution of the amidites 10, 11 and 15 in anhydrous
acetonitrile was used for the synthesis of modified
oligonucleotides. Standard phosphoramidates were used
for incorporation of A, G, C and T residues. The
oligonucleotides were synthesized on solid support on a
solid-phase DNA synthesizer. The solid supports 19, 20 and
21 were used when necessary. For incorporation of 10, 11,
and 15, phosphoramidite solutions were delivered in two
portions, each followed by a 5 min coupling wait time. All
other steps in the protocol supplied by a manufacturer were
used without modification. Oxidation of the internucleotide
phosphite to the phosphate was carried out using CSO ([1-S-
(þ)-(10-camphorsulfonyl)oxaziridine], 0.5 M in aceto-
nitrile) and 4 min oxidation wait time. The coupling
efficiencies were more than 97%. After completion of the
synthesis, the solid support-bound oligonucleotides 23–25
(2 mmol, Table 1) and 27–29 (2 mmol, Table 1) was
suspended in aqueous ammonium hydroxide (2 mL,
30 wt%) and kept at room temperature for 2 h. The solid
support was filtered and the filtrate was heated at 558C for
6 h to complete the removal of all protecting groups. The
solid support bound oligonucleotide 31 (2 mmol) was
suspended in aqueous ammonium hydroxide (1.8 mL,
30 wt%) and kept at room temperature for 2 h. To this
aqueous methylamine (0.2 mL, 40 wt%) was added to
facilitate the deprotection of phthalimodo group and
filtered. The filtrate was heated at 558C for 6 h. Crude
oligonucleotides were purified by HPLC (C-4 column,
Waters, 7.8£300 mm, A¼100 mM ammonium acetate, pH
6.5–7, B¼acetonitrile, 5–60% of B in 55 min, flow
2.5 mL min21, l 260 nm). Oligonucleotides were detrityl-
ated with aqueous 80% acetic acid at room temperature for
1 h. Purification and desalting by HPLC gave 20-modified
oligonucleotides in 30–40% isolated yield. The oligo-
nucleotides were characterized by ES MS and purity was
assessed by HPLC and capillary gel electrophoresis.
3.4. Nuclease stability assay
The nuclease stability of the 20-modified oligonucleotides
was evaluated using SVPD assay as described previously.12
Oligonucleotides were 50 end-labeled using [g232P]ATP
and T4 polynucleotide kinase. After the labeling reaction,
the T4 polynucleotide kinase was heat inactivated at 958C
for 3 min and oligonucleotides were used without further
purification. The SVPD assay was performed at 378C using
1 mM oligonucleotide in a buffer of 50 mM Tris–HCl, pH
8.5, 72 mM NaCl and 14 mM MgCl2. The SVPD (USB,
Cleveland,
OH)
enzyme
concentration
was
5£1023 U mL21. The enzyme was shown to maintain its
activity under these condition for 24 h. Aliquots of the
reaction mixture were removed at the indicated times,
quenched by addition to an equal volume of 80% formamide
gel loading buffer containing tracking dyes, heated for 2 min
at 958C and then stored until analysis by denaturing
polyacrylamide electrophoresis. Quantitation was per-
formed on a Molecular Dynamics PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
3.5. Binding of oligonucleotides to human serum
albumin
The equilibrium constants, Kd, of the oligonucleotides
albumin binding were determined by following the
procedure reported in the literature.13,14b
Acknowledgements
3.3. Tm analysis
We thank our colleagues S. R. Owens for the nuclease
stability assays, Dr. B. S. Ross and Dr. A. P. Guzaev for
helpful discussions and valuable comments and Dr. P. Dan
Cook for his enthusiastic support of this research and
initiating and leading the Medicinal Chemistry program at
Isis.
The thermal stability of the duplexes formed by oligo-
nucleotides with the 20-modified residues and comple-
mentary RNA and DNA was studied by measuring the UV
absorbance versus temperature curves as described pre-
viously.16 Absorbance versus temperature curves were

7422
T. P. Prakash et al. / Tetrahedron 59 (2003) 7413–7422
8. Manoharan, M.; Lu, Y.; Casper, M. D.; Just, G. Org. Lett.
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