Synthesis of 2′-O-modified adenosine building blocks and application for RNA interference

Article abstract of DOI:10.1016/j.bmc.2007.09.019

RNA-Interference has been recognized as a powerful tool to control gene function and has been used for gene silencing by knocking down mRNA. Chemically modified RNAs, especially 2′-O-modification, successfully improved their physicochemical and pharmaceut

Full text of DOI:10.1016/j.bmc.2007.09.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 518–529
Synthesis of 20-O-modified adenosine building blocks
and application for RNA interference
Dalibor Odadzic,a Jesper B. Bramsen,b Romualdas Smicius,a Claus Bus,b
Jørgen Kjemsb and Joachim W. Engelsa,*
aInstitute for Organic Chemistry, and Chemical Biology, Johann Wolfgang Goethe-University,
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany
bInterdisiplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus,
C.F. Mollers Alle´, 8000C Aarhus, Denmark
Received 3 July 2007; revised 29 August 2007; accepted 11 September 2007
Available online 15 September 2007
Abstract—RNA-Interference has been recognized as a powerful tool to control gene function and has been used for gene silencing
by knocking down mRNA. Chemically modified RNAs, especially 20-O-modification, successfully improved their physicochemical
and pharmaceutical properties such as stability, nuclease resistance and delivery. Here, we report the synthesis of adenosine building
blocks with different 20-tethered modifications like aminoethyl and guanidinoethyl and show that they are compatible with RNAi
function. They enhance the half life of the siRNA in serum suggesting that these modifications can enhance the pharmacokinetic
properties and knock down activity of siRNAs in vivo.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
the degradation of the target mRNA is mediated by
the Agonaute 2 protein associated with RISC. The tar-
get mRNA is cleaved in the complementary region at
the phosphodiester bond that lies across from nucleo-
tides 10 and 11 of the 50-end of the siRNA.5 For
RNAi-mediated mRNA cleavage and degradation to
be successful, a 50-phosphate must be present on the
antisense strand, and the double helical antisense-target
mRNA duplex must be in the A-form.6
Since the recognition of RNA interference (RNAi) in
1998, the process by which specific mRNAs are targeted
and degraded by complementary short-interfering
RNAs so called siRNA became a powerful tool to con-
trol gene function and has great hopes as therapeutic
approach for gene silencing.1,2 For developing the most
effective siRNA construct it is necessary to understand
the mechanism of RNAi. The generally accepted mech-
anism of RNAi can be divided into two main steps. In
the first double stranded RNA (dsRNA) is cleaved into
short 21–24 nt siRNAs. This process is catalysed by
Dicer, an endonuclease of the RNase III family.3 The
resultant siRNAs duplexes have 30-overhangs of 2 nt
with 30-hydroxyl termini and a 50-phosphate at both
ends. In the second step, siRNAs are incorporated into
the RNA-induced silencing complex (RISC). A helicase
in RISC unwinds the duplex siRNA, which than pairs to
messenger RNAs (mRNAs) that bear a high degree of
sequence complementarity to the siRNA.4 In humans
The main challenge for therapeutic applications are the
rapid degradation of RNA by extra- and intra-cellular
enzymes and the delivery inside to the relevant cells in
whole organism.
Chemically modified nucleosides have shown to be of
great importance for antisense strategies and are now
being applied for RNA interference mediated gene
silencing. The rationale for the synthesis of 20-O-cationic
modified nucleosides is on one hand a higher expected
nuclease resistance and on the other hand a potentially
better cellular uptake due to an overall reduced negative
charge based on internal charge compensation.
Keywords: RNAi; Nuclease resistence; 20-O-Modified building blocks,
eGFP knock down.
*
A large number of oligonucleotide derivatives bearing
20-O-tethered modifications are reported.7–10 As a result
Corresponding author. Tel.: +49 69 798 29151; fax: +49 69 798
29148; e-mail: joachim.engels@chemie.uni-frankfurt.de
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.019

D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
519
of the higher nuclease resistance and affinity for
complementary oligonucleotides of 20-O-aminopropyl11
and 20-O-guanidinoethyl,12 we decided to synthesise
20-O-modified adenosine with cationic modifications
and to analyse their stabilising effect on the duplex.
Here, we report the preparation of adenosine building
blocks carrying as 20-modifications either cationic
groups like [aminoethyl(D1), guanidinoethyl(D2)] or
neutral ones like [cyanoethyl(D3), allyl(D4)] (Scheme 1).
base N6 we choose the dimethylformamidine group to
provide 6 in 86% yield. The final step of Scheme 1 is
the phosphitylation of nucleoside 6 using 2-cya-
noethyldiisopropylchloro-phosphoramidite to yield the
adenosine tethered phthalimidoethyl nucleoside phos-
phoramidite 7.
The synthesis of compound 14 is shown in Scheme 3.
Adenosine 1 was alkylated at the 20-O-position under
the same conditions reported for compound 2.Nucleo-
side 2 was protected at the 50- and 30-position with
tert-butyl-dimethylsilyl (TBDMS) to provide 8 in 92%
yield. Nucleoside 8 was reduced with LiAlH4 to produce
the 20-O-hydroxyethyl nucleoside 9 in 86% yield. For the
incorporation of the guanidino group we used triboc-
guanidin, which we had synthesised before,15 in a Mits-
unobu type reaction first under conventional conditions
heating for 8 h to produce nucleoside 10 in 78% yield.
The microwave assisted method was even more efficient.
We chose a power of 150 W at 80 ꢁC for 1.5 h to provide
nucleoside 10 in 92% yield.
These modified building blocks were synthesised and
incorporated into RNA in order to test their effects on
RNA stability and siRNA silencing. 12mer RNA oligo-
nucleotides were synthesised for measuring thermal sta-
bility as well as their CD spectra. 21mer RNA
oligonucleotides were prepared for siRNA silencing
and measuring nuclease resistance.
2. Synthesis
Preparation of the adenosine nucleoside phosphorami-
dites with an aminoethyl modification on the 20-OH
group is shown in Scheme 2. Different strategies for an
efficient method to introduce the aminoethyl group on
the 20-O-position were reported earlier.13
Removal of the 30- and 50-silyl groups with TBAF, gave
11 in 90% yield and was followed by 50-DMTr protec-
tion to produce 12 in 75% yield and the protection of
the N6 position of the base with DMF to provide 13
in 86% yield. The final step was the phosphitylation
yielding 14 in 65%.
Adenosine 1 was alkylated at the 20-position with NaH
and bromoacetate14 to provide 2 in 47% yield. The
nucleoside 2 could be separated easily from the isomers
by silica gel chromatography.
The synthesis of the neutral building blocks 20 and 23 is
shown in Scheme 4.
Nucleoside 2 was protected at the 50-position with
dimethoxytriphenylmethyl (DMTr) to produce 3 in
75%. Nucleoside 3 was reduced with LiBH4 to provide
the 20-(hydroxyethyl) nucleoside 4 in 80% yield. Reduc-
tion using LiAlH4 instead of LiBH4 was not so efficient
because of a side reaction producing 20-vinyl adenosine
in 20%. The nitrogen was introduced by phthalimide
in a Mitsunobu reaction to yield the 20-(phthalimidoeth-
yl) nucleoside 5 in 89%. For protection of the nucleo-
We protected the exocyclic amino group of the base with
dimethylformamidine to avoid undesired derivatization
on N6 in the alkylation step and obtained compound
15 in 99% yield. The protection of the 50- and 30-position
with TIPDS produced nucleoside 16 in 95% yield. For
the 20-O-allyl modified building block we chose the pal-
ladium-catalyzed allylation16 to provide 17 in 80% yield.
A Michael addition with acrylonitrile and Cs2CO3 in
tert-butanol7 produced nucleoside 21 in 70% yield. The
TIPDS 30,5-protecting group was removed with
NEt3*3HF, producing 18 and 22 with 90% yield. Selec-
tive reprotection of the 50-hydroxyl group with DMTr
led to nucleosides 19 and 23 followed by phosphitylation
providing 20 and 24 in a yield of 52%.
NH2
NH2
N
N
N
N
N
N
N
N
O
O
O
O
3. Results and discussion
NH2
NH2
O
O
O
O
NH3
The route of our synthesis from 1 to 7 takes 6 steps with
6 chromatographic purifications in a 14% overall yield.
For the nucleoside 14 it takes 8 steps with 7 chromato-
graphic purifications in a 13% overall yield. The
synthesis from 1 to 20 and 24 takes 6 steps with
5 chromatographic purifications in a 35% overall yield
for 20 and 31% for 24.
N
H
D1
D2
NH2
NH2
N
N
N
N
N
N
N
N
O
O
O
O
All the synthesized nucleoside phosphoramidites 7, 14, 20
and 24 were incorporated into 12mer RNA oligonucleo-
tides and their thermal stability as well as their CD spec-
tra measured. The sequence of the RNA 12mer, the Tm
values and the resulting DTm are shown in Scheme 5.
O
O
O
O
CN
D3
D4
Scheme 1. Synthesised 20-O-modified building blocks.

520
D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
A
A
A
DMTrO
HO
DMTrO
O
O
a
b
O
c
Adenosine
O
O
1
OH
3
75%
O
OH
2
47%
O
OH
O
OMe
OMe
OH
4
80%
ADMF
A
DMTrO
DMTrO
O
OH
O
O
O
e; f
d
O
O
O
O
N
5
N
P
89%
N
O
O
CN
7
56%
Scheme 2. Synthesis of the aminoethyl modified building block 7. Reagents and conditions: (a) NaH, BrCH2COOMe, DMF, rt 8 h; (b) DMTr-Cl,
NEt3, pyridine, rt, 16 h; (c) LiBH4, THF/MeOH, 0 ꢁC, 3 h; (d) phthalimide, PPh3, DEAD, THF, rt, 1.5 h; (e) DMF-dimethyl-acetale, DMF, 60 ꢁC,
1 h; (f) CEP-Cl, sym collidine, 1-methylimidazole, acetonitrile, 0 ꢁC, 0.5 h, rt, 0.25 h.
A
A
TBSO
A
TBSO
O
DMTrO
a; b
O
c; d
Adenosine
1
O
O
e; f
Boc
NH
Boc
NH
O
N
N
OMe
TBSO
O
8
43%
TBSO
HO
O
10
N
12
N
Boc
Boc
79%
Boc
Boc
67%
ADMF
DMTrO
O
Boc
g;h
N
O
P
O
O
NH
Boc
N
Boc
N
CN
14
56%
Scheme 3. Synthesis of the guanidinoethyl modified building block 14. Reagents and conditions: (a) NaH, BrCH2COOMe, DMF, rt 8 h; (b)
TBDMS-Cl, imidazole, DMF, rt, 16 h; (c) LiAlH4, ether, 0 ꢁC, 0.5 h ; (d) N,N0,N00-triboc-guanidin, PPh3, DEAD, THF, rt, 1.5 h, p:1 bar, P: 150 W;
(e) TBAF/THF(1 mol), THF, rt, 16 h; (f) DMTr-Cl, NEt3, pyridine, rt, 16 h; (g) DMF-dimethyl-acetale, DMF, 60 ꢁC, 1 h; (h) CEP-Cl, sym collidine,
1-methylimidazole, acetonitrile, 0 ꢁC, 0.75 h.
The results of the Tm and CD measurement show that
the aminoethyl and guanidinoethyl modified building
blocks D1 and D2 stabilize the 12mer duplex. Guanidi-
noethyl modified building block D2 gives the highest Tm
value and therefore the highest stabilization
(DTm:+3.1 ꢁC). The aminoethyl modified building
block D1 has a stabilization of (DTm:+1 ꢁC). The
zneutral modified building blocks were synthesized to
improve the effect of the positive charge in combination
with increasing the lipophilicity.
eGFP mRNA17 with modified building blocks at differ-
ent positions in the sense strand and two antisense
strands, see Scheme 7.
The 30-terminal additional U was included for synthetic
reasons and was shown to have no effect on siRNA
knock down.18 The synthesis of the modified building
blocks C and G will be published (manuscript in prepa-
ration). These sense RNAs DO002-4 were annealed to
the antisense RNA modified oligos JE1001 and
JE1002, to obtain siRNAs.
The results indicate that the allyl modification D4 has
the lowest stabilization, whereas the cyanoethyl modi-
fied building block D3 shows
a
stabilisation of
4. Biological test
(DTm:+0.9 ꢁC). The CD spectra depicted in Scheme 6
support the A-helix geometry of the duplex and confirm
the results of the Tm measurements.
Incorporation of unnatural nucleotides in the siRNA
will presumably inhibit the activity of RNases and
extent the halflife of the duplex in vivo. To test this
hypothesis siRNA duplexes were incubated at 37 ꢁC in
80% fetal calf serum to mimic in vivo conditions and fol-
lowed by taking time aliquots. The integrity of the siR-
NAs was subsequently analyzed by staining the RNA
after size fractionation on a 15% native polyacrylamide
The oligonucleotides with the above 20-O-modifications
were used for biological tests including stability in serum
and inhibitory activity in RNA interference. For this
purpose we synthesized four 21mer siRNAs, directed to-
wards a previously established functional target in the

D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
521
ADMF
ADMF
ADMF
O
DMTrO
HO
Si
O
O
O
O
a; b
e; f
c(1); d
Adenosine
Si
O
OH
1
O
O
OH
18
72%
O
P
16
N
O
94%
CN
20
52%
ADMF
ADMF
ADMF
O
HO
DMTrO
Si
O
O
O
O
a; b
c(2); d
e; f
Adenosine
Si
O
OH
OH
22
63%
O
1
O
O
CN
CN
P
16
N
O
94%
CN
24
52%
Scheme 4. Synthesis of the neutral modified building blocks 20 and 23. Reagents and conditions: a DMF-dimethyl-acetale, DMF, 60 ꢁC, 1 h b
TIPDS-Cl, pyridine, 20 h, rt c(1) allyl ethyl carbonate, Pd2(dpa)2, dppb, THF, 1 h, 70 ꢁC c(2) Acrylonitrile, Cs2CO3, tert-butanol, rt, 16 h d
NEt3*3HF, THF, rt, 0.5 h e DMTr-Cl, NEt3, pyridine, rt, 16 h f CEP-Cl, sym collidine, 1-methylimidazole, acetonitrile, 0 ꢁC, 0.5 h.
46
Base
U
Tm
Tm
45
44
43
42
41
40
1
2
3
4
A
43.1
45.2
42.5
43.0
42.1
+1.0
+3.1
+0.4
+0.9
-------
1
2
3
4
A
U
U
U
U
U
*
Tm +/- 0.2
5’-AAG-AAX-GAA-AAG-3’
3’-UUC-UUU-CUU-UUC-5’
1:X=Aminoethyl 2: X= Guanidinoethyl 3: X= Ally 4: X= Cyanoethyl
Scheme 5. RNA 12mer sequence and the resulting Tm values.
ingly, we also found that 2-O-aminoethyladenosine
modified siRNA (DO0003) and 20-O-guanidinoethylad-
enosine (DO004) were more resistant than siRNA con-
taining 20-O-allyladenosine (DO0002) modification
(Scheme 8; compare panels a–f and g) with JE1001 as
antisense strand.
The biological activity of the chemically modified siR-
NA were tested together with unmodified and mis-
matched control siRNAs by transfecting them into a
H1299 lung carcinoma cell line that stably expresses
destabilized EGFP, and the level of EGFP protein
expression was monitored on the basis of flow cytometry
(Scheme 9). Treating the cells with a single dose of
10 nM of the various siRNAs gave a comparable 80%
knock down in the average EGFP expression for all con-
structs containing the lightly-modified JE1001 antisense
strand 48 h after transfection and the decline in protein
level correlated closely with the level of eGFP
mRNA expression (Scheme 9, lanes 1–3). This result
Scheme 6. CD spectra of the modified and unmodified 12mer RNA
oligonucleotides.
gel at the indicated time points. All modified siRNA
duplexes exhibited prolonged half life compared to
unmodified siRNA, with the 50-terminal modified
siRNA (containing JE1002) being most stable. Interest-

522
D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
was indistinguishable from the knockdown obtained
siRNA:
with unmodified RNA suggesting that the substitutions
were fully compatible with siRNA function (Scheme 9,
lane7). In contrast, all siRNAs containing the 5-terminal
antisense JE1002 gave only small (10-20%), but repro-
ducible, knock down in protein and mRNA expression
(Scheme 9, lanes 4–6), implying that a 50-terminal mod-
ification in the antisense strand interferes with RNAi
activity.
5`-
GAC GUA AAC GGC CAC AAG UUC-3`
3`-CG CUG CAU UUG CCG GUG UUC A -5`
Sense A Allyl:
5`-GAC GUA AAC GGC CAC AAG UUC-3`
Sense A Aminoethyl:
DO0002
DO0003
DO0004
The knock down efficiencies between different siRNA
(denoted below) were compared by targeting eGFP
mRNA. The eGFP protein expression was measured
by flow cytometry (mean fluorescence of approximately
50.000 cells) and the eGFP mRNA levels were measured
by Northern blotting. The siRNA mismatch represents a
siRNA that contains four mismatches to the eGFP tar-
get and green cells denote untreated cells.
5`-GAC GUA AAC GGC CAC AAG UUC-3`
Sense A Guanidinoethyl:
5`-GAC GUA AAC GGC CAC AAG UUC-3`
Antisense C,G Aminoethyl:
3`-UCG CUG CAU UUG CCG GUG UUC A-5` JE1001
Antisense C,G, A Aminoethyl:
5. Conclusion
3`-UCG CUG CAU UUG CCG GUG UUC A-5` JE1002
We have described a set of experimental procedures for
synthesizing 20-O-cationic and neutral modified adeno-
Scheme 7. siRNA sequences and tested 21mers.
Scheme 8. Serum stability of siRNAs and density scan.

D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
523
Scheme 9. RNAi test.
sine building blocks. Furthermore we have shown that
our cationic modifications have a stabilizing effect on a
RNA 12mer duplex. The modifications led generally to
higher stability in serum, an effect that was strongest
for the 20-O-aminoethyladenosine modification. As long
as the modifications were restricted to the 30-end of the
antisense strand the siRNA duplexes were all fully active
in RNA interference when tested in cell lines. This sug-
gests that the new modifications are compatible with
RNAi function and may prove advantageous for
in vivo applications.
Each melting curve was determined twice. The tempera-
ture range was 0–60 ꢁC with a heating rate 0.5 ꢁC. The
thermodynamic data were extracted from the melting
curves by means of a two state model for the transition
from duplex to single strands.
CD spectra. CD spectra of RNA duplexes were recorded
at 350–190 nm with oligonucleotide concentration of
2 lM for each strand in a phosphate buffer containing
NaCl (140 mM, pH 7.0). The measurements were per-
formed at 10 ꢁC to ensure that only duplex RNA was
present.
6. Experimental
Transfection. The human lung cancer cell line H1299
produced to stably express EGFP (EGFP half-life
2 h; a gift from Dr Anne Chauchereau, CNRS, Ville-
juif, France) were plated in 6-well plates and grown
in RPMI-1640 containing 10% FBS, 1% penicillin/
streptomycin to 60–80% confluence. Immediately be-
fore transfection, the cells were replenished in 1.25 ml
of complete growth media per well. Sense and anti-
sense strands where mixed in annealing buffer
(10 mM Tris–HCl, pH 7.3, 50 mM NaCl) at 20 mM
equimolar concentration and incubated at 95 ꢁC for
1 min and at 1 h at 37 ꢁC. Per well in a 6-well plate,
the following solution was prepared: 6 ll of TransIT-
TKO in 250 ll serum free RPMI media. siRNA was
added, mixed carefully, incubated for 20 min at rt,
and applied to the cells at a final concentration of
10 nM. After 24 h incubation at 37 ꢁC, the media
was changed and the cells were incubated for another
24 h at 37 ꢁC. The cells were removed by trypsination
and the expression of EGFP protein was quantified by
flow cytometric analysis counting approximately
50,000 cells and averaged (mean fluorescence).
6.1. Materials and methods
Oligonucleotides synthesis. The RNA oligomers were
synthesized on Expedite synthesizer by phosphoamidite
chemistry with coupling time for modified monomers
of 12 min.19 The RNA oligomers were cleaved
from the controlled-pore-glass (CPG) support with
ethanol:ammonia solution (1:3) at 40 ꢁC for 24 h. The
20-TBDMS groups were deprotected with triethylamine,
N-methylpyrrolidinone and Et3N Æ 3HF mixture for
1.5 h at 65 ꢁC.20 The RNA oligomers were precipitated
with BuOH at ꢀ80 ꢁC over 30 min and purified by anion
exchange HPLC (NucleoPac-PA-100) and desalted
(Sephadex-G25). All ribonucleotides were characterized
by MALDI-TOF-MS.
UV Melting curves. UV Melting profiles of the RNA du-
plexes were recorded in a phosphate buffer containing
NaCl (140 mM, pH 7.0) at oligonucleotide concentra-
tions 2 lM for each strand at wavelength 260 nm.21

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D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
siRNA stability assay. siRNA sense and antisense
strands where mixed in annealing buffer (10 mM Tris–
HCl, pH 7.3, 50 mM NaCl) at 20 lM equimolar concen-
tration and incubated at 95 ꢁC for 1 min and at 1 h at
37 ꢁC. siRNA duplexes were incubated at 37 ꢁC in
80% fetal calf serum in DMEM (Gibco). Aliquots of
5 ll (each containing 20 pmol of siRNA) were diluted
in 25 ll 1.2· TBE loading buffer (1.2· TBE, 10% glyc-
erol, bromophenol blue) and snap-frozen on dry ice
immediately upon sample taking. Samples were run on
a 15% native polyacrylamide gel and stained using
SYBR Goldꢂ (Invitrogen).
tracted with CH2Cl2. The combined organic extracts
were dried over MgSO4 and concentrated under vac-
uum. The resulting orange oil was purified by chroma-
tography with 10% MeOH in CH2Cl2 as the eluent to
produce 1.34 g (73%) 3 as a white foam. 1H NMR
(250 MHz, DMSO-d6) d [ppm] 8.29 (s, 1H, H2), 8.11
(s, 1H, H8), 7.37 (s, 2H, NH2), 7.35 (m, 8H, HDMTr),
6.85 (m, 5H, HDMTr), 6.11 (s, 1H, 10H), 5.38 (m, 1H,
30OH), 4.78 (m, 1H, 20H), 4.49 (m, 1H, 30H), 4.24 (m,
3H, 40H, 50H), 4.08 (m, 2H, 200H), 3.73 (s, 6H, OCH3),
3.53 (s, 3H, OCH3). 13C NMR (62.9 MHz, DMSO-d6)
d[ppm] 170.42 (C@O), 156.67 (C6), 152.41 (C8), 149.57
(C5) 144.81 (CDMTr) 139.72 (C2), 135.5–123.9(CDMTr),
119.7 (C4), 113.6 (CDMTr), 85.84 (C10), 85.49 (C40),
80.65 (C20), 69.04 (C30), 67.02 (C200), 63.51 (C50), 54.94
(OCH3DMTr), 51.82 (O–CH3). MALDI-MS: Calcd
C34H35N5O8: 641.34. Found: 642.34 [M+H]+.
6.2. General procedures
1
All H and 13C NMR spectra were measured on Bruker
AM 250 (250 MHz) or AMX 400 (400 MHz) spectrom-
eters. Chemical shifts (d) are reported in parts per mil-
lion (ppm). For peak multiplicity are used: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; b, broad-
ened. J values are given in Hz. MALDI mass spectra
were recorded on a Fisons VG Tofspec spectrometer
and ESI mass spectra on a Fisons VG Plattform II spec-
trometer. The microwave-supported synthesis was
accomplished in equipment of the company CEM, model
Discover. All solvents and reagents were obtained
from commercial sources used without further
purifications.
6.3.3. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-(hydroxy-
ethyl)-adenosine (4). Lithium borhydride (0.39 g,
17.52 mmol) was added to a solution of 3 (1.41 g,
2.19 mmol) in 60 ml THF/MeOH (4:1) cooled to
0 ꢁC, the reaction mixture was stirred for 3 h at 0 ꢁC
and then saturated aqueous NH4Cl was added drop-
wise. The solution was warmed to room temperature
and extracted with CH2Cl2. The combined organic
extracts were dried over MgSO4 and concentrated un-
der vacuum. The resulting oil was purified by chroma-
tography with 10% MeOH in CH2Cl2 as the eluent to
produce 0.89 g (66%) 4 as a white foam. 1H NMR
(400 MHz, DMSO-d6) d [ppm] 8.31 (s, 1H, H2), 8.15
(s, 1H, H8), 7.42 (s, 2H, NH2), 7.41 (m, 8H, HDMTr),
6.89 (m, 5H, HDMTr), 6.10 (d, 1H, J = 5.8 Hz, 10H),
5.21 (d, 1H, J = 6.02 Hz, 30OH), 4.77 (t, 1H,
J = 5.33 Hz, 300OH), 4.70 (m, 1H, 20H), 4.48 (m, 1H,
30H), 4.12 (m, 1H, 40H), 3.77 (s, 6H, OCH3), 3.66
(m, 2H, 200H), 3.62 (m, 2H, 300H), 3.27 (m, 2H, 50H).
13C NMR (100.62 MHz, DMSO-d6) d [ppm] 156.57
(C6), 153.14 (C8), 149.70 (C5), 145.29 (CDMTr),
140.06 (C2), 136.13–127.12 (CDMTr), 119.65 (C4),
113.69 (CDMTr) 86.46 (C10), 83.72 (C40), 81.13 (C20),
72.33 (C200), 69.82 (C30), 64.01 (C50), 60.68 (C300),
55.48(O–CH3). MALDI-MS: Calcd C33H35N5O7:
613.33. Found: 614.33 [M+H]+.
6.3. Chemistry
6.3.1. 20-O-(Methoxycarbonylmethyl)-adenosine (2).
Sodium hydride 0.25 g (10.5 mmol) was added to a sus-
pension of adenosine (2 g, 7.5 mmol) in 40 ml DMF at
0 ꢁC. The suspension was stirred at 0 ꢁC for 1 h and
methyl bromoacetate (1.1 ml, 11.25 mmol) was added.
This mixture was warmed to room temperature and
stirred for 8 h. After the reaction was complete, the sus-
pension was quenched with 1 ml acetic acid and 3 ml
methanol. The solvents were concentrated under vac-
uum, and the residue was purified by a dry-load silica
gel chromatography with 10% MeOH in CH2Cl2 as
the eluant to provide 1.2 g (47%) 2 as a white foam.
1H NMR (400 MHz, DMSO-d6) d [ppm] 8.35 (s, 1H,
H2), 8.12 (s, 1H, H8), 7.32 (s, 2H, NH2), 6.06 (d,
1H, J = 6.3 Hz, 10H), 5.39 (m,1H, 50OH), 5.28 (d, 1H,
J = 4.77 Hz, 30OH), 4.67 (m, 1H, 20H), 4.39 (m, 1H,
30H): 4.29 (m, 2H, 200H), 4.08 (m, 1H, 40H), 3.68 (m,
2H, 50H), 3.52 (s, 3H, OCH3).13C NMR (100.62 MHz,
DMSO-d6) d [ppm]: 170.79 (C@O), 156.63 (C6), 152.91
(C8), 149.58 (C5),140.33 (C2), 119.72 (C4), 86.73
(C10), 86.22 (C40), 81.56 (C20), 69.37 (C30), 67.24
(C200), 61.93 (C50), 51.81 (O–CH3). ESI(+)-MS: Calcd
C13H17N5O6: 339.03. Found: 340.03 [M+H]+.
6.3.4. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-(phtha-
limidoethyl)-adenosine (5). To a solution of 4 (0.15 g,
0.24 mmol) in 10 ml THF, triphenylphosphine
(0.093 g, 0.36 mmol) and phthalimide (0.036 g,
0.24 mmol) were added at room temperature, stirred
for 5 min and then diethylazodicarboxylate (0.063 g,
0.36 mmol) was added dropwise to the mixture. The
mixture was stirred for 1 h at room temperature, and
quenched with 3 ml saturated aqueous NaHCO3. The
mixture was extracted with CH2Cl2. The combined or-
ganic extracts were dried over MgSO4 and concentrated
under vacuum. The resulting orange oil was purified by
chromatography with 10% MeOH in CH2Cl2 as the elu-
ent to produce 0.16 g (90%) 5 as a white foam.1H NMR
(400 MHz, DMSO-d6) d [ppm] 8.16 (s, 1H, H2), 7.96 (s,
1H, H8), 7.78 (m, 4H, Hphthalimide), 7.34 (s, 2H, NH2),
7.25 (m, 8H, HDMTr), 6.83 (m, 5H, HDMTr), 5.91 (d,
1H, J = 5.5 Hz, 10H), 5.19 (d, 1H, J = 4.92 Hz, 30OH),
6.3.2. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-(meth-
oxycarbonylmethyl)-adenosine (3).
A portion of 2
(0.98 g, 2.88 mmol) was dissolved in 15 ml pyridine. Tri-
ethylamine (0.64 ml, 4.32 mmol) and 4,40-dimethoxytri-
tyl chloride (1.16 g, 3.45 mmol) were added at room
temperature. The mixture was stirred for 16 h at room
temperature, and quenched with 5 ml methanol and
10 ml saturated aqueous NaHCO3. The mixture was ex-

D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
525
4.66 (m, 1H, 20H), 4.40 (m, 1H, 30H), 3.98 (m, 1H, 40H),
3.84 (m, 4H, 200H, 300H), 3.72 (s, 6H, OCH3), 3.16 (m,
2H, 50H). 13C NMR (100.62 MHz, DMSO-d6) d [ppm]
168.18 ( C@O), 158.48 (Cphthalimide), 156.57 (C6),
153.14 (C8), 149.70 (C5), 145.29 (CDMTr), 140.06 (C2),
vents were removed under vacuum, and the resulting
paste was partitioned between water and CH2Cl2. The
combined organic extracts were washed with saturated
aqueous NaCl, dried over MgSO4 and concentrated un-
der vacuum. The resulting oil was purified by chroma-
tography with 5% MeOH in CH2Cl2 as the eluent to
produce 0.77 g (92%) 8 as a white solid. 1H NMR
(250 MHz, DMSO-d6) d [ppm] 8.16 (s, 1H, H2), 7.98
(s, 1H, H8), 7.17 (s, 2H, NH2), 5.94 (d, 1H,
J = 6.2 Hz, 10H), 4.65 (m, 1H, 20H), 4.51(m, 1H, 30H),
4.07 (m, 2H, 200H), 3.72 (m, 3H, 40H, 50H), 3.41 (s,
3H, OCH3), 0.94 (m, 18H, Htert-butyl), 0.09 (m, 12H,
Hdimethylsilyl). 13C NMR (62,90 MHz, DMSO-d6) [d
ppm ]: 169.85 (C@O), 156.03 (C6), 152.54 (C8), 149.24
(C5),139.52 (C2), 119.04 (C4), 85.17 (C10), 84.75 (C40),
80.29 (C20), 70.24 (C30), 66.91 (C200), 62.02 (C50), 51.36
(O–CH3), 25.76 (CTBS), 17.76 (CTBS), -5.12 (CTBS).
ESI(+)-MS: Calcd C25H46N5O6Si2: 567.29. Found:
568.30 [M+H]+.
136.13–127.18 (CDMTr), 119.65 (C4), 113.69 (CDMTr
)
86.46 (C10), 83.71 (C40), 81.13 (C20), 72.33 (C200), 69.82
(C30), 64.01 (C50), 60.68 (C300), 55.48(O–CH3). MAL-
DI-MS: Calcd C41H38N6O8: 742.41. Found: 743.52
[M+H]+.
6.3.5. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-(phtha-
limidoethyl)-N6-dimethylformamidine adenosine (6). Dim-
ethylformamidine acetal (0.15 ml, 1.07 mmol) was
added to a solution of 5 (0.12 g, 0.16 mmol) in 10 ml
DMF. The mixture was stirred for 1 h at 60 ꢁC and
the solvents were concentrated under vacuum. The
resulting oil was purified by chromatography with 10%
MeOH in CH2Cl2 as the eluent to produce 0.11 g
(86%) 6 as a white foam.
6.3.8. 50,30-O-(tert-Butyldimethylsilyl)-20-O-(hydroxyeth-
yl)-adenosine (9). A portion of 8 (0.77 g, 1.35 mmol)
was dissolved in 10 ml ether and cooled to 0 ꢁC. After
adding Lithium aluminium hydride (0.14 g, 3.38 mmol)
the solution was stirred for 0.5 h at 0 ꢁC and quenched
by addition of saturated aqueous NH4Cl. After the
solution was warmed to room temperature, it was di-
luted with ether. The combined organic extracts were
washed with saturated aqueous NaCl, dried over
MgSO4 and concentrated under vacuum. The crude
product was purified by chromatography in 5% MeOH
in CH2Cl2 as the eluent to produce 0.61 g (86%) 9 as
fine needeld crystalls. 1H NMR (400 MHz, DMSO-
d6) d [ppm] 8.20 (s, 1H, H2), 8.01 (s, 1H, H8), 7.18
(s, 2H, NH2), 5.89 (d, 1H, J = 4.8 Hz, 10H), 4.52 (m,
1H, 20H), 4.45(m, 2H, 30H, OH), 3.82 (m, 2H, 200H),
3.38 (m, 3H, 40H, 50H), 0.96 (m, 18H, Htert-butyl),
0.08 (m, 12H, Hdimethylsilyl). 13C NMR (62,90 MHz,
DMSO-d6) d [ppm]: 156.07 (C6), 152.61 (C8), 149.18
(C5), 139.38 (C2), 119.08 (C4), 85.59 (C10), 84.52
(C40), 80.29 (C20), 71.79 (C30), 70.11 (C300), 61.97
(C200), 60.02 (C50), 25.76 (CTBS), 17.76 (CTBS), -5.12
(CTBS). MALDI-MS: Calcd C24H46N5O5Si2: 539.30.
Found: 540.08 [M+H]+.
1H NMR (400 MHz, DMSO-d6) d [ppm] 8.87 (s, 1H,
Hamidine), 8.27 (s, 1H, H2), 8.23 (s, 1H, H8), 7.76
(m, 4H, Hphthalimide), 7.35 (m, 8H, HDMTr), 6.79 (m,
5H, HDMTr), 5.98 (d, 1H, J = 5.7 Hz, 10H), 5.22 (d,
1H, J = 5.12 Hz, 30OH), 4.70 (t, 1H, J = 4.7 Hz,
20H), 4.42 (m, 1H, 30H), 3.91 (m, 3H, 40H, 50H),
3.72 (s, 6H, OCH3), 3.31 (m, 10H, 200H, 300H,
N(CH3)2). 13C NMR (100.62 MHz, DMSO-d6)
d
[ppm] 168.18 (C@O), 157,89 (Camidine), 156.57 (C6),
153.14 (C8), 149.70 (C5), 145.29 (CDMTr), 140.06
(C2), 136.13 (CDMTr), 130.17 (Cphthalimide), 128.21
(CDMTr), 119.65 (C4), 113.69 (CDMTr), 86.46 (C10),
83.72 (C40), 81.13 (C20), 72.33 (C200), 69.82 (C30),
64.01 (C50), 60.68 (C300), 55.48(O–CH3), 34.53
(N(CH3)2). MALDI-MS: Calcd C44H43N7O8: 797.44.
Found: 798.48 [M+H]+.
6.3.6. 30-O-(2-Cyanoethoxydiisoproylphosphine)-50-O-(4,40-
dimethoxytriphenylmethyl)-20-O-(phthalimidoethyl)-N6-dim-
ethylformamidine adenosine (7). Sym.-collidine (0.16 ml,
1.2 mmol) and 1-methylimidazole (5 ll, 0.062 mmol)
were added dropwise to a solution of 6 (0.1 g, 0.12
mmol) in 5 ml acetonitrile. After the mixture was cooled
to 0 ꢁC, 2-cyanoethyldiisopropylchloro-phosphorami-
dite (0.04 ml, 0.18 mmol) was added dropwise. The mix-
ture was stirred for 30 min at 0 ꢁC, 15 min at room
temperature and quenched with 1.5 ml saturated aque-
ous NaHCO3 and extracted with CH2Cl2. The combined
organic extracts were washed with 0.01 M citric acid,
dried over MgSO4 and concentrated under vacuum.
The resulting oil was purified by chromatography with
2% MeOH in CH2Cl2 as the eluent to produce 78 mg
(65%) 7 as a white foam. 31P NMR (400 MHz, CDCl3)
d [ppm] 141.55, 140.89 (ratio 1:1.5). MALDI-MS: Calcd
C53H60N9O9P: 997.53. Found: 998.56 [M+H]+.
6.3.9. 50,30-O-(tert-Butyldimethylsilyl)-20-O-(N,N0,N00-tri-
boc-guanidinoethyl)-adenosine (10).
A portion of 9
(0.7 g, 1.3 mmol) was dissolved in 45 ml of THF. Tri-
phenylphosphine (0.6 g, 1.95 mmol) and N,N0,N00tri-
boc-guanidine (1.4 g, 3.9 mmol) were added and cooled
to ꢀ5 ꢁC. After diethylazodicarboxylate (0.35 g,
1.95 mmol) was added dropwise to the mixture, it was
refluxed for 1.5 h at 80 ꢁC with a power P: 150W under
microwave conditions. The reaction was quenched by
adding saturated aqueous NaHCO3 and extracted with
CH2Cl2. The combined organic extracts washed with
saturated aqueous NaCl, dried over MgSO4 and concen-
trated under vacuum. The resulting oil was purified by
chromatography with 5% MeOH in CH2Cl2 as the elu-
ent to produce 1.05 g (92%) 10 as a white foam. 1H
NMR (250 MHz, DMSO-d6) d [ppm] 10.03 (br s, 1H,
NH), 8.15 (s, 1H, H2), 7.99 (s, 1H, H8), 7.18 (s, 2H,
6.3.7. 50,30-O-(tert-Butyldimethylsilyl)-20-O-(methoxy-
carbonylmethyl)-adenosine (8). tert-Butyldimethylsilyl
chloride (0.71 g, 4.7 mmol) and imidazole (0.31 g,
2.2 mmol) were added to a stirred solution of nucleoside
2 (0.5 g, 1.47 mmol) in 10 ml DMF. After 16 h, the sol-

526
D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
NH2), 5.88 (s, 1H, 10H), 4.51 (m, 2H, 20H, 30H), 3.78
(m, 2H, 200H), 3.54 (m, 5H, 40H, 50H, 300H), 1.22 (m,
27H, HBoc), 0.89 (m, 18H, Htert-butyl), 0.06 (m, 12H,
Hdimethylsilyl). 13C NMR (62,90 MHz, DMSO-d6) d
[ppm]: 159.67 (C@O), 156.07 (C6), 152.51 (C8), 151.89
(CGua), 148.99 (C5), 139.32 (C2), 119.18 (C4), 85.94
(C10), 84.08 (C40), 82.24 (C20), 70.80 (C30), 69.14
(C300), 61.93 (C200), 60.09 (C50), 30.66 (CBoc), 27.70
(CBoc), 25.76 (CTBS), 17.76 (CTBS), ꢀ5.12 (CTBS) MAL-
DI-MS: Calcd C40H72N8O10Si2: 880.49. Found: 881.58
[M+H]+.
6.3.12. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-(N,N0,N00-
tri-boc-guanidinoethyl)-N6-dimethylformamidine-adeno-
sine (13). Dimethylformamidine acetal (0.19 ml,
1.41 mmol) was added to a solution of 12 (0.2 g,
0.21 mmol) in 15 ml DMF. The mixture was stirred
for 1 h at 60 ꢁC and the solvents were removed under
vacuum. The resulting oil was purified by chromatog-
raphy with 5% MeOH in CH2Cl2 as the eluent to pro-
duce 0.18 g (86%) 13 as a white foam. 1H NMR
(250 MHz, Acetone-d6) d [ppm] 8.81 (s, 1H, Hamidine),
8.29 (s, 1H, H2), 8.09 (s, 1H, H8), 7.25 (m, 8H,
HDMTr), 6.71 (m, 5H, HDMTr), 6.09 (s, 1H, 10H),
4.55 (m, 2H, 20H, 30H), 3.98 (m, 5H, 40H, 50H,
200H,), 3.68 (s, 6H, OCH3), 3.31 (m, 2H, 300H), 3.13
(s, 3H, NCH3), 3.08 (s, 3H, NCH3), 1.32 (m, 27H,
HBoc). 13C NMR (62,90 MHz, Acetone-d6) d [ppm]
159.17 (Camidine), 158.95 (C@O), 156.15 (C6), 153.59
(C8), 153.06 (CGua), 146.15 (C5), 141.56 (CDMTr),
139.58 (C2), 135.54–126.60 (CDMTr), 118.38 (C4),
113.86 (CDMTr), 87.95 (C10), 86.95 (C40), 83.33 (C20),
69.34 (C30), 68.14 (C300), 64.51 (C200), 62.51 (C50),
55.49 (OCH3), 34.86 (N(CH3)2), 30.76 (CBoc), 27.75
(CBoc). MALDI-MS: Calcd C52H67N9O12: 1010.14.
Found: 1010.59.
6.3.10. 20-O-(N,N0,N00-tri-boc-guanidinoethyl)-adenosine
(11). TBAF (1 M) in THF (3.26 ml, 11.3 mmol) was
added dropwise to a solution of 10 (1 g, 1.13 mmol) in
25 ml THF cooled to 0 ꢁC. The solution was warmed
to room temperature and stirred for 20 h. The solvents
were removed under vacuum and the resulting oil was
purified by chromatography with 10% MeOH in CH2Cl2
as the eluent to produce 0.66 g (90%) 11 as a white foam.
1H NMR (250 MHz, DMSO-d6) d [ppm] 10.17 (br s, 1H,
NH), 8.32 (s, 1H, H2), 8.11 (s, 1H, H8), 7.32 (br s, 2H,
NH2), 5.99 (d, 1H, J = 5.05 Hz, 10H), 5.34 (m, 1H,
50OH), 5.07 (d, 1H, J = 5.37 Hz, 30OH), 4.44 (t, 1H,
J = 5.05 Hz, 20H), 4.33 (m, 1H, 30H), 3.94 (m, 1H,
40H), 3.60 (m, 6H, 50H, 200H, 300H), 1.35 (m, 27H, HBoc).
13C NMR (62,90 MHz, DMSO-d6) d [ppm] 159.67
(C@O), 156.16 (C6), 152.44 (C8), 152.03 (CGua),
148.78 (C5), 139.56 (C2), 119.35 (C4), 86.52 (C10),
85.42 (C40), 81.86 (C20), 69.31 (C30), 68.14 (C300), 61.93
(C200), 61.12 (C50), 30.65 (CBoc), 27.70 (CBoc). MALDI-
MS: Calcd C28H44N8O10: 652.70. Found: 653.82
[M+H]+.
6.3.13. 30-O-(2-Cyanoethoxydiisoproylphosphine)-50-O-
(4,40dimethoxytriphenylmethyl)-20-O-(N,N0, N00-tri-boc-
guanidinoethyl)-N6-dimethylformamidine-adenosine (14).
Sym. Collidine (0.13 ml, 1 mmol) and 1-methylimidazole
(4.2 ll, 0.052 mmol) were added dropwise to a solution
of 13 (0.1 g, 0.1 mmol) in 5 ml acetonitrile. After the
mixture was cooled to 0 ꢁC, 2-cyanoethyldiisopropyl-
chloro-phosphoramidite (0.03 ml, 0.15 mmol) was
added slowly and dropwise. The mixture was stirred
for 30 min at 0 ꢁC, 15 min at room temperature
quenched with 1.5 ml saturated aqueous NaHCO3 and
extracted with CH2Cl2. The combined organic extracts
were washed with 0.01 M citric acid, dried over MgSO4
and concentrated under vacuum. The resulting oil was
purified by chromatography with 2% MeOH in CH2Cl2
as the eluent to produce 78 mg (65%) 14 as a white foam.
31P NMR(400 MHz, CDCl3) d [ppm] 141.55, 140.89
(ratio 1:1,5). MALDI-MS: Calcd C61H84N11O13P:
1210.85. Found: 1210.98.
6.3.11. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-
(N,N0,N00-tri-boc-guanidinoethyl)
adenosine (12).
4,40Dimethoxytrityl-chloride (0.41 g, 1.17 mmol) and
NEt3 (0.24 ml, 1.47 mmol) were added to a solution
of 11 (0.64 g, 0.98mmol) in 10 ml pyridine cooled to
0 ꢁC. The solution was warmed to room temperature
and stirred for 24 h. The reaction was quenched by
adding saturated aqueous NaHCO3 and MeOH. The
resulting solution was extracted with CH2Cl2 and
the combined organic extracts were washed with satu-
rated aqueous NaCl, dried over MgSO4 and concen-
trated under vacuum. The residual yellow oil was
coevaporated two times with toluene and purified by
chromatography with 5% MeOH in CH2Cl2 as the
6.3.14. N6-dimethylformamidine-adenosine (15). Dimeth-
ylformamidine acetal (10 ml, 75 mmol) was added to a
solution of 1 (3 g, 11.25 mmol) in 50 ml DMF. The
mixture was stirred for 1h at 60 ꢁC and the solvents
were removed under vacuum to produce 3.58 g (99%)
1
eluent to produce 0.7 g (75%) 12 as a white foam. H
NMR (250 MHz, DMSO-d6) d [ppm] 10.20 (br s, 1H,
NH), 8.21 (s, 1H, H2), 8.10 (s, 1H, H8), 7.32 (m, 2H,
NH2), 7.20 (m, 8H, HDMTr), 6.82 (m, 5H, HDMTr),
6.07 (m, 1H, 10H), 5.10 (d, 1H, J = 6.32 Hz, 30OH),
4.55 (m, 2H, 20H, 30H), 4.06 (m, 2H, 200H), 3.68 (m,
9H, 40H, 50H, OCH3), 3.22 (m, 2H, 300H), 1.33 (m,
27H, HBoc)13C NMR (62,90 MHz, DMSO-d6) d [ppm]
158.55 (C@O), 156.10 (C6), 152.58 (C8), 152.08 (CGua),
148.78 (C5), 144.82 (CDMTr), 139.54 (C2), 135.54–
126.60 (CDMTr), 119.28 (C4), 113.05 (CDMTr), 85.37
(C10), 81.83 (C40), 80.88 (C20), 69.34 (C30), 68.14
(C300), 61.93 (C200), 61.12 (C50), 30.66 (CBoc), 27.75
(CBoc). MALDI-MS: Calcd C49H62N8O12: 954.45.
Found: 953.91 [M-H]+.
1
15 as a white solid. H NMR (250 MHz, DMSO-d6)
d [ppm] 8.97 (s, 1H, Hamidine), 8.53 (s, 1H, H2), 8.47
(s, 1H, H8), 5.97 (d, 1H, J = 6.0 Hz, 10H), 5.52 (d,
1H, J = 6.3 Hz, 20OH), 5.35 (t, 1H, J = 4.7 Hz,
50OH), 5.25(d, 1H, J = 4.7 Hz, 30OH), 4.67 (m, 1H,
20H), 4.22 (m, 1H, 30H), 4.02 (m, 1H, 40H), 3.65 (m,
2H, 50H), 3.26-3.19 (m, 6H, CH3). 13C NMR
(62.9 MHz, DMSO-d6)
d [ppm] 159.29 (Camidine),
158.14 (C6), 151.73 (C8), 151.18 (C5), 141.54 (C2),
125.92 (C4), 87.69 (C10), 85.73 (C40), 73.42 (C20),
70.52 (C30), 61.53 (C50), 34.55 ((CH3)2N) MALDI-
MS: Calcd C14H19N5O4: 321.33. Found: 322.35
[M+H]+.

D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
527
6.3.15. 50,30-O-(Tetraisopropyldisiloxane-1,3-diyl)-N6-dim-
ethylformamidine-adenosine (16). TIPDS-Cl2 (3.9 ml,
11.95 mmol) were added dropwise to a solution of 15
(3.5 g, 10.86 mmol) in 25 ml pyridine at 0 ꢁC. The solu-
tion was warmed to room temperature and stirred for
24 h, quenched with water and extracted with CH2Cl2.
The combined organic extracts were washed with satu-
rated aqueous NaCl, dried over MgSO4 and concen-
trated under vacuum. The residual oil was
coevaporated two times with toluene and purified by
chromatography with 5% MeOH in CH2Cl2 as the elu-
ent to produce 5.8 g (95%) 16 as a white foam. 1H
NMR (250 MHz, DMSO-d6) d [ppm] 8.91 (s, 1H,
Hamidine), 8.43 (s, 1H, H2), 8.39 (s, 1H, H8), 5.92 (s,
1H, 10H), 5.62 (d, 1H, J = 6.1 Hz, 20OH), 4.80 (m, 1H,
20H), 4.55 (m, 1H, 30H), 3.96 (m, 3H, 40H, 50H), 3.20
(s, 3H, CH3), 3.10 (s, 3H, CH3), 1.08 (m, 27H,
SiCH(CH3)2). 13C NMR ( 62.9 MHz, DMSO-d6) d
[ppm] 159.24 (Camidine), 157.92 (C6), 151.74 (C8), 150.62
(C5), 141.13 (C2), 125.94 (C4), 86.69 (C10), 80.78 (C40),
73.51 (C20), 69.85 (C30), 61.50 (C50), 34.57 ((CH3)2N),
16.89 (SiCHCH3), 12.24 (SiCHCH3) MALDI-MS: Calcd
C26H45N5O5Si2: 563.84. Found: 565.61 [M+2H]+.
NMR (62.9MHz, DMSO-d6) d [ppm] 159.30 (Camidine),
158.03 (C6), 151.81 (C8), 151.03 (C5), 141.30 (C2),
134.63 (C300), 125.75 (C4), 116.64 (C400), 86.21 (C10),
85.94 (C40), 80.33 (C20), 70.19 (C200), 68.94 (C30), 61.30
(C50),
C16H20N6O4: 360.16. Found: 361.27 [M+H]+.
34.55
((CH3)2N).
MALDI-MS:
Calcd
6.3.18. 50-O-(4,40-Dimethoxytriphenylmethyl)-20-O-(allyl)-
N6-dimethylformamidine-adenosine (19). 4,40Dimethoxy-
trityl-chloride (0.6 g, 1.73 mmol) and NEt3 (0.35 ml,
2.16 mmol) were added to a solution of 18 (0.52 g,
1.44 mmol) in 10 ml pyridine cooled to 0 ꢁC. The solu-
tion was warmed to room temperature and stirred for
24 h. The reaction was quenched by adding saturated
aqueous NaHCO3 and MeOH. The resulting solution
was extracted with CH2Cl2 and the combined organic
extracts were washed with saturated aqueous NaCl,
dried over MgSO4 and concentrated under vacuum.
The residual yellow oil was coevaporated two times with
toluene and purified via chromatography with 5%
MeOH in CH2Cl2 as the eluent to produce 0.73 g
(75%) 19 as a white foam. 1H NMR (250 MHz,
DMSO-d6) d [ppm] 8.90 (s, 1H, Hamidine), 8.35 (m, 2H,
H2, H8), 7.29 (m, 9H, HDMTr), 6.28 (m, 4H, HDMTr),
6.12 (d, 1H, J = 6.2Hz, 10H), 5.82 (m, 1H, 300H), 5.31
(d, 1H, J = 5.8 Hz, 30OH), 5.12 (m, 2H, 400H), 4.65 (m,
1H, 20H), 4.45 (m, 1H, 30H), 4.12 (m, 3H, 40H, 200H),
3.7 (m, 6H, OCH3), 3.33 (m, 2H, 50H), 3.21 (s, 3H,
CH3), 3.14 (s, 3H, CH3). 13C NMR (62.9 MHz,
DMSO-d6) d [ppm] 159.22 (Camidine), 158.03 (C6),
151.94 (C8), 151.15 (C5), 144.71 (CDMTr), 141.29 (C2),
134.46 (C300), 129.62–126.60 (CDMTr), 125.71 (C4),
116.76 (C400), 113.08 (CDMTr), 86.03 (C10), 85.47 (C40),
83.48 (C20), 70.41 (C200), 69.18 (C30), 63.46 (C50), 52.95
(OCH3) 34.55 ((CH3)2N). MALDI-MS: Calcd
C37H39N6O6: 663.37. Found: 664.21 [M+H]+.
6.3.16. 50,30-O-(Tetraisopropyldisiloxane-1,3-diyl)-20-O-
(allyl)-N6-dimethylformamidine-adenosine (17). A por-
tion of 16 (1.3 g, 2.3 mmol) was dissolved in a solution
of allyl-ethyl-carbonate (0.58 ml, 4.6 mmol) in 10 ml
THF. The resulting solution was added dropwise to a
suspension composed of tris(dibenzylidinacetone)-dipal-
ladium(0) (24.6 mg, 0.023 mmol) and 1,4- bis-
diphenylphosphino-butane (39.17 mg, 0.092 mmol) in
5 ml THF and stirred for 1 h at 70 ꢁC. The solution
was removed under vacuum and the residual green oil
was purified by chromatography with 5% MeOH in
1
CH2Cl2 to produce 1.1 g (79%) 17 as a white foam. H
NMR (250 MHz, DMSO-d6) d [ppm] 8.89 (s, 1H,
Hamidine), 8.32 (s, 1H, H2), 8.29 (s, 1H, H8), 6.02 (s,
1H, 10H), 5.91 (m, 1H, 300H), 5.21 (m, 2H, 400H), 4.51
(m, 1H, 30H), 4.32 (m, 2H, 200H), 3.89 (m, 3H, 40H,
50H), 3.21 (s, 3H, CH3), 3.12 (s, 3H, CH3), 1.12 (m,
27H, SiCH(CH3)2). 13C NMR (62.9 MHz, DMSO-d6)
d [ppm] 159.24 (Camidine), 157.90 (C6), 151.75 (C8),
150.48 (C5), 141.14 (C2), 134.15 (C300), 125.93 (C4),
116.41 (C400), 87.71 (C10), 80.67 (C40), 80.14 (C20),
70.97 (C200), 69.98 (C30), 59.93 (C50), 34.55 ((CH3)2N),
17.06 (SiCHCH3), 12.19 (SiCHCH3). MALDI-MS:
Calcd C29H49N5O5Si2: 603.90. Found: 606.08.
6.3.19. 30-O-(2-Cyanoethoxydiisoproylphosphine)-50-O-
(4, 40dimethoxytriphenylmethyl)-20-O-(allyl)-N6-dimeth-
ylformamidine-adenosine (20). Sym. Collidine (0.4 ml,
3 mmol) and 1-methylimidazole (12.6 ll, 0.16 mmol)
were added dropwise to a solution of 19 (0.2 g,
0.3 mmol) in 8 ml acetonitrile. After the mixture was
cooled to 0 ꢁC, 2-cyanoethyldiisopropylchloro-phospho-
ramidite (0.1 ml, 0.45 mmol) was added dropwise. The
mixture was stirred for 30 min at 0 ꢁC, quenched with
1.5 ml saturated aqueous NaHCO3 and extracted with
CH2Cl2. The combined organic extracts were washed
with 0.01 M citric acid, dried over MgSO4 and concen-
trated under vacuum. The resulting oil was purified by
chromatography with 2% MeOH in CH2Cl2 as the elu-
ent to produce 0.18 g (69%) 20 as a white foam. 31P
NMR(400 MHz, CDCl3) d [ppm] 150.75, 149.98 (ratio
1:1,5). MALDI-MS: Calcd C46H56N8O7P: 863.43.
Found: 864.43 [M+H]+.
6.3.17. 20-O-(allyl)-N6-dimethylformamidine-adenosine
(18). NEt3* 3HF (1.1 ml, 6.37 mmol) were added drop-
wise to a solution of 17 (1 g, 1.65 mmol) and NEt3
(0.37 ml, 2.73 mmol) in 20 ml THF and stirred for 1 h
at room temperature. The solvent was concentrated un-
der vacuum and the resulting oil was purified by chro-
matography with 10% MeOH in CH2Cl2 as the eluent
1
to produce 0.53 g (90%) 18 as a white foam. H NMR
6.3.20. 50,30-O-(Tetraisopropyldisiloxane-1,3-diyl)-20-O-
(cyanoethyl)-N6-dimethylformamidine-adenosine (21). A
portion of 16 (1 g, 1.77 mmol) was dissolved in 10 ml
tert-butanol. Acrylnitrile (1.9 ml, 35.4 mmol) and
Cs2CO3 (0.58 g, 1.77 mmol) were added to the solution
and stirred for 24 h at room temperature. After the reac-
tion was done the solution was filtrated over celite and
(250 MHz, DMSO-d6) d [ppm] 8.89 (s, 1H, Hamidine),
8.51 (s, 1H, H2), 8.42 (s, 1H, H8), 6.09 (d, 1H,
J = 6.3 Hz, 10H), 5.75 (m, 1H, 300H), 5.41 (m, 2H,
30OH, 50OH), 5.12 (m, 2H, 400H), 4.51 (m, 1H, 20H),
4.32 (m, 1H, 30H), 4.13 (m, 3H, 40H, 200H), 3.70 (m,
2H, 50H), 3.22 (s, 3H, CH3), 3.12 (s, 3H, CH3). 13C

528
D. Odadzic et al. / Bioorg. Med. Chem. 16 (2008) 518–529
washed with CH2Cl2 and concentrated under vacuum.
The resulting oil was purified by chromatography with
5% MeOH in CH2Cl2 as the eluent to produce 0.76 g
(70%) 17 as a white foam. 1H NMR (250 MHz,
DMSO-d6) d [ppm] 8.89 (s, 1H, Hamidine), 8.56 (s, 1H,
H2), 8.44 (s, 1H, H8), 6.06 (d, 1H, J = 5.6 Hz, 10H),
5.02 (m, 1H, 20H), 4.61 (m, 1H, 30H), 3.91 (m, 5H,
40H, 50H, 200H), 3.23 (s, 3H, CH3), 3.12 (s, 3H, CH3),
2.82 (m, 2H, 300H), 1.04 (m, 28H, SiCH(CH3)2). 13C
NMR (62.9 MHz, DMSO-d6) d [ppm] 159.21 (Camidine),
157.89 (C6), 151.72 (C8), 150.51 (C5), 141.23 (C2),
125.96 (C4), 118.87 (CN), 87.61(C10), 80.52 (C40),
70.11 (C20), 65.68 (C30), 65.04 (C200), 60.08 (C50), 34.55
((CH3)2N), 18.11 (C300), 17.25 (17.06 (SiCHCH3), 12.33
(SiCHCH3). MALDI-MS: Calcd C28H47N7O5Si2:
617.44. Found: 618.95 [M+H]+.
113.85 (CDMTr), 87.88 (C10), 86.97 (C40), 82.33 (C20),
70.80 (C30), 66.61 (C200), 64.39 (C50), 55.48 (OCH3),
34.88 ((CH3)2N), 19.10 (C300). MALDI-MS: Calcd
C37H39N7O6: 677.37. Found: 678.52 [M+H]+.
6.3.23. 30-O-(2-cyanoethoxydiisoproylphosphine)-50-O-(4,
40dimethoxytriphenylmethyl)-20-O-(cyanoethyl)-N6-dimet-
hylformamidine-adenosine (24). Sym. Collidine (0.31 ml,
2.36 mmol) and 1-methylimidazole (9.9 ll, 0.123 mmol)
were added dropwise to a solution of 23 (0.16 g,
0.24 mmol) in 8 ml acetonitrile. After the mixture was
cooled to 0 ꢁC, 2-cyanoethyldiisopropylchloro-phospho-
ramidite (0.08 ml, 0.35 mmol) was added dropwise. The
mixture was stirred for 30 min at 0 ꢁC, quenched with
1.5 ml saturated aqueous NaHCO3 and extracted with
CH2Cl2. The combined organic extracts were washed
with 0.01 M citric acid, dried over MgSO4 and concen-
trated under vacuum. The resulting oil was purified by
chromatography with 5% MeOH in CH2Cl2 as the elu-
ent to produce 0.14 g (69%) 24 as a white foam. 31P
NMR(400 MHz, CDCl3) d [ppm] 150.10, 149.89 (ratio
1:1,5) MALDI-MS: Calcd C46H56N9O7P: 877.43.
Found: 878.43 [M+H]+.
6.3.21. 20-O-(Cyanoethyl)-N6-dimethylformamidine-aden-
osine (22). NEt3* 3HF (1.07 ml, 6.23 mmol) were added
dropwise to a solution of 21 (1.1 g, 1.78 mmol) and NEt3
(0.36 ml, 2.67 mmol) in 20 ml THF and stirred for 1 h at
room temperature. The solvent was removed under vac-
uum and the resulting oil was purified by chromatogra-
phy with 10% MeOH in CH2Cl2 as the eluent to produce
1
0.6 g (90%) 22 as a white foam. H NMR (250 MHz,
DMSO-d6) d [ppm] 8.91 (s,1H,Hamidine), 8.57 (s, 1H,
H2), 8.42 (s, 1H, H8), 6.06 (d, 1H, J = 5.5 Hz, 10H),
5.35 (m, 2H, 30OH, 50OH), 4.61 (m, 1H, 20H), 4.35
(m, 1H, 30H), 3.98 (m, 1H, 40H), 3.72 (m, 4H, 50H,
200H), 3.21 (s, 3H, CH3), 3.10 (s, 3H, CH3), 2.82 (m,
2H, 300H). 13C NMR (62.9 MHz, DMSO-d6) d [ppm]
159.90 (Camidine), 158.04 (C6), 151.87 (C8), 151.10
(C5), 141.25 (C2), 125.77 (C4), 118.83 (CN), 85.93
(C10), 85.75 (C40), 80.99 (C20), 68.74 (C30), 64.70
(C200), 61.15 (C50), 34.55 ((CH3)2N), 18.11 (C300). MAL-
DI-MS: Calcd C16H21N7O4: 375.16. Found: 376.59
[M+H]+.
Acknowledgment
We thank the ‘‘Sonderforschungsbereich SFB579’’ and
the RIGHT EU-Project No. LSHB-CT-2004-005276
for financial support.
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