Synthesis of 2′-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virus

Article abstract of DOI:10.1016/j.bmc.2011.12.024

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3′ ends. To im

Full text of DOI:10.1016/j.bmc.2011.12.024

Bioorganic & Medicinal Chemistry 20 (2012) 1594–1606
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 20-O-guanidinopropyl-modified nucleoside phosphoramidites
and their incorporation into siRNAs targeting hepatitis B virus
Jolanta Brzezinska a, Jennifer D’Onofrio a, Maximilian C.R. Buff a, Justin Hean b, Abdullah Ely b,
Musa Marimani b, Patrick Arbuthnot b, Joachim W. Engels a,
⇑
a Goethe-University, Institute of Organic Chemistry & Chemical Biology, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany
b Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of
pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approxi-
mately 21 bp with 2 nt overhangs at the 30 ends. To improve efficacy of siRNAs, chemical modification
at the 20-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immu-
nostimulation have been demonstrated using this approach. Although promising, efficient and controlled
delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the
potential utility of introducing positively charged groups at the 20 position, our investigations aimed at
assessing efficacy of novel siRNAs containing 20-O-guanidinopropyl (GP) moieties. We describe the for-
mation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acry-
lonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to
generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demon-
strated that the GP modifications improved silencing. Moreover, thermodynamic stability was not
affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide
strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that
modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic
application of synthetic RNAi activators.
Received 1 November 2011
Revised 12 December 2011
Accepted 13 December 2011
Available online 21 December 2011
Keywords:
siRNA
RNA synthesis
20-Modification
HBV
Guanidinopropyl
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
oligocationic compounds such as spermidine.8 However, success
using these methods has been variable. To overcome difficulties of
Use of synthetic small interfering RNAs (siRNAs) to trigger RNA
interference- (RNAi-) mediated gene silencing has shown consider-
able potential for therapeutic application.1–3 Typically, siRNAs are
synthetic mimics of natural Dicer products and comprise 21–25 nt
duplexes with 2 nt 30 overhangs. Progress with use of synthetic siR-
NAs has profited from vast experience gained from developing anti-
sense RNA molecules. Consequently advances have been rapid and
improving siRNA efficacy has benefited from valuable biological
and synthetic chemistry insights. Advantages of synthetic siRNAs
over expressed RNAi activators are that they are amenable to chem-
ical modification to improve stability, safety and specificity.4,5 Also,
controlled large scale preparation necessary for clinical use is feasi-
ble with chemical synthetic procedures. Nevertheless, despite sig-
nificant advances, the delivery of these polyanionic nucleic acids
across lipid-rich cell membranes remains problematic. Vectors used
to transport synthetic RNAi activators to target cells have included
cationic lipid-containing lipoplexes,6 conjugations to peptides7 or
the excessive negative charge of nucleic acids, while at the same
time improving thermal and serum stability, we previously
investigated an approach that entailed 20-modification of ribose
with cationic groups.9,10 Initially we generated always 20-O-amino-
ethyl-adenosine and 20-O-aminoethyl uridine. Synthesis entailed
initial alkylation by methyl bromoacetate, which was followed by
a series of transformation reactions. Using a luciferase reporter as-
say to measure knockdown, it was demonstrated that the 20-O-ami-
noethyl modifications were at least as efficient as 20-OMe siRNA
modifications. An important property of the 20-O-aminoethyl deriv-
atives was their ability to rescue less active siRNAs when the chem-
ical modifications were placed at the 30 end of the siRNA passenger
strand.11 Subsequently this approach was advanced by developing
methods that enabled successful alkylation of all four ribonucleo-
sides.12 This was achieved using phalimidoethyltriflate as an alkyl-
ating agent and with this methodology all four phosphoramidites
bearing 20-O-aminoethyl side chains were formed. Although
encouraging, a problem of using these siRNA reagents is that the
yields of the multistep chemical synthesis are typically low. More-
over scaling up the synthesis reaction is difficult. To address these
⇑
Corresponding author. Tel.: +49 69 798 29150; fax: +49 69 798 29148.
E-mail address: Joachim.Engels@chemie.uni-frankfurt.de (J.W. Engels).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.024

J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
1595
concerns, we have investigated utility of an alternative 20-O-guani-
dinopropyl (GP) nucleoside modification method. Using the novel
approach reported here, we describe the formation of all four GP-
modified nucleosides using the synthesis sequence of Michael addi-
tion with acrylonitrile13–15 followed by Raney-Ni reduction16 and
guanidinylation. Efficiency of the GP siRNAs was assessed in a cell
culture model of hepatitis B virus (HBV) replication using target se-
quences that have previously been shown to be suitable for RNAi-
based inhibition of viral replication.17–20 Results demonstrate more
effective silencing of markers of viral replication than unmodified
counterparts. Moreover, the GP-modified siRNAs were more stable
to serum conditions than the unmodified controls.
with dimethylaminomethylene groups employing standard condi-
tions and a benzoyl group was attached to N3-position of U using
the two phase system reported by Sekine.22 The resulting nucleo-
tide precursors (1a–4a) were then subjected to the first crucial step
of the 20-O-guanidinopropyl derivatisation. Employing the proce-
dure reported by Sekine et al.,23 a Michael addition under mild
conditions (Cs2CO3, tert-butanol, room temperature) was per-
formed using acrylonitrile to obtain the 20-O-cyanoethyl deriva-
tives. In a subsequent step the dimethylaminomethylene group
of the A and C derivatives was removed with hydrazine to form
the 20-O-cyanoethyl derivatives 1b and 2b. This additional depro-
tection step was necessary to avoid formation of a mixture of
dimethylaminomethylene protected and unprotected derivatives
that result from proceeding directly to the next reduction step.
For the uridine derivative 3b no intermediate deprotection of the
N3-benzoyl group was necessary. This is because the benzoyl group
was completely removed under the ammonia conditions of the fol-
lowing step. The O6-TPS group of the guanosine derivative was re-
moved without further purification of the Michael reaction
product. This was achieved after filtration and evaporation of sol-
vents using formic acid in a mixture of dioxane and water to yield
the 20-O-cyanoethyl-guanosine derivative 4b.
2. Results
2.1. Synthesis of the four 20-O-guanidinopropyl-nucleoside-
phosphoramidites
Each of the four 20-O-guanidinopropyl-nucleoside phospho-
ramidites was synthesised using essentially analogous methodol-
ogy. The synthesis of the adenosine (A), cytidine (C) and uridine
(U) derivatives is depicted in Scheme 1. Since a different protecting
group strategy was employed to synthesise the guanosine (G)
derivative, it is shown in a separate scheme (Scheme 2). Each
synthesis was initiated by simultaneous protection of 50- and the
30-OH-groups with 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPS)
(for A,C and U) or di-tert-butylsilanediyl (DTBS) for guanosine.
DTBS was selected for protection of G as this group has been
reported to improve selectivity for the subsequent 2,4,6-tri-
isopropylbenzenesulfonyl (TPS) protection of O6-position of guano-
sine.21 The exocyclic amino functions of A and C were protected
In the next step, the 20-O-cyanoethyl group was transformed
into a 20-O-aminopropyl group. Reduction with hydrogen (30 bar)
with Raney-nickel as catalyst in ammonia and methanol was used
to achieve this according to a procedure we previously described.24
The hydrogenation step was sensitive to reaction conditions such
as the ratio of amount of starting material to catalyst, the size of
the autoclave employed and reaction time. Under optimised condi-
tions, yields from reduction of each nucleotide derivative were
moderate (about 50%). A loss of the desired product was also
Scheme 1. Synthesis of the 20-O-guanidinopropyl adenosine-, cytidine- and uridine- phosphoramidites for oligoribonucleotide synthesis. (i) acrylonitrile, Cs2CO3, tert-butyl
alcohol, rt; (ii) H2N–NH2ꢀH2O, methanol, rt (adenosine and cytidine derivative); no deprotection of the uridine derivative; (iii) H2 (30 bar), NH3, methanol, 30–60 min, rt; (iv)
N,N0-di-Boc-N00-triflylguanidine, Et3N, CH2Cl2, 0 °C (30 min) to rt (30 min); (v) DMF-dimethyl diacetale, methanol, rt (adenosine derivative); benzoyl chloride, pyridine, 0 °C
(30 min) to rt (30 min) (cytidine derivative); no protection group was applied to the uridine derivative; (vi) Et3Nꢀ3HF, THF, rt; (vii) 4,40-dimethoxytrityl chloride, pyridine, rt;
(viii) 2-cyanoethyl N,N,N0,N0-tetraisopropyl phosphane, 4,5-dicyanoimidazole, CH2Cl2, rt.
Scheme 2. Synthesis of the 20-O-guanidinopropyl guanosine phosphoramidite for oligoribonucleotide synthesis. (i) Acrylonitrile, Cs2CO3, tert-butyl alcohol, rt; (ii) formic acid
(70%), dioxane/water; (iii) H2 (30 bar), NH3, methanol, 30–60 min, rt; (iv) N,N0-di-Boc-N00-triflylguanidine, Et3N, CH2Cl2, 0 °C (30 min) to rt (30 min); (v) isobutyryl chloride,
pyridine, 0 °C (1 h) to rt (1 h); (vi) Et3Nꢀ3HF, THF, rt; (vii) 4,40-dimethoxytrityl chloride, pyridine, rt; (viii) 2-cyanoethyl N,N,N0,N0-tetraisopropyl phosphane, 4,5-
dicyanoimidazole, CH2Cl2, rt.

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the 20-O-guanidinopropyl uridine phosphoramidite was performed
in eight steps with an overall yield of 11.8% (3d).
Table 1
Synthesised GP modified oligonucleotides
The obtained phosphoramidites were used for synthesis of the
GP-modified siRNA antisense strands depicted in Table 1 and the
GP-modified oligonucleotides for melting point studies depicted
in Table 2. For the modified phosphoramidites a prolonged cou-
pling step of 25 min was used. To ensure complete cleavage of
the boc groups, an additional deprotection step of 30 min with
3% trichloroacetic acid in dichloromethane was applied after com-
pletion of oligonucleotide synthesis. The boc group was chosen for
protection of the guanidine moiety due to its compatibility con-
cerning the protecting group strategy that was used for synthesis
of the monomers. Although partial cleavage of the boc groups dur-
ing RNA-synthesis is expected in consequence of the acidic depro-
tection step of each cycle, no side reaction of the free guanidino
groups was observed. According to the anion-exchange chromato-
grams of the deprotected RNA (an exemplary chromatogram is
shown in the Supplementary data), only one main product has
formed. After purification the oligonucleotides were obtained with
Name
Sequence
GP2 siRNA3
GP3 siRNA3
GP4 siRNA3
GP5 siRNA3
GP6 siRNA3
GP7 siRNA3
GP8 siRNA3
GP13 siRNA3
5-UUGP G AAG UAU GCC UCA AGG UCG-30
5-UUGGP AAG UAU GCC UCA AGG UCG-30
5-UUG AGP AG UAU GCC UCA AGG UCG-30
5-UUG AAGP G UAU GCC UCA AGG UCG-30
5-UUG AAGGP UAU GCC UCA AGG UCG-30
5-UUG AAG UGP AU GCC UCA AGG UCG-30
5-UUG AAG UAGP U GCC UCA AGG UCG-30
5-UUG AAG UAU GCC UGP CA AGG UCG-30
Table 2
Effect of GP modification on duplex stability with complementary RNA (50-GGCAU-
ACUUCAA-30)
Oligo
Sequence
Tm [°C]
D
Tm [°C]
ON 1
ON 2
ON 3
ON 4
ON 5
ON 6
50-UUG AAG UAU GCC-30
54.9
54.5
52.5
54.4
54.6
54.4
—
50-UUGGP AAG UAU GCC-30
50-UUG AAGGP UAU GCC-30
50-UUG AAG UAUGP GCC-30
50-UUGGP AAGGP UAU GCC-30
50-UUGGP AAGGP UAUGP GCC-30
ꢁ0.4
ꢁ2.4
ꢁ0.5
ꢁ0.3
ꢁ0.5
10% to 40% yield (1 lmol synthesis scale).
2.2. Hybridization studies
The influence of GP-modified nucleosides on thermal stability
of different RNA duplexes was examined. For this purpose, the
GGP and UGP modified phosphoramidites were inserted into
12mer RNA (ON2–ON6) and the duplex melting point measured.
As shown in Table 2, the presence of GP group in oligoribonucleo-
tides did not significantly affect the stability of duplexes, although
slightly destabilized them. Guanidinopropyl modified building
blocks give almost the same Tm value for single, double and triple
substituted oligonucleotides.
confirmed by the observation that part of the amino compound
was not released from the catalyst during filtration, despite being
subjected to several washes with methanol. To minimise losses
the crude unpurified 20-O-aminopropyl compounds were used to
introduce the guanidino groups. N,N0-di-Boc-N00-triflylguanidine
was employed as guanidinylation agent. The procedure we em-
ployed was initially reported by Goodman et al. in 1998 and the re-
agent is now commercially available.25 Our previous studies
showed that the boc groups are cleaved under the repetitive depro-
tection conditions during oligonucleotide synthesis when employ-
ing the TBDMS-phosphoramidite method. Also, the guanidino
group undergoes no side reaction during the solid phase synthe-
sis.26 The guanidinylation took place with good yields (70% for 1c
(A), 60% for 2c (C), around 60% for 3c (U) and approximately 90%
for 4c (G)).
After successful guanidinylation, established reaction condi-
tions were applied to synthesize the desired phosphoramidites
(1d– 4d). This entailed use of protection groups that were suitable
for the TBDMS method of oligoribonucleotide synthesis. The A
derivative was protected with dimethylaminomethylene at the
N6-position, and the exocyclic amino function of the C derivative
was protected with a benzoyl group. The N2-position of the G
derivative was protected with an isobutyryl group. However, under
the reaction conditions we employed, a mixture of the desired G
derivative product, as well as a compound with an additional iso-
butyryl group on the non-boc-protected nitrogen of the guanidino
group, were obtained. It was very difficult to separate this double
isobutyryl modified compound using chromatography. However,
since it would be cleaved during the ammonia deprotection step
at the completion of oligonucleotide synthesis, we utilised this
mixture of 4d and 4d⁄ for solid phase oligonucleotide synthesis.
To synthesise U derivatives, no further protection was necessary.
For synthesis of all of the 20-O-guanidinopropyl phosphoramidites,
removal of silyl protecting groups was achieved with Et3Nꢀ3HF. The
50-OH-group was protected with a 4,40-dimethoxytrityl group and
in a last step the 30-OH group was converted to a phosphoramidite
using 2-cyanoethyl N,N,N0,N0-tetraisopropylamino phosphane and
4,5-dicyanoimidazole as activator. Starting with the adenosine,
cytidine and guanosine nucleosides, synthesis of the 20-O-guanidi-
nopropyl phosphoramidites took place in 10 steps and provided
overall yields of 15.4% (1d), 6.3% (2d) and 7.5% (4d). Synthesis of
Interestingly, in the case when a GP modification of G was
placed in a central position, the Tm decreased more significantly
(D
Tm = ꢁ2.4 °C).
The results indicate that the thermodynamic effect of GP group
is more or less independent on the placement of the modification
and modified nucleoside. While more modifications were incorpo-
rated, substitutions were not adjacent, additional destabilizing ef-
fect was not observed (ON5 and ON6, Table 2). Moreover, for the
modified oligonucleotides bearing more than one GP residue, high
binding affinity to the complementary strand was unaffected.
2.3. Inhibition of Firefly luciferase activity in transfected cells
Initially, to measure knockdown efficiency of GP-modified siR-
NAs in situ, HEK293 cells were co-transfected with RNAi activators
together with a reporter gene plasmid (psiCHECK-HBx)20 (Fig. 1).
The siRNAs targeted a single sequence of the X open reading frame
(ORF) of HBV (HBx) that has previously been shown to be an effec-
tive cognate for RNAi-based silencing.27 Each of the siRNAs differed
with respect to location of the GP modification, and these were
within the seed region or at nucleotide 13 of the antisense strand
of the siRNA duplex. siRNAs have been named according to the
positioning of the GP modifications from the 50 end of the intended
guide strand. In psiCHECK-HBx, the viral target sequence was lo-
cated in the Renilla transcript but downstream of the reporter
ORF (Fig. 1A). Expression of Firefly luciferase is constitutively ac-
tive to enable correction for variations in transfection efficiency.
The ratio of Renilla to Firefly luciferase activity was used to assess
knockdown efficacy. Compared to a scrambled siRNA control, anal-
ysis showed that the Firefly luciferase activity was diminished by
approximately 70% when co-transfected with the unmodified siR-
NA (Fig. 1B). There was some variation in the efficacy of the inhibi-
tion of reporter gene activity that was dependent on the position of
the chemically modified siRNAs. Knockdown efficacy was weakest

J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
1597
Figure 1. Dual luciferase assay to determine efficacy of GP antiHBV siRNAs. (A)
Schematic illustration of dual luciferase reporter plasmid. The HBx target sequence
was inserted downstream of the hRLuc ORF. Renilla luciferase activity was used as
an indicator of target silencing and efficacy was determined relative to activity of
constitutively expressed Firefly luciferase. (B) Ratio of Renilla to Firefly luciferase
activity following cotransfection with indicated siRNAs together with dual lucifer-
ase reporter plasmid. Controls included a mock transfection in which inert plasmid
DNA was substituted for siRNA as well as a scrambled siRNA that did not have
complementary sequences to the HBx target. Data are represented as mean ratios of
Renilla to Firefly luciferase activity ( SEM) and are normalised relative to the mock
treated cells. Differences were considered statistical significant when the p value,
determined according to the Student’s 2 tailed paired t-test, was less than 0.05.
Figure 2. Inhibition of HBV replication by antiHBV siRNAs in cultured cells. A.
Illustration of the HBV replication competent plasmid, pCH-9/3091, which was used
to transfect liver-derived Huh7 cells in culture. (B) The concentration of HBsAg was
measured in cell culture supernatants following co-transfection GP-modified
siRNAs. Values are given as relative OD readings from the ELISA assay. Unmodified
siRNA did not include GP residues. Controls included a mock transfection in which
inert plasmid DNA was substituted for siRNA as well as a scrambled siRNA that did
not have complementary sequences to the HBx target. Data are represented as mean
ratios of Renilla to Firefly luciferase activity ( SEM) and are normalised relative to
the mock treated cells. Differences were considered statistical significant when the
p value, determined according to the Student’s 2 tailed paired t-test, was less than
0.05.
with GP2 siRNA3, when the GP modification was placed at nucleo-
tide 2 of the siRNA antisense sequence. siRNAs with the modifica-
tion at positions 5 and 6 (GP5 siRNA3 and GP6 siRNA3) achieved
most effective knockdown of reporter gene expression which was
similar to that of the unmodified siRNA. A siRNA with the GP mod-
ification placed outside of the seed region at nucleotide 13 also
achieved knockdown of 75%. GP modifications in antiHBV siRNA
sequences are therefore compatible with effective target silencing,
but position within the seed of the antisense guide influences
efficacy.
is unclear but may result from better GP2 siRNA3 target accessibil-
ity in the context of the natural HBV transcripts. Overall, these data
support the notion that seed region GP modifications are compat-
ible with target silencing that is similar or more effective than
unmodified siRNAs.
2.5. Stability of GP-modified siRNAs in 80% FCS
2.4. Inhibition of HBV surface antigen (HBsAg) secretion from
transfected cells by GP-modified siRNAs
siRNAs containing GP modifications were incubated in the pres-
ence or absence of 80% foetal calf serum (FCS) for time intervals of
0–24 h to assess their stability (Fig. 3). siRNAs were detected using
polyacrylamide gel electrophoresis and staining with ethidium
bromide. Bands corresponding to siRNAs were quantified to deter-
mine stability and FCS resistance. Analysis revealed that unmodi-
fied siRNA3 was stable for 24 h when maintained in DMEM
tissue culture medium that did not include FCS. However, rapid
degradation of siRNA occurred in the presence of FCS, and approx-
imately 10% of the input siRNA remained at 5 h. Analysis of stabil-
ity of GP2 siRNA3, GP3 siRNA3, GP4 siRNA3, GP5 siRNA3 and GP6
siRNA3 showed a similar rapid degradation. When the GP modifi-
cations were placed further from the 50 end of the antisense strand
of the siRNA (GP7 siRNA3, GP8 siRNA3 and GP13 siRNA3) slower
degradation of the siRNAs was observed. With these siRNAs, at
the time point of 5 h approximately 40% of the input siRNA re-
mained intact. Stability is therefore improved by including GP
To assess efficacy against HBV replication in vitro, Huh7 liver-
derived cells were co-transfected with siRNAs together with the
pCH-9/3091 HBV replication competent target plasmid
(Fig. 2A).28 Compared to HBsAg concentration in the culture super-
natant of cells treated with scrambled siRNA, knockdown of up to
85% of viral antigen secretion was achieved by GP-modified siRNAs
(Fig. 2B). The unmodified siRNA was slightly less effective than the
siRNAs containing GP moieties. Of the modified siRNAs, positioning
of the GP residue at nucleotides 5 or 6 (GP5 siRNA3 and GP6 siR-
NA3) resulted in the most effective suppression of HBsAg secretion
(approximately 90%). These data correlate with observations using
the reporter gene knockdown assay. Interestingly, GP2 siRNA3
inhibited HBsAg secretion from transfected cells more effectively
than it did Renilla luciferase activity. The reason for this difference

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J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
based on Michael addition of suitably protected nucleosides with
acrylonitrile. The following Raney-Ni reduction results in the pro-
pylamino derivatives that are finally guanidinylated by triflylgua-
nidine. Different chemical structures of each of the bases require
that the protecting group strategies differ slightly between the four
nucleosides. Standard phosphitylation provides the appropriate
phosphoramidites 1d–4d, which are ready for solid phase oligonu-
cleotide synthesis (see Tables 1 and 2). Using this reaction se-
quence, this is the first time that a full set of phosphoramidites
modified with guanidino groups at the 20 position is provided for
incorporation in synthetic oligonucleotides. Synthesis of modified
oligonucleotides was carried out under standard conditions of
phosphoramidite chemistry. Moreover coupling efficiency of the
GP-modified phosphoramidites was as good as for the unmodified
phosphoramidites.
An additional advantage of the synthetic procedures described
here is that it is possible to introduce diversification at the
20-O-aminopropyl site of our compounds. With common peptide-
coupling reagents, such as carbodiimides and 1-hydroxybenzo-
triazoles, the 20-O-aminopropyl group can readily be modified with
carboxylic acid derivatives. These include amino acids, fatty acids
or carboxy-modified spermine to obtain more cationic or more
lipophilic oligonucleotides.24 Also, protection of the amino group
with a trifluoroacetyl group during oligonucleotide solid phase
synthesis would enable postsynthetic labelling with amino-reac-
tive fluorophore derivatives (e.g., NHS-esters or isothiocyanates)
or reaction with cross linkers to connect nucleic acids to carrier
molecules, for example, cell penetrating peptides.
Figure 3. Assessment of stability of GP-modified siRNAs. The panel of GP-modified
siRNAs was incubated with DMEM alone, or DMEM with 80% foetal calf serum, for
times ranging from 0 to 24 h. Thereafter degradation of siRNAs was assessed using
polyacrylamide gel electrophoresis with ethidium bromide staining.
The melting behaviour of the duplex oligonucleotides showed
that the melting temperature was independent of the placement
and number of GP modifications (Table 2). For this analysis, we
chose a 12-mer with a sequence that was identical to the seed se-
quence of our siRNA. This shorter sequence, with a Tm of approxi-
mately 55 °C, was better for demonstrating smaller changes in
melting properties. The results concur with hybridization analysis
of oligonucleotides containing 20-O-aminopropyl (AP) groups.30
Incorporation of one AP unit at the 30-end or in the middle of an oli-
gomer reduces the Tm of a RNA duplex. Duplex stabilization was
observed in RNA containing AP groups at adjacent sites or within
an entire strand. Molecular dynamic and NMR data indicate that
no strong electrostatic interaction or hydrogen bonding is formed
as a result of flexibility of aminoalkyl chains.30 Another possibility
that has been proposed is that some stabilizing effect might be
conferred by hydration of the AP.31,32 For the guanidino group re-
ported here, there are three nitrogen atoms in a plane which are
protonated over a wide pH range. The individual contributions of
GP-RNA to thermodynamic stability are not clear yet but nanosec-
ond molecular modelling points to high flexibility in water. (Villa
and Stock, unpublished)
When investigating the potential therapeutic utility of a new
chemical modification to siRNAs, it is desirable to verify that
silencing is maintained in a clinically relevant model. In this study,
we assessed the efficacy in a model of HBV infection. Persistence of
HBV is an important global cause of public health problems (re-
viewed in Ref. 33). Cirrhosis and liver cancer, which are potentially
fatal complications of HBV persistence, occur frequently. There is a
need to develop more effective antiHBV treatments to eliminate
these problems and harnessing the RNAi pathway to counter the
infection has shown promise.34 To assess silencing by GP-modified
siRNAs, a panel of silencing sequences was tested against a previ-
ously described susceptible HBV target.27 Evidence from this study
shows that incorporation of GP groups at the 20 ribose position en-
hances HBV silencing, while also improving stability. Interaction of
sequences within the guide seed region and the target are critically
important to effect silencing. Chemical modification in the guide
seed potentially compromises the interaction of the guide with
modifications, but location of these moieties to central regions of
the siRNAs is important to confer this property.
3. Discussion
Successful preparation of GP phosphoramidite nucleosides for
incorporation into siRNAs presents a novel approach to utilising
chemical modification for enhancement of efficacy of synthetic siR-
NAs. Ideally, for gene silencing sequences to be therapeutically
applicable they should be stable, amenable to efficient delivery
to target tissues, effective and specific to their targets. Alterations
to siRNAs have typically involved 20-OH modification of the ribose
groups (11 and reviewed in Refs. 4,5). Changes at this site are useful
for improving overall siRNA efficacy for several reasons. The 20-OH
may cause nucleophilic attack and removal of the hydroxyl group
limits susceptibility of siRNAs to degradation. Alterations at the
20-OH of ribose may also attenuate toxic innate immune responses
of duplex RNA by preventing interaction with intracellular Toll Like
Receptors (TLRs). Furthermore, 20-OH modification may influence
nucleotide stacking to enhance interaction of the siRNA guide with
its target. Alterations to the 20-OH that have been reported to date
have included 20-F, 20-methoxy ribosyl (20-OMe) and locked nucleic
acid modifications.5,11 Recently a 20-azido modification was re-
ported, that not only replaces the 20-OH group but can also be used
for postsynthetic labelling of RNA.29 Although improvements in
siRNA stability, efficacy and immunostimulatory effects have been
reported, difficulties with efficient regulation of nucleic acid deliv-
ery remain. The GP modification reported here presents a novel ap-
proach that has distinct properties that potentially have
advantages over previously described 20-OH modifications. The
three amino groups of the GP moiety together with the three car-
bon atom propyl linker provide a flexible charge-neutralising
group.
Chemical synthesis of GP-modified nucleosides is straightfor-
ward and comprises the following crucial steps. Cyanoethylation

J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
1599
its cognate. Our data demonstrate that silencing efficacy of the in-
tended target is retained after incorporating the GP residues into
each of the nucleotides comprising the guide strand seed. This
observation is in accordance with previous reports showing mini-
mal attenuation of intended target silencing caused by modifica-
tion in the seed region.35,36 Complete base pair matching within
the remainder of the siRNA guide and intended target compensates
for disruption that may occur in the seed.
stirred vigorously at room temperature for 3 h. The reaction mix-
ture was filtered and the residue was washed with dichlorometh-
ane. The filtrate was evaporated and the residue was purified using
column chromatography with ethyl acetate/methanol (99:1–95:5,
v/v) to give 3.28 g (87%) of the product. 1H NMR (400 MHz,
DMSO-d6) d [ppm] 8.90 (s, 1H, admidine-H), 8.34 (s, 1H, H2 or
H8), 8.32 (s, 1H, H2 or H8), 6.02–6.01 (m, 1H, H10), 5.05–5.01 (m,
1H, H30), 4.64–4.62 (m, 1H, H20), 4.08–3.84 (m, 5H, H40, 2 ꢂ H50,
O–CH2–CH2–CN), 3.20 (s, 3H, N–CH3), 3.13 (s, 3H, N–CH3), 2.83–
2.80 (m, 2H, O–CH2–CH2–CN), 1.10–1.00 (m, 28H, tetraisopropyl-
CH and -CH3); MS (ESI) was calculated to be 618.3 for C28H48N7O5-
Si2 (M+H+), and found to be 618.8.
The novel 20-GP modification that is described here potentially
has several advantages for therapeutic application. Our data dem-
onstrate that changes in single nucleotides achieve enhanced
silencing efficacy and stability of siRNAs. Modification of more
than one nucleotide in a siRNA is feasible and multiple PG moieties
could further augment the efficacy of therapeutic sequences. Cur-
rent investigations are addressing this as well as the role of GP
modifications on off target seed interaction and improvement in
in vivo delivery of GP-modified siRNA by non-viral vectors.
4.2.1.2. 20-O-Cyanoethyl-30,50-O-(tetraisopropyldisiloxane-1,3-
diyl)-adenosine
(1b).
N6-Dimethylaminomethylene-20-O-
cyanoethyl-30,50-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine
(1e) (1.0 g, 1.62 mmol) was dissolved in methanol (20 mL) then
hydrazine hydrate (H2N–NH2ꢀH2O; 500
lL, 10.3 mmol) was added.
The reaction solution was stirred at room temperature for 3 h. The
solvents were evaporated and the residue was purified using a sil-
ica gel column with ethylacetate as eluent to give 773 mg (87%) of
1b. 1H NMR (400 MHz, DMSO-d6) d [ppm] 8.21 (s, 1H, H2 or H8),
8.07 (s, 1H, H2 or H8), 7.33 (bs, 2H, NH2), 5.98–5.96 (m, 1H, H10),
5.03–4.99 (m, 1H, H30), 4.59–4.57 (m, 1H, H20), 4.08–3.83 (m, 5H,
H40, 2 ꢂ H50, O–CH2–CH2–CN), 2.84–2.80 (m, 2H, O–CH2–CH2–
CN), 1.09–0.97 (m, 28H, tetraisopropyl-CH and -CH3); 13C NMR
(101 MHz, DMSO-d6) d [ppm] 156.01, 152.41 (C2 or C8), 148.46,
139.26 (C2 or C8), 119.20, 118.83, 87.47 (C10), 81.11 (C20), 80.45
(C40), 70.04 (C30), 65.62 (O–CH2–CH2–CN), 60.09 (C50), 18.38 (O–
CH2–CH2–CN), {17.20, 17.06, 17.05, 17.01, 16.98, 16.85, 16.81,
16.71} (tetraisopropyl-CH3), {12.60, 12.28, 12.09, 12.04} (tetraiso-
propyl-CH); MS (MALDI) was calculated to be 563.8 for
4. Experimental
4.1. Material and methods
All reagents were of analytical reagent grade, obtained from
commercial resources and used without further purification. For
synthesis, solvents with quality pro analysis were used. Dry
solvents were kept over molecular sieve and for column chroma-
tography technical solvents were distiled before use.
All NMR spectra were measured on Bruker AM250
(1H: 250 MHz, 13C: 63 MHz), AV300 (1H: 300 MHz, 13C: 75 MHz,
31P: 121 MHz) and AV400 (1H: 400 MHz, 13C: 101 MHz, 31P:
162 MHz) instruments. Chemical shifts (d) are reported in parts
per million (ppm). The following annotations were used with peak
multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; b, broadened. J values are given in Hz.
C
25H43N6O5Si2 (M+H+) and found to be 564.0.
4.2.1.3. 20-O-Aminopropyl-30,50-O-(tetraisopropyldisiloxane-1,3-
diyl)-adenosine (1f). Compound 1b (1.0 g, 1.78 mmol) was
MALDI mass spectra were recorded on a Fisons VG Tofspec spec-
trometer and ESI mass spectra on a Fisons VG Plattform II spec-
dissolved in 10 mL of methanol in a glass tube suitable for use in
an autoclave. Approximately 0.5 mL of the Raney-nickel slurry
was rinsed thoroughly with dry methanol and then washed into
the glass tube with the solution of 1b. After addition of 5 mL meth-
anol saturated with ammonia, the mixture was stirred for 1 h at
room temperature under a hydrogen atmosphere (30 bar). The
reaction mixture was filtered and the catalyst was washed several
times with methanol. The filtrate was evaporated and the residue
was purified using column chromatography with ethyl acetate/
methanol/triethylamine (70:25:5, v/v/v) to yield 503 mg (50%) of
the desired compound. When this reaction was repeated, the crude
product was used for the next step without further purification. 1H
NMR (400 MHz, DMSO-d6) d [ppm] 8.20 (s, 1H, H2 or H8), 8.07 (s,
1H, H2 or H8), 7.32 (bs, 2H, NH2), 5.95–5.94 (m, 1H, H10), 4.95–4.90
(m, 1H, H30), 4.41–4.39 (m, 1H, H20), 4.08–3.90 (m, 3H, H40,
2 ꢂ H50), 3.86–3.70 (m, 2H, O–CH2–CH2–CH2–NH2), 2.66–2.61 (m,
2H, O–CH2–CH2–CH2–NH2), 1.65–1.58 (m, 2H, O–CH2–CH2–CH2–
NH2), 1.08–0.96 (m, 28H, tetraisopropyl-CH and -CH3); MS (MALDI)
was calculated to be 567.9 for C25H47N6O5Si2 (M+H+), and found to
be 567.9.
trometer. High resolution mass spectra were acquired on
Thermo MALDI Orbitrap XL.
UV-melting curves were measured on a JASCO V-650 spectro-
photometer. Melting profiles of the RNA duplexes were recorded
in a phosphate buffer containing NaCl (100 mM, pH 7) at oligonu-
a
cleotide concentrations
2 lM for each strand at wavelength
260 nm. Each melting curve was determined triply. The tempera-
ture range was 5–95 °C with a heating rate 0.5 °C. The thermody-
namic data were extracted from the melting curves by means of
a two state model for the transition from duplex to single strands.
Statistical
analysis: Data have been expressed as the
mean standard error of the mean. Statistical difference was con-
sidered significant when P <0.05 and was determined according to
the student’s t-test and calculated with the GraphPad Prism soft-
ware package (GraphPad Software Inc., CA, USA).
4.2. Chemistry
4.2.1. Synthesis of the 20-O-guanidinopropyl adenosine
phosphoramidite
30,50-O-(Tetraisopropyldisiloxane-1,3-diyl)-N6-dimethylamino-
methylene adenosine (1a) was synthesised as previously
described.16
4.2.1.4.
isopropyldisiloxane-1,3-diyl)-adenosine (1c).
N00-triflyl guanidine (280 mg, 0.72 mmol) was dissolved in 5 mL
dichloromethane then triethylamine (100 L) was added. After
20-O-(N,N0-Di-boc-guanidinopropyl)-30,50-O-(tetra-
N,N0-Di-boc-
l
4.2.1.1. N6 -Dimethylaminomethylene-20-O-cyanoethyl-30,50-O-
cooling to 0 °C, 20-O-aminopropyl-30,50-O-(tetraisopropyldisilox-
ane-1,3-diyl)-adenosine (1f) (400 mg, 0.71 mmol) was added and
the mixture was stirred for 1 h at 0 °C then for 1 h at room temper-
ature. The reaction was diluted with dichloromethane and washed
with saturated sodium bicarbonate solution and brine. The organic
(tetraisopropyldisiloxane-1,3-diyl)-adenosine (1e).
To
a
solution of compound 1a (3.0 g, 5.31 mmol) in tert-butanol
(25 mL), freshly distiled acrylonitrile (6.7 mL, 102 mmol) and
cesium carbonate (1.6 g, 4.9 mmol) were added. The mixture was

1600
J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
layer was dried over Na2SO4 and the solvent was evaporated. The
residue was purified using column chromatography with dichloro-
methane/methanol (98:2, v/v) to give a yield of 402 mg (70%) of 1c.
1H NMR (400 MHz, DMSO-d6) d [ppm] 11.50 (s, 1H, NH-boc), 8.45–
8.41 (m, 1H, NH–CH2–), 8.17 (s, 1H, H2 or H8), 8.06 (s, 1H, H2 or
H8), 7.31 (bs, 2H, NH2), 6.02–5.99 (m, 1H, H10), 4.96–4.91 (m, 1H,
H30), 4.43–4.40 (m, 1H, H20), 4.06–3.70 (m, 5H, H40, 2 ꢂ H50, O–
CH2–CH2–CH2–NH–), 3.51–3.32 (m, 2H, O–CH2–CH2–CH2–NH–),
1.84–1.78 (m, 2H, O–CH2–CH2–CH2–NH–), 1.44 (s, 9H, C(CH3)3),
1.37 (s, 9H, C(CH3)3), 1.07–0.99 (m, 28H, tetraisopropyl-CH and -
CH3); 13C NMR (101 MHz, DMSO-d6) d [ppm] 163.00, 156.00,
155.07, 152.40 (C2 or C8), 151.96, 148.48, 139.04 (C2 or C8),
119.21, 87.69 (C10), 82.70, 81.26 (C20), 80.41 (C40), 77.87, 69.99
(C30), 69.63 (O–CH2–CH2–CH2–NH–), 60.13 (C50), 38.41 (O–CH2–
CH2–CH2–NH–), 28.71 (O–CH2–CH2–CH2–NH–), 27.85 (C(CH3)3),
27.44 (C(CH3)3), {17.19, 17.05, 17.03, 17.00, 16.95, 16.82, 16.74,
16.68} (tetraisopropyl-CH3), {12.59, 12.28, 12.09, 12.01} (tetraiso-
propyl-CH); MS (MALDI) was calculated to be 810.1 for
(m, 4H, DMTr), 6.14–6.13 (m, 1H, H10), 5.18–5.15 (m, 1H, 30-OH),
4.57–4.54 (m, 1H, H20), 4.47–4.42 (m, 1H, H30), 4.14–4.08 (m, 1H,
H40), 3.72–3.71 (m, 6H, 2 ꢂ OCH3), 3.70–3.56 (m, 2H, O–CH2–
CH2–CH2–NH–), 3.37–3.32 (m, 2H, O–CH2–CH2–CH2–NH–), 3.24–
3.21 (m, 2H, 2 ꢂ H50), 3.19 (s, 3H, N–CH3), 3.12 (s, 3H, N–CH3),
1.77–1.70 (m, 2H, O–CH2–CH2–CH2–NH–), 1.44 (s, 9H, C(CH3)3),
1.35 (s, 9H, C(CH3)3); 13C NMR (101 MHz, DMSO-d6) d [ppm]
162.98, 159.15, 157.97, 157.94, 157.91, 157.85 (N6 = CH–NMe2),
155.09, 151.88 (C2 or C8), 151.06, 144.73, 141.18 (C2 or C8),
135.44, 135.37, {129.60, 129.56, 127.64, 127.59, 126.53} (DMTr),
125.70, 112.99 (DMTr), 86.14 (C10), 85.34, 82.97 (C40), 82.70,
80.36 (C20), 77.96, 69.08 (C30), 68.21 (O–CH2–CH2–CH2–NH–),
63.40 (C50), 54.88 (OCH3), 40.54 (N–CH3), 37.97 (O–CH2–CH2–
CH2–NH–), 34.44 (N–CH3), 28.56 (O–CH2–CH2–CH2–NH–), 27.84
(C(CH3)3), 27.50 (C(CH3)3); MS (MALDI) was calculated to be
925.1 for C48H62N9O10 (M+H+), and found to be 924.9.
4.2.1.8. N6 -Dimethylaminomethylene-20-O-(N,N0-di-boc-guani-
C
36H65N8O9Si2 (M+H+), and found to be 808.3.
dinopropyl)-50-O-(4,40-dimethoxytrityl)-adenosine
30-(cyano-
ethyl)-N,N-diisopropyl phosphoramidite (1d).
N6-Dimeth-
4.2.1.5. N6 -Dimethylaminomethylene-20-O-(N,N0-di-boc-guani-
dinopropyl)-30,50-O-(tetraisopropyldisiloxane-1,3-diyl)-adeno-
ylaminomethylene-20-O-(N,N0-di-boc-guanidinopropyl)-50-O-(4,40-
dimethoxytrityl)-adenosine (1i) (320 mg, 346
in dichloromethane (8 mL). 2-cyanoethyl N,N,N0,N0-tetraisopro-
pylamino phosphane (132 L, 415 mol) and 4,5-dicyanoimidaz-
ole (47 mg, 398 mol) were added. The mixture was stirred at
lmol) was dissolved
sine (1g).
in methanol (5 mL) and N,N-dimethylformamide dimethyl acetale
(500 L, 3.7 mmol) was added. The reaction was stirred at room
Compound 1c (500 mg, 0.61 mmol) was dissolved
l
l
l
l
temperature overnight and the solvents were evaporated. The
crude product was used for further reactions without purification.
room temperature. After 3 h, TLC revealed that some starting mate-
rial did not react. An additional 0.6 equiv of the phosphitylating
agent as well as the catalyst were therefore added. After 4 h the
reaction was complete. The mixture was diluted with dichloro-
methane, washed with saturated sodium bicarbonate solution
and the organic layer was dried over MgSO4. The solvent was evap-
orated and the residue dissolved in a small amount of dichloro-
methane (ca. 5 mL). This solution was added dropwise into a
flask with hexane (500 mL) to form a white precipitate. Two thirds
of the solvent were evaporated and the remaining solvent was dec-
anted from the solid. The precipitated product was redissolved in
benzene and lyophilised to give 329 mg (84%) of 1d as a white
powder. 1H NMR (300 MHz, acetone-d6) d [ppm] 11.65 (s, 1H,
NH-boc) 8.95–8.93 (m, 1H, N6 = CH–NMe2), 8.42–8.27 (m, 3H, H2,
H3, NH–CH2–), 7.50–7.46 (m, 2H, DMTr), 7.38–7.17 (m, 7H, DMTr),
6.87–6.80 (m, 4H, DMTr), 6.28–6.26 (m, 1H, H10), 4.96–4.79 (m, 2H,
H20, H30) 4.45–4.37 (m, 1H, H40), 4.05–3.35 (m, 16H), 3.25 (s, 3H, N–
CH3), 3.18 (s, 3H, N–CH3), 2.85 (m, 1H, cyanoethyl), 2.64–2.60 (m,
1H, cyanoethyl), 1.90–1.82 (m, 2H, O–CH2–CH2–CH2–NH–), 1.50–
1.49 (m, 9H, C(CH3)3), 1.42–1.40 (m, 9H, C(CH3)3), 1.25–1.10 (m,
12H, iPr-CH3); 31P NMR (121 MHz, acetone-d6) d [ppm] 149.6,
4.2.1.6. N6 -Dimethylaminomethylene-20-O-(N,N0-di-boc-guani-
dinopropyl)-adenosine (1h).
mmol) was dissolved in tetrahydrofurane (5 mL) and triethylam-
monium trihydrofluoride (Et3Nꢀ3HF; 330 L, 2.0 mmol) was added.
Compound 1g (500 mg, 0.58
l
The mixture was stirred at room temperature for 1.5 h, then the
solvent was evaporated. The residue was purified by column chro-
matography with ethyl acetate/methanol (98:2–9:1, v/v) giving
300 mg (83%) of the desired product. 1H NMR (400 MHz, DMSO-
d6) d [ppm] 11.47 (s, 1H, NH-boc), 8.92 (s, 1H, N6 = CH–NMe2),
8.50 (s, 1H, H2 or H8), 8.41 (s, 1H, H2 or H8), 8.33–8.29 (m, 1H,
NH–CH2–), 6.11–6.09 (m, 1H, H10), 5.28–5.24 (m, 1H, 50-OH),
5.18–5.16 (m, 1H, 30-OH), 4.46–4.43 (m, 1H, H20), 4.36–4.32 (m,
1H, H30), 4.01–3.98 (m, 1H, H40), 3.72–3.46 (4H, 2 ꢂ H50, O–CH2–
CH2–CH2–NH–), 3.33–3.28 (m, 2H, O–CH2–CH2–CH2–NH–), 3.20
(s, 3H, N–CH3), 3.13 (s, 3H, N–CH3), 1.74–1.68 (m, 2H, O–CH2–
CH2–CH2–NH–), 1.45 (s, 9H, C(CH3)3), 1.37 (s, 9H, C(CH3)3); 13C
NMR (101 MHz, DMSO-d6)
d [ppm] 162.97, 159.22, 157.97
(N6 = CH–NMe2), 155.09, 151.89, 151.77 (C2 or C8), 151.00,
141.08 (C2 or C8), 125.70, 85.91 (C10), 85.74 (C40), 82.72, 81.02
(C20), 77.99, 68.72 (C30), 67.88 (O–CH2–CH2–CH2–NH–), 61.04
(C50), 40.56 (N–CH3), 37.86 (O–CH2–CH2–CH2–NH–), 34.45 (N–
CH3), 28.57 (O–CH2–CH2–CH2–NH–), 27.87 (C(CH3)3), 27.51
(C(CH3)3); MS (MALDI) was calculated to be 622.7 for C27H44N9O8
(M+H+), and found to be 624.6.
149.3; MS (ESI) was calculated to be 1125.3 for C57H79N11O11
P
(M+H+), and found to be 1125.7.
4.2.2. Synthesis of the 20-O-guanidinopropyl cytidine
phosphoramidite
N4-Dimethylaminomethylene-30,50-O-(tetraisopropyldisilox-
ane-1,3-diyl)-cytidine (2a) was synthesised according to a previ-
4.2.1.7. N6 -Dimethylaminomethylene-20-O-(N,N0-di-boc-guani-
dinopropyl)-50-O-(4,40-dimethoxytrityl)-adenosine
ously described procedure.37
(1i).
Compound 1h (1.0 g, 1.6 mmol) was dissolved in dry pyr-
4.2.2.1. N4 -Dimethylaminomethylene-20-O-cyanoethyl-30,50-O-
idine (20 mL). 4,40-Dimethoxytrityl chloride (660 mg, 1.95 mmol)
was added and the reaction was stirred at room temperature over-
night. The solution was diluted with dichloromethane and washed
with saturated sodium bicarbonate solution. After evaporation of
the solvents the residue was purified on a silica gel column with
dichloromethane/methanol (98:2, v/v) containing 0.5% triethyl-
amine, and 1.32 g (90%) of the tritylated compound was obtained.
1H NMR (400 MHz, DMSO-d6) d [ppm] 11.48 (s, 1H, NH-boc), 8.90
(s, 1H, N6 = CH–NMe2), 8.38–8.34 (m, 3H, H2, H3, NH–CH2–),
7.37–7.34 (m, 2H, DMTr), 7.27–7.17 (m, 7H, DMTr), 6.84–6.79
(tetraisopropyldisiloxane-1,3-diyl)-cytidine
(2e).
Com-
pound 2a (4 g, 7.39 mmol) was dissolved in acrylonitrile (8 mL,
122 mmol) and tert-Butanol (35 mL). Cesium carbonate (1.8 g,
5.52 mmol) was added and the reaction was stirred for 2.5 h at
room temperature. The mixture was filtered over celite, the sol-
vents evaporated, and then the residue was purified using column
chromatography. Ethyl acetate was initially used as solvent then
changed to ethyl acetate/methanol (9:1, v/v) after the unpolar
impurities had passed through the column. A yield of 3.78 g
(86%) of the product were obtained. 1H NMR (400 MHz,

J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
1601
DMSO-d6) d [ppm] 8.62 (s, 1H, N4 = CH–NMe2), 7.88 (d, 1H,
J = 7.3 Hz, H6), 5.90 (d, 1H, J = 7.3 Hz, H5), 5.65 (s, 1H, H10), 4.22–
3.91 (m, 7H), 3.17 (s, 3H, N–CH3), 3.04 (s, 3H, N–CH3), 2.86–2.82
(m, 2H, O–CH2–CH2–CN), 1.07–0.96 (m, 28H, tetraisopropyl-CH
and -CH3); 13C NMR (101 MHz, DMSO-d6) d [ppm] 171.21 (C4),
157.77 (N4 = CH–NMe2), 154.57 (C2), 140.61 (C6), 118.86 (O–
CH2–CH2–CN), 101.14 (C5), 88.99 (C10), 81.42, 80.69, 67.83, 65.22
(O–CH2–CH2–CN), 59.39 (C50), 40.79 (N–CH3),34.71 (N–CH3),
18.18 (O–CH2–CH2–CN), {17.22, 17.11, 17.04, 16.97, 16.84, 16.72,
16.69, 16.61} (tetraisopropyl-CH3), {12.60, 12.20, 11.88} (tetraiso-
propyl-CH); MS (ESI) was calculated to be 594.9 for C27H48N5O6Si2
(M+H+) and found to be 594.9.
7.19 (bs, 2H, NH2), 5.68 (d, 1H, J = 7.4 Hz, H5), 5.63 (s, 1H, H10),
4.17–3.78 (m, 7H), 3.49–3.33 (m, 2H, O–CH2–CH2–CH2–NH–),
1.84–1.77 (m, 2H, O–CH2–CH2–CH2–NH–), 1.45 (m, 9H, C(CH3)3),
1.38 (m, 9H, C(CH3)3), 1.06–0.96 (m, 28 H, tetraisopropyl-CH and
-CH3); 13C NMR (101 MHz, DMSO-d6) d [ppm] 165.61, 162.99,
155.04, 154.52, 151.94, 139.45 (C6), 93.21 (C5), 88.97 (C10),
82.66, 81.76 (C20), 80.36 (C40), 77.86, 69.11 (O–CH2–CH2–CH2–
NH–), 68.27 (C30), 59.51 (C50), 38.28 (O–CH2–CH2–CH2–NH–),
28.61 (O–CH2–CH2–CH2–NH–), 27.86 (C(CH3)3), 27.44 (C(CH3)3),
{17.22, 17.10, 17.03, 16.96, 16.83, 16.70, 16.68, 16.60} (tetraisopro-
pyl-CH3), {12.59, 12.26, 12.21, 11.87} (tetraisopropyl-CH); MS
(MALDI) was calculated to be 786.1 for C35H65N6O10Si2 (M+H+)
and found to be 786.4.
4.2.2.2. 20-O-Cyanoethyl-30,50-O-(tetraisopropyldisiloxane-1,3-
diyl)-cytidine (2b).
noethyl-30,50-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine
(1.0 g, 1.68 mmol) was dissolved in methanol (10 mL) and hydra-
zine hydrate (500 L, 10.3 mmol) was added. The mixture was stir-
N4-Dimethylaminomethylene-20-O-cya-
4.2.2.5. N4 -Benzoyl-20-O-(N,N0-di-boc-guanidinopropyl)-30,50-O-
(2e)
(tetraisopropyldisiloxane-1,3-diyl)-cytidine
(2g).
Com-
pound 2c (1.0 g, 1.27 mmol) was dissolved in dry pyridine
(10 mL) and the solution was cooled in an ice bath. Benzoyl chlo-
l
red for 1 h at room temperature and then the solvents were
evaporated. The residue was purified on a silica gel column with
ethyl acetate/methanol (95:5, v/v) to give 745 mg (82%) of 2b. 1H
NMR (400 MHz, DMSO-d6) d [ppm] 7.69 (d, 1H, J = 7.4 Hz, H6),
7.21 (s, 2H, NH2), 5.69 (d, 1H, J = 7.4 Hz, H5), 5.61 (s, 1H, H10);
4.19–3.90 (m, 7H), 2.90–2.76 (m, 2H, O–CH2–CH2–CN), 1.07–0.97
(m, 28 H, tetraisopropyl-CH and -CH3); 13C NMR (101 MHz,
DMSO-d6) d [ppm] 165.70, 154.60, 139.36 (C6), 118.89, 93.30
(C5), 88.66 (C10), 81.55 (C20), 80.49, 67.92, 65.19 (O–CH2–CH2–
CN), 59.44 (C50), 18.20 (O–CH2–CH2–CN), {17.23, 17.11, 17.05,
16.98, 16.85, 16.73, 16.72, 16.63} (tetraisopropyl-CH3), {12.62,
12.28, 12.21, 11.88} (tetraisopropyl-CH); MS (ESI) was calculated
to be 539.8 for C24H43N4O6Si2 (M+H+) and found to be 540.0.
ride (240 lL, 2.06 mmol) was added and the reaction solution
was stirred at 0 °C for 1 h. The reaction was quenched with water
and ammonia (25% in water; 3 mL) was added. The mixture was
then stirred for 30 min at room temperature. The solvents were
evaporated and the residue was dissolved in dichloromethane
and washed with saturated sodium bicarbonate solution. The or-
ganic layer was dried over Na2SO4 and after evaporating the sol-
vent, the residue was purified by column chromatography using
dichloromethane/methanol (98:2, v/v) and 950 mg (84%) of the
product were obtained. 1H NMR (400 MHz, DMSO-d6) d [ppm]
11.50 (s, 1H, NH), 11.31 (s, 1H, NH), 8.40–8.37 (m, 1H, NH–CH2–),
8.15 (d, 1H, J = 7.3 Hz, H6), 8.03–7.99 (m, 2H, benzoyl), 7.65–7.60
(m, 1H, benzoyl), 7.53–7.49 (m, 2H, benzoyl), 7.38 (d, 1H,
J = 7.3 Hz, H5), 5.73 (s, 1H, H10), 4.24–4.13 (m, 3H, H30, H40, H50),
4.02–4.03 (m, 1H, H20), 3.97–3.92 (m, 1H, H50), 3.87–3.83 (m, 2H,
O–CH2–CH2–CH2–NH–), 3.52–3.35 (m, 2H, O–CH2–CH2–CH2–NH–
), 1.87–1.80 (m, 2H, O–CH2–CH2–CH2–NH–), 1.45 (m, 9H,
C(CH3)3), 1.38 (m, 9H, C(CH3)3), 1.08–0.95 (m, 28H, tetraisopro-
pyl-CH and -CH3); 13C NMR (101 MHz, DMSO-d6) d [ppm] 167.21,
163.14, 163.00, 155.06, 154.01, 151.97, 143.37 (C6), 132.97,
132.64, 128.31, 95.61 (C5), 89.46 (C10), 82.67, 81.30 (C20), 80.86
(C40), 77.86, 69.26 (O–CH2–CH2–CH2–NH–), 67.95 (C30), 59.38
(C50), 38.28 (O–CH2–CH2–CH2–NH–), 28.62 (O–CH2–CH2–CH2–
NH–), 27.86 (C(CH3)3), 27.43 (C(CH3)3), {17.22, 17.11, 17.04,
16.97, 16.89, 16.73, 16.71, 16.66} (tetraisopropyl-CH3), {12.56,
12.29, 12.22, 11.86} (tetraisopropyl-CH); MS (ESI) was calculated
to be 890.2 for C42H69N6O11Si2 (M+H+), and found to be 890.4.
4.2.2.3. 20-O-Aminopropyl-30,50-O-(tetraisopropyldisiloxane-1,3-
diyl)-cytidine (2f).
Compound 2b (500 mg, 928 lmol) was
dissolved in 10 mL of methanol in a glass tube. Approximately
0.5 mL of the Raney-nickel sediment was washed thoroughly with
dry methanol and was rinsed into the glass tube with the solution
of 2b. After addition of 5 mL methanol saturated with ammonia,
the mixture was stirred for 1 h at room temperature under a
hydrogen atmosphere (30 bar). The reaction mixture was filtered
through celite and the catalyst was washed several times with
methanol. The solvent was evaporated and the residue was puri-
fied on a silica gel column using ethyl acetate/methanol/triethyl-
amine (60:35:5) to give 251 mg (50%) of the product. When this
procedure was repeated, the crude material after filtration and
evaporation was used in further reactions without purification.
1H NMR (400 MHz, DMSO-d6) d [ppm] 7.69 (d, 1H, J = 7.2 Hz, H6),
7.18 (bs, 2H, ar. NH2), 5.68 (d, 1H, J = 7.5 Hz, H5), 5.60 (s, 1H,
H10), 4.18–3.76 (m, 7H), 2.70–2.66 (m, 2H, O–CH2–CH2–CH2–
NH2), 1.68–1.61 (m, 2H, O–CH2–CH2–CH2–NH2), 1.07–0.95 (m, 28
H, tetraisopropyl-CH and -CH3); MS (MALDI) was calculated to be
643.8 for C24H47N4O6Si2 (M+H+) and found to be 544.6.
4.2.2.6. N4-Benzoyl-20-O-(N,N0-di-boc-guanidinopropyl)-cytidine
(2h).
(tetraisopropyldisiloxane-1,3-diyl)-cytidine
1.01 mmol) was dissolved in tetrahydrofurane (20 mL). Triethyl-
amine trihydrofluoride (Et3Nꢀ3HF; 560 L, 3.54 mmol) was added
N4-Benzoyl-20-O-(N,N0-di-boc-guanidinopropyl)-30,50-O-
(2g)
(900 mg,
l
and the solution was stirred at room temperature for 2 h. The sol-
vent was evaporated and the residue was purified using column
chromatography with dichloromethane/methanol (98:2–97:3, v/
v) to give 607 mg (93%) of the product as a pale yellow foam. 1H
NMR (300 MHz, DMSO-d6) d [ppm] 11.50 (s, 1H, NH), 11.28 (bs,
1H, NH), 8.57 (d, 1H, J = 7.5 Hz, H6), 8.40–8.35 (m, 1H, NH–CH2–),
8.02–7.98 (m, 2H, benzoyl), 7.66–7.60 (m, 1H, benzoyl), 7.54–
7.48 (m, 2H, benzoyl), 7.34 (d, 1H, J = 7.2 Hz, H5), 5.86–5.85 (m,
1H, H10), 5.24 (t, 1H, J = 5.0 Hz, 50-OH), 4.98 (d, 1H, J = 6.8 Hz,
30-OH), 4.12–3.60 (m, 7H), 3.44–3.37 (m, 2H, O–CH2–CH2–CH2–
NH–), 1.85–1.76 (m, 2H, O–CH2–CH2–CH2–NH–), 1.46 (m, 9H,
C(CH3)3), 1.38 (m, 9H, C(CH3)3); 13C NMR (75 MHz, acetone-d6) d
[ppm] 169.22, 165.59, 164.82, 157.83, 156.15, 154.80, 147.10
(C6), 135.58, 134.60, 130.46, 130.05, 97.67 (C5), 91.39 (C10),
86.19 (C40), 84.69 (C(CH3)3), 84.62 (C20), 79.88 (C(CH3)3), 70.71
4.2.2.4.
isopropyldisiloxane-1,3-diyl)-cytidine (2c).
triflyl guanidine (360 mg, 920 mol) was dissolved in 5 mL dichlo-
romethane and triethylamine (125 L) then added. After cooling to
0 °C, compound 2f (500 mg, 922 mol) was added and the solution
20-O-(N,N0-Di-boc-guanidinopropyl)-30,50-O-(tetra-
N,N0-Di-boc-N00-
l
l
l
was stirred for 1 h at 0 °C and then 1 h at room temperature. The
reaction was diluted with dichloromethane and washed with satu-
rated sodium bicarbonate solution and brine. The combined organ-
ic layers were dried over Na2SO4 and after evaporating the solvent
the residue was purified using column chromatography with
dichloromethane/methanol (98:2–95:5, v/v) to give 434 mg (60%)
of 2c. 1H NMR (400 MHz, DMSO-d6) d [ppm] 11.48 (s, 1H, NH-
boc), 8.38–8.35 (m, 1H, NH–CH2–), 7.67 (d, 1H, J = 7.4 Hz, H6),

1602
J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
(O–CH2–CH2–CH2–NH–), 69.63 (C30), 61.46 (C50), 40.29 (O–CH2–
CH2–CH2–NH–), 30.86 (O–CH2–CH2–CH2–NH–), 29.46 (C(CH3)3),
29.14 (C(CH3)3); HRMS (MALDI) was calculated to be 647.3035
for C30H43N6O10 (M+H+), and found to be 647.3031.
(The signal of the hydrolysed phosphitylation reagent appears at
13.9 ppm); HRMS (MALDI) was calculated to be 1149.5421 for
C
60H78N8O13P (M+H+), was found to be 1149.5447.
4.2.3. Synthesis of the 20-O-guanidinopropyl uridine
phosphoramidite
4.2.2.7. N4-Benzoyl-20-O-(N,N 0-di-boc-guanidinopropyl)-50-O-
(4,40-dimethoxytrityl)-cytidine (2i).
N4-Benzoyl-20-O-(N,N0-
N3-Benzoyl-30,50-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine
di-boc-guanidinopropyl)-cytidine (2h) (516 mg, 798
l
mol) was
(3a) was synthesised according to a previously described
dissolved in dry pyridine (20 mL) and the solution was cooled in
an ice bath. 4,40-Dimethoxytrityl chloride (515 mg, 1.52 mmol)
was added and the mixture was stirred overnight while the bath
came up to room temperature. The reaction was quenched with
methanol (10 mL) and the solvents were evaporated. The residue
was purified by column chromatography using dichloromethane/
methanol (99:1–98:2, v/v). The column was packed with solvent
containing 1% triethylamine to yield 715 mg (94%) of the product
as a pale yellow foam. 1H NMR (400 MHz, DMSO-d6) d [ppm]
11.50 (s, 1H, NH), 11.29 (bs, 1H, NH), 8.43–8.37 (m, 2H, H6, NH–
CH2–), 8.02–7.99 (m, 2H, benzoyl), 7.65–7.60 (m, 1H, benzoyl),
7.54–7.50 (m, 2H, benzoyl), 7.43–7.25 (m, 9H, DMTr), 7.18–7.15
(m, 1H, H5), 6.94–6.91 (m, 4H, DMTr), 5.88 (s, 1H, H10), 5.04 (d,
1H, J = 7.3 Hz, 30-OH), 4.34–4.28 (m, 1H, H30), 4.13–4.10 (m, 1H,
H40), 3.94–3.87 (m, 2H, H20, 1 ꢂ O–CH2–CH2–CH2–NH–), 3.76 (s,
6H, 2 ꢂ OCH3), 3.76–3.70 (m, 1H, 1 ꢂ O–CH2–CH2–CH2–NH–),
3.46–3.36 (m, 4H, 2 ꢂ H50, O–CH2–CH2–CH2–NH–), 1.86–1.80 (m,
2H, O–CH2–CH2–CH2–NH–), 1.42 (m, 9H, C(CH3)3), 1.36 (m, 9H,
C(CH3)3); 13C NMR (75 MHz, DMSO-d6) d [ppm] 167.19, 163.02,
158.11, 158.08, 155.12, 154.07, 151.93, 144.24 (C6), 135.47,
135.11, 133.06, 132.62, 129.70, 129.55, 128.35, 127.85, 127.73,
126.78, 113.19, 95.93 (C5), 88.99 (C10), 85.90, 82.67 (C(CH3)3),
81.93 (C20), 81.44 (C40), 77.94 (C(CH3)3), 68.44 (O–CH2–CH2–CH2–
NH–), 67.62 (C30), 61.36 (C50), 54.91 (OCH3), 54.90 (OCH3), 38.17
(O–CH2–CH2–CH2–NH–), 28.59 (O–CH2–CH2–CH2–NH–), 27.87
(C(CH3)3), 27.48 (C(CH3)3); HRMS (MALDI) was calculated to be
971.4161for C51H60N6O12Na (M+Na+), and found to be 971.4181.
procedure.22
4.2.3.1.
disiloxane-1,3-diyl)-uridine (3b).
N3-Benzoyl-20-O-cyanoethyl-30,50-O-(tetraisopropyl-
Compound 3a (1.14 g,
1.93 mmol) was dissolved in 9.6 mL of tert-butanol. Freshly dist-
iled acrylonitrile (2.5 mL, 38.6 mmol) was added. After addition
of cesium carbonate (645 mg, 1.98 mmol) the reaction was stirred
for 4 h at room temperature. The reaction solution was filtered
over celite. The residue was washed with 100 mL of dichlorometh-
ane. The filtrate was evaporated in vacuum. Purification via
column chromatography in dichloromethane/ethyl acetate
(99:1–95:5, v/v) yielded 746 mg (60%) of the desired product as
a white powder. 1H NMR (250 MHz, acetone-d6) d [ppm] 8.03–
7.99 (m, 3H, H6, benzoyl), 7.79–7.72 (m, 1H, benzoyl), 7.61–7.55
(m, 2H, benzoyl), 5.80–5.74 (m, 2H, H5, H10), 4.50–3.94 (m, 7H),
2.80–2.75 (m, 2H, O–CH2–CH2–CN), 1.17–1.07 (m, 28H, tetraiso-
propyl-CH and -CH3); 13C NMR (63 MHz, acetone-d6) d [ppm]
171.11, 163.78, 151.06, 141.48, 136.94, 133.92, 132.20, 131.13,
119.80, 102.75, 91.47, 84.05, 83.69, 70.65, 68.08, 61.60, 20.35,
18.92, 18.91, 18.75, 18.73, 18.64, 18.50, 18.46, 18.40, 15.23,
14.79, 14.72, 14.43; HRMS (XXX) was calculated to be 666.2637
for C31H45N3O8Si2Na (M+Na+) and found to be 666.2647.
4.2.3.2. 20-O-(Aminopropyl)-30,50-O-(tetraisopropyldisiloxane-
1,3-diyl)-uridine (3e).
Compound 3b (500 mg, 0.78 mmol)
was dissolved in 10 mL of methanol in a glass tube suitable for
the applied autoclave. Approximately 0.5 mL of the Raney-nickel
slurry was put on a glass filter, washed thoroughly with dry meth-
anol and rinsed into the glass tube with the solution of 3b. After
addition of 5 mL methanol saturated with ammonia, the mixture
was stirred for 1 h at room temperature in an autoclave under a
hydrogen atmosphere (30 bar). The reaction solution was decanted
from the catalyst into a glass filter. The catalyst was washed sev-
eral times with methanol and the solvent was removed from the
combined filtrates under reduced pressure. The product was puri-
fied on a silica gel column initially using dichloromethane/ethyl
acetate (7:3–0:1, v/v) and thereafter ethyl acetate/methanol/tri-
ethylamine (6: 3.5: 0.5, v/v/v) to obtain 253 mg (60%) of a white
powder. When we repeated the reduction we used the crude prod-
uct after filtration and evaporation for further derivatisation. 1H
NMR (250 MHz, acetone-d6) d [ppm] 7.81 (d, 1H, J = 8.1 Hz, H6),
5.71 (s, 1H, H10), 5.53 (d, 1H, J = 8.1 Hz, H5), 4.39–4.34 (m, 1H,
H30), 4.28–4.23 (m, 1H, H50), 4.14–4.03 (m, 3H, H20, H40, H50),
3.97–3.81 (m, 2H, O–CH2–CH2–CH2–NH2), 3.37–3.25 (m, 2H, O–
CH2–CH2–CH2–NH2), 1.92–1.82 (m, 2H, O–CH2–CH2–CH2–NH2),
1.14–1.05 (m, 28H, tetraisopropyl-CH and -CH3); 13C NMR
(63 MHz, acetone-d6) d [ppm] 167.18, 164.59, 151.93, 141.033,
102.82, 91.15, 83.77, 83.50, 70.88, 70.85, 61.78, 49.36, 33.03,
18.91, 18.90, 18.74, 18.61, 18.49, 18.47, 18.40, 15.19, 14.82,
14.71, 14.41; HRMS (MALDI) was calculated to be 544.2869 for
4.2.2.8. N4-Benzoyl-20-O-(N,N 0-di-boc-guanidinopropyl)-50-O-
(4,40-dimethoxytrityl)-cytidine 30-(cyanoethyl)-N,N-diisopropyl
phosphoramidite (2d).
was dissolved in dichloromethane (15 mL). 2-cyanoethyl
N,N,N0,N0-tetraisopropylamino phosphane (274
L, 864 mol) and
4,5-dicyanoimidazole (98 mg, 828 mol) were added. After stirring
Compound 2i (683 mg, 720 lmol)
l
l
l
at room temperature for 5 h, TLC revealed that some starting mate-
rial had not reacted. Therefore 10 mg of 4,5-dicyanoimidazole and
30 lL of the phosphitylation agent were added and the reaction
was stirred at room temperature overnight. The solution was di-
luted with dichloromethane and washed with saturated sodium
bicarbonate solution. After drying the organic layer over MgSO4
the solvent was evaporated and the residue was dissolved in a
small amount (5 mL) of dichloromethane. This solution was
dripped into a flask with hexane (500 mL) to form a white precip-
itate. Two thirds of the solvent was evaporated and the residual
solvent was decanted carefully. The precipitate was redissolved
in benzene and lyophilised to give 738 mg (89%) of 2d. According
to 31P NMR spectrum the product was still containing a small
amount of the hydrolysed phosphitylation reagent but this did
not interfere with the oligonucleotide synthesis. 1H NMR
(400 MHz, DMSO-d6) d [ppm] 11.50–11.48 (m, 1H, NH), 11.25
(bs, 1H, NH), 8.52–8.45 (m, 1H, H6), 8.39–8.34 (m, 1H, NH–CH2–),
8.01–7.98 (m, 2H, benzoyl), 7.66–7.61 (m, 1H, benzoyl), 7.53–
7.49 (m, 2H, benzoyl), 7.45–7.25 (m, 9H, DMTr), 7.13–7.09 (m,
1H, H5), 6.93–6.89 (m, 4H, DMTr), 5.95–5.92 (m, 1H, H10), 4.56–
4.38 (m, 1H, H30), 4.31–4.28 (m, 1H, H40), 4.07–3.29 (m, 17H),
2.90–2.57 (m, 2H, cyanoethyl), 1.86–1.78 (m, 2H, O–CH2–CH2–
CH2–NH–), 1.40–1.35 (m, 18H, 2 ꢂ C(CH3)3), 1.20–0.93 (m, 12H,
iPr-CH3); 31P NMR (162 MHz, DMSO-d6) d [ppm] 148.4, 148.0
C
24H46N3O7Si2 (M+H+), and found to be 544.2880.
4.2.3.3.
isopropyldisiloxane-1,3-diyl)-uridine (3c).
triflyl guanidine (320 mg, 0.82 mmol) was dissolved in 3.6 mL
20-O-(N,N 0-Di-boc-guanidinopropyl)-30,50-O-(tetra-
N,N0-Di-boc-N00-
dichloromethane and triethylamine (150 lL) was added. The solu-
tion was cooled in an ice bath and 20-O-(aminopropyl)-30,50-O-(tet-
raisopropyldisiloxane-1,3-diyl)-uridine (3e) (490 mg, 0.9 mmol)

J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
1603
was added. After 15 min the reaction mixture was removed from
the ice bath was and stirred for 2.5 h at room temperature. The
reaction solution was washed with saturated sodium bicarbonate
solution and brine. After drying over Na2SO4 the solvent was evap-
orated in vacuum. The crude product was purified using column
chromatography with dichloromethane/methanol (96:4–94:6, v/
v). 410 mg (58%) of compound 3c were obtained. 1H NMR
(400 MHz, DMSO-d6) d [ppm] 11.49 (s, 1H, NH), 11.37 (m, 1H,
NHuridine), 8.40–8.37 (m, 1H, NH–CH2–), 7.64 (d, 1H, J = 7.9 Hz,
H6), 5.64 (s, 1H, H10), 5.53 (d, 1H, J = 7.9 Hz, H5), 4.25–4.22 (H30),
4.13–4.09 (m, 1H, H50), 4.06–4.05 (m, 1H, H20), 4.03–4.00 (m, 1H,
H40), 3.93–3.89 (m, 1H, H50), 3.84–3.70 (m, 2H, O–CH2–CH2–CH2–
NH–), 3.49–3.32 (m, 2H, O–CH2–CH2–CH2–NH–), 1.83–1.77 (m,
2H, O–CH2–CH2–CH2–NH–), 1.45 (s, 9H, C(CH3)3), 1.38 (s, 9H,
C(CH3)3), 1.06–0.97 (m, 28H, tetraisopropyl-CH and -CH3); HRMS
(MALDI) was calculated to be 808.3955 for C35H63N5O11Si2Na
(M+Na+), and found to be 808.3991.
acetate 1:1 (v:v), containing 0.5% triethylamine). Because the reac-
tion was not complete after 2 h, an additional 0.3 equiv of the re-
agents were added and the reaction was completed after
additional 40 min. The resulting solution was washed twice with
saturated sodium bicarbonate solution (2 ꢂ 100 mL) and once with
brine (200 mL). After drying over Na2SO4, the solvent was evapo-
rated and the residue was purified on a silica gel column with
dichloromethane/ethyl acetate (6:4–1:1, v/v) containing 0.5% tri-
ethylamine. The mixture of the two diastereomers was obtained
as a light yellow foam (762 mg, 83%). 1H NMR (400 MHz, DMSO-
d6) d [ppm] 11.50–11.48 (m, 1H, NH), 11.35 (bs, 1H, NH), 8.39–
8.33 (m, 1H, NH–CH2–), 7.87–7.80 (m, 1H, H6), 7.41–7.22 (m, 9H,
DMTr), 6.91–6.86 (m, 4H, DMTr), 5.86–5.84 (m, 1H, H10), 5.23–
5.18 (m, 1H, H5), 4.46–4.32 (m, 1H), 4.21–4.16 (m, 1H), 4.09–4.03
(m, 1H), 3.83–3.26 (m, 16H), 2.80–2.59 (m, 2H, –O–CH2–CH2–CN),
1.81–1.74 (m, 2H, O–CH2–CH2–CH2–NH–), 1.42–1.36 (m, 18H,
C(CH3)3), 1.13–0.94 (m, 12H, iPr-CH3); 31P NMR (121 MHz, DMSO-
d6) d [ppm] 150.0, 148.6; HRMS (MALDI) was calculated to be
1046,4999 for C53H73N7O13P (M+H+), and found to be 1046,5021.
4.2.3.4. 20-O-(N,N0-Di-boc-guanidinopropyl)-uridine (3f).
a solution of compound 3c (910 mg, 1.16 mmol) and triethylamine
(240 L, 4.3 mmol)
L) in 13 mL tetrahydrofurane NEt3ꢀ3HF (700
To
l
l
4.2.4. Synthesis of the 20-O-guanidinopropyl guanosine
phosphoramidite
was added. The reaction mixture was stirred for 1 h at room
temperature. The solvents were evaporated and the residue was
purified on a silica gel column using dichloromethane/methanol
(93:7–92:8, v/v) to give 629 mg (97%) of a white foam. 1H NMR
(250 MHz, acetone-d6) d [ppm] 11.67 (bs, 1H, NH), 10.03 (bs, 1H,
NH), 8.46–8.41 (m, 1H, NH–CH2–), 8.10 (d, 1H, J = 8.2 Hz, H6),
5.99–5.97 (m, 1H, H10), 5.58 (d, 1H, J = 8.2 Hz, H5), 4.39–3.46 (m,
11H), 1.95–1.85 (m, 2H, O–CH2–CH2–CH2–NH–), 1.51 (s, 9H,
C(CH3)3), 1.43 (s, 9H, C(CH3)3); 13C NMR (63 MHz, acetone-d6) d
[ppm] 165.64, 164.59, 157.88, 154.86, 152.37, 142.33, 103.31,
89.82, 86.67, 84.77, 84.74, 84.39, 79.91, 70.73, 70.69, 62.45,
40.20, 30.96, 29.51, 29.19; MS (ESI) was calculated to be 566.2
for C23H37N5O10Na (M+Na+), and found to be 567.0.
O6-(2,4,6-Triisopropylbenzenesulfonyl)-30,50-O-di-tert-buty-
lsilanediyl guanosine (4a) was synthesised according to a previ-
ously described procedure.21
4.2.4.1. 20-O-(2-Cyanoethyl)-30,50-O-di-tert-butylsilanediyl gua-
nosine (4b).
Compound 4a (2.28 g, 3.3 mmol) was dissolved
in tert-butanol (17 mL). Freshly distiled acrylonitrile (4.25 mL,
66 mmol) and cesium carbonate (1.16 g, 3.3 mmol) were added
to the solution. After vigorous stirring at room temperature for
2–3 h, the mixture was filtered through celite. The solvent and ex-
cess reagents were removed in vacuum. The crude material was
used for the next reaction without further purification. The residue
was dissolved in 4 mL of a mixture of formic acid/dioxane/water
(70:24:6, v/v/v). After stirring at room temperature for 1 h, water
(150 mL) was added to the mixture and the solution extracted with
dichloromethane. The organic layer was dried over Na2SO4 and the
solvent was evaporated. The residue was purified using column
chromatography with dichloromethane/methanol (9:1, v/v) to give
1.1 g (70% over two steps) of 4b as a colourless foam. 1H NMR
(250 MHz, DMSO-d6) d [ppm] 10.71 (bs, 1H, NH), 7.89 (s, 1H, H8),
6.45 (bs, 2H, NH2), 5.81 (s, 1H, H10), 4.45–4.33 (m, 3H), 4.05–3.81
(m, 4H), 2.83–2.76 (m, 2H, O–CH2–CH2–CN), 1.06 (s, 9H, C(CH3)3),
1.01 (s, 9H, C(CH3)3); 13C NMR (63 MHz, DMSO-d6) d [ppm]
156.51, 153.69, 150.50, 135.36, 118.71, 116.53, 87.25, 80.31,
76.35, 73.80, 66.64, 65.14, 27.12, 26.80, 22.07, 19.82, 18.29; MS
(ESI) was calculated to be 477.2 for C21H33N6O5Si (M+H+), and
found to be 477.5.
4.2.3.5. 20-O-(N,N 0-Di-boc-guanidinopropyl)-50-O-(4,40-dime-
thoxytrityl)-uridine (3g).
20-O-(N,N0-Di-boc-guanidinopro-
pyl)-uridine (3f) (588 mg, 1.08 mmol) was dissolved in 11.4 mL of
dry pyridine and 4,40-dimethoxytrityl chloride (460 mg, 1.36 mmol)
was added. The reaction solution was stirred at room temperature
for 5 h. The reaction mixture was quenched with water and the sol-
vents were evaporated. The residue was dissolved in dichlorometh-
ane, washed twice with saturated sodium bicarbonate solution
(2 ꢂ 50 mL) and then twice with brine (2 ꢂ 50 mL). The organic
layer was dried over Na2SO4 and the solvent was removed under re-
duced pressure. After purification using column chromatography
with dichloromethane/methanol (97:3, v/v) containing 0.5% trieth-
ylamine, 785 mg (86%) of a yellow powder was obtained. The yellow
impurity could not be separated on the column. 1H NMR (250 MHz,
DMSO-d6) d [ppm] 11.49 (s, 1H, NH), 11.37 (m, 1H, NH), 8.41–8.36
(m, 1H, NH–CH2–), 7.75 (d, 1H, J = 8.1 Hz, H6), 7.40–7.23 (m, 9H,
DMTr), 6.92–6.88 (m, 4H, DMTr), 5.83–5.82 (m, 1H, H10), 5.29–
5.25 (m, 1H, H5), 5.09–5.06 (m, 1H, 30-OH), 4.23–3.88 (m, 3H),
3.74 (s, 6H, 2 ꢂ O–CH3), 3.68–3.63 (m, 2H), 3.43–3.20 (m, 4H),
1.82–1.72 (m, 2H, O–CH2–CH2–CH2–NH–), 1.44 (s, 9H, C(CH3)3),
1.37 (s, 9H, C(CH3)3); HRMS (MALDI) was calculated to be
846.3920 for C44H56N5O12 (M+H+), and found to be 846.3946.
4.2.4.2.
guanosine (4e).
20-O-(2-Aminopropyl)-30,50-O-di-tert-butylsilanediyl
Compound 4b (500 mg, 1.06 mmol) was dis-
solved in dry methanol (5 mL). Raney nickel (ca. 0.5 mL of the
methanol-washed sediment) and methanol (5 mL) saturated with
ammonia were then added. The mixture was hydrogenated at
30 bar hydrogen-pressure for 1 h at room temperature. Thereafter
the mixture was filtered through a glass filter and the catalyst was
washed several times with methanol and a methanol/water mix-
ture. The solvents were evaporated from the filtrate and the resi-
due was used without further purification for the next reaction.
MS (ESI) was calculated to be 481.3 for C21H37N6O5Si (M+H+),
and found to be 481.8.
4.2.3.6. 20-O-(N,N 0-Di-boc-guanidinopropyl)-50-O-(4,40-dime-
thoxytrityl)-uridine 30-(cyanoethyl)-N,N-diisopropyl phospho-
ramidite
(3d).
20-O-(N,N0-Di-boc-guanidinopropyl)-50-O-
(4,40-dimethoxytrityl)-uridine (3g) (770 mg, 0.9 mmol) was dis-
solved in dichloromethane (11 mL). To this solution, 2-cyanoethyl
N,N,N0,N0-tetraisopropylamino phosphane (400
lL, 1.26 mmol)
4.2.4.3. 20-O-(N,N0-Di-boc-guanidinopropyl)-30,50-O-di-tert-buty-
lsilanediyl guanosine (4c).
(163 mg, 0.415 mmol) was dissolved in dichloromethane (2.1 mL)
and 4,5-dicyanoimidazole (130 mg, 1.1 mmol) were added. The
reaction progress was observed with TLC (dichloromethane/ethyl
N,N0-Di-boc-N00-triflyl guanidine

1604
J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
and triethylamine (54
l
L) was then added. The solution was cooled
J = 4.8 Hz, 30-OH), 5.06–5.03 (m, 1H, 50-OH), 4.36–4.29 (m, 2H,
H20, H30), 3.95–3.93 (m, 1H, H40), 3.67–3.46 (m, 4H, 2 ꢂ H50, O–
CH2–CH2–CH2–NH–), 3.33–3.28 (m, 2H, O–CH2–CH2–CH2–NH–),
2.77 (sep, 1H, J = 6.8 Hz, –CH(CH3)2), 1.75–1.67 (m, 2H, O–CH2–
CH2–CH2–NH–), 1.45 (s, 9H, –CO–C(CH3)3), 1.37 (s, 9H, –CO–
C(CH3)3), 1.12 (d, 6H, J = 6.8 Hz, –CH(CH3)2); 13C NMR (63 MHz,
CDCl3) d [ppm] 178.72, 163.52, 156.12, 155.16, 153.39, 147.73,
147.05, 138.81, 122.49, 88.47, 86.74, 83.65, 82.28, 79.58, 70.69,
69.87, 62.66, 38.87, 36.39, 29.32, 28.28, 28.11, 18.96, 18.89; HRMS
(MALDI) was calculated to be 653.3253 for C28H45N8O10 (M+H+),
and found to be 653.3274.
in an ice bath and then 20-O-(2-Aminopropyl)-30,50-O-di-tert-buty-
lsilanediyl guanosine (4e) (200 mg, 0.42 mmol) was added. After
30 min the reaction mixture was removed from the ice bath then
stirred for an additional 30 min at room temperature. The reaction
solution was washed with saturated sodium bicarbonate solution
and brine. After drying over Na2SO4 the solvent was evaporated.
The residue was purified by column chromatography using dichlo-
romethane/methanol (9:1, v/v) to give 270 mg (89%) of 4c. 1H NMR
(400 MHz, DMSO-d6) d [ppm] 11.49 (bs, 1H, NH), 10.66 (bs, 1H,
NH), 8.56–8.53 (m, 1H, NH–CH2–), 7.87 (s, 1H, H8), 6.39 (bs, 2H,
NH2), 5.86 (s, 1H, H10), 4.42–4.38 (m, 1H, H30), 4.30–4.27 (m, 2H,
H20, H50), 4.06–3.93 (m, 3H, H40, H50, ½ ꢂ O–CH2–CH2–CH2–NH–),
3.72–3.67 (m, 1H, ½ ꢂ O–CH2–CH2–CH2–NH–), 3.51–3.30 (m, 2H,
O–CH2–CH2–CH2–NH–), 1.84–1.77 (m, 2H, O–CH2–CH2–CH2–NH–
), 1.46 (s, 9H, –CO–C(CH3)3), 1.39 (s, 9H, –CO–C(CH3)3), 1.06 (s,
9H, –Si–C(CH3)3), 0.97 (s, 9H, –Si–C(CH3)3); HRMS (MALDI) was
calculated to be 723.3856 for C32H55N8O9Si (M+H+), and found to
be 723.3880.
4.2.4.6. N2-Isobutyryl-20-O-(N,N 0-di-boc-guanidinopropyl)-50-O-
(4,40-dimethoxytrityl)-guanosine (4h) and N2-Isobutyryl-20-O-
(N,N 0-di-boc-N00-isobutyryl-guanidinopropyl)-50-O-(4,40-dime-
thoxytrityl)-guanosine (4h⁄).
A mixture of N2-Isobutyryl-20-
O-(N,N0-di-boc-guanidinopropyl)-guanosine (4g) and N2-Isobuty-
ryl-20-O-(N,N0-di-boc-N00-isobutyryl-guanidinopropyl)-guanosine
(4g⁄) (400 mg, ca. 583
lmol) was dissolved in dry pyridine (11 mL).
4,40-Dimethoxytrityl chloride (280 mg, 0.82 mmol) was added and
the solution was stirred for 3 h at room temperature. TLC revealed
that some starting material remained at this time and an additional
0.3 equiv of 4,40-Dimethoxytrityl chloride were therefore added.
When TLC demonstrated that the starting material had been con-
sumed, the reaction was quenched with water and the solvents
evaporated. The residue was purified by column chromatography
using dichloromethane/methanol (98:2, v/v) containing 0.5% tri-
ethylamine to give 427 mg (ca. 74%) of the desired products. 1H
NMR (400 MHz, DMSO-d6) d [ppm] 12.09 (s, 1H, NH), 11.58 (s,
1H, NH), 11.47 (s, 0.5H, NH-boc), 10.50 (s, 0.5H, NH–boc⁄), 8.33–
8.30 (m, 0.5H, 20-O–CH2–CH2–CH2–NH–), 8.15–8.12 (m, 1H, H8),
7.35–7.18 (m, 9H, DMTr), 6.84–6.80 (m, 4H, DMTr), 5.97–5.94
(m, 1H, H10), 5.15–5.13 (m, 1H, 30-OH), 4.42–4.37 (m, 1H, H20),
4.35–4.30 (m, 1H, H30), 4.09–4.03 (m, 1H, H40), 3.72 (s, 6H,
2 ꢂ O–CH3), 3.69–3.47 (m, 2H, 20-O–CH2–CH2–CH2–NH–), 3.37–
3.26 (m, 3H, 20-O–CH2–CH2–CH2–NH–, H50), 3.17–3.13 (m, 1H,
H50), 2.79–2.73 (m, 1.5H, –CH(CH3)2), 1.77–1.67 (m, 2H, 20-O–
CH2–CH2–CH2–NH–), 1.43–1.35 (m, 18H, 2 ꢂ –CO–C(CH3)3), 1.13–
1.10 (m, 6H, –CH(CH3)2), 1.00–0.98 (m, 3H, –CH(CH3)2⁄). As a result
of the mixture comprising mono- and diisobutyryl derivatives,
some of the integrals could not be given as whole numbers. Thus,
signals that depend only on the diisobutyryl compound are marked
with an asterisk. MS (ESI) was calculated to be 955.5 for
4.2.4.4. N2-Isobutyryl-20-O-(N,N0-di-boc-guanidinopropyl)-30,50-
O-di-tert-butylsilanediyl guanosine (4f) and N2-Isobutyryl-20-
O-(N,N0-di-boc-N00-isobutyryl-guanidinopropyl)-30,50-O-di-tert-
butylsilanediyl guanosine (4f⁄).
A solution of compound 4c
(400 mg, 0.55 mmol) in 3.6 mL of pyridine was cooled in an ice
bath and isobutyryl chloride (145 L, 1.37 mmol) was then added
l
dropwise. The mixture was stirred at 0 °C for 1 h, then at room
temperature for 1 h and evaporated to dryness. The residue was
dissolved in 40 mL dichloromethane and washed twice with sat-
urated sodium bicarbonate solution (2 ꢂ 60 mL) and once with
brine (60 mL). The organic phase was dried over Na2SO4 and the
solvent was evaporated. The residue was purified by column
chromatography using dichloromethane/methanol (95:5–90:10,
v/v) to give 318 mg (ca. 70%) of a mixture of mono- and di-isobu-
tyryl derivative. 1H NMR (250 MHz, DMSO-d6) d [ppm] 12.12 (s,
1H, NH), 11.57–11.51 (m, NH, NH-boc), 10.53 (s, NH-boc⁄),
8.54–8.49 (m, 20-O–CH2–CH2–CH2–NH–), 8.25–8.22 (m, 1H, H8),
5.90–5.88 (m, 1H, H10), 4.42–3.42 (m, 9H), 2.85–2.72 (m, 1.5H, –
CH(CH3)2), 1.99–1.73 (m, 2H, 20-O–CH2–CH2–CH2–NH–), 1.47–
1.33 (m, 18H, 2 ꢂ –CO–C(CH3)3), 1.15–0.99 (m, 27H, 2 ꢂ –Si–
C(CH3)3, –CH(CH3)2, –CH(CH3)2⁄). As a result of the mixture com-
prising mono- and diisobutyryl derivatives, some of the integrals
could not be given as whole numbers. Thus, signals that depend
only on the diisobutyryl compound are marked with an asterisk.
MS (ESI) was calculated to be 793.4 for C36H61N8O10Si (M+H+),
and found to be 794.6.
C
49H63N8O12 (M+H+), and found to be 956.5.
4.2.4.7. N2 -Isobutyryl-20-O-(N,N0-di-boc-guanidinopropyl)-50-O-
(4,40-dimethoxytrityl)-guanosine 30-(cyanoethyl)-N,N-diisopro-
pyl phosphoramidite (4d) and N2-Isobutyryl-20-O-(N,N0-di-
boc-N00-isobutyryl-guanidinopropyl)-50-O-(4,40-dimethoxytrityl)-
guanosine 30-(cyanoethyl)-N,N-diisopropyl phosphoramidite
2
0
4.2.4.5. N -Isobutyryl-20-O-(N,N -di-boc-guanidinopropyl)-gua-
nosine (4g) and N2-Isobutyryl-20-O-(N,N0-di-boc-N00-isobutyryl-
guanidinopropyl)-guanosine (4g⁄).
A mixture of N2-Isobuty-
ryl-20-O-(N,N0-di-boc-guanidinopropyl)-30,50-O-di-tert-buty-
lsilanediyl guanosine (4f) and N2-Isobutyryl-20-O-(N,N0-di-boc-N00-
isobutyryl-guanidinopropyl)-30,50-O-di-tert-butylsilanediyl guano-
(4d⁄).
384
noethyl
A mixture of compounds 4h and 4h⁄ (380 mg, ca.
l
mol) was dissolved in dichloromethane (8 mL), then 2-cya-
N,N,N0,N0-tetraisopropylamino
phosphane
(160 lL,
sine (4f⁄) (490 mg, ca. 592
furane (7 mL). Triethylamine (165
(352 L, 2.16 mmol) were then added. After stirring at room tem-
l
mol) was dissolved in dry tetrahydro-
L, 1.11 mmol) and Et3Nꢀ3HF
0.52 mmol) and 4,5-dicyanoimidazole (57 mg, 0.5 mmol) were
added. After 2 h TLC showed complete consumption of the starting
material. The reaction solution was then washed twice with satu-
rated sodium bicarbonate solution (2 ꢂ 50 mL) and once with
brine (100 mL). After drying over Na2SO4 the solvent was evapo-
rated and the residue was purified using column chromatography
with dichloromethane/ethyl acetate (8:2, v/v) containing 0.5% tri-
ethylamine to give 350 mg (ca. 76%) of the two diastereomers of
4d and 4d⁄. 1H NMR (400 MHz, DMSO-d6) d [ppm] 12.11 (bs, 1H,
NH), 11.61–11.57 (m, 1H, NH), 11.46–11.44 (m, 0.5H, NH-boc),
10.50–10.46 (m, 0.5H, NH-boc⁄), 8.31–8.27 (m, 0.5H, 20-O–CH2–
CH2–CH2–NH–), 8.18–8.14 (m, 1H, H8), 7.36–7.19 (m, 9H, DMTr),
6.85–6.80 (m, 4H, DMTr), 5.97–5.88 (m, 1H, H10), 4.64–4.61 (m,
l
l
perature for 1 h the solvent was evaporated. The residue was puri-
fied using column chromatography with dichloromethane/
methanol (9:1, v/v) to give 322 mg (ca. 79%) of a mixture of N2-iso-
butyryl-20-O-(N,N0-di-boc-guanidinopropyl)-guanosine and N2-Iso-
butyryl-20-O-(N,N0-di-boc-N00-isobutyryl-guanidinopropyl)-guano-
sine as white foam. A small sample of the mixture was separated
for NMR spectroscopy. NMR data is given for the mono-isobutyryl
compound. 1H NMR (400 MHz, DMSO-d6) d [ppm] 12.08 (s, 1H,
NH), 11.65 (s, 1H, NH), 11.46 (s, 1H, NH), 8.29 (s, 1H, H8), 8.28–
8.25 (m, 1H, NH–CH2–), 5.91 (d, 1H, J = 6.0 Hz, H10), 5.16 (d, 1H,

J. Brzezinska et al. / Bioorg. Med. Chem. 20 (2012) 1594–1606
1605
1H, H20), 4.44–4.37 (m, 1H, H30), 4.27–4.12 (m, 1H, H40), 3.79–3.18
(m, 10H), 3.72 (s, 6H, 2 ꢂ OCH3), 2.81–2.70 (m, 2.5H, –CH(CH3)2),
2.60–2.47 (m, 2H, –P–O–CH2–CH2–CN), 1.75–1.65 (m, 2H, 20-O–
CH2–CH2–CH2–NH–), 1.41–1.34 (m, 18H, 2 ꢂ –CO–C(CH3)3), 1.15–
1.10 (m, 1⁄8)H; , –N((CH(CH3)2)2, –CO–CH(CH3)2), 1.00–0.96 (m, 3H,
down of HBV replication in a liver-derived line, Huh7 cells were
transfected with 100 ng pCH-9/309128 and 32.5 ng siRNA. Forty-
eight hours after transfection, growth medium was harvested
and HBsAg concentration was measured by ELISA using the MON-
OLISAÒ HBs Ag ULTRA kit (Bio-Rad, CA, USA). Each experiment was
repeated at least in triplicate.
–CH(CH3)2
31P NMR (162 MHz, DMSO-d6)
d [ppm] 149.59,
149.44, 149.52, 149.19. As a result of the mixture comprising
mono- and diisobutyryl derivatives, some of the integrals could
not be given as whole numbers. Thus, signals that depend only
on the diisobutyryl compound are marked with an asterisk. MS
(ESI) was calculated to be 1155.6 for C58H80N10O13P (M+H+), and
found to be 1157.3.
4.4.2. siRNA serum stability assessment
Annealed siRNAs were diluted in 80% FCS to a final concentra-
tion of 5 lM and incubated at 37 °C. At time points ranging from
1 to 24 h, aliquots were removed and snap frozen using liquid
nitrogen. Twenty picomoles of the samples were subjected to elec-
trophoresis through a 10% denaturing polyacrylamide gel then
stained with ethidium bromide.
4.3. Oligonucleotide synthesis
Modified oligonucleotides were synthesised on 500 Å CPG
material on an Expedite 8909 synthesiser using phosphoramidite
chemistry. The GP modified nucleosides were inserted into the
HBV antisense strand (intended guide, 50 UUG AAG UAU GCC
UCA AGG UCG 30) at each of positions 2, 3, 4, 5, 6, 7 and 13 from
the 50 end. The sense strand oligonucleotide (50 ACC UUG AAG
CAU ACU UCA ATT 30) did not include modifications. The duplex
HBV siRNA3 targeted HBV genotype A coordinates 1693 to 1711.
Control siRNA with scrambled unmodified sequences comprised
50-UAUUGGGUGUGCGGUCACGGT-30 (antisense) and 50-CGU-
Acknowledgments
Financial support for this work from the South African National
Research Foundation, Poliomyelitis Research Foundation and Med-
ical Research Council and from the German Research Foundation
(DFG) is gratefully acknowledged.
We would like to thank Stefan Bernhardt for his valuable help
with oligonucleotide synthesis and purification and Diana Knapp
for scientific discussion about conception of the modified
oligonucleotides.
GACCGCACACCCAAUATT-30
(sense).
5-Ethylthio-1H-tetrazole
(0.25 M in acetonitrile) was used as activator. Unmodified 20-
TBDMS-phorphoramidites were benzoyl- (A), isobutyryl- (G) or
acetyl- (C) protected. Coupling time for the modified phosphorami-
dites was 25 min. After completion of synthesis, 30 min of depro-
tection in 3% trichloroacetic acid in dichloromethane was carried
out to ensure complete cleavage of the boc groups. The RNA oligo-
mers were cleaved from the controlled-pore-glass (CPG) support
by incubation at 40 °C for 24 h using an ethanol:ammonia solution
(1:3). The 20-TBDMS groups were deprotected by incubation for
90 min at 65 °C with a triethylamine, N-methylpyrrolidinone and
Et3Nꢀ3HF mixture. RNA oligomers were precipitated with BuOH
at 80 °C for 30 min and purified by anion exchange HPLC using a
Dionex DNA-Pac 100 column(1 M LiCl, water, gradient: 0–70% LiCl
solution in 40 min, flow: 1 mL/min). Oligonucleotides were
desalted in a subsequent reverse phase HPLC step (colunm: phe-
nomenex Jupiter 4u Proteo 90A 250 mm ꢂ 15 mm, 0.1 M triethyl-
ammonium acetate pH 7, acetonitrile, gradient: 0% acetonitrile
for 2 min, 0–37% acetonitrile in 28 min). Identity was confirmed
by mass spectroscopy on a Bruker micrOTOF-Q.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.024.
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siRNA (5 nM final concentration) at ratios of 1:1 and 1:3 (ml:mg),
respectively. The psiCHECK-HBx reporter vector contains the HBx
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