Synthesis of RNA by the rapid phosphotriester method using azido-based 2'-O-protecting groups

Article abstract of DOI:10.1080/15257770903170286

The azidomethyl and 2-(azidomethyl)benzoyl as 2'-OH protecting groups are reported for preparation of oligoribonucleotides by the phosphotriester solid-phase method using O-nucleophilic intramolecular catalysis. The procedures for the synthesis of the corresponding monomer synthons were developed and the usefulness of the application of 2'-O-azidomethyl and 2'-O-2-(azidomethyl) benzoyl groups was examined in the synthesis of different RNA fragments with a chain length of 15-22 nucleotides. The azidomethyl group was found to be more preferable for effective synthesis of oligoribonucleotides. Hybridization properties of RNAs toward their complementary oligonucleotides were examined before and after the removal of 2'-O-azidomethyl groups.

Full text of DOI:10.1080/15257770903170286

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Synthesis Of RNA by the Rapid
Phosphotriester Method Using Azido-
Based 2′-O-Protecting Groups
V. A. Efimov a , A. V. Aralov a , V. N. Klykov a & O. G.
Chakhmakhcheva a
a Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian
Academy of Sciences, Moscow, Russia
Available online: 04 Sep 2009
To cite this article: V. A. Efimov, A. V. Aralov, V. N. Klykov & O. G. Chakhmakhcheva (2009): Synthesis
Of RNA by the Rapid Phosphotriester Method Using Azido-Based 2′-O-Protecting Groups, Nucleosides,
Nucleotides and Nucleic Acids, 28:9, 846-865
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Nucleosides, Nucleotides and Nucleic Acids, 28:846–865, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903170286
SYNTHESIS OF RNA BY THE RAPID PHOSPHOTRIESTER METHOD
USING AZIDO-BASED 2ꢀ-O-PROTECTING GROUPS
V. A. Efimov, A. V. Aralov, V. N. Klykov, and O. G. Chakhmakhcheva
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Moscow, Russia
The azidomethyl and 2-(azidomethyl)benzoyl as 2ꢁ-OH protecting groups are reported for
2
preparation of oligoribonucleotides by the phosphotriester solid-phase method using O-nucleophilic
intramolecular catalysis. The procedures for the synthesis of the corresponding monomer syn-
thons were developed and the usefulness of the application of 2ꢁ-O-azidomethyl and 2ꢁ-O-2-
(azidomethyl)benzoyl groups was examined in the synthesis of different RNA fragments with a chain
length of 15–22 nucleotides. The azidomethyl group was found to be more preferable
for effective synthesis of oligoribonucleotides. Hybridization properties of RNAs toward
their complementary oligonucleotides were examined before and after the removal of
2ꢁ-O-azidomethyl groups.
Keywords Oligoribonucleotides; synthesis; phosphotriester method; azidomethyl and
2-(azidomethyl)benzoyl groups
INTRODUCTION
Natural and modified oligonucleotides are universal tools in a variety
of studies in molecular biology, biotechnology, and medicine. They are
widely used in chip technology, gene diagnostics and therapy, functional
genomics, and nanotechnology because of their hybridization affinity for
target DNA or RNA molecules. Thus, synthetic RNA fragments such as ri-
bozymes and small interfering RNAs (siRNAs) as well as chemically modified
oligonucleotides have attracted increasing interest for molecular biologists
and medical researchers. In response to the current increased demand for
natural RNA fragments and their analogues with predetermined properties,
Received 6 April 2009; accepted 30 June 2009.
This work is supported by Russian Foundation for Basic Reseach (07–04-00455).
Address correspondence to V. A. Efimov, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
RAS, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia. E-mail: eva@mx.ibch.ru
846

2ꢁ-O-Azido-Based Protecting Groups
847
much attention is paid to the development of new strategies for their
chemical synthesis.
Recently, the phosphoramidite method is routinely used for the synthe-
sis of oligoribonucleotides.[1] However, by virtue of its chemical peculiari-
ties, it cannot be used for the synthesis of some modified oligonucleotides,
particularly derivatives containing strong electrophilic centers (e.g., azide
and N-oxide groups) due to the proceeding side reactions. Also, it cannot
be used for the synthesis of stereospecific phosphorothioate analogues of
nucleic acids.
As ribonucleotides contain an additional 2ꢁ-hydroxyl compared with
deoxyribonucleotides, the chemical synthesis of RNA requires an ad-
ditional protecting group for this function. The selection of suitable
protecting groups for the 2ꢁ-OH function of the ribose is one of the
most demanding problems in RNA synthesis. A protective group for 2ꢁ-
hydroxyl must be stable both in the process of chain elongation and
during the removal of other blocking groups, and it must be read-
ily removable at the last step under mild conditions preventing degra-
dation of the resulting oligoribonucleotide. With this connection, sev-
eral problems have been pointed out, including the necessity of pro-
longed coupling times due to the steric hindrance of the 2ꢁ-O-protecting
group, difficulties in its deprotection, 3ꢁ-2ꢁ migration of internucleo-
tidic phosphate groups, the necessity of purification of 2ꢁ-O-protected
oligonucleotide intermediates, and instability of RNA fragments. Various
kinds of 2ꢁ-OH protecting groups have been reported for the phospho-
ramidite method, particularly a tert-butyldimethylsilyl (TBDMS),[2] triiso-
propylsilyloxymethyl (TOM),[3] and bis(2-acetoxyethoxy)methyl (ACE),[4]
t-butyldithiomethyl (DTM),[5] 2-(4-tolylsulfonyl)ethoxymethyl (TEM),[6]
cyanoethyl,[7] 2-cyanoethoxymethyl (CEM),[8] and monomers containing
some of these blocking groups are commercially available. Although known
protecting groups represent improvements in the synthesis of oligoribonu-
cleotides, there are still many points to be resolved in the field of RNA
synthesis.
Earlier, we reported the rapid phosphotriester method involving O-
nucleophilic intramolecular catalysis at the stage of internucleotide bond
formation as an alternative approach to the phosphoramidite oligonu-
cleotide synthesis.[9] It was successfully applied for the synthesis of natural
oligodeoxyribonucleotides as well as modified oligonucleotides containing
2ꢁ-azido- and 2ꢁ-amino-2ꢁ-deoxyuridine residues.[10] Moreover, the potential
of this method for preparation of stereospecific oligonucleotide phospho-
rothioate analogues has been demonstrated.[11,12] In the process of the
further improvement of this approach to the synthesis of natural and mod-
ified RNA fragments, we screened various blocking groups for 2ꢁ-hydroxyls
of ribonucleotides.[12] In the present study, we have examined the useful-
ness of azido-based protecting groups, particularly 2-(azidomethyl)benzoyl

848
V. A. Efimov et al.
(AZMB) and azidomethyl (AZM) groups, as 2ꢁ-O-protective in the synthesis
of oligonucleotides by the phosphotriester method under intramolecular
O-nucleophilic catalysis.
RESULTS AND DISCUSSION
Synthesis of Monomer Building Blocks
The preparation and properties of several types of nucleoside derivatives
containing azido-based protecting groups were described earlier,[13–16] but
they were not applied to the phosphoramidite method of oligonucleotide
synthesis. It was shown that the azido function had a negative influence
on the coupling behavior of the 3ꢁ-O-phosphoramidites, presumably due to
iminophosphorane formation by Staudinger reaction.[17,18]
At first, we developed a synthetic route for the ribonucleotide
monomeric synthons 9, which contained 2ꢁ-O-AZM group, 5ꢁ-O-
4,4ꢁ-dimethoxytrityl (DMTr) group, and O-nucleophilic catalytic
4-methoxy-1-oxido-2-picolyl phosphate protective group. In this route,
shown in Scheme 1, we introduce O-methylthiomethyl group (MTM)
into the 2ꢁ-position of a 3ꢁ,5ꢁ-protected nucleoside 1 by the reaction with
a mixture of dimethylsulfoxide, acetic anhydride and acetic acid.[19]
Then, the MTM derivative of the nucleoside 2 was converted into the
azidomethyl compound 5. The earlier described procedure included the
treatment of MTM derivative 2 with sulfuryl chloride (or Br2) followed
by the action of lithium azide solution in dimethylformamide.[16,20]
SCHEME 1

2ꢁ-O-Azido-Based Protecting Groups
849
However, we observed that a side reaction of halogenation of uracil
and cytosine C-5 positions took place in the presence of Br2, or sulfuryl
chloride, and the yields of undesired halogenation products achieved up
to 50%. We found a solution to this problem, when a milder reagent,
2-nitrobenzenesulfenyl chloride (NBSCl), was used at this stage.[21] As
NBSCl cannot interact with free hydroxyl groups in the absence of bases, a
nucleoside 3ꢁ,5ꢁ-protective group was removed from the derivative 2 before
the treatment with NBSCl. The following in situ addition of lithium azide
allowed us to obtain the pyrimidine nucleoside derivatives 5 with high
yields. For preparation of the purine derivatives 5, NBSC1 was added in
the presence of trifluoromethanesulfonic acid to avoid the side reaction of
the interaction of the 2ꢁ-O-chloromethyl intermediate 4 with the N -3 atom
of a purine nucleobase with the formation of a cyclonucleoside.[21]
After the introduction of 5ꢁ-OH protective dimethoxytrityl group,
the preparation of 3ꢁ-phosphodiester
9
containing the catalytic
4-methoxy-1-oxido-2-picolyl group was performed using a modified
procedure.
Earlier, we described a two-step preparation of the nucleotide phos-
photriesters of type 8, which included the phosphorylation of the 3ꢁ-
hydroxyl of a protected nucleoside with bis(triazolyl)arylphosphate followed
by the treatment with an aqueous triethylammonium bicarbonate solution
(TEAB) to obtain the corresponding nucleoside phosphodiester, which
was converted into the fully protected nucleoside phosphotriester by the
action of 1-oxido-4-methoxy-2-pyridinemethanol in the presence of TPSCl
and MeIm.[21] In this study, we developed a simplified procedure based on
the conversion of the 2ꢁ,5ꢁ-protected nucleoside 6 into the phosphotriester
8 by the action of the preliminary prepared phosphodiester 7 in the
presence of a condensing agent. The following removal of the chlorophenyl
protecting group by the action of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
in aqueous acetonitrile gave the desired synthon 9.
The other group tested by us for the protection of ribonucleotide 2ꢁ-
hydroxyls was the AZMB group. In contrast to the usual protecting groups of
acyl type, which require basic conditions for their removal, AZMB group can
be removed from nucleosides in neutral conditions.[13–15] The introduction
of an AZMB group into 5ꢁ- and 3ꢁ-hydroxyls of deoxyribonucleosides by
the action of (2-azidomethyl)benzoyl chloride was described earlier.[13]
Using a similar procedure, we have synthesized the 2ꢁ-O-AZMB uridine
derivative 11 as shown in Scheme 2. After protection of the 5ꢁ-OH group,
phosphorylation of the compound 12 was performed by the action of the
phosphodiester 13 in the presence of a condensing agent. The removal
of the methyl protecting group from the phosphotriester 14 by treatment
with piperidine gave the monomeric synthon 15. Chemical structures of all
monomers obtained in this study were confirmed by mass spectrometry and
NMR spectroscopy data.

850
V. A. Efimov et al.
SCHEME 2
Automated Phosphotriester RNA Synthesis and Postsynthetic
Deprotection Treatment
To verify the potential of 2ꢁ-O-azido-based protective groups for auto-
mated solid phase synthesis, we have undertaken the preparation of olig-
oribonucleotides with chain lengths of 15–22 units. At first, we synthesized
a set of U15 oligomers to examine the effect of 2ꢁ-O-protecting groups
on the kinetics of the internucleotide bond formation. The synthesis was
˚
performed on a 1 µmol scale using the 1000 A CPG support containing
uridine derivatives 6, or 12. For coupling reactions, monomer synthons
were used as 0.05 M solutions in acetonitrile-pyridine (3:1, v/v), and TPSCl
(0.15 M) was used as the condensing reagent. The average stepwise yield,
obtained by a detritylation assay, was used to evaluate the synthesis efficiency.
It was found that in the case of monomers with an AZM 2ꢁ-O-blocking group
practically quantitative yields were achieved in 2 min, whereas in the case of
monomers with a bulky AZMB blocking group a longer coupling time was
needed (up to 5 minutes; see Figure 1). The conditions of the optimized
synthetic cycle are shown in Table 1.
After the chain elongation was completed, oligonucleotide-anchored
solid supports were treated to remove phosphate protecting groups. Earlier,
we described the use of piperidine (10 hours, 20◦C), or thiophenol-
dioxane-triethylamine (1:2:2, v/v, 3–5 hours, 20◦C) for this purpose. In this
study, we used 1 M lithium iodide solution in acetonitrile (3 hours, room
temperature) for the removal of 1-oxido-4-methoxy-2-picolyl groups from
internucleotide phosphate residues and found that it is a very effective and
convenient reagent. Cleavage from the solid support was then carried out by
the treatment with a mixture of 28% ammonia water solution and ethanol

2ꢁ-O-Azido-Based Protecting Groups
851
FIGURE 1 Mobility shift analysis of crude U15 oligonucleotides by a 15% denaturing PAGE. Deprotec-
tion of oligonucleotides was carried out by the treatment with 1 M lithium iodide solution in acetonitrile
for 3 hours to remove phosphate blocking groups. Then, oligonucleotides were cleaved from the support
by the treatment with a mixture of 28% ammonia water solution and ethanol (3:1, v/v) for 2 hours.
2ꢁ-O-AZM blocking groups were removed by the action of 0.1 M triphenylphosphine in acetonitrile-water
(9:1, v/v) for 2 hours, and 2ꢁ-O-AZMB groups were removed in the same conditions for 5 hours.
Lane 1 shows the oligonucleotide before the removal of 2ꢁ-O-AZM blocking groups (coupling time in
internucleotide condensations was 2 minutes). Lane 2 shows the same fully deprotected oligonucleotide.
Lanes 3–5 show crude oligonucleotides before the removal of 2ꢁ-O-AZMB blocking groups synthesized
using 5 minutes (lane 3), 2 minutes (lane 4), and 1 minute (lane 5) coupling times in the reactions of
the internucleotide bond formation. Visualization was by UV-shadowing at 254 nm.
(3:1, v/v) at room temperature for 1.5–2 hours. As it can be expected
from the literature data,[16] 2ꢁ-O-AZM group remained intact in an aqueous
ammonia solution for a long time at room temperature. The preliminary ex-
amination of the 2ꢁ-O-AZMB group stability to the action of ammonia using
the monomer 15 and control by TLC revealed that it was fully stable under
these conditions for more than 5 hours (data not shown). The resulting
homo-U oligonucleotides carrying 2ꢁ-O-protective groups were isolated by
gel filtration on a NAP-10 column, and the crude products were subjected
to PAGE analysis. As it followed from HPLC data and PAGE analysis (e.g.,
see Figure 1, lane 3), the treatment with ammonia did not influence the
stability of the oligonucleotide 2ꢁ-O-AZMB groups and the stability of an
TABLE 1 Elongation cycle for the solid phase phosphotriester synthesis of oligoribonucleotides using
2ꢁ-O-AZM and 2ꢁ-O-AZMB blocking groups
Step
Solvents and reagents
Time (min)
1. Detritylation 2% Trichloroacetic acid in dichloromethane
1.0
2. Wash
Acetonitrile
1.0
3. Wash
4. Coupling
Acetonitrile—pyridine (3:1, v/v)
0.05 M P-component; 0.15 M TPSCl in acetonitrile—pyridine (3:1,
0.5
2.0(5.0∗)
v/v)
5. Wash
6. Capping
7. Wash
Acetonitrile-pyridine
Ac2O - 1-methylimidazole - pyridine - acetonitrile (1:1:2:6, v/v/v/v)
Acetonitrile
0.5
0.5
1.5
∗The reaction time for internucleotide condensations with monomers containing 2ꢁ-O-AZMB groups.

852
V. A. Efimov et al.
oligonucleotide itself during 2 hours. The next step was removal of the
2ꢁ-O-blocking groups. In model experiments, the efficiency of the removal
of 2ꢁ-O–azido-based groups by the action of 0.1 M triphenylphosphine in
acetonitrile-water (9:1, v/v), or methyldiphenylphosphine in dioxane-water
(4:1, v/v) was checked using all four monomers and the control by TLC.
It was found that 2ꢁ-O-AZM group can be completely removed from the
monomers during 5–10 min, whereas the removal of 2ꢁ-O-AZMB group
from the monomer 15 required about 30 min under the same conditions.
Then, the aliquots of homo-U oligonucleotides carrying 2ꢁ-O-protective
groups were subjected to the treatment with the same reagents to remove
azido-based 2ꢁ-O-blocking groups for 1, 2, 3, and 5 hours with the following
analysis by PAGE. The results obtained have shown that the removal of
2ꢁ-O-AZM groups from oligonucleotide by 0.1 M triphenylphosphine in
acetonitrile-water (9:1, v/v), or by methyldiphenylphosphine in dioxane-
water (4:1, v/v) is complete in 2 hours and 30 minutes, respectively. The
removal of 2ꢁ-O-AZMB groups from oligonucleotides required more time
(1 hour with methyldiphenylphosphine and 3–5 hours with triphenylphos-
phine), probably due to spatial hindrances, and some degradation of the
oligonucleotide chain was observed during this deprotection step (data not
shown).
So, we concluded that 2ꢁ-O-AZM blocking group is more preferable for
the effective synthesis of oligoribonucleotides, and further demonstration
of the efficacy of the automatic solid phase synthesis using our strategy
was carried out on the examples of preparation of several RNAs with
mixed nucleobase sequences using 2ꢁ-O-AZM protected monomer synthons.
The average yields at the coupling steps exceeded 98%. The resulting
oligonucleotides were deprotected as described above. Cleavage from the
solid support and removal of blocking groups from heterocycles were
carried out by the treatment with ammonia. In all cases, reverse-phase
and anion-exchange HPLC as well as PAGE analysis of the final oligomers
were used to confirm the purity of the RNA obtained (Figure 2). For
comparison, oligoribonucleotides with the same sequences were obtained
by the phosphoramidite method using commercially available monomers
and the standard procedure.
Enzymatic Digestion and Hybridization Properties of Synthetic
Oligoribonucleotides
Examination of synthetic RNAs obtained by the phosphotriester method
has shown that they exhibited the same properties as those obtained by the
phosphoramidite method. They demonstrated similar cleavage results when
subjected to RNase H and phosphodiesterase digestion. Figure 3 shows
the analysis of cleavage efficiency with RNase H of oligoribonucleotides.
The results obtained allow us to conclude that oligoribonucleotides

2ꢁ-O-Azido-Based Protecting Groups
853
FIGURE 2 Analysis of an oligoribonucleotide CAGACAAAAGAAAGCGCA by a 15% denaturing PAGE.
Visualization was by UV-shadowing at 254 nm. Lane 1 shows crude oligonucleotide synthesized by the
phosphotriester method before the removal of 2ꢁ-O-AZM blocking group; lane 2 shows the same fully
deprotected crude oligonucleotide; lane 3 shows the control iso-sequential fully deprotected crude
oligoribonucleotide obtained by the phosphoramidite method; lanes 4 and 5 show the oligonucleotides
from lanes 2 and 3 after purification by HPLC.
synthesized by our procedure are pure enough for biological
studies.
At the same time, oligonucleotides containing 2ꢁ-O-AZM groups were
more resistant to the action of nucleases than fully deprotected oligonu-
cleotides. We have tested the stability of oligonucleotides containing 2ꢁ-
O-AZM groups towards RNA-specific nucleases, particularly RNase A and
RNase T1, and found that 2ꢁ-O-AZM RNA is stable to the action of
these enzymes in concentrations that produced total digestion of natural
oligoribonucleotides. Moreover, S1 nuclease, mung bean nuclease and
exonuclease III were also unable to cleave 2ꢁ-O-AZM RNA (data not shown).
At the same time, snake venom phosphodiesterase and bovine spleen
phosphodiesterase were able to cleave 2ꢁ-O-modified oligoribonucleotides,
but the resistance of 2ꢁ-O-AZM RNA to these enzymes was higher then the
resistance of natural oligonucleotides (Figure 4). The data on the human
plasma stability and other biological properties of 2ꢁ-O-AZM RNA will be
presented in detail elsewhere.
Also, we observed a high stability of duplexes formed by 2ꢁ-O-AZM RNAs
with the complementary targets. A comparison of melting temperatures
(Tm) of duplexes formed by fully deprotected RNAs and those containing
2ꢁ-O-AZM groups with complementary synthetic oligonucleotides has shown
that the Tm values of both types of duplexes are very close. Thus, the pres-
ence of 2ꢁ-O-AZM groups in oligoribonucleotides did not significantly affect
the stability of duplexes, although it slightly destabilized them (∼0.25◦C per
base pair; see Table 2).

854
V. A. Efimov et al.
FIGURE 3 Analysis of the RNase H digestion of 32P-labeled oligoribonucleotides by denaturing
PAGE. A) RNase H cleavage of oligonucleotide r(CAUGGAGAGCGACGAGAGC)dTT obtained by the
phosphotriester method. Lanes 1–5 represent digestion of RNA after 2, 5, 10, 30, and 60 minutes
of incubation with the enzyme. Lane 6 shows the oligonucleotide before the treatment. B) RNase
H cleavage of RNA CGCUCUCGUCGCUCUCCAUGU. Lanes 1 shows oligonucleotide synthesized by
phosphotriester method before the addition of the nuclease; lane 2 shows the control iso-sequential
oligonucleotide synthesized by the phosphoramidite method before the addition of the nuclease; lanes
3 and 4 represent digestion of the same oligonucleotides after the incubation with the enzyme for 2
hours. Visualization by autoradiography.
CONCLUSIONS
In this study, we developed a highly effective rapid phosphotriester-
based method for the synthesis of oligoribonucleotides using the AZM
group as a protecting for the 2ꢁ-hydroxyl of ribonucleotides. With this
methodology, RNA synthesis can be carried out on a standard solid-phase
synthesizer with high coupling yields and a coupling time of only 2 minutes.
As the 2ꢁ-O-AZM protective group is stable with ammonia treatment, stan-
dard acyl N-blocking groups and supports can be used. The postsynthesis

2ꢁ-O-Azido-Based Protecting Groups
855
FIGURE 4 Nuclease stability of 2ꢁ-O-AZM oligouridylate. Time course of degradation of 2ꢁ-O-AZM
homo-U15 and natural homo-U15 by venom (VPDE) and spleen (SPDE) phosphodiesterases.
deprotection procedure is simple and gives products of high purity. The
developed method opens prospects for the effective solid phase synthesis
of stereospecific phosphorothioate oligoribonucleotide analogues.[12] Also,
our results demonstrated that the 2ꢁ-O-AZM RNA can be used as a synthetic
intermediate for natural-type RNA as well as a 2ꢁ-O-modified functional
RNA. As the azido group can be converted to other functional groups by a
variety of chemical reactions, single incorporations of 2ꢁ-O-AZM monomers
into oligoribonucleotides may be useful precursors for further postsynthetic
RNA modification by the introduction of various labels and functional
groups via the azido function. Further development of the strategy using
azido-based protection groups for RNA synthesis will be reported in the near
future.
EXPERIMENTAL
Solvents and reagents were obtained from commercial suppliers and
1
were used without further purification. H spectra were recorded on a
Avance III-800 (Bruker, USA) and DPX-300 (Bruker, Germany) spectrom-
eters, and 31P NMR spectra were recorded on a Bruker DPX-300 spectrom-
eter. Chemical shifts are given in p.p.m. relative to tetramethylsilane (1H),
or H3PO4 (31P). Mass spectra were recorded using Ultraflex II MALDI-TOF
(Bruker Daltonics, Germany) mass spectrometer. TLC was carried out on
Merck Silica Gel 60 F254 plates in CHCl3-CH3OH (3:1, v/v; solvent A), or
CHCl3-CH3OH (9:1, v/v; solvent B). Silica gel column chromatography
was performed using Merck silica gel 60. Synthesis of oligonucleotides was

856

2ꢁ-O-Azido-Based Protecting Groups
857
performed on a1 µmol scale using Applied Biosystems Synthesizer 381A.
Electrophoresis of oligomers was performed using polyacrylamide gel/7 M
urea with 0.1 M Tris–borate/EDTA buffer (pH 8.3).
General Procedure for the Synthesis of
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-(azidomethyl)nucleosides (6)
The 2ꢁ-O-methylthiomethyl derivatives 3 were synthesized starting from
the 3ꢁ,5ꢁ-protected nucleosides 1 essentially as it was described by Pechenov
et al.[19] Then, the compound 3 (1 mmol) was treated with 1.5 mmol of 2-
nitrobenzenesulfenyl chloride in 5 ml of dry DMF at 0◦C. For the 2ꢁ-O-MTM
derivatives of adenosine and guanosine, trifluoromethane sulfonic acid (1.2
mmol) was added to the reaction mixture also. After 5 minutes, 5 ml of 1
M solution of lithium azide in DMF was added. After the incubation during
5 minutes, the reaction was terminated by the addition of 0.25 M TEAB
(25 ml). In the case of the uridine derivative 5, the mixture obtained was
washed with chloroform (2 × 20 ml), and the aqueous solution containing
2ꢁ-azidomethyluridine was evaporated to dryness. In all other cases, the
reaction mixture was extracted with chloroform—1-butanol (7:3, v/v, 3 ×
20 ml), and combined organic fractions were evaporated. The residue
containing the crude product was dried by co-evaporation with toluene, and
a 2ꢁ-O-AZM nucleoside (5) was isolated by silica gel column chromatography
using a gradient of methanol (0–10%) in chloroform. Fractions containing
the desired product were combined, concentrated to a gum and dried in
vacuo. The analytical data obtained for compounds 5 were in accordance
with the literature data.[16] The following 5ꢁ-protection of 2ꢁO-azidomethyl
nucleosides was performed by the action 4,4ꢁ-dimethoxytritylchloride
(1.2 equiv.) in pyridine during 1.5 hours. The reaction was stopped by the
addition of 0.5 M TEAB (20 ml), the crude product was extracted with chlo-
roform (2 × 20 ml). The combined organic fractions were evaporated to a
gum. The residue was dissolved in chloroform, and a 5ꢁ-O-dimethoxytrityl
-2ꢁ-O-azidomethyl nucleoside 6 was isolated by silica gel column chromatog-
raphy using a gradient of methanol (0–5%) in chloroform containing 0.1%
of triethylamine (TEA). The fractions containing the desired derivative (6)
were evaporated to a gum and dried in vacuo.
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-(azidomethyl)uridine. Yield 85%, Rf (sol-
1
vent B) = 0.45. H-NMR (300 MHz, CDCl3): δ 8.01 (1H, d, J = 8.1 Hz,
Ura-H-6), 7.82–7.43 (13H, m, DMTr-H-Ar), 5.98 (1H, d, J = 5.4 Hz, H-1ꢁ),
5.33 (1H, d, J = 8.1 Hz, Ura-H-5), 5.03 (1H, d, J = 9.0 Hz, OCHαN), 4.99
(1H, d, J = 9.0 Hz, OCHβN), 4.56–4.52 (1H, m, H-2ꢁ), 4.33–4.28 (1H, m,
H-3ꢁ), 4.10–4.08 (1H, m, H-4ꢁ), 3.81 (6H, s, DMTr-OCH3), 3.64–3.52 (2H,
m, H-5ꢁ). MS: 602.20, calc. for C31H31N5O8 (M +H+) 602.22.
N4-Benzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-(azidomethyl)cytidine. Yield 77%,
1
Rf (solvent B) = 0.55. H-NMR (800 MHz, CDCl3): δ 8.58 (1H, d, J = 7.3

858
V. A. Efimov et al.
Hz, Cyt-H-6), 7.94–7.50 (5H, m, Bz-H-Ar), 7.48–6.89 (14H, m, DMTr-H-Ar,
Cyt-H-5), 5.99 (1H, s, H-1ꢁ), 5.20 (1H, d, J = 8.7 Hz, OCHαN), 5.15 (1H,
d, J = 8.7 Hz, OCHβN), 4.58–4.54 (1H, m, H-3ꢁ), 4.33 (1H, d, J = 5.6 Hz,
H-2ꢁ), 4.15 (1H, dt, J = 8.8 Hz, J = 2.3 Hz, H-4ꢁ), 3.85 (6H, d, J = 1.6 Hz,
DMTr-OCH3), 3.66 (1H, dd, J = 10.8 Hz, J = 2.3 Hz, Hα-5ꢁ), 3.61 (1H, dd, J
= 10.8 Hz, J = 2.3 Hz, Hβ-5ꢁ). MS: 727.30, calc. for C38H36N6O8 (M +Na+)
727.25.
N6-Benzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-(azidomethyl)adenosine.Yield
1
63%, Rf (solvent B) = 0.55. H-NMR (800 MHz, CDCl3): δ 8.82 (1H, s,
Ade-H-8), 8.09 (1H, s, Ade-H-2), 8.06–7.54 (5H, m, Bz-H-Ar), 7.35–6.84
(13H, m, DMTr-H-Ar), 6.05 (1H, d, J = 7.6 Hz, H-1ꢁ), 5.05–5.03 (1H, m,
H-3ꢁ), 4.73 (1H, d, J = 9.1 Hz, OCHαN), 4.69 (1H, d, J = 9.1 Hz, OCHβN),
4.64–4.63 (1H m, H-2ꢁ), 4.41 (1H, m, H-4ꢁ), 4.03–4.00 (1H, m, Hα-5ꢁ),
3.82 (6H, s, DMTr-OCH3), 3.81–3.80 (1H, m, Hβ-5ꢁ). MS: 729.20, calc. for
C39H36N8O7 (M +H+) 729.28.
N2-Isobutyryl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-(azidomethyl)guanosine. Yield
1
57%, Rf (solvent B) = 0.40. H-NMR (800 MHz, CDCl3): δ 7.87 (1H, s,
Gua-H-8), 7.38–6.84 (13H, m, DMTr-H-Ar), 5.99 (1H, d, J = 5.9 Hz, H-1ꢁ),
4.89 (1H, d, J = 9.1 Hz, OCHαN), 4.85 (1H, d, J = 9.1 Hz, OCHβN),
4.72–4.69 (1H, m, H-3ꢁ), 4.28–4.25 (1H, m, H-2ꢁ), 3.97–3.94 (1H, m, H-4ꢁ),
3.82 (6H, s, DMTr-OCH3), 3.56–3.53 (1H, m, Hα-5ꢁ), 3.26–3.23 (1H, m,
Hβ-5ꢁ), 2.61 (1H, m, iBu-CH), 1.10, 1.06 (6H, d, J = 7.2 Hz, iBu-CH3). MS:
711.30, calc. for C36H38N8O8 (M +H+) 711.29.
Triethylammonium (4-Chlorophenyl)-(1-oxido-4-methoxy-2-
picolyl) phosphate (7)
1-Oxido-4-methoxy-2-pyridinemethanol (3.1 g, 20 mmol) was dissolved
in CH2Cl2 (100 ml), TEA (3.5 ml, 25 mmol) was added, and the solution
was cooled to 0◦C. Then, bis(2-chlorophenyl) chlorophosphate (5.28 ml,
22 mmol) was slowly added under stirring. After 20 minutes, CH2Cl2 (200
ml) was added, and the reaction mixture was washed with 0.5 M TEAB (2
× 100 ml). The organic fraction was evaporated, and the phosphotriester
obtained was isolated by silica gel column chromatography in a gradient of
methanol (1–2%) in chloroform. Fractions containing the desired product
[Rf (solvent B) = 0.7] were evaporated. The residue was dissolved in 75
ml of acetonitrile, and 11 ml of water and 14 ml of TEA were added. The
solution obtained was incubated at 50◦C for 2 hours. Then, water (100 ml)
was added, and the mixture was washed with CH2Cl2 (2 × 100 ml). The
water solution was evaporated to dryness, and the residue was co-evaporated
with pyridine. The gum obtained was dissolved in 50 ml of pyridine and
precipitated with dry ether (500 ml). The precipitate of the compound 7
[Rf (solvent A) = 0.3] was collected by centrifugation. The yield was 8.85 g
(81%). 1H-NMR (300 MHz, CDCl3): δ 8.05 (1H, d, J = 7.2 Hz, picolyl-H-6),

2ꢁ-O-Azido-Based Protecting Groups
859
7.71–7.10 (4H, m, H-Ar), 7.25 (1H, d, J = 3.4 Hz, picolyl-H-3), 6.69 (1H, dd,
J = 7.2 Hz, J = 3.4 Hz, picolyl-H-5), 5.27 (2H, d, J = 7.4 Hz, P-OCH2), 3.73
(3H, s, picolyl-OCH3), 3.05 (6H, dq, J = 7.3 Hz, J = 3.5 Hz, +HNEt3-CH2),
+
1.31 (9H, t, J = 7.3 Hz, HNEt3-CH3). 31P NMR (121.5 MHz, CDCl3) δ =
4.92. MS: 346.05, calc. for C13H13ClNO6P (M +H+) 346.02.
General Method for the Synthesis of Ribonucleotide Monomers
(9) Containing 2ꢁ-O-AZM Protecting Groups
To introduce a phosphate residue bearing the catalytic protective group,
the compound 6 (1 mmol) was allowed to react with (4-chlorophenyl)-(1-
oxido-4-methoxy-2-picolyl) phosphate 7 (2 mmol) in 10 ml of dry pyridine
in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl) (3
mmol) for 15 minutes. The reaction was terminated by the addition of 1
M TEAB (20 ml). The solution obtained was extracted with chloroform
(2 × 20 ml), and the combined organic fractions were evaporated to
dryness. To remove the traces of pyridine, the residue was evaporated with
toluene. The gum obtained was treated with 10 ml of 0.25 M solution
of DBU in CH3CN-H2O (9:1, v/v) for 16 hours at room temperature.
After the following treatment with 0.5 M TEAB (10 ml), the reaction
mixture was extracted with chloroform (2 × 15 ml). The combined organic
fractions were washed with 0.5 M TEAB (20 ml), evaporated to dryness
and co-evaporated with toluene. The product was isolated by silica gel
column chromatography using 0–15% gradient of methanol in chloroform
with 1% of TEA. The fractions containing the desired compound 9 were
concentrated by evaporation to a gum, which was dissolved in chloro-
form (20 ml), washed with 20 ml of 0.5 M 1,8-Diazabicyclo[5.4.0]undec-
7-enium hydrogen carbonate. After the concentration of chloroform so-
lution under reduced pressure, foam obtained was additionally dried in
vacuo.
1,8-Diazabicyclo[5.4.0]undec-7-enium 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-(azido-
methyl)uridine 3ꢁ-O-(1-oxido-4-methoxy-2-picolyl)phosphate. Yield 67.5%, Rf (sol-
1
vent A) = 0.25. H-NMR (300 MHz, CDCl3): δ 8.10 (1H, d, J = 7.2 Hz,
picolyl-H-6), 7.74 (1H, d, J = 8.1 Hz, Ura-H-6), 7.37–6.77 (14H, m, DMTr-
H-Ar, picolyl-H-3), 6.71 (1H, dd, J = 7.2 Hz, J = 3.4 Hz, picolyl-H-5), 6.12
(1H, d, J = 5.4 Hz, H-1ꢁ), 5.20–5.07 (2H, m, P-OCH2), 5.16 (1H, d, J = 8.1
Hz, Ura-H-5), 5.05–4.99 (2H, m, OCHαN, H-3ꢁ), 4.77 (1H, d, J = 9.0 Hz,
OCHβN), 4.65–4.59 (1H, m, H-2ꢁ), 4.41–4.35 (1H, m, H-4ꢁ), 3.79 (3H, s,
picolyl-OCH3), 3.77 (6H, d, J = 1.8 Hz, DMTr-OCH3), 3.57–3.36 (8H, m,
H-5ꢁ, DBU-CH2), 2.83–2.86 (2H, m, DBU-CH2), 1.96 (2H, q, J = 5.6 Hz,
DBU-CH2), 1.71–1.64 (6H, m, DBU-CH2). 31P NMR (121.5 MHz, CDCl3) δ
= 0.35. MS: 993.40, calc. for C47H55N8O13P (M +Na+) 993.35.
1,8-Diazabicyclo[5.4.0]undec-7-enium N4-benzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
2ꢁ-O-(azidomethyl)cytidine 3ꢁ-O-(1-oxido-4-methoxy-2-picolyl)phosphate. Yield
1
64%, Rf (solvent A) = 0.3. H-NMR (800 MHz, CDCl3): 8.06 (1H, d,

860
V. A. Efimov et al.
J = 7.0 Hz, picolyl-H-6), 7.93 (1H, m, Cyt-H-6), 7.61–7.41 (5H, m, Bz-H-Ar),
7.34–6.85 (15H, m, DMTr-H-Ar, Cyt-H-6, picolyl-H-3), 6.71 (1H, dd, J =
7.0 Hz, J = 3.4 Hz, picolyl-H-5), 6.15 (1H, d, J = 3.2 Hz, H-1ꢁ), 5.17 (1H, dd,
J = 16.7 Hz, J = 7.3 Hz, P-OCHα), 5.11 (1H, d, J = 8.8 Hz, OCHαN), 5.06
(1H, dd, J = 16.7 Hz, J = 7.3 Hz, P-OCHβ), 5.01–4.98 (1H, m, H-3ꢁ), 4.98
(1H, d, J = 8.8 Hz, OCHβN), 4.62–4.60 (1H, m, H-2ꢁ), 4.46–4.44 (1H, m,
H-4ꢁ), 3.81 (6H, d, J = 2.3 Hz, DMTr-OCH3), 3.79 (3H, s, picolyl-OCH3),
3.51–3.35 (8H, m, H-5ꢁ, DBU-CH2), 2.81 (2H, m, DBU-CH2), 1.97 (2H, q,
J = 5.6 Hz, DBU-CH2), 1.71 (4H, m, DBU-CH2), 1.66 (2H, m, DBU-CH2).
31P NMR (121.5 MHz, CDCl3) δ = 0.08. MS: 1074.40, calc. for C54H60N9O13P
(M +H+) 1074.41.
1,8-Diazabicyclo[5.4.0]undec-7-enium N6-benzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
2ꢁ-O-(azidomethyl)adenosine 3ꢁ-O-(1-oxido-4-methoxy-2-picolyl)phosphate. Yield
1
62%, Rf (solvent A) = 0.3. H-NMR (800 MHz, CDCl3): δ 8.72 (1H, s,
Ade-H-8), 8.18 (1H, s, Ade-H-2), 8.08 (1H, d, J = 7.3 Hz, picolyl-H-6),
8.06–7.52 (5H, m, Bz-H-Ar), 7.43–6.77 (14H, m, DMTr-H-Ar, picolyl-H-3),
6.74 (1H, dd, J = 7.3 Hz, J = 3.4 Hz, picolyl-H-5), 6.34 (1H, d, J =
6.4 Hz, H-1ꢁ), 5.32–5.30 (1H, m, H-3ꢁ), 5.20 (1H, dd, J = 17.0 Hz, J =
7.0 Hz, P-OCHα), 5.16–5.12 (2H, m, P-OCHβ, H-2ꢁ), 5.01 (1H, d, J = 9.1
Hz, OCHαN), 4.80 (1H, d, J = 9.1 Hz, OCHβN), 4.60–4.58 (1H, m, H-4ꢁ),
3.81 (3H, s, picolyl-OCH3), 3.78 (6H, s, DMTr-OCH3), 3.53 (1H, dd, J =
10.8 Hz, J = 2.6 Hz, Hα-5ꢁ) 3.48 (1H, dd, J = 10.8 Hz, J = 2.6 Hz, Hβ-5ꢁ),
3.44–3.36 (6H, m, DBU-CH2), 2.83 (2H, m, DBU-CH2), 1.97 (2H, q, J =
5.6 Hz, DBU-CH2), 1.73 (4H, m, DBU-CH2), 1.66 (2H, m, DBU-CH2). 31P
NMR (121.5 MHz, CDCl3) δ = 0.01. MS: 1098.40, calc. for C55H60N11O12P
(M +H+) 1098.42.
1,8-Diazabicyclo[5.4.0]undec-7-enium N2-isobutyryl-5ꢁ-O-(4,4ꢁ-dimethoxytri-
tyl)-2ꢁ-O-(azidomethyl)guanosine
3ꢁ-O-(1-oxido-4-methoxy-2-picolyl)phosphate.
1
Yield 60%, Rf (solvent A) = 0.2. H-NMR (800 MHz, CDCl3): δ 8.03 (1H,
d, J = 7.0 Hz, picolyl-H-6), 7.76 (1H, s, Gua-H-8), 7.38–6.71 (14H, m,
DMTr-H-Ar, picolyl-H-3), 6.73 (1H, dd, J = 7.0 Hz, J = 3.4 Hz, picolyl-H-5),
6.01 (1H, d, J = 5.6 Hz, H-1ꢁ), 5.42–5.39 (1H, m, H-3ꢁ), 5.22–5.16 (2H,
m, P-OCH2), 5.14–5.11 (1H, m, H-2ꢁ), 4.91 (1H, d, J = 9.1 Hz, OCHαN),
4.72 (1H, d, J = 9.1 Hz, OCHβN), 4.48–4.45 (1H, m, H-4ꢁ), 3.79 (3H, s,
picolyl-OCH3), 3.77 (6H, d, J = 3.2 Hz, DMTr-OCH3), 3.48–3.26 (8H, m,
H-5ꢁ, DBU-CH2), 2.78–2.62 (2H, m, DBU-CH2), 2.51–2.44 (1H, m, iBu-CH),
1.95 (2H, q, J = 5.6 Hz, DBU-CH2), 1.78–1.58 (6H, m, DBU-CH2), 1.11,
1.03 (6H, d, J = 7.3 Hz, iBu-CH3). 31P NMR (121.5 MHz, CDCl3) δ = 0.76.
MS: 1080.40, calc. for C52H62N11O13P (M +H+) 1080.
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-[2-(azidomethyl)benzoyl]uridine
(12)
5ꢁ,3ꢁ-O-(1,1,3,3-tetraisopropyldisilox-1,3-diyl)uridine (0.98 g, 2 mmol)
was dissolved in 10 ml of dichloromethane and allowed to react with

2ꢁ-O-Azido-Based Protecting Groups
861
2-(azidomethyl)benzoyl chloride (0.59 g, 3 mmol) in the presence of 0.5
ml of 1-methylimidazole for 30 minutes. The reaction was terminated by the
addition of 15 ml of 0.5 M TEAB. Then, the organic fraction was isolated
and washed with 10% solution of citric acid (15 ml). Dichloromethane so-
lution of 2ꢁ-O-2-(azidomethyl)benzoyl)-5ꢁ,3ꢁ-O-(1,1,3,3-tetraisopropyldisilox-
1,3-diyl)uridine 10 was evaporated and co-evaporated with acetonitrile.
The residue was dissolved in THF (20 ml) and treated with 1 M solution
of TBAF (5 ml) in THF for 2 hours. After the evaporation, crude 2ꢁ-O-
(2-azidomethyl)benzoyl uridine 11 [Rf (solvent B) = 0.65] was dissolved
in chloroform and purified by silica gel column chromatography in a
gradient of methanol (0–10%) in chloroform. The dimethoxytritylation of
compound 11 was performed as described for the derivative 6. The product
12 [Rf (solvent B) = 0.8] was obtained with the yield 0.93 g (66%). 1H-NMR
(800 MHz, CDCl3): δ 8.01 (1H, d, J = 7.3 Hz, Ura-H-6), 7.94–7.45 (17H, m,
DMTr-H-Ar, AZMB-H-Ar), 6.12 (1H, d, J = 5.4 Hz, H-1ꢁ), 5.56–5.54 (1H,
m, H-2ꢁ), 5.35 (1H, d, J = 7.3 Hz, Ura-H-5), 4.91(1H, d, J = 15.0 Hz,
AZMB-CHαN), 4.79 (1H,d, J = 15.0 Hz, AZMB-CHβN), 4.37–4.35 (1H,m,
H-3ꢁ), 4.12–4.09 (1H, m, H-4ꢁ), 3.81 (6H,s, DMTr-O-CH3), 3.66–3.57 (2H,
m, H-5ꢁ). MS: 728.20, calc. for C38H35N5O9 (M +Na+) 728.23.
Triethylammonium Methyl-(1-oxido-4-methoxy-2-picolyl)-
phosphate (13)
1-Oxido-4-methoxy-2-pyridinemethanol (1.55 g, 10 mmol) was dried by
co-evaporation with pyridine. Then, CH2Cl2 (25 ml) and TEA (3.5 ml,
25 mmol) were added, and the suspension obtained was cooled to 0◦C.
Dimethylchlorophosphate (11 ml of 1 M solution in CH2Cl2) was slowly
added to the suspension under the stirring. After 30 minutes, the reaction
was terminated by the addition of 1 M solution of TEAB (30 ml). The crude
product was extracted with 1-butanol-chloroform (3:7, v/v, 2 × 30 ml). The
organic fractions were combined and evaporated to a gum. It was dissolved
in acetonitrile (20 ml) and treated with piperidine (4 ml) for 16 hours
at room temperature to remove a methyl group. After evaporation, the
crude compound 13 was dissolved in 20 ml of chloroform, and TEA (5 ml)
was added. After 10 minutes, the solution was evaporated to dryness. The
residue was dissolved in pyridine (10 ml) and the product 13 [Rf (solvent
A) = 0.22] was precipitated with dry ether (100 ml). The precipitate was
collected by centrifugation and dried in vacuo. The yield was 1.97 g (56%).
1H-NMR (300 MHz, CDCl3): δ 8.12 (1H, d, J = 7.2 Hz, picolyl-H-6), 7.37
(1H, d, J = 3.4 Hz, picolyl-H-3), 6.81 (1H, dd, J = 7.2 Hz, J = 3.4 Hz,
picolyl-H-5), 5.28 (2H, d, J = 7.4 Hz, P-OCH2), 3.81 (3H, s, picolyl-OCH3),
3.65 (3H, s, P-OCH3), 3.05 (6H, q, J = 7.3 Hz, +HNEt3-CH2), 1.31 (9H, t, J
= 7.3 Hz, +HNEt3-CH3). 31P NMR (121.5 MHz, CDCl3) δ = 0.1. MS: 252.10,
calc. for C8H14NO6P (M +H+) 252.06.

862
V. A. Efimov et al.
1,8-Diazabicyclo[5.4.0]undec-7-enium 5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-
2ꢁ-O-[2-(azidomethyl)benzoyl]uridine 3ꢁ-O-(1-oxido-4-methoxy-2-
picolyl)phosphate (15)
The synthesis and isolation of the compound 14 was accomplished
starting from the derivative 12 (0.71 g, 1 mmol) and methyl-(1-oxido-4-
methoxy-2-picolyl) phosphate 13 (0.7 g, 2 mmol) according the method
described above for 8. The removal of methyl group from the phosphate
residue of the nucleoside phosphotriester 14 was performed by the action
of piperidine (4 ml) in acetonitrile (10 ml) during 3 hours at room
temperature. The yield of compound 15 was 0.59 g (55%), Rf (solvent A)
= 0.2. 1H-NMR (800 MHz, CDCl3): δ 8.03 (1H, d, J = 7.2, picolyl-H-6), 7.76
(1H, m, Ura-H-6), 7.56–6.78 (18H, m, DMTr-H-Ar, AZMB-H-Ar, picolyl-H-
3), 6.71 (1H, dd, J = 7.2 and 3.4 Hz, picolyl-H-5), 6.27 (1H, d, J = 4.3
Hz, H-1ꢁ), 5.68–5.64 (1H, m, H-2ꢁ), 5.37 (1H, d, J = 8.1 Hz, Ura-H-5),
5.25–5.20 (1H,m, H-3ꢁ), 5.11–5.03 (2H, m, P-CH2), 4.87 (1H, d, J = 15.0
Hz, AZMB-CHα-N), 4.74 (1H,d, J = 15.0 Hz, AZMB-CHβN), 4.44–4.39 (1H,
m, H-4ꢁ), 3.77 (6H,d, J = 7.0 Hz, DMTr-O-CH3), 3.72 (3H, s, picolyl-OCH3),
3.57–3.37 (8H, m, H-5ꢁ, DBU-CH2), 2.83–2.86 (2H, m, DBU-CH2), 1.96
(2H, q, J = 5.6 Hz, DBU-CH2), 1.71–1.65 (6H, m, DBU-CH2). 31P NMR
(121.5 MHz, CDCl3) δ = 0.309. MS: 1075.35, calc. for C54H59N8O14P (M
+H+) 1075.40.
Synthesis and Deprotection of Oligonucleotides
The synthesis of oligodeoxyribonucleotides by the phosphoramidite
method was performed using commercially available monomers and stan-
dard protocols. Synthesis of oligoribonucleotides was carried out by the
automated phosphoramidite method using 1-phenyl-1H-tetrazole-5-thiol as
the activation agent as described.[22] Chain elongation in the solid phase
synthesis of oligoribonucleotides by the phosphotriester method was carried
out at 1 µmol scale according to conditions given in Table 1. Solid
supports derivatized with nucleosides (6) and (12) were prepared from
LCAA CPG using the procedure described earlier.[23] The deprotection of
oligonucleotides included the removal of catalytic P-protective groups by
the action of 1 M solution of LiI in acetonitrile (1 ml) at room temperature
for 3 hours with the following washing the support-bound oligonucleotide
with acetonitrile. The removal of N-protecting groups from heterocycles
and cleavage from the support were achieved by the treatment with con-
centrated aqueous ammonia for 48–62 hours at room temperature. Then,
oligonucleotides were desalted by gel filtration using Pharmacia NAP-10
columns, and the fractions containing oligonucleotides were lyophilized.
The powder of 2ꢁ-O-protected oligonucleotide obtained was treated with 0.5
ml of 0.1 M solution of triphenylphosphine in acetonitrile-water (9:1, v/v)

2ꢁ-O-Azido-Based Protecting Groups
863
containing 25 mM Tris-HCl (pH 7.0) for 2 hours, or with 0.1 M solution
of methyldiphenylphosphine in dioxane-water (4:1, v/v) containing 25 mM
Tris-HCl (pH 7.0) for 20–30 minutes at room temperature. The mixture
was diluted with 0.5 ml of 0.1 M TEAB (pH 7) and washed with 1 ml
of the ether. The water fraction was purified by anion-exchange HPLC
(high performance liquid chromatography) and/or gel-electrophoresis as
described.[9,22] The identity of the oligomers obtained was confirmed by
MALDI-TOF MS.
Thermal Denaturation and Enzyme Degradation Experiments
Absorbance (260 nm) versus temperature curves of duplexes formed
by RNA and DNA oligomers were measured using a Pharmacia Ultrospec
2000 (Pharmacia, Sweden) UV-VIS spectrophotometer equipped with a
heated sample holder and a Peltier temperature control accessory. Solutions
contained 3–5 µM of each oligomer in 150 mMNaCl/10 mM Tris–HCl (pH
7–8)/5 mM EDTA/10 mM MgCl2. Data were collected with the help of the
corresponding software from 20 to 95◦C using the heating rate 0.5◦C/min
with an equilibration time 0.2 minute at each temperature.
In assays for enzymatic stability, each RNA sample (5–10 pmol) was
5ꢁ-end labeled with 32P using T4 polynucleotide kinase and [γ -32P]ATP
by standard procedure and mixed with the corresponding unlabeled RNA
(1 OD unit at 260 nm). The digestions of synthetic oligonucleotides with
phosphodiesterase I (0.5 U) were performed in 20 mM Tris-HCl (pH
8.0)/50 mM NaCl/10 mM MgCl2 with a total reaction volume of 100 µl.
Assays for bovine spleen phosphodiesterase stability were performed in 30
mM NaOAc (pH 6.0) (pH 7.5)/10mM supplemented with the enzyme (0.4
U). A reaction mixture was incubated at 37◦C. At appropriate periods, 5 µl
aliquots of the reaction mixture were taken, and the enzyme was deactivated
by heating at 100◦C for 2 minutes. The mixtures were then analyzed by 20%
denaturing PAGE, and radioactive densities of the gel were visualized by a
bio-imaging analyzer.
RNase H digestion of RNAs was carried out according to the following
procedure: oligonucleotide (0.1 µM, 25 µl), which was preliminary 5ꢁ-
labeled using T4 polynucleotide kinase and [γ -32P]ATP by the standard
procedure, and the complementary DNA (0.5 µM, 25 µl) in 20 mM
Tris-HCl(pH 8)/0.1 M KCl/0.1M EDTA/10 mM MgCl2 were annealed by
heating at 90◦C for 5 minutes with the following slow cooling to 20◦C. After
the addition of DTT to 0.1 mM, the solution was incubated with 1 U RNase
H (Gibco BRL) at 37◦C. Aliquots (5 µl) were taken after 2, 5, 10, 30, and
60 minutes, and the reactions were terminated by mixing with 10 µl of 0.05
M EDTA, 0.05% (w/v) xylene cyanole FF and 0.05% (w/v) bromophenol
blue in 80% formamide. The digestion products were analyzed by a 15%
denaturing PAGE.

864
V. A. Efimov et al.
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