2-(4-Tolylsulfonyl)ethoxymethyl (TEM) - A new 2′-OH protecting group for solid-supported RNA synthesis

Article abstract of DOI:10.1039/b614210a

The 2-(4-tolylsulfonyl)ethoxymethyl (TEM) as a new 2′-OH protecting group is reported for solid-supported RNA synthesis using phosphoramidite chemistry. The usefulness of the 2′-O-TEM group is exemplified by the synthesis of 12 different oligo-RNAs of var

Full text of DOI:10.1039/b614210a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
2-(4-Tolylsulfonyl)ethoxymethyl (TEM)—a new 2ꢀ-OH protecting group for
solid-supported RNA synthesis†
Chuanzheng Zhou, Dmytro Honcharenko and Jyoti Chattopadhyaya*
Received 29th September 2006, Accepted 14th November 2006
First published as an Advance Article on the web 28th November 2006
DOI: 10.1039/b614210a
The 2-(4-tolylsulfonyl)ethoxymethyl (TEM) as a new 2ꢀ-OH protecting group is reported for
solid-supported RNA synthesis using phosphoramidite chemistry. The usefulness of the 2ꢀ-O-TEM
group is exemplified by the synthesis of 12 different oligo-RNAs of various sizes (14–38 nucleotides
long). The stepwise coupling yield varied from 97–99% with an optimized coupling time of 120 s. The
synthesis of all four pure phosphoramidite building blocks is also described. Two new reliable
ꢀ
ꢀ
ꢀ
ꢀ
parameters, dC2 − dC3 and dH2 − dH3 , have been suggested for the characterization of isomeric
2ꢀ-O-TEM and 3ꢀ-O-TEM as well as other isomeric mono 2ꢀ/3ꢀ-protected ribonucleoside derivatives.
The most striking feature of this strategy is that the crude RNA prepared using our 2ꢀ-O-TEM strategy
is sufficiently pure (>90%) for molecular biology research without any additional purification step,
thereby making oligo-RNAs easily available at a relatively low cost, saving both time and lab resources.
Introduction
Recent development of RNA interference (RNAi),1 and evidence
that short interfering RNA (siRNA)2 can effectively silence gene
expression, have highlighted the need for dependable methodolo-
gies for the chemical synthesis of oligo-RNA sequences 20–25-
nucleotides long. The most convenient way to obtain oligo-RNA
is solid-supported synthesis using phorsphoramidite building
blocks.3 For this, the correct choice of a 2ꢀ-OH protecting group is
necessary for the assembly of the desired RNA sequence, because
the nature of this protecting group can influence considerably
the coupling reaction time4 and the coupling yield between the
3ꢀ-O-phosphoramidite monomer block and the 5ꢀ-OH terminal
of the ribonucleotide chain on the solid support, as well as the
purity of the final product. With the aim of circumventing this,
the sterically less hindered and more reactive 3ꢀ-(2-cyanoethyl-
N-ethyl-N-methylamino phosphoramidite), in conjunction with a
2ꢀ-O-tBDMS group, has been tried, but without favorable results.4
Therefore, the design of an ideal 2ꢀ-OH protecting group for
Scheme 1 The 2ꢀ-OH protecting groups used in oligo-RNA synthesis.
improving RNA synthesis has been a key issue during past 30 years
or so.5 The currently available 2ꢀ-OH protecting groups can be
classified into the following types, depending upon the unblocking
conditions (as shown in Scheme 1): (1) photosensitive groups such
as Nbn6 and Nbom,7 (2) acid-labile acetal derivatives such as
Mthp,8 MDMP,9,10 Ctmp and Fpmp,11 (3) base-labile groups such
as Npes,12 Fnebe and Nebe,13 (4) the reductively removable DTM
group with a labile S–S bond,14 (5) fluoride-labile groups such as
tBDMS,15–17 SEM,18 or CEE,19 which have been found to be useful
in the solid-phase oligo-RNA synthesis.
An ideal 2ꢀ-OH protecting group should be:20 (1) easy to
introduce, (2) achiral, (3) unable to migrate to vicinal 3ꢀ-OH, (4)
completely stable under the conditions required for the assembly
of the fully desired RNA sequence (as well as for its subsequent
unblocking and release from the solid support), (5) removable
under conditions under which RNA is completely stable (the 3ꢀ→5ꢀ
phosphodiester should also not isomerize to a 2ꢀ→5ꢀ linkage).
Though the 2ꢀ-OH protecting groups shown in Scheme 1 are
widely used, none of them completely fulfils the above criteria. For
example, tBDMS, the most widely used 2ꢀ-OH protecting group,
is unstable21 in ammonia solution, giving chain cleavage22 and the
product of migration to 3ꢀ-OH,23 as well as giving a relatively poor
coupling yield after the required coupling time of ca. 10 min.24 The
recent appearance of 2ꢀ-O-ACE25 (Scheme 1) gives considerable
improvements in the synthesis of oligo-RNA compared to the
2ꢀ-O-tBDMS group, but this strategy is not compatible with a
conventional automated synthesizer, and also makes it impossible
to monitor the coupling reactions in a standard solid-phase
synthesizer with a UV detector.25
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala
University, S-751 23, Uppsala, Sweden. E-mail: jyoti@boc.uu.se
† Electronic supplementary information (ESI) available: Additional exper-
imental information. See DOI: 10.1039/b614210a
Recently, some new 2ꢀ-OH protecting groups with a formalde-
hyde acetal linker such as (triisopropylsilyloxy)methyl (TOM)26
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and 2-cyanoethoxymethyl (CEM)27 have been developed for solid-
phase RNA synthesis. Using the 2ꢀ-O-TOM or 2ꢀ-O-CEM groups,
an average coupling yield of 99% can be obtained after a
coupling time of 60–150 s. Presumably, the formaldehyde acetal
linker has relatively less steric hindrance toward the vicinal 3ꢀ-
phosphoramidite, thereby giving a much higher coupling yield in
a relatively short coupling time. As for the 2ꢀ-O-CEM, it can be
unblocked through a b-elimination process with fluoride ion as
the base. As expected, it is also cleaved to some extent during
the ammonia treatment, which increases the possibility of chain
cleavage, and may limit its further use for synthesis of larger oligo-
RNAs. Clearly, reducing the acidity of the proton a to the -CN
group in 2ꢀ-O-CEM (2ꢀ-O–CH2OCH2CH2CN)27 will increase its
stability in the ammonia deprotection step.
We report here our efforts to modulate the base-lability of the
a-proton by employing an appropriate substituent (X) in the aro-
matic moiety of 2-arylsulfonylethoxymethyl group as a potential
2ꢀ-OH protecting group (Scheme 2). Given the fact that the pKa of
the RCH2SO2Ph proton (pKa = 27.9 in DMSO, R = PhO) is very
similar to that of RCH2CN (pKa = 28.1 in DMSO, R = PhO),28
introducing a para-methyl substituent (X = p-Me in Scheme 2)
to the phenyl, as in para-tolylsulfonylethoxymethyl (TEM), is
expected to reduce the acidity of the a-proton.29 This electronic
tuning may make TEM a more stable 2ꢀ-OH protecting group
than CEM. This paper demonstrates the solid-supported chemical
synthesis of RNA using TEM as a new 2ꢀ-OH protecting group.
We also report the synthesis of all four native phosphoramidite
building blocks, and their use in automated RNA synthesis, as
well as the deblocking conditions to prepare pure oligo-RNA.
Scheme 3 Reagents and conditions: (i) 2-chloroethanol, 10 M aq. NaOH,
ethanol, reflux, 2 h; (ii) 35% hydrogen peroxide, HOAc, H2O, reflux, 20 min;
(iii) DMSO, HOAc, Ac2O, r.t., 48 h; (iv) SO2Cl2, CH2Cl2, r.t., 1 h.
OH by the TEM group by the procedure optimized by Pitsch
and co-workers26 to give a mixture of two 2ꢀ- and 3ꢀ-isomeric
ribonucleosides 7 and 8 (Scheme 4) by reacting 6a/b/c/d with
Bu2SnCl2/iPr2NEt in 1,2-dichloroethane at room temperature to
form activated cyclic 2ꢀ-O,3ꢀ-O-dibutylstannylidene intermediate,
foll◦owed by treatment with 1.3 equivalents of TEM-Cl (5) at
80 C for 1 h. The two isomeric 2ꢀ-O- and 3ꢀ-O-TEM derivatives
can be isolated by silica gel column chromatography. The first
isomer eluting from the column was the predominant product, 2ꢀ-
O-TEM ribonucleoside 7a/b/c/d in 26–38% yield. The second-
eluting isomer, 8a/b/c/d, was isolated in 20–27% yield. These
two isomers have been identified by NMR (see Section B).
Compound 7a/b/c/d was further converted to the correspond-
ing 5ꢀ-O-DMTr-2ꢀ-O-TEM-ribonucleoside 3ꢀ-(2-cyanoethyl N,N-
diisopropylphosphoramidite) 9a/b/c/d (in 79/68/60/54% yield
respectively) by using a literature procedure34 (see the Experimen-
tal section).
(B) Identification of isomeric 2ꢀ-O-TEM and 3ꢀ-O-TEM
Scheme 2 The electronic nature of X dictates the fragmentation reactivity.
ribonucleosides by NMR
Results and discussion
When ribonucleoside 6a/b/c/d was alkylated with TEM-Cl 5,
two monoalkylated 2ꢀ/3ꢀ-O-TEM isomers were obtained. The
isomer with lower Rf was converted to corresponding pimelate
(A) Synthesis of the phosphoramidite building blocks
1
The reagent, TEM-Cl (5), was synthesized using the strategy
shown in Scheme 3: 2-(4-tolylthio)ethanol (2) was synthesized
from 4-methylbenzenethiol (1).30 The thioether 2 was first oxidized
to sulfone 3 in 96% yield by 35% hydrogen peroxide in aqueous
acetic acid (H2O–AcOH, 1 : 1, reflux, 20 min). Then sulfone 3 was
treated with a mixture of DMSO, acetic acid and acetic anhydride
at room temperature for two days, followed by purification by
silica gel column chromatography to give O,S-acetal 4 (74%).
Finally, the reagent 5 was generated immediately before use by
treatment of 4 with SO2Cl2 (1 equiv.) in CH2Cl2. This conversion
of 4 → 5 was a quantitative reaction, and no further purification
step was necessary.
Acetyl (Ac), dimethylaminomethylene (Dmf) and phenoxy-
acetyl (Pac) were used to protect N4 of cytosine, N2 of guanosine
and N6 ofadenosine, respectively. These exocyclic amino-protected
building blocks and uridine were converted to their respective 5ꢀ-
O-DMTr derivatives according to the published procedures.31–33
The appropriately protected ribonucleoside was protected at 2ꢀ-
10a/b/c/d, which showed a downfield shift of H-2ꢀ in the H
NMR spectrum (data listed in Table 1), thereby showing that the
compound with lower Rf is the 3ꢀ-O-TEM derivative.
1
The H and 13C NMR data of isomerically pure 2ꢀ- and 3ꢀ-O-
TEM derivatives 7a–d and 8a–d, the 2ꢀ- and 3ꢀ-O-CEM derivatives
11a–c and 12a–c, as well as the 2ꢀ- and 3ꢀ-O-tBDMS ribonucleoside
derivatives 13a–14c, are listed in Table 1. It can be seen from
Table 1, regardless of alkylation or silylation at the 2ꢀ-OH or
3ꢀ-OH, the dH1 values do not follow any systematic upfield or
ꢀ
ꢀ
downfield shift, and in fact the difference between the dH1 values
3
ꢀ
ꢀ
of the two isomers is negligible. On the other hand, the J1 ,2
values of all alkylated compounds 7a–12c follow Reese’s rule,35
3
in that the J1 ,2 values of the 2ꢀ-isomers are smaller than those
ꢀ
ꢀ
of the 3ꢀ-isomers, while this is not the case for the 2ꢀ,3ꢀ-isomeric
ꢀ
ꢀ
silylated compounds 13a–14c. A comparison of dH2 and dH3
showed that for all the mono-2ꢀ/3ꢀ-O-alkylated compounds 7a–
12c, alkylation causes an upfield shift of the proton attached to
the site of 2ꢀ- or 3ꢀ-O-alkylation. The value of dH2 − dH3 for the
ꢀ
ꢀ
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Scheme
4
Reagents and conditions: (i) (a) Bu2SnCl2, iPr2NEt, ClCH2CH2Cl, r.t.,
1
h, (b) compound 5, 80 ◦C,
1
h, (ii) 2-cya-
noethyl-N,N-diisopropylchlorophosphoramidite, iPr2NEt, CH2Cl2, r.t., 2 h, (iii) pimelic acid, EDAC, DMAP, pyridine, r.t., 4 h.
2ꢀ-isomer is always smaller than that of the 3ꢀ-isomer. In contrast,
a completely opposite rule can be drawn for the isomeric 2ꢀ/3ꢀ-O-
silylated compounds 13a–14c.
convenient identification parameter. The value of dC2 − dC3 for the
ꢀ
ꢀ
2ꢀ-isomer is always larger than that of the corresponding 3ꢀ-isomer.
13C NMR signals were also used by Ogilvie et al. to identify
2ꢀ-O- and 3ꢀ-O-silylated isomers.36 They found that silylation at a
sugar hydroxyl leads to a downfield shift of the sugar carbon to
which it is attached. As shown in Table 1, we find that this rule
also applies to all the isomeric 2ꢀ/3ꢀ-O-alkylated compounds 7a–
(C) Preparation of the solid support
Initially, we anchored the ribonucleoside to LCAA-CPG support
through the succinate linker. Just as described by Pitsch,26 the
coupling yields for the first seven coupling steps were unsatis-
factory. Pitsch and his co-workers have overcome this problem
ꢀ
ꢀ
12c. Alternatively, the value of dC2 − dC3 can be used as a more
Table 1 1H and 13C NMR chemical shifts of isomeric 2ꢀ/3ꢀ-protected ribonucleoside derivatives
dH1 (3JH1 ,H2
)
dH2
dH3
dH2 − dH3
dC2 − dC3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
dC2
ꢀ
dC3
ꢀ
ꢀ
7a
5.90 (1.84)
5.90 (3.70)
5.88b
4.21
4.34
4.18
4.33
4.86
4.91
4.66
4.80
4.29
4.47
4.30
4.39
4.92
4.93
4.34
4.16
5.03
4.48
5.26
4.85
5.37
5.39
5.83
5.92
4.48
4.27
4.44
4.18
4.56
4.44
4.47
4.36
4.50
4.38
4.47
4.28
4.58
4.50
4.33
4.39
4.37
4.60
4.34
4.46
4.43
4.40
4.71
4.51
−0.27
0.07
−0.26
0.15
0.30
0.47
0.19
0.44
−0.21
0.09
−0.17
0.11
0.34
0.43
0.01
−0.23
0.66
0.28
0.92
0.39
—
80.18
74.53
80.11
75.37
80.46
74.35
80.03
74.04
79.99
74.18
79.73
75.16
80.14
74.36
76.32
71.30
75.91
74.79
74.32
73.60
73.97
74.31
74.37
74.13
68.72
75.90
67.81
75.61
70.32
77.25
70.41
76.81
68.87
75.07
67.93
75.10
70.38
76.90
74.46
75.29
71.76
72.32
71.32
71.65
73.54
72.42
73.96
73.51
11.46
−1.37
12.3
−0.24
10.14
−2.90
9.62
−2.77
11.12
−0.89
11.8
0.06
9.76
−2.54
1.86
−3.99
4.15
2.47
3.00
1.95
—
8a
7b
8b
5.93 (2.10)
6.21 (3.18)
6.04 (5.52)
6.05 (4.81)
5.91 (5.33)
5.98 (2.15)
6.03 (3.52)
5.95b
5.93 (2.34)
6.25 (3.90)
6.06 (5.71)
5.95 (2.65)
5.96 (4.17)
6.11 (5.34)
6.07 (4.93)
5.74 (7.42)
5.71 (5.97)
—
7c
8c
7d
8d
11aa
12aa
11ba
12ba
11ca
12ca
13aa
14aa
13ba
14ba
13ca
14ca
10a
10b
10c
10d
—
—
—
—
—
—
—
—
—
a Compounds 11a–14c were synthesized according to the literature procedure;27,37 their structures are shown in Scheme 5. b Broad singlet.
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(BTT) (pKa = 4.08)40 as the activator with coupling time of 120 s;
ETT gave the best result. Since an activator acts as an acid to
protonate, and thereby activate, the phosphoramidites, the more
acidic the activator is, the faster the coupling reaction should
be.41 On the other hand, as the activator becomes increasingly
acidic, it can also lead to some concomitant loss of the 5ꢀ-O-
DMTr protecting group during coupling, which explains why
ETT, despite being less acidic than BTT, gives a better coupling
yield in the RNA synthesis. Next, with ETT as the activator,
we tried different coupling times (60, 90 and 120 s), and found
that the ASWY improved with longer coupling time. However, a
coupling time of 150 s or 200 s did not improve the yields. We
therefore settled for 120 s as the coupling time for the synthesis.
During the final optimization experiments, we found the first
7 couplings always gave a poor coupling yield, and after that
the ASWY increased steadily. A pimelated CPG (with a chain
three methylenes longer) instead of the conventional succinate
(Scheme 4) improved the coupling yield only slightly.
Scheme 5 Structures of isomeric mono-2ꢀ/3ꢀ-protected ribonucleosides.
On the basis of the above experiments, 12 different oligo-RNAs
(sequences shown in Table 2) were synthesized to test the efficacy
of TEM as a 2ꢀ-OH protecting group. The poly-U synthesis
always gave a perfect coupling yield, whereas the synthesis of
mixed RNA sequences (ON3–12), particularly the one involving
G-phosphoramidite, generally gave poorer yields. The average
stepwise yield for each synthesis is also listed in Table 2.
by employing a longer linker. We also adopted this strategy,
but a more convenient synthetic route was developed: pimelic
acid was reacted directly with the protected ribonucleosides 8a–d
in the presence of 2 equivalents of N-(3-dimethylaminopropyl)-
Nꢀ-ethylcarbodiimide hydrochloride (EDAC) and DMAP in dry
pyridine. This quick and clean reaction, completed in 4 h, was
worked-up and purified by silica gel column chromatography to
give the corresponding pimelates 10a–d in 65–75% yield. The
pimelates were subsequently immobilized to the LCAA-CPG sup-
port by a modification of the general procedure of Pon and Yu,38
which involved shaking the mixture of pimelates, LCAA-CPG,
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexfluo-
rophosphate (BOP), N-hydroxybenzotriazole (HOBt) and diiso-
propylethylamine (DIPEA) in acetonitrile for 2 h to give good
loadings (20–25 lmol g−1). Prolonging the reaction time overnight
gave improved loadings (35 lmol g−1).
(E) Post-synthesis deprotection treatment
Prior to deprotection of the oligo-RNAs, we tested the stability
of 2ꢀ-O-TEM in ammonia. Compound 7a was treated with 3%
Cl2CHCOOH in CH2Cl2 to give compound 15 (Scheme 6).
Compound 15 was first treated with 33% MeNH2 in EtOH at
room temperature for 24 h. The reaction was monitored by
TLC, and it was found about 10% loss of 2ꢀ-O-TEM has taken
place (the TLC is shown in the Supporting information). This
suggests that TEM is not stable under highly alkaline conditions
involving MeNH2/EtOH. Anhydrous methanolic ammonia (25%
NH3/MeOH)22 was then tried. The reaction mixture was kept at
55 ◦C for 21 h, and very small amount of TEM cleavage was
observed. If the reaction was allowed to proceed at room temper-
ature, the amount of 2ꢀ-O-TEM cleavage was very low. Therefore
NH3/MeOH was chosen for exocyclic amino deprotection. In view
of the fact that about 5% cleavage of 2ꢀ-O-CEM is found to take
place under the same conditions, it is concluded that 2ꢀ-O-TEM is
more stable in ammonia solution, just as we expected at the outset.
In order to find an optimal condition to remove 2ꢀ-O-TEM,
compound 15 was treated separately with neat Et3N·3HF and
1 M TBAF/THF. It was found that only 1 M TBAF/THF is an
(D) Automated RNA synthesis
We first synthesized U12 under different conditions to optimize
the synthesis cycle. This oligonucleotide was synthesized on an
Applied Biosystems 392 DNA/RNA synthesizer with common
DNA synthesis reagents. 2ꢀ-O-Succinate-8a CPG was used as
the solid support and a 1.0 lmol RNA synthesis cycle was
applied but with a modified coupling time. The average stepwise
yield (ASWY), obtained by a detritylation assay, was used to
evaluate the synthesis efficiency. We first tried synthesis with 4,5-
dicarbonitrile-1H-imidazole (DCI) (pKa = 5.2),39 5-(ethylthio)-
1H-tetrazole (ETT) (pKa = 4.28) and 5-(benzylthio)-1H-tetrazole
Scheme 6 Stability of 2ꢀ-O-TEM upon ammonia and fluoride treatment.
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Table 2 Average stepwise and overall yields and mass of synthesized RNAs using the 2ꢀ-O-TEM protecting group
Oligo-RNAs
ON1c
RNA sequence
[MH]+ (calcd)
6062.42
[MH]+ (found)
6064.33
Yield (%)a
99.1
Crude productb (Overall yield (%))
102 OD (53%)
ON2c
11683.37 [M + 5Na]+
6719.16
11682.17 [M + 5Na]+
6720.07
98.4
144 OD (40%)
ON3c
97.8
112 OD (54%)
ON4c
ON5d
6643.00
5779.53
6643.92
5777.13
98.0
97.5
124 OD (64%)
21 OD (65%)
ON6d
ON7d
ON8d
4453.74
4523.84
5786.57
4451.56
4521.69
5784.14
97.0
97.0
97.6
17 OD (69%)
20 OD (78%)
18 OD (53%)
ON9d
5481.39
5152.18
5479.18
5150.11
97.2
97.4
21 OD (64%)
19 OD (63%)
ON10d
ON11d
ON12d
4493.76
4918.14
4491.68
4915.51
97.3
97.0
17 OD(68%)
24 OD(68%)
a Average stepwise coupling yield. b Overall crude product yields were measured at 260 nm UV absorption. c Oligonucleotides were synthesized on a 1
lmol scale. d Oligonucleotides were synthesized on a 0.2 lmol scale.
efficient reagent for the deprotection of the 2ꢀ-O-TEM group, in
that the deprotection was found to be complete in 5 min at room
temperature.
Therefore, the oligonucleotide-anchored solid supports were
first treated with 25% NH3/MeOH at room temperature for 20 h,
and then at 40 ◦C for 4 h to ensure the complete removal of the ex-
ocyclic amino protecting groups. After solvent removal and drying
by co-evaporation with dry THF, the samples were treated with
1 M TBAF/THF for 20 h at room temperature to make sure that
all the 2ꢀ-O-TEM groups were removed. After desalting through
a NAP-10 column and Sep-Pak column, the crude products were
subjected to PAGE or HPLC analysis. The PAGE diagrams (see
Fig. 1) together with HPLC profiles (see Fig. S1.2†) show that the
crude products of U20 and U38 are more than 80% pure.
Scheme 7 The mechanism of adduct formation.
However, just as in the CEM strategy, deprotection of the mixed
oligo-RNA sequence using the same deprotection procedure did
not produce satisfactory results. This was because of the formation
of the adducts owing to the side-reaction of the purine bases as
nucleophiles (at N7, N3, and N1) with the a,b-unsaturated sulfone
16, generated by fluoride ion treatment (Scheme 7). The formation
of these adducts has been evidenced by denatured PAGE as well
as by MALDI-TOF MS analysis.
In the CEM strategy,27 10% n-propylamine and 1% bis(2-
mercaptoethyl) ether was used as scavenger for the active a,b-
unsaturated compound (acrylonitrile), and thereby adduct for-
mation was suppressed efficiently. However, it should be kept in
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Table 3 Conditions used for deblocking ON3 and ON4atreatment
Entry
Oligonucleotide
Step I: NH3
Step II: 2ꢀ-Deprotection
Isolated yield (%)
HPLC profileb
1
2
3
4
5
6
7
8
9
ON4
ON4
ON3
ON4
ON3
ON4
ON4
ON4
ON4
ON3
ON4
ON3
Condition 1
Condition 1
Condition 1
Condition 1
Condition 1
Condition 1
Condition 1
Condition 1
Condition 1
Condition 1
Condition 2
Condition 3
Condition 4
Condition 5
Condition 5
Condition 6
Condition 7
Condition 8
Condition 9
Condition 10
Condition 11
Condition 12
Condition 6
Condition 6
53.7
48.6
57.4
50.4
43.7
Fig. S3.1
Fig. S3.2
Fig. S3.11
Fig. S3.3
Fig. S3.4
Fig. S3.5
Fig. S3.6
Fig. S3.7
Fig. S3.8
Fig. S3.9
Fig. S3.10
Fig. S3.12
27.9
c
c
c
10
11
12
49.1
34.9
52.1
a Condition 1: 25% NH3/MeOH, r.t., 20 h, 40 ◦C, 4 h; Condition 2: 25% NH3/MeOH (with 1% CH3NO2), r.t., 20 h, 40 ◦C, 4 h; Condition 3:
25% NH3/MeOH, 55 ◦C, 3 h; Condition 4: 1 M TBAF/THF (with 10% n-propylamine and 1% bis(2-mecaptoethyl) ether), 24 h; Condition 5: 1 M
TBAF/THF (with 10% n-propylamine and 1% bis(2-mecaptoethyl) ether), 4 h; Condition 6: 1 M TBAF/THF (with 10% n-propylamine and 1%
CH3NO2), 4 h; Condition 7: 1 M TBAF/THF (with 10% DBU and 1% CH3NO2), 4 h; Condition 8: 1 M TBAF/THF (with 10% piperidine and 1%
CH3NO2), 4 h; Condition 9: 1 M TBAF/THF (with 10% CH3NO2), 4 h; Condition 10: 1 M TBAF/THF (with 1% n-propylamine and 10% CH3NO2),
4 h; Condition 11: 1 M TBAF/THF (with 10% n-propylamine and 10% CH3NO2), 4 h; Condition 12: 1 M TBAF/THF (with 10% n-propylamine), 4 h.
b HPLC conditions: RP-column, 0–40 min, buffer C → C/D 8 : 2. All of the HPLC profiles can be found in Supporting information.† c No pure product
obtained.
(2) Amine-promoted chain cleavage is significant. When more
basic amines such as DBU or piperidine were used, considerably
more cleavage was observed.
(3) n-Propylamine itself is an efficient scavenger. In the absence
of bis(2-mercaptoethyl)ether, the deprotecting reaction is cleaner.
(4) CH3NO2 was reported as a scavenger for acrylonitrile in the
presence of amines such as NH3/MeCN, MeNH2/H2O–EtOH–
MeCN,42 but this is not the case in the presence of TBAF in
THF. On the contrary, when CH3NO2 was used in conjunction
with n-propylamine, it can inhibit the activity of n-propylamine.
(5) The 2ꢀ-O-TEM group is found to be stable in ammonia.
Treating the oligonucleotide with NH3/MeOH at room tempera-
◦
◦
ture for 20 h, then 40 C for 4 h (or 55 C for 3 h) gave similar
results, which again proves the enhanced stability of 2ꢀ-O-TEM in
NH3/MeOH.
(6) Up until now, 1 M TBAF/THF with 10% n-propylamine
and 1% bis(2-mercaptoethyl) ether has remained the preferred
reagent for deprotecting the 2ꢀ-O-TEM group from oligo-RNAs.
However, recent unpublished data from this lab suggests that 1 M
TBAF/THF with 10% morpholine at room temperature seems to
be a better deprotection agent. This will be reported in due course.
We then deprotected all of our oligo-RNAs, ON3–12, in the
following manner: (i) treatment with methanolic ammonia (RT,
20 h followed by 40 ◦C for 4 h) as described above, and (ii)
treatment with 1 M TBAF/THF containing 10% n-propylamine
and 1% bis(2-mercaptoethyl) ether for 20 h at room temperature
to give full-length oligo-RNAs in good purity. The PAGE pictures
of the crude products (>90% pure, which are acceptable for
enzymological work, see below) are shown in Fig. 1. These oligo-
RNAs were subsequently examined by MALDI-TOF MS, which
confirmed the chemical integrity of all the synthesized RNAs (see
Table 2 and Fig. S1.3–13†).
Fig. 1 20%-Denatured PAGE diagrams of the crude products directly
after deprotection. a: Xylene cyanol blue. b: Bromophenol blue.
mind that n-propylamine also acts as a moderate base and can
promote chain cleavage. So, in the hope of developing an improved
scavenger, we tested several different deprotecting conditions for
2ꢀ-O-TEM with ON3 and ON4 as the substrates. The results are
listed in Table 3. Comparison of these data suggests the following:
(1) Deprotecting 2ꢀ-O-TEM with TBAF/THF is a fast process.
Reaction periods of 4 h and 24 h give nearly the same yields.
(F) Enzymatic digestion of the crude RNA directly after
deprotection without involving any purification step
RNA synthesis with the TEM strategy described above can indeed
give products with high purity. To see whether the crude product
338 | Org. Biomol. Chem., 2007, 5, 333–343
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is pure enough for biological research, RNA 15 nucleotides long
(ON12 in Table 2) was synthesized and, after deprotecting and
desalting (see Fig. 1 for purity of the crude product), applied
directly in an RNase H digestion study (Fig. S4†). It may be
noted that the synthesized crude RNA (compare its relative purity
(∼90%) with that of the purified RNA in Fig. S5†) has nearly the
same cleavage efficiency as the pure RNA (compare the RNase
H promoted relative degradation rates in Fig. 2). This suggests
that the crude RNA prepared by our TEM strategy is satisfactory
for most biological experiments. To our knowledge, it is the first
approach that can provide crude RNA for biological studies
directly without a laborious, time-consuming purification step.
Conclusions
The conclusions of our study are as follows:
ꢀ
ꢀ
ꢀ
ꢀ
,
(1) A new set of NMR trends, involving dH2 − dH3 and dC2 − dC3
was identified to characterize monomeric 2ꢀ-O- and 3ꢀ-O-alkylated
or silylated ribonucleosides. The proposed parameters are more
regular and reliable than earlier reported parameters such as dH1
ꢀ
,
ꢀ
ꢀ
ꢀ
ꢀ
.
J
H1 ,H2 , dC3 and dC2
(2) TEM has been developed as a new 2ꢀ-OH protecting group
for solid-supported oligo-RNA synthesis. With this methodology,
RNA synthesis can be carried out on a standard solid-support
synthesizer with high average coupling yield and a coupling time
of only 120 s.
(3) The advantages of our 2ꢀ-O-TEM-based strategy for RNA
synthesis are the stability of the 2ꢀ-O-TEM group upon ammonia
treatment, the simpler post-synthesis deprotection procedure and
the higher purity of the crude product than that of RNA prepared
using the 2ꢀ-O-cyanoethoxymethyl (2ꢀ-O-CEM) group.
(4) Furthermore, the crude RNA obtained by our 2ꢀ-O-TEM-
based strategy is of high purity, which, after desalting, can be
directly used in biological research without further purification,
which is an important advantage over other strategies based on
either 2ꢀ-O-tBDMS,15–17 2ꢀ-O-TOM,26 2ꢀ-O-CEM,27 2ꢀ-O-ACE,25 or
2ꢀ-O-Fpmp.20
Experimental
Chromatographic separations were performed on Merck G60
silica gel. Thin layer chromatography (TLC) was performed on
Merck pre-coated silica gel 60 F254 glass-backed plates. 1H NMR
spectra were recorded at 270.1 MHz and 500 MHz respectively,
using TMS (0.0 ppm) as internal standards. 13C NMR spectra were
recorded at 67.9 MHz, 125.7 MHz and 150.9 MHz respectively,
using the central peak of CDCl3 (76.9 ppm) as an internal
standard. 31P NMR spectra were recorded at 109.4 MHz using
85% phosphoric acid as an external standard. Chemical shifts
are reported in ppm (d scale). MALDI-TOF mass spectra were
recorded in the positive ion mode for oligonucleotides and
for other compounds as indicated. The mass spectrometer was
externally calibrated with a peptide mixture using a-cyano-4-
hydroxycinnamic acid as matrix. Anion exchange (AE) HPLC:
Fig. 2 RNA cleavage efficiency of pure RNA (Fig. S5†) and crude RNA
(ON12 in Fig. 1) with RNase H (for the purity of pure RNA see the PAGE
picture in Fig. S5). Conditions of cleavage reactions: pure or crude RNA
(0.1 lM) and complementary DNA (1 lM) in buffer containing 20 mM
Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl2 and 0.1 mM DTT at 21 ◦C;
0.06 U of RNase H in a total reaction volume of 30 lL.
The crude ON12 was also subjected to digestion by phosphodi-
esterase I (from Crotalus adamanteus venom) and shrimp alkaline
phosphatase. The products were analyzed by RP-HPLC, and the
profile is shown in Fig. 3. The crude products are completely
digested to give pure nucleosides, which strongly suggests that the
oligo-RNA is in a biologically active form and that no modified
base is present.
−1
˚
Luna 5 l, NH2, 100 A, 150 × 4.6 mm. Flow 1 ml min at room
temperature, UV detector with detecting wavelength of 260 nm.
Buffer A: 20 mM LiClO4, 20 mM NaOAc in H2O–CH3CN (9 : 1),
pH 6.5 with AcOH. Buffer B: 600 mM LiClO4, 20 mM NaOAc
in H2O–CH3CN (9 : 1), pH 6.5 with AcOH. Reversed-phase (RP)
HPLC: Kromasil 100, C18, 5 l, 100 × 4.6 mm. Flow 1 ml min−1
at room temperature, UV detector with detecting wavelength of
260 nm. Buffer C: 0.1 M TEAA in H2O–CH3CN (95 : 5). Buffer
D: 0.1 M TEAA in H2O–CH3CN (50 : 50). Polyacrylamide gel
electrophoresis (PAGE): 20% acrylamide (acrylamide/bis-
acrylamide 29 : 1), 7 M urea, TBE buffer.
2-(4-Tolylthio)ethanol (2)
Fig. 3 Reversed-phase HPLC profile of the products obtained by
digestion of crude ON12 (Rt = 14.12 min) in Fig 1 with a mixture of
phosphodiesterase I and alkaline phosphatase. The peaks correspond to
uridine (3.83 min), guanosine (4.17 min) and adenosine (5.79 min). HPLC
conditions: C18 RP column, 100 × 4.6 mm, 1 ml min−1, r.t., 0–10 min,
buffer C → C/D 9 : 1, 10–35 min, buffer C/D 9 : 1 → C/D 2 : 8.
To a solution of 4-methylbenzenethiol (1, 50 g, 0.394 mol) and
2-chloroethanol (47.6 ml, 0.71 mol) in ethanol (400 ml) was added
dropwise aqueous 10 M NaOH (39.4 ml, 0.394 mol) over 1 h. The
reaction mixture was refluxed for 2 h, cooled and concentrated
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in vacuo. The residue was diluted with AcOEt, washed with H2O
for 1 hour. The mixture was allowed to cool down and saturated
NaHCO3 was added. After shaking vigourously, the resulting tur-
bid solution was filtered through a Celite pad. CH2Cl2 was added
to the filtrate, and the organic layer was separated and dried over
MgSO4. The residue, after evaporation of the solvent, was applied
chromatographed on a short column (CH2Cl2 with 1% Et3N, ethyl
acetate from 0 to 40%). The first-eluted isomer was 7a (1.83 g,
34.4%). 1H NMR (270 MHz, CDCl3 + DABCO): d 2.43 (s, 3H),
3.40 (t, J = 5.8 Hz, 2H), 3.54 (t, J = 2.7 Hz, 2H), 3.79 (s, 6H), 3.94
(m, 1H), 4.05–4.16 (m, 2H), 4.21 (dd, J = 1.8, 2.0 Hz, 1H, H-2ꢀ),
4.48 (dd, J = 5.4, 5.5 Hz, 1H, H-3ꢀ), 4.80 (d, J = 6.8, 1H), 4.98 (d, J
= 6.8, 1H), 5.28 (d, J = 6.3, 1H), 5.90 (d, J = 1.8, 1H, H-1ꢀ), 6.84 (d,
J = 8.9, 4H), 7.23–7.40 (m, 11H), 7.78 (d, J = 8.3, 2H), 7.96 (d, J
= 8.2, 1H). 13C NMR (67.9 MHz, CDCl3 + DABCO): d 21.6, 55.3,
56.2, 61.4, 61.8, 68.7 (C-3ꢀ), 80.2 (C-2ꢀ), 83.2, 87.1, 88.1, 95.1, 102.2,
113.3, 127.2, 127.9, 128.0, 128.2, 130.0, 130.1, 130.2, 135.1, 135.3,
136.8, 140.0, 144.4, 145.0, 150.1, 158.8, 163.0. MALDI-TOF MS:
[M + Na]+ 781.20, calcd 781.25. The second-eluted isomer was 8a
(1.07 g, 20.2%). 1H NMR (270 MHz, CDCl3 + DABCO): d 2.41
(s, 3H), 3.30–3.39 (m, 3H), 3.57 (dd, J = 2.5, 2.3 Hz, 1H), 3.79 (s,
6H), 3.87 (t, J = 5.2 Hz, 1H), 3.99 (t, J = 6.1 Hz, 1H), 4.22 (t, J =
5.3 Hz, 1H), 4.27 (t, J = 5.0 Hz, 1H, H-3ꢀ), 4.34 (d, J = 3.9, 1H,
H-2ꢀ), 4.72 (s, 2H), 5.35 (d, J = 8.1, 1H), 5.90 (d, J = 3.7, 1H, H-1ꢀ),
6.84 (d, J = 8.2, 4H), 7.23–7.37 (m, 11H), 7.75 (d, J = 8.3, 2H),
7.83 (d, J = 8.1, 1H). 13C NMR (67.9 MHz, CDCl3 + DABCO):
d 21.6, 55.3, 56.1, 61.9, 62.0, 74.5 (C-2ꢀ), 75.9 (C-3ꢀ), 81.7, 87.1,
89.8, 95.6, 102.4, 113.3, 127.2, 127.9, 128.0, 128.2, 129.9, 130.1,
130.2, 135.1, 135.2, 136.7, 140.0, 144.2, 145.0, 150.6, 158.8, 163.0.
MALDI-TOF MS: [M + Na]+ 781.21, calcd 781.25.
and dried over MgSO4. After filtration, the filtrate was evaporated
1
in vacuo to give 76.6 g (97%) of 2 as a colorless oil. H NMR
(270 MHz, CDCl3): d 2.32 (s, 3H), 3.06 (t, J = 5.9 Hz, 2H), 3.70 (t,
J = 5.9 Hz, 2H), 7.11 (d, J = 9.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H).
2-(4-Tolylsulfonyl)ethanol (3)
To a solution of 2 (20 g, 0.119 mol) in AcOH (50 ml) and H2O
(50 ml) in an ice bath was added hydrogen peroxide (30%, 36.5 ml)
dropwise over half an hour. The mixture was then refluxed for
20 min and cooled. NaHCO3 was added to the reaction mixture to
neutralize this solution, followed by AcOEt. The organic layer was
separated, washed with brine and water, and concentrated in vacuo
to give 22.9 g (96.3%) of 3 as a white solid. 1H NMR (270 MHz,
CDCl3): d 2.46 (s, 3H), 3.33 (t, J = 5.4 Hz, 2H), 3.99 (t, J = 5.4 Hz,
2H), 7.39 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.3 Hz, 2H). 13C NMR
(67.9 MHz, CDCl3): d 21.7, 56.4, 58.3, 128.0, 130.1, 136.0, 145.2.
4-Tolylsulfonylethyl methylthiomethyl ether (4)
A solution of 3 (2 g, 10 mmol) in dimethyl sulfoxide (28 ml,
40 mmol) was treated with acetic anhydride (11.4 ml, 20 mmol)
and acetic acid (19 ml, 20 mmol). The mixture was stirred at room
temperature for 48 h and then added dropwise to an aqueous
solution of NaHCO3. After stirring for 1 h, the reaction mixture
was extracted with ethyl acetate. The organic layer was separated,
washed with saturated aqueous NaCl solution and dried over
MgSO4. The solvent was removed under reduced pressure and
the residual oil was chromatographed on a short column (ethyl
acetate–cyclohexane, 1 : 5 to 2 : 5) to give 1.935 g (74%) of 4 as
a colorless oil. 1H NMR (270 MHz, CDCl3): d 2.08 (s, 3H), 2.46
(s, 3H), 3.40 (t, J = 6.3 Hz, 2H), 3.88 (t, J = 6.3 Hz, 2H), 4.54 (s,
3H), 7.36 (d, J = 7.9 Hz, 2H), 7.81 (d, J = 8.3 Hz, 2H). 13C NMR
(67.9 MHz, CDCl3): d 14.1, 21.7, 56.2, 61.4, 75.6, 128.1, 129.9,
136.8, 144.9.
N6-Acetyl-5ꢀ-O-(dimethoxytrityl)-2ꢀ-O-[2-(4-tolylsulfonyl)ethoxy-
methyl]cytidine (7b) and N6-acetyl-5ꢀ-O-(dimethoxytrityl)-3ꢀ-O-[2-
(4-tolylsulfonyl)ethoxymethyl]lcytidine (8b)
N4-Acetyl-5ꢀ-O-DMTr-cytidine (3.95 g, 6.7 mmol) was treated as
described for 7a and 8a to give 7b (2.04 g, 38.1%) and 8b (1.08 g,
20.1%). Compound 7b: 1H NMR (270 MHz, CDCl3 + DABCO):
d 2.20 (s, 3H), 2.41 (s, 3H), 2.96 (broad, 1H), 3.43 (m, 2H), 3.54
(dd, J = 2.4 Hz, 2.4 Hz, 1H), 3.61 (dd, J = 2.0 Hz, 1.6 Hz, 1H),
3.81 (s, 6H), 3.92 (q, J = 5.8 Hz, 1H), 4.07–4.13 (m, 2H), 4.18 (d,
J = 5.1 Hz, 1H, H-2ꢀ), 4.44 (t, 1H, H-3ꢀ), 4.83 (d, J = 6.6 Hz, 1H),
5.13 (d, J = 6.6 Hz, 1H), 5.88 (s, 1H, H-1ꢀ), 6.86 (d, J = 8.9 Hz,
4H), 7.09 (d, J = 7.9 Hz, 1H), 7.30–7.44 (m, 11H), 7.78 (d, J =
8.3 Hz, 2H), 8.47 (d, J = 7.5 Hz, 1H), 9.17 (s, 1H). 13C NMR
(67.9 MHz, CDCl3 + DABCO): d 21.6, 24.9, 55.3, 56.3, 60.9, 61.7,
67.8 (C-3ꢀ), 80.1 (C-2ꢀ), 82.9, 87.1, 89.8, 94.9, 96.5, 113.3, 127.2,
128.0, 128.1, 129.9, 130.1, 135.3, 135.5, 136.8, 144,3, 144.7, 144.9,
155.0, 158.7162.6, 170.2. MALDI-TOF MS: [MH]+ 800.21, calcd
800.28. Compound 8b:1H NMR (270 MHz, CDCl3 + DABCO):
d 2.21 (s, 3H), 2.42 (s, 3H), 3.35 (dd, J = 2.9 Hz, 2.2 Hz, 1H), 3.44
(q, J = 3.9 Hz, 2H), 3.60 (dd, J = 2.0 Hz, 2.3 Hz, 1H), 3.81 (s, 7H),
4.03 (q, J = 4.7 Hz, 1H), 4.18 (dd, J = 5.0, 5.4 Hz, 1H, H-3ꢀ), 4.33
(m, 2H, H-2ꢀ, 4ꢀ), 4.61 (d, J = 7.0 Hz, 1H), 5.68 (d, J = 7.0 Hz,
1H), 5.93 (d, J = 2.1 Hz, 1H, H-1ꢀ), 6.84 (d, J = 8.8 Hz, 4H),
7.17 (d, J = 7.5 Hz, 1H), 7.23–7.34 (m, 11H), 7.78 (d, J = 8.3 Hz,
2H), 8.35 (d, J = 7.5 Hz, 1H), 8.99 (s, 1H). 13C NMR (67.9 MHz,
CDCl3 + DABCO): d 21.7, 25.0, 55.3, 56.1, 61.6, 61.9, 75.4, 75.6,
82.1, 87.1, 92.6, 95.7, 96.7, 113.4, 127.3, 128.0, 128.1, 128.2, 130.0,
130.2, 135.2, 135.3, 136.7, 144,2, 144.7, 144.9, 156.0, 158.8, 162.5,
170.2. MALDI-TOF MS: [MH]+ 800.23, calcd 800.28.
4-Tolylsulfonylethoxymethyl chloride (5)
4-Tolylsulfonylethyl methylthiomethyl ether (4, 1.78 g, 6.8 mmol)
was dissolved in CH2Cl2 (20 ml). Keeping the solution in an ice-
bath, SO2Cl2 (0.95 g, 6.8 mmol) was added dropwise, and the
reaction was allowed to proceed for 2 h at room temperature. After
evaporation of the solvent under reduced pressure, 1.69 g (99%)
of 5 was obtained as a colorless oil. This was used for next step
of the synthesis without further purification. 1H NMR (270 MHz,
CDCl3): d 2.46 (s, 3H), 3.44 (t, J = 6.2 Hz, 2H), 4.03 (t, J = 6.2 Hz,
2H), 5.35 (s, 3H), 7.36 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 7.5 Hz,
2H). 13C NMR (67.9 MHz, CDCl3): d 21.7, 55.7, 63.7, 82.0, 128.1,
129.9, 136.5, 145.0.
5ꢀ-O-(Dimethoxytrityl)-2ꢀ-O-[2-(4-tolylsulfonyl)ethoxymethyl]-
uridine (7a) and 5ꢀ-O-(dimethoxytrityl)-3ꢀ-O-[2-(4-tolyl-
sulfonyl)ethoxymethyl]uridine (8a)
To a solution of 5ꢀ-O-DMTr-uridine (3.83 g, 7 mmol) in CH2Cl2
(30 ml) was added diisopropylethylamine (4.2 ml, 24.7 mmol) and
dibutyltin dichloride (2.55 g, 8.4 mmol), and the reaction was
allowed to proceed at room temperature for 1 h. The mixture
was then heated to 80 ◦C, 4-tolylsulfonylethoxymethyl chloride (5)
(2.5 g, 9.1 mmol) added dropwise, and the mixture stirred at 80 ◦C
340 | Org. Biomol. Chem., 2007, 5, 333–343
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N6-Phenoxyacetyl-5ꢀ-O-(dimethoxytrityl)-2ꢀ-O-[2-(4-tolyl-
sulfonyl)ethoxymethyl]adenosine (7c) and N6-phenoxyacetyl-5ꢀ-O-
(dimethoxytrityl)-3ꢀ-O-[2-(4-tolylsulfonyl)ethoxymethyl]adenosine
(8c)
5ꢀ-O-(Dimethoxytrityl)-2ꢀ-O-[2-(4-tolylsulfonyl)ethoxymethyl]-
uridine 3ꢀ-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (9a)
A solution of compound 7a (544 mg, 0.72 mmol) in dichloro-
methane (5 ml) was treated with N,N-diisopropylethylamine
(0.3 ml, 1.79 mmol) and 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite (0.24 ml, 1.1 mmol) was added dropwise. The
reaction was allowed to proceed for 2 h at room temperature.
After quenched with MeOH (0.5 ml), the mixture was diluted
with dichloromethane and washed with saturated NaHCO3 so-
lution, dried over MgSO4, chromatographed on a short column
(cyclohexane with 1% Et3N, ethyl acetate from 10% to 50%) to give
the phosphoramidite 9a (0.54 g, 78.6%). 31P NMR (109.4 MHz,
CDCl3 + DABCO): 149.90, 151.00. MALDI-TOF: [MH]+ 959.16,
calcd 959.36.
N6-Phenoxyacetyl-5ꢀ-O-DMTr-adenosine (0.9 g, 1.2 mmol) was
treated as described for 7a and 8a to give 7c (0.31 g, 26.4%) and
8c (0.265 g, 22.8%). Compound 7c: 1H NMR (270 MHz, CDCl3
+ DABCO): d 2.41 (s, 3H), 3.28 (t, J = 3.0 Hz, 2H), 3.40–3.54 (m,
2H), 3.76–3.86 (m, 7H), 3.99–4.04 (m, 1H), 4.25 (t, 1H), 4,56 (t,
1H, H-3ꢀ), 4.76–4.86 (m, 5H, H-2ꢀ included), 6.21 (d, J = 3.2 Hz,
1H, H-1ꢀ), 6.78 (d, J = 8.9 Hz, 4H), 7.07 (m, 3H), 7.23–7.41 (m,
13H), 7.74 (d, J = 8.2 Hz, 2H), 8.23 (s, 1H), 8.69 (s, 1H). 13C
NMR (67.9 MHz, CDCl3 + DABCO): d 21.6, 55.2, 56.1, 61.9,
63.0, 68.1, 70.3 (C-3ꢀ), 80.5 (C-2ꢀ), 84.1, 86.7, 87.3, 96.0, 113.2,
115.0, 122.5, 127.0, 127.8, 127.9, 128.2, 129.9, 130.1, 135.6, 136.7,
142.2, 144.5, 145.0, 148.4, 152.6, 157.0, 158.6, 166.6. MALDI-
N6-Acetyl-5ꢀ-O-(dimethoxytrityl)-2ꢀ-O-[2-(4-tolylsulfonyl)-
ethoxymethyl]cytidine 3ꢀ-(2-cyanoethyl-N,N-diisopropyl-
phosphoramidite) (9b)
1
TOF MS: [MH]+ 916.18, calcd 916.01. Compound 8c: H NMR
(270 MHz, CDCl3 + DABCO): d 2.42 (s, 3H), 3.27–3.34 (m, 3H),
3.47 (dd, J = 4.0, 4.2 Hz, 1H), 3.77 (s, 6H), 3.87–4.03 (m, 2H), 4.32
(t, J = 3.7 Hz, 1H), 4.44 (t, J = 4.4 Hz, 1H, H-3ꢀ), 4,72–4.86 (m,
4H), 4.91 (t, J = 5.2 Hz, 1H, H-2ꢀ) 6.04 (d, J = 5.5 Hz, 1H, H-1ꢀ),
6.78 (d, J = 8.9 Hz, 4H), 7.03–7.37 (m, 16H), 7.75 (d, J = 8.2 Hz,
2H), 8.22 (s, 1H), 8.72 (s, 1H). 13C NMR (67.9 MHz, CDCl3 +
DABCO): d 21.6, 55.2, 56.1, 61.7, 63.0, 68.1, 74.3 (C-2ꢀ), 77.2 (C-
3ꢀ), 83.2, 86.7, 89.5, 95.5, 113.2, 115.0, 122.5, 127.0, 127.9, 128.0,
129.8, 130.0, 135.4, 135.5, 136.7, 142.1, 144.4, 145.0, 148.4, 151.5,
152.4, 157.0, 158.6, 166.6. MALDI-TOF MS: [MH]+ 916.30,
calcd 916.01.
Compound 7b (1.23 g, 1.54 mmol) was treated as described for
9a. Short column chromatography (cyclohexane with 1% Et3N,
acetone from 20% to 50%) gave 9b (1.08 g, 67.9%). 31P NMR
(109.4 MHz, CDCl3 + DABCO): 150.16, 151.69. MALDI-TOF
MS: [MH]+ 1000.29, calcd 1000.39.
N6-Phenoxyacetyl-5ꢀ-O-(dimethoxytrityl)-2ꢀ-O-[2-(4-tolyl-
sulfonyl)ethoxymethyl]adenosine 3ꢀ-(2-cyanoethyl-
N,N-diisopropylphosphoramidite) (9c)
Compound 7c (0.246 g, 0.26 mmol) was treated as described for
9a. Short column chromatography (petroleum ether with 1% Et3N,
acetone from 20% to 40%) gave 9c (0.18 g, 60.0%). 31P NMR
(109.4 MHz, CDCl3 + DABCO): 148.66, 148.79. MALDI-TOF:
[MH]+ 1115.99, calcd 1116.42.
N2-(N,N-Dimethylaminomethylene)-5ꢀ-O-(dimethoxytrityl)-2ꢀ-O-
[2-(4-tolylsulfonyl)ethoxymethyl]guanosine (7d) and N2-(N,N-
dimethylaminomethylene)-5ꢀ-O-(dimethoxytrityl)-3ꢀ-O-[2-(4-tolyl-
sulfonyl)ethoxymethyl]guanosine (8d)
N2-(N,N-Dimethylaminomethylene)-5ꢀ-O-(dimethoxytrityl)-2ꢀ-O-
[2-(4-tolylsulfonyl)ethoxymethyl]guanosine 3ꢀ-(2-cyanoethyl-N,N-
diisopropylphosphoramidite) (9d)
N2-(N,N-Dimethylaminomethylene)-5ꢀ-O-DMTr-guanosine (4.5 g,
7 mmol) was treated as described for 7a and 8a to give 7d (1.88 g,
31.4%) and 8d (1.60 g, 26.7%). Compound 7d: 1H NMR (270 MHz,
CDCl3 + DABCO): d 2.38 (s, 3H), 3.03 (s, 3H), 3.07 (s, 3H), 3.27
(t, J = 5.2 Hz, 2H), 3.42 (broad, 2H), 3.71–3.76 (m, 7H), 4.0 (q,
J = 5.1 Hz, 1H), 4.23 (broad, 1H), 4.47 (broad, 1H, H-3ꢀ), 4.66
(t, J = 5.0 Hz, 1H, H-2ꢀ), 4.82 (dd, J = 6.9, 7.0 Hz, 2H), 6.05
(d, J = 4.8 Hz, 1H, H-1ꢀ), 6.80 (d, J = 8.6 Hz, 4H), 7.18–7.74
(m, 14H), 8.52 (s, 1H), 9.25 (broad, 1H). 13C NMR (67.9 MHz,
CDCl3 + DABCO): d 21.6, 35.1, 41.3, 53.4, 55.2, 61.8, 63.6, 70.4
(C-3ꢀ), 80.0 (C-2ꢀ), 83.7, 85.6, 86.7, 95.3, 113.3, 120.6, 127.7, 127.8,
127.9, 128.1, 129.8, 130.0, 130.1, 135.5, 135.6, 136.0, 136.7, 144.5,
145.0, 150.4, 157.0, 157.9, 158.4, 158.6. MALDI-TOF: [MH]+
853.33, calcd 853.32. Compound 8d: 1H NMR (270 MHz, CDCl3
+ DABCO): d 2.38 (s, 3H), 2.99 (s, 3H), 3.03 (s, 3H), 3.28–3.39
(m, 4H), 3.76 (s, 6H), 3.85–4.05 (m, 2H), 4.22 (t, J = 3.9 Hz,
1H), 4.36 (t, J = 4.8 Hz, 1H, H-3ꢀ), 4.70–4.82 (m, 3H, H-2ꢀ,
OCH2O), 5.91 (d, J = 5.3 Hz, 1H, H-1ꢀ), 6.78 (d, J = 8.7 Hz, 4H),
7.17–7.75 (m, 14H), 8.44 (s, 1H). 13C NMR (67.9 MHz, CDCl3
+ DABCO): d 21.6, 35.1, 41.3, 55.2, 56.2, 61.7, 63.2, 74.0 (C-
2ꢀ), 76.8 (C-3ꢀ), 82.2, 86.5, 88.2, 95.5, 113.2, 120.4, 127.8, 127.9,
128.1, 129.8, 129.9, 130.0, 135.5, 135.6, 136.5, 136.7, 144.5, 144.9,
150.2, 156.7, 157.9, 158.1, 158.6. MALDI-TOF: [MH]+ 853.30,
calcd 853.32.
Compound 7d (0.965 g, 1.13 mmol) was treated as described for
9a. Short column chromatography (CH2Cl2 with 1% Et3N, acetone
from 10% to 30%) gave 9d (0.64 g, 53.9%). 31P NMR (109.4 MHz,
CDCl3 + DABCO): 148.67, 148.90. MALDI-TOF MS: [MH]+
1053.38, calcd 1054.17.
5ꢀ-O-(Dimethoxytrityl)-3ꢀ-O-[2-(4-tolylsulfonyl)ethoxymethyl]-
uridine 2ꢀ-O-pimelate (10a)
To a solution of 8a (0.5 g, 0.66 mmol) in dry pyridine
(10 ml) was added pimelic acid (258 mg, 1.61 mmol), N-
(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide
hydrochloride
(EDAC, 284 mg, 1.48 mmol) and 4-(dimethylamino)pyridine
(82 mg, 0.67 mmol). The reaction was allowed to proceed at room
temperature for 4 h. After evaporation of solvent, the residue
was diluted with CH2Cl2, washed with H2O twice, saturated
(NH4)2CO3 once, H2O once, dried over MgSO4 and chromato-
graphed on a short column (dichloromethane with 1% Et3N,
methanol from 1% to 5%) to give 10a (448 mg, 75.4%). 1H NMR
(270 MHz, CDCl3 + DABCO): d 1.32 (m, 2H), 1.63 (m, 4H), 2.21
(t, J = 6.9 Hz, 2H), 2.36 (t, J = 6.6 Hz, 2H), 2.43 (s, 3H), 3.19 (t,
J = 3.8 Hz, 2H), 3.37 (dd, J = 1.9 Hz, 1H), 3.58 (dd, J = 1.8 Hz,
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1H), 3.73 (t, J = 4.7 Hz, 2H), 3.79 (s, 6H), 4.14 (t, J = 2.4 Hz, 1H),
4.43 (t, J = 5.1 Hz, 1H, H3ꢀ), 4.54 (dd, J = 7.1 Hz, 2H), 5.29 (d, J
= 8.1 Hz, 1H), 5.37 (t, J = 4.8 Hz, 1H, H2ꢀ), 6.11 (d, J = 4.7 Hz,
1H), 6.84 (d, J = 8.0 Hz, 4H), 7.22–7.34 (m, 11H), 7.72 (m, 3H).
13C NMR (67.9 MHz, CDCl3 + DABCO): d 21.7, 24.7, 25.7, 28.9,
33.8, 36.8, 55.3, 56.0, 61.6, 62.2, 73.5 (C3ꢀ), 74.0 (C2ꢀ), 82.3, 86.6,
87.2, 94.9, 102.7, 113.3, 127.3, 127.9, 128.0, 128.3, 129.9, 130.1,
130.2, 135.0, 139.5, 144.0, 144.8, 150.3, 158.8, 163.2, 172.6, 179.8.
MALDI-TOF MS: [M + Na]+ 1023.16, calcd 1023.31.
156.9, 157.6, 158.5, 158.6, 172.6, 179.6. MALDI-TOF MS: [M]+
995.37, calcd 995.10.
2ꢀ-O-[2-(4-Tolylsulfonyl)ethoxymethyl]uridine (15)
Compound 7a (115 mg, 0.15 mmol) was treated with 3%
DCA/DCM (5 ml), and the reaction was allowed to proceed
at room temperature for 5 min. The mixture was then diluted
with ethyl acetate, washed with saturated NaHCO3, and dried
over MgSO4. Short column chromatography (CH2Cl2–MeOH, 9
: 1, v/v), gave compound 15 (23 mg, 33%). 1H NMR (270 MHz,
CD3OD): d 2.42 (s, 3H), 3.50 (t, J = 5.7 Hz, 2H), 3.73 (dd, J =
2.6 Hz, 1H), 3.83–3.97 (m, 4H), 4.09 (t, J = 4.2 Hz, 1H), 4.19
(t, J = 5.4 Hz, 1H), 4.71 (dd, J = 7.0 Hz, 1H), 5.67 (d, J =
8.1 Hz, 1H), 5.88 (d, J = 3.7 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H),
7.77 (d, J = 8.0 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H). 13C NMR
(67.9 MHz, CD3OD): d 20.28, 55.76, 60.38, 61.87, 68.85, 79.20,
84.72, 88.01, 94.72, 101.30, 127.90, 129.57, 137.09, 141.11, 144.95,
150.82, 164.83. MALDI-TOF MS: [MH]+ 457.22, calcd 457.12.
N6-Acetyl-5ꢀ-O-(dimethoxytrityl)-3ꢀ-O-[2-(4-tolylsulfonyl)ethoxy-
methyl]cytidine 2ꢀ-O-pimelate (10b)
Compound 8b (150 mg, 0.19 mmol) was treated as described for
1
10a to give 10b (123 mg, 70.0%). H NMR (270 MHz, CDCl3 +
DABCO): d 1.37 (m, 1H), 1.64 (m, 2H), 2.21 (s, 3H), 2.31 (t, J =
6.6 Hz, 1H), 2.37 (t, J = 3.4 Hz, 1H), 2.42 (s, 3H), 3.16 (t, J =
3.7 Hz, 2H), 3.39 (dd, J = 2.0 Hz, 1H), 3.67 (m, 3H), 3.81 (s, 6H),
4.19 (t, J = 6.9 Hz, 1H), 4.40 (m, 2H), 4.53 (d, J = 7.1 Hz, 1H),
5.39 (t, J = 4.3 Hz, 1H, H2ꢀ), 6.1 (d, J = 2.1 Hz, 1H, H1ꢀ), 6.85 (d,
J = 7.4 Hz, 4H), 7.02 (d, J = 7.5, 1H), 7.71 (d, J = 8.2, 2H), 8.27
(d, J = 7.6, 1H). 13C NMR (67.9 MHz, CDCl3 + DABCO): d 21.7,
24.7, 24.8, 24.9, 28.6, 33.8, 34.8, 55.3, 56.1, 61.4, 61.7, 72.4 (C3ꢀ),
74.3 (C2ꢀ), 81.7, 87.3, 88.8, 94.9, 96.8, 113.4, 123.8, 127.5, 128.0,
128.1, 128.4, 130,0, 130.2, 130.3, 135.1, 136.1, 136.9, 143.9, 144.7,
144.9, 149.8, 158.9, 162.8, 170.6, 172.2, 178.4. MALDI-TOF MS:
[M + Na]+ 964.31, calcd 965.04.
Preparation of the solid support
To
a solution of 10a–d (0.1 mmol) in dry acetoni-
trile (20 ml) was added LCAA-CPG (1 g, Biotech com-
pany), and then diisopropylethylamine (1.74 ml, 10 mmol),
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexfluo-
rophosphate (BOP, 84 mg, 0.2 mmol) and N-hydroxybenzotriazole
(27 mg, 0.2 mmol). After shaking at room temperature for 2 h, this
reaction mixture was filtered, washed in turn with acetonitrile,
CH2Cl2, methanol, CH2Cl2 and diethyl ether. The solid was then
suspended in dry pyridine (20 ml) with acetic anhydride (2.25 ml)
and 4-(dimethylamino)pyridine (DMAP, 465 mg), and shaken for
2 h at room temperature. After filtration, the solids were washed in
turn with pyridine, toluene, CH2Cl2, methanol, CH2Cl2 and diethyl
ether, and dried over P2O5 under high vacuum. The loadings,
determined by detritylation assays, were 20–25 lmol g−1.
N6-Phenoxyacetyl-5ꢀ-O-(dimethoxytrityl)-3ꢀ-O-[2-(4-tolyl-
sulfonyl)ethoxymethyl]adenosine 2ꢀ-O-pimelate (10c)
Compound 8c (214 mg, 0.23 mmol) was treated as described for
1
10a to give 10c (160 mg, 64.8%). H NMR (270 MHz, CDCl3 +
DABCO): d 1.32 (m, 1H), 1.61 (m, 2H), 2.29–2.39 (m, 2H), 2.42 (s,
3H), 3.22 (t, J = 5.8 Hz, 2H), 3.33 (dd, J = 3.8 Hz, 1H), 3.56 (dd,
J = 3.1 Hz, 1H), 3.77 (m, 8H), 4.27 (m, 1H), 4.57 (s, 2H), 4.71 (t,
J = 5.5 Hz, 1H, H3ꢀ), 4.86 (s, 2H), 5.83 (t, J = 4.4 Hz, 1H, H2ꢀ),
6.26 (d, J = 4.1 Hz, 1H, H1ꢀ), 6,79 (d, J = 8.8 Hz, 1H), 7.04 (m,
3H), 7.15–7.39 (m, 13H), 7.73 (d, J = 8.3 Hz, 2H), 8.27 (s, 1H),
8.74 (s, 1H). 13C NMR (67.9 MHz, CDCl3 + DABCO): d 21.6,
24.3, 24.4, 28.2, 33.6, 33.7, 55.4, 56.0, 61.7, 62.5, 68.2, 73.9 (C3ꢀ),
74.4 (C2ꢀ), 82.7, 86.6, 86.8, 95.2, 113.2, 115.2, 123.0, 127.8, 128.1,
129.8, 130.1, 130.3, 135.4, 135.5, 136.8, 141.8, 144.3, 145.0, 148.5,
151.4, 153.0, 157.1, 158.7, 166.8, 172.4, 176.6. MALDI-TOF MS:
[M]+ 1058.36, calcd 1058.38
RNA synthesis and purification
All the RNAs were assembled on a Applied Biosystems 392
DNA/RNA synthesizer. All syntheses were carried out in trityl-
off mode; the synthesis cycle and reagents can be found in
the Supplementary information†. After the assembly, the solid
supports were removed from the cartridges and treated with
25% NH3/MeOH (4 ml) at room temperature for 20 h, and
then at 40 ◦C for 4 h. The supernatant solutions were then
separated from the solid supports, and evaporated to dryness.
After co-evaporation with dry THF twice, they were treated with
1 M tetrabutylammonium fluoride in THF (this solution was
N2-(N,N-Dimethylaminomethylene)-5ꢀ-O-(dimethoxytrityl)-3ꢀ-O-
[2-(4-tolylsulfonyl)ethoxymethyl]guanosine 2ꢀ-O-pimelate (10d)
˚
dried with 4 A molecular sieves overnight before use) containing
10% n-propylamine and 1% bis(2-mercaptoethyl) ether at room
temperature for 20 h. The reactions were quenched by the addition
of an equal volume of doubly distilled water and applied to a
NAP-10 column according to the manufacturer’s instructions,
with doubly distilled water as the eluting buffer. To ensure the
complete removal of fluoride, the products were passed through a
Sep-Pak cartridge.
Compound 8d (165 mg, 0.19 mmol) was treated as described for
10a to give 10d (126 mg, 65.6%). H NMR (270 MHz, CDCl3 +
1
DABCO): d 1.35 (m, 1H), 1.61 (m, 2H), 2.20 (t, J = 7.3 Hz, 1H),
2.38 (m, 4H), 3.07 (s, 3H), 3.11 (s, 3H), 3.15 (t, J = 2.4 Hz, 2H),
3.26 (dd, J = 2.6 Hz, 1H), 3.43 (dd, J = 2.2 Hz, 1H), 3.71–3.77 (m,
8H), 4.21 (m, 1H), 4.51 (m, 3H), 5.92 (t, J = 3.2 Hz, 1H, H2ꢀ), 5.98
(d, J = 2.3 Hz, 1H, H1ꢀ), 6.79 (m, 4H), 7.21–7.38 (m, 11 H), 7.71
(m, 3H), 8.58 (s, 1H). 13C NMR (67.9 MHz, CDCl3 + DABCO): d
21.6, 24.8, 25.8, 29.0, 33.9, 35.2, 36.9, 41.3, 55.3, 55.9, 61.5, 62.7,
73.5 (C3ꢀ), 74.1 (C2ꢀ), 81.7, 86.5, 86.6, 95.0, 113.2, 127.0, 127.8,
127.9, 128.1, 129.9, 130.0, 135.5, 136.5, 136.8, 144.3, 145.5, 152.2,
RNase H digestion assays
Escherichia coli RNase H (5 units lL−1, specific activity
420 000 units mg−1, molecular weight 21 000 g mol−1), T4
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polynucleotide Kinase (30 units lL−1) and [c-32P]ATP were
purchased from Amersham Pharmacia Biotech (Sweden). The
pure 15-mer RNA (ON12) was from IBA BioTAGnology (received
in crude form and purified by PAGE; the purity is shown in
Fig. S5†). Synthesis of the complementary DNA was carried out
as previously described.43 ON12 was synthesized, deprotected,
desalted by NAP-10 column and Sep-Pak cartridge as in the
description above, to give the crude ON12, which was used directly
for RNase H digestion. The RNA was 5ꢀ-end labeled with 32P using
T4 polynucleotide kinase, [c-32P]ATP by standard procedure.
The RNase H digestion of synthesized crude RNA and pure
RNA was carried out according to the following procedure:
target pure RNA (0.1 lM) or crude RNA (0.1 lM) (specific
activity 70000 cpm) and 10-fold excess of complementary DNA
(1 lM) were incubated in a buffer, containing 20 mM Tris-HCl
(pH 8.0), 20 mM KCl, 10 mM MgCl2, 0.1 mM EDTA and
D. E. Bergstrom, G. D. Glick and R. A. Jones, John Wiley & Sons,
New York, 2000.
6 S. G. Chaulk and A. M. MacMillan, Nucleic Acids Res., 1998, 26,
3173–3178.
7 M. E. Schwartz, R. R. Breaker, G. T. Asteriadis, J. S. de Bear and G. R.
Gough, Bioorg. Med. Chem. Lett., 1992, 2, 1019–1024.
8 C. B. Reese, R. Saffhill and J. E. Sulston, J. Am. Chem. Soc., 1967, 89,
3366–3368.
9 A. Sandstroem, M. Kwiatkowski and J. Chattopadhyaya, Acta Chem.
Scand., Ser. B, 1985, 39, 273–290.
10 A. Sandstroem, M. Kwiatkowski and J. Chattopadhyaya, Nucleosides
Nucleotides, 1985, 4, 177–181.
11 W. Lloyd, C. B. Reese, Q. Song, A. M. Vandersteen, C. Vistina and P. Z.
Zhang, J. Chem. Soc., Perkin Trans. 1, 2000, 165–176.
12 M. Pfister, H. Schirmeister, M. Mohr, S. Farkas, K. P. Stengele, T.
Reiner, M. Dunkel, S. Gokhale, R. Charubala and W. Pfleiderer, Helv.
Chim. Acta, 1995, 78, 1705–1737.
13 S. Matysiak and W. Pfleiderer, Helv. Chim. Acta, 2001, 84, 1066–1085.
14 A. Semenyuk, A. Foldesi, T. Johansson, C. Estmer-Nilsson, P. Blom-
gren, M. Brannvall, L. A. Kirsebom and M. Kwiatkowski, J. Am. Chem.
Soc., 2006, 128, 12356–12357.
15 K. K. Ogilvie, K. L. Sadana, E. A. Thompson, M. A. Quilliam and
J. B. Westmore, Tetrahedron Lett., 1974, 33, 2861–2863.
16 K. K. Ogilvie, N. Theriault and K. L. Sadana, J. Am. Chem. Soc., 1977,
99, 7741–7743.
17 N. Usman, K. K. Ogilvie, M. Y. Jiang and R. J. Cedergren, J. Am.
Chem. Soc., 1987, 109, 7845–7854.
18 T. Wada, M. Tobe, T. Nagayama, K. Furusawa and M. Sekine,
Tetrahedron Lett., 1995, 36, 1683–1684.
19 T. Umemoto and T. Wada, Tetrahedron Lett., 2004, 45, 9529–9531.
20 M. V. Rao, C. B. Reese, V. Schehlmann and P. S. Yu, J. Chem. Soc.,
Perkin Trans. 1, 1993, 43–55.
21 J. Stawinski, R. Stro¨mberg, M. Thelin and E. Westman, Nucleic Acids
Res., 1988, 16, 9285–9308.
◦
0.1 mM DTT at 21 C in the presence of 0.06 U E. coli RNase
H. Prior to the addition of the enzyme reaction components
◦
were pre-annealed in the reaction buffer by heating at 80 C for
4 min followed by 1.5 h equilibration at 21 ◦C. Total reaction
volume was 30 lL. Aliquots of 3 lL were taken after 2, 5, 10,
15, 25, 40 and 60 min and the reactions were terminated by
mixing with stop solution (7 lL), containing 0.05 M EDTA,
0.05% (w/v) bromophenol blue and 0.05% (w/v) xylene cyanole
in 80% formamide. The samples were subjected to 20% 7 M
urea PAGE and visualized by autoradiography. Quantitation of
cleavage products was performed using a Molecular Dynamics
PhosphorImager.
22 T. Wu, K. K. Ogilvie and R. T. Pon, Nucleic Acids Res., 1989, 17,
3501–3517.
23 S. S. Jones and C. B. Reese, J. Chem. Soc., Perkin Trans. 1, 1979, 2762–
2764.
Phosphodiesterase digestion assays
24 S. A. Scaringe, C. Francklyn and N. Usman, Nucleic Acids Res., 1990,
18, 5433–5441.
25 S. A. Scaringe, F. E. Wincott and M. H. Caruthers, J. Am. Chem. Soc.,
1998, 120, 11820–11821.
26 S. Pitsch, P. A. Weiss, L. Jenny, A. Stutz and X. Wu, Helv. Chim. Acta,
2001, 84, 3773–3795.
27 T. Ohgi, Y. Masutomi, K. Ishiyama, H. Kitagawa, Y. Shiba and J. Yano,
Org. Lett., 2005, 7, 3477–3480.
Shrimp Alkaline Phosphatase and Phosphodiesterase I (Crotalus
adamanteus Venom) are from Amersham Pharmacia Biotech,
Sweden. A reaction mixture containing 20 mM Tris-HCl (pH 8.0),
10 ml MgCl2, Shrimp Alkaline Phosphatase (0.5 unit) and
Phosphodiesterase I (Crotalus adamanteus Venom) (0.4 unit) was
added to crude ON12 (1 OD unit at 260 nm) with a total reaction
volume of 30 lL. The reaction mixture was incubated at 37 ◦C
for 24 h and subjected to HPLC analysis directly (0.2 OD per
injection).
28 F. G. Bordwell, Acc. Chem. Res., 1988, 21, 456–463.
29 S. Josephson, N. Balgobin and J. Chattopadhyaya, Tetrahedron Lett.,
1981, 22, 4537–4540.
30 M. Jain, J. Fan, N. Z. Baturay and C. H. Kwon, J. Med. Chem., 2004,
47, 3843–3852.
31 K. A. Watanabe and J. J. Fox, Angew. Chem., Int. Ed. Engl., 1966, 5,
579–580.
Acknowledgements
32 T. L. Sheppard, A. T. Rosenblatt and R. Breslow, J. Org. Chem., 1994,
59, 7243–7248.
33 C. Chaix, A. M. Duplaa, D. Molko and R. Te´oule, Nucleic Acids Res.,
1989, 17, 7381–7393.
Generous financial support from the Swedish Natural Science
Research Council (Vetenskapsra˚det), the Swedish Foundation for
Strategic Research (Stiftelsen fo¨r Strategisk Forskning) and the
EU-FP6-funded RIGHT project (project no. LSHB-CT-2004-
005276) is gratefully acknowledged.
34 L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 1983, 24, 245–
248.
35 H. P. M. Fromageot, B. E. Griffin, C. B. Reese, J. E. Sulston and D. R.
Trentham, Tetrahedron, 1966, 22, 705–710.
36 K. K. Ogilvie, A. L. Schifman and C. L. Penney, Can. J. Chem., 1979,
57, 2230–2238.
37 J. Milecki, E. Zamaratski, T. V. Maltseva, A. Fo¨ldesi, R. Adamiak and
J. Chattopadhyaya, Tetrahedron Lett., 1999, 55, 6603–6622.
38 R. T. Pon and S. Yu, Tetrahedron Lett., 1997, 38, 3331–3334.
39 C. Vargeese, J. Carter, J. Yegge, S. Krivjansky, A. Settle, E. Kropp, K.
Peterson and W. Pieken, Nucleic Acids Res., 1998, 26, 1046–1050.
40 E. Lieber and T. Enkoji, J. Org. Chem., 1961, 4472–4479.
41 B. H. Dahl, J. Nielsen and O. Dahl, Nucleic Acids Res., 1987, 15, 1729–
1743.
References
1 A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C.
Mello, Nature, 1998, 391, 808–811.
2 S. M. Elbashir, J. Harborth, W. Lendechel, A. Yalcin, K. Weber and T.
Tuschl, Nature, 2001, 411, 494–498.
3 C. B. Reese, Org. Biomol. Chem., 2005, 3, 3851–3868.
4 D. Gasparutto, D. Molko and R. Te´oule, Nucleosides Nucleotides, 1990,
9, 1087–1098.
42 T. Umemoto and T. Wada, Tetrahedron Lett., 2005, 46, 4251–4253.
43 P. I. Pradeepkumar, E. Zamaratski, A. Fo¨ldesi and J. Chattopadhyaya,
J. Chem. Soc., Perkin Trans. 2, 2001, 402–408.
5 C. B. Reese, ‘Protection of 2ꢀ-hydroxy functions of ribonucleosides’
in Current Protocols in Nucleic Acid Chemistry, ed. S. L. Beaucage,
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 333–343 | 343
©