A post-synthetic approach for the synthesis of 2′-O- methyldithiomethyl-modified oligonucleotides responsive to a reducing environment

Article abstract of DOI:10.1039/c3cc43725f

Based on a novel concept, a Reducing-Environment-Dependent Uncatalyzed Chemical Transforming RNA, REDUCT RNA , we established a post-synthetic approach for the synthesis of 2′-O-methyldithiomethyl- modified oligonucleotides from 2′-O-(2,4,6-trimethoxybenzylthiomethyl)- oligonucleotides by treatment with dimethyl(methylthio)sulfonium tetrafluoroborate. 2′-O-methyldithiomethyl oligonucleotides were easily converted into 2′-hydroxy oligonucleotides under reducing conditions, such as those found in the intracellular environment. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc43725f

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A post-synthetic approach for the synthesis of
20-O-methyldithiomethyl-modified oligonucleotides
responsive to a reducing environment†
Cite this: Chem. Commun., 2013,
49, 7620
Received 17th May 2013,
Accepted 25th June 2013
Yosuke Ochi, Osamu Nakagawa, Katsunori Sakaguchi, Shun-ichi Wada and
Hidehito Urata*
DOI: 10.1039/c3cc43725f
www.rsc.org/chemcomm
Based on a novel concept, a Reducing-Environment-Dependent
Uncatalyzed Chemical Transforming RNA, ‘‘REDUCT RNA’’, we established
a post-synthetic approach for the synthesis of 20-O-methyldithiomethyl-
modified oligonucleotides from 20-O-(2,4,6-trimethoxybenzylthiomethyl)-
oligonucleotides by treatment with dimethyl(methylthio)sulfonium
tetrafluoroborate. 20-O-methyldithiomethyl oligonucleotides were
easily converted into 20-hydroxy oligonucleotides under reducing
conditions, such as those found in the intracellular environment.
Scheme 1 Conversion of 20-O-alkyldithiomethyl-modified nucleotides into
native nucleotides in a reducing environment.
cells to liberate native RNA 3 via unstable thiohemiacetal derivative 2
Technologies based on ribozymes1 and RNA interference2 (RNAi) (Scheme 1). As far as we know, there is only one report of the synthesis
provide attractive gene silencing strategies different from small mole- of 20-O-alkyldithiomethyl RNA, in which the t-butyldithiomethyl group
cule approaches. However, such gene silencing technologies need to is employed as a novel protecting group for the 20-hydroxy groups in
overcome some hurdles before they can be used in practical applica- the chemical synthesis of RNA.14 However, the instability of the
tions. Exogenous unmodified RNAs are rapidly degraded by nucleases phosphoramidite units bearing the alkyldithiomethyl group would
and exhibit poor cell permeation. To circumvent these problems, limit their application, because the trivalent phosphorus atom on
numerous chemical modifications of RNA have been reported. Among such phosphoramidites is capable of allowing intramolecular attack
them, the 20-O-modifications conferred efficient resistance to nucleases on the disulfide bond.14,15 Thus, the development of a novel approach
on oligonucleotides3 but usually decreased the activities of small for the synthesis of 20-O-alkyldithiomethyl-modified RNA is required
interfering RNAs (siRNAs) and ribozymes.4,5 Indeed, siRNA activity for its practical use.
virtually disappeared with the full 20-O-methyl modification of oligo-
Here, we report a post-synthetic approach for the synthesis of
ribonucleotides.4,6 These drawbacks of the modifications would be 20-O-methyldithiomethyluridine-containing oligonucleotides. We also
circumvented with prodrug-type RNA bearing transient modifications show the functional basis of the 20-O-methyldithiomethyl-modified
as biolabile groups. Based on this idea, Debart et al. reported oligo- oligonucleotides, which are efficiently converted into the 20-hydroxy
nucleotides bearing 20-O-acyloxymethyl groups that were designed to be oligonucleotides under reducing conditions, namely Reducing-
cleaved by intracellular esterases to liberate native RNA.7,8 In mammals, Environment-Dependent Uncatalyzed Chemical Transforming
however, high carboxyesterase activities in the serum are found9 and RNA (REDUCT RNA).
this may lead to cleavage of the promoieties to liberate native RNA
before delivery of the modified RNA into target cells.
Originally, we focused on the 4-methoxybenzylthio group, which
was reported to be easily converted into the methyldithio group by
It is known that the intracellular concentration of glutathione is treatment with dimethyl(methylthio)sulfonium tetrafluoroborate
very high (1–10 mM) compared to the plasma concentration (E2 mM), (DMTSF).16 Thus, we synthesized 20-O-(4-methoxybenzyl)thiomethyl
creating a reducing environment inside cells.10–13 Therefore, we uridine derivative 4a and attempted to convert it into 20-O-methyl-
designed 20-O-alkyldithiomethyl RNA 1, whose promoiety’s disulfide dithiomethyl derivative 6 (Scheme 2). However, the reaction gave
bond was expected to be cleaved in the reducing environment inside 20-O-fluoromethyl uridine derivative 8 as the major product. This
result should be due to the stronger electron-donating ability of the
20-oxygen atom to the methylthiosulfonium moiety of 5a than the
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki,
4-methoxybenzyl group. Thus, we designed a novel 20-O-promoiety,
Osaka 569-1094, Japan. E-mail: urata@gly.oups.ac.jp; Fax: +81 72 690 1089;
the 2,4,6-trimethoxybenzylthiomethyl (TMBTM) group, which
Tel: +81 72 690 1089
has
a stronger electron-donating characteristic than the
† Electronic supplementary information (ESI) available: Experimental details and
characterization data. See DOI: 10.1039/c3cc43725f
4-methoxybenzylthiomethyl group. Indeed, TMBTM-containing
c
7620 Chem. Commun., 2013, 49, 7620--7622
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Table 1 Synthesized oligonucleotides bearing 20-O-TMBTM groupsa
MALDI-TOF mass
ODN
Sequence (50–30)
Calcd [M ꢁ H]ꢁ
3860.6
4088.9
4317.2
4317.2
Found
14a
15a
16a
17a
d(GCGTTXTTTGCT)a
d(GCGTTXTXTGCT)a
d(GCGXTXTXTGCT)a
d(GCGTTXXXTGCT)a
3859.1
4090.0
4316.4
4318.4
a
X: 20-O-TMBTM-uridine.
5-Ethylthio-1H-tetrazole (ETT) was employed as an activator for the
coupling reaction and the coupling time was extended to 900 s.
Oxidation of the trivalent phosphorus was conducted by using
0.02 M iodine solution. The synthesized oligonucleotides were
deprotected and cleaved from the support with concentrated
aqueous ammonia at 55 1C for 10 h. A nearly single peak for the
crude oligonucleotides bearing the 50-O-DMTr group was observed
using HPLC. A typical chromatogram of the crude 50-O-dimethoxy-
tritylated oligonucleotide is shown in Fig. S2 (ESI†). After HPLC
purification followed by detritylation, the structures were character-
ized using MALDI-TOF mass spectrometry (Table 1).
Scheme 2 Conversion of 20-O-benzylthiomethyluridine derivatives into a 20-O-methyl-
dithiomethyl or a 20-O-fluoromethyl derivative.
Post-synthetic modifications of oligonucleotides 14a–17a into the
corresponding 20-O-methyldithiomethyl oligonucleotides 14b–17b
were conducted by treatment with DMTSF. A 0.1 mM solution of
the oligonucleotide in 200 mM sodium acetate buffer (pH 4) was
treated with DMTSF (final concentration: 30 mM). Fig. 1 shows the
HPLC analysis of the conversion of 15a into 15b. The reaction
efficiently proceeded without the generation of much by-product
within 3 h. Without the buffer, depurination took place due to the
strong acidity of DMTSF (data not shown). Depurination was well
prevented by appropriate pH control to pH 4 with sodium acetate
buffer, although the reaction rate was somewhat low compared with
the non-buffered system. Other oligonucleotides bearing 20-O-TMBTM
Scheme 3 Synthesis of a 20-O-TMBTM uridine amidite unit. Conditions: (i) DTBS-(OTf)2,
DMF, 0 1C, 1 h; (ii) DMSO, Ac2O, AcOH, r.t., 24 h, 74% (from 9); (iii) SO2Cl2, CH2Cl2,
r.t., 0.75 h; (iv) 2,4,6-(MeO)3BnSH, NaH, DMF, 0 1C, 2 h, 72% (from 10); (v) Et3Nꢀ3HF,
THF, r.t., 0.3 h, 89%; (vi) DMTr-Cl, pyridine, r.t., 1.5 h, 95%; (vii) [(iPr)2N]2POCE,
diisopropylammonium tetrazolide, CH2Cl2, r.t., 15 h, 92%.
uridine derivative 4b was converted into methyldithiomethyl- (14a, 16a, and 17a) were similarly converted into 20-O-methyldithio-
containing uridine derivative 6 in excellent yield. methyl oligonucleotides 14b, 16b, and 17b by DMTSF treatment. The
To evaluate the conversion of the 20-O-TMBTM group into the structures of 14b–17b were confirmed using MALDI-TOF mass
methyldithiomethyl group in oligonucleotides, we synthesized ami- spectrometry (Table S1, ESI†). Thus, our post-synthetic approach
dite building block 13 bearing the 20-O-TMBTM groups (Scheme 3). successfully provided 20-O-methyldithiomethyl oligonucleotides in
We initially protected the 30,50-hydroxy groups of uridine with the an easy and practical manner.
di-tert-butylsilanediyl (DTBS) group and subsequently introduced the
To evaluate the resistance of 20-O-methyldithiomethyl oligo-
methylthiomethyl (MTM) group into the 20-hydroxyl group in 74% nucleotides to 30-exonuclease, we synthesized oligonucleotide
yield. The resulting 20-O-MTM derivative 10 was treated with sulfuryl 18 50-d(TTTTTTTTUMDTMT)-30 (UMDTM: 20-O-methyldithiomethyl-
chloride to afford the 20-O-chloromethyl derivative,17 which was uridine) as well as thymidine 10-mer 19 as control. Each 10-mer
treated with 2,4,6-trimethoxybenzylmercaptan18 in the presence of was incubated with snake venom phosphodiesterase (SVPDE) and
sodium hydride to give 20-O-TMBTM-derivative 4b in 72% yield.
Removal of the DTBS group with Et3Nꢀ3HF afforded 20-O-TMBTM-
uridine 11 in 89% yield. The 50-hydroxy group of 11 was protected by
the 4,40-dimethoxytrityl (DMTr) group, and finally the 30-hydroxy
group was phosphitylated to afford phosphoramidite unit 13 in 87%
yield. The overall yield of 13 obtained from uridine 9 in seven steps
was 42%. Amidite 13 was completely stable for several months
at ꢁ20 1C and also showed excellent stability even in solution.
Dissolving 13 in acetonitrile resulted in less than 2% degradation
within one week (Fig. S1, ESI†). This is in marked contrast to
20-O-t-butyldithiomethyl-modified amidites.14
By using amidite unit 13, oligonucleotides bearing one to
three 20-O-TMBTM groups shown in Table 1 were synthesized.
Fig. 1 HPLC analysis of the conversion of 15a into 15b by treatment with DMTSF.
c
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as the promoiety of nuclease resistant prodrug-type RNA
(REDUCT RNA). Indeed, oligonucleotides containing the
20-O-methyldithiomethyl group were rapidly and efficiently
converted into the corresponding 20-hydroxy oligonucleotides.
The results demonstrated that the REDUCT RNA would be
highly beneficial for in vivo stabilization and activation in cells
for the widespread application of functional RNAs. The second
concept is a novel post-synthetic approach for the synthesis
of oligonucleotides containing the 20-O-methyldithiomethyl
group. To avoid utilization of unstable amidite units containing
the 20-O-alkyldithiomethyl group, the novel post-synthetic
approach was developed. This efficient synthetic method would
make practical applications of oligonucleotides containing the
alkyldithiomethyl group possible. The synthesis of TMBTM-
modified amidite units other than the uridine derivative and the
conversion of TMBTM-modified oligonucleotides consisting of
four natural bases into the 20-O-methyldithiomethyl oligonucleo-
tides are ongoing.
Fig. 2 Kinetics of degradation of oligonucleotides 50-d(TTTTTTTTUMDTMT)-30 18
and 50-d(TTTTTTTTTT)-30 19 with SVPDE.
This work was supported in part by a Grant-in-Aid for Young
Scientists (B), No. 24790121 (to O.N.), from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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