Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

Article abstract of DOI:10.1134/S106816201102004X

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligo-nucleotide phosphotriester method using O-nucleophilic intramole

Full text of DOI:10.1134/S106816201102004X

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 2, pp. 254–258. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © V.A. Efimov, A.V. Aralov, O.G. Chakhmakhcheva, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 2, pp. 284–288.
LETTER
TO THE EDITOR
Methoxymethyl and (pꢀNitrobenzyloxy)methyl Groups
in Synthesis of Oligoribunucleotides
by the Phosphotriester Method
V. A. Efimov†, A. V. Aralov, and O. G. Chakhmakhcheva1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. MiklukhoꢀMaklaya 16/10, Moscow, 117997, Russia
Received October 1, 2010; in final form, November 1, 2010
Abstract—An efficient method to synthesize monomer ribonucleotide synthons containing 2'ꢀOꢀmethꢀ
oxymethyl and 2'ꢀ
nucleotide phosphotriester method using
Oꢀ(pꢀnitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligoꢀ
O
ꢀnucleophilic intramolecular catalysis at the stage of the internuꢀ
cleotide bond formation. The former synthons may be used for the automatic synthesis of 2'ꢀmodified oligoꢀ
nucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high
yields.
Keywords: oligoribinucleotides, phosphotriester method, Oꢀnucleophilic catalysis, methoxymethyl and pꢀnitrobenꢀ
zyloxymethyl groups
DOI: 10.1134/S106816201102004X
1†
The phosphotriester method of oligonucleotide
For the first time, the synthesis of uridine monoꢀ
mer containing the 4ꢀNBOM group and its use for
the synthesis of oligo(U) by the phosphoroamidite
method was described by Gough et al. [8]. It was
further shown that this protecting group along
synthesis with the use of
Оꢀnucleophilic catalysis at
the stage of the internucleotide bond formation is an
alternative to the phosphoroamidite approach. The
wellꢀdeveloped phosphotriester method was successꢀ
fully used for the synthesis of both native and modified
oligonucleotides [1, 2]. Moreover, it was shown that
this method can be used for the preparation of steꢀ
reospecific phosphorothioate oligonucleotide analogs
[3, 4].
with (оꢀnitrobenzyloxy)ꢀ, [4ꢀ(Nꢀmethylamino)benꢀ
zyloxy]ꢀ, and [4ꢀ(
N
ꢀdichloroacetylꢀ(
N
ꢀmethylꢀ
amino)benzyloxy]methyl groups can be applied for
the efficient synthesis of RNA fragments by the
phosphoroamidite method [9–11]. However, the
synthesis of 2'ꢀ
method led to the formation of the hardꢀtoꢀseparate
mixture of 2'ꢀ and 3'ꢀ ꢀ(4ꢀNBOM) derivatives
Оꢀ(4ꢀNBOM)ꢀuridine by the Gough
To develop the efficient phosphotriester method of
the synthesis of native and modified oligoribonucleꢀ
otides, we examined a number of protecting and modꢀ
ifying groups [5–7]. In continuation of the study, we
elaborated schemes of the synthesis of monomer synꢀ
thons for the solidꢀphase synthesis of RNA fragments
О
ꢀ
О
and, thereby, to the low yield of the target comꢀ
pound.
We used the alternative scheme of regioselective
introduction of the 4ꢀNBOM group at the 2'ꢀposiꢀ
tion (Scheme 1). At the first stage, 3'ꢀ and 5'ꢀOH
functions of nucleosides were blocked by the treatꢀ
ment with 1,3ꢀdichloroꢀ1,1,3,3ꢀtetraisopropyldisiꢀ
loxane (TIPDSꢀCl2) [12] to prepare derivatives
containing modifying 2'ꢀ
Оꢀmethoxymethyl (MOM)
and protecting 2'ꢀ ꢀpꢀnitrobenzyloxymethyl (4ꢀ
О
NBOM) groups. The synthesis of oligoribonucleotides
was performed using these synthons.
(
IV). At the same time,
successively converted into methyl ester of
ꢀnitrobenzyl)oxyacetic acid ( ), ( ꢀnitrobenꢀ
zyl)oxyacetic acid (II), and acetoxymethylꢀ
pꢀnitrobenzyl alcohol was
Abbreviations: DBU, 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀen;
DMTr,
4ꢀNBOM,
4,4'ꢀdimethoxytrityl;
MOM,
methoxymethyl;
Nꢀiodosuccinimide;
(
p
I
p
pꢀnitrobenzyloxymethyl; NIS,
TPS, 2,4,6ꢀtriisopropylbenzeneꢀsulfonyl; TBAF, tetrabutylamꢀ
monium fluoride.
Deceased.
Corresponding author: phone: +7(495) 336ꢀ5911; fax: +7(495)
330ꢀ6738; eꢀmail: eva@mx.ibch.ru.
p
ꢀ
nitrobenzyl ester (III), which was used for the introꢀ
duction of the 4ꢀNBOM group into the 2'ꢀposition
of nucleosides (IV).
†
1
254

METHOXYMETHYL AND (
p
ꢀNITROBENZYLOXY)METHYL GROUPS IN SYNTHESIS
255
Cs CO
2
NaOH
COOCH3
CH OH
3
3
+
COOCH3
OH
O2N
O
Br
O2N
DMF
(I)
Pb(OAc)
DMF
4
O2N
O
COOH
O2N
O
OAc
(II
)
(III)
Base
H
Base
Base
O
HO
O
O
O
O
Si
Si
(
III)/SnCl
KF
CH OH
3
DMTrꢀCl
Py
4
H
H
O
H
H
O
H
H
C H Cl
2
2
4
H
H
H
O
O
H
OH
H
O
OH
O
Si
Si
O
O
(IV)
(
V
)
(VI)
NO2
NO2
Cl
O
ᮎ
O
1.
H3CO
P
O
O
+
TPSꢀCl
Base
H
Base
H
DMTrO
DMTrO
N
O
O
O
H
H
O
H
O
H
2. DBU
CH CN/H O
H
H
3
2
ᮎ
O
O
OH
P
O
O
O
O
H3CO
(VII)
N
O
NO2
(VIII)
NO2
Base = Ura, CytBz, AdeBz, GuaiBu
Scheme 1.
The 3'ꢀ and 5'ꢀfunctions of resultant compounds nucleoside and nucleotide chemistry for the protecꢀ
V) should be deprotected. It was previously shown tion of hydroxyl functions due to the simplicity of its
(
that ammonium fluoride in methanol may be used for introduction and stability in both strong alkaline and
this purpose [10]. We found that the 2'ꢀ ꢀ(4ꢀNBOM) moderate acidic conditions, as well as under condiꢀ
O
group was also not touched by potassium fluoride in tions used for the removal of silyl, alkoxyacetyl, and
methanol; on the other hand, it can be easily benzyl groups [13–15].
removed from compounds (V) along with the TIPDS
2'ꢀОꢀMOM nucleotide derivatives (XII) were synꢀ
group by treatment with tetrabutylammonium fluoꢀ
ride (TBAF) in THF. The further introduction of
the 4,4'ꢀdimethoxytrityl protecting group at the
5'ꢀhydroxyl function of nucleosides (VI) and
thesized according to successive reactions presented in
Scheme 2. Uridine, cytidine, and adenosine derivaꢀ
tives (IV) with protected 3'ꢀ and 5'ꢀhydroxyls and
amino groups of heterocyclic bases (in the cases of Cyt
and Ade) were treated with an excess of
dimethoxymethane in dichloroethane in the presence
of boron trifluoride etherate at room temperature for
the preparation of 3'ꢀphosphate derivatives (VIII
containing 4ꢀmethoxyꢀ1ꢀoxideꢀ2ꢀpicolyl ꢀprotectꢀ
ing group were carried out according to [6, 7].
)
P
The methoxymethyl protecting group found wide 2 h. Completely protected nucleosides (
application in both classical organic synthesis and lated, and TIPDS groups were removed with 1 M
X) were isoꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 2
2011

256
EFIMOV et al.
TBAF in THF for 1 h followed by tritylation of the amount of the fluorescent side product. In this case,
resultant MOM derivatives. The prepared compound
XI) was treated with (4ꢀchlorophenyl)ꢀ(1ꢀoxidoꢀ4ꢀ
therefore, the synthesis of the 2'ꢀ
ОꢀМОМ derivative
(
(
X) was carried out starting from the methylthiomethyl
methoxyꢀ2ꢀpicolyl)phosphate in the presence of
TPSꢀCl and then with DBU in aqueous acetonitrile
[6, 7] to introduce the phosphoric acid residue conꢀ
derivative (IX) (its preparation can be seen in [16]) by
its treating with trifluoromethane sulfonic acid and
NIS in a THF–methanol mixture (25 : 1) for 20 min
taining the 4ꢀmethoxyꢀ1ꢀoxidoꢀ2ꢀpicolyl
group at the 3'ꢀhydroxyl group. In the case of guaꢀ
nosine derivative, the treatment of nucleoside (IV
with dimethoxymethane in the presence of boron triꢀ
fluoride etherate led to the formation of a large NMR spectroscopy methods.
Рꢀprotected
at –40
ate compounds and target monomer synthons (VIII
and (XII) were confirmed by mass spectrometry and
°С. In all cases, the structures of the intermediꢀ
)
)
GuaiBu
Base
H
O
O
O
Si
O
Si
H
O
H
H
O
H
O
H
O
H
O
H
S
OH
Si
Si
(
IV)
(IX)
Base = Ura, CytBz, AdeBz
CH OCH OCH
2
CH OH, NIS, CF SO H
3
3
BF
3
3
THF
3
⋅
Et O
2
3
C H Cl
2
4
2
Base
O
Base
DMTrO
1. TBAF/THF
2. DMTrꢀCl/Py
O
Si
O
H
O
H
O
H
H
O
H
O
H
H
O
H
OH
Si
O
(
X)
(XI)
Base = Ura, CytBz, AdeBz, GuaiBu
Cl
O
Base
DMTrO
O
–
+
P
O
O
1.
TPSꢀCl
O
H
O
H
O
H3CO
H
N
H
O
–O
O
P
2. DBU
CH CN/H O
O
O
3
2
H3CO
N
O
(XII
)
Scheme 2.
Monomers (VIII) and (XII) were used for the solid support as described in [7, 17] using TPSꢀCl as a conꢀ
phase synthesis of 15–25ꢀmer oligoribonucleotides. densing agent for the formation of the internucleotide
The synthesis was carried out on the standard CPGꢀ bond. The yields at the chain elongation steps were
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

METHOXYMETHYL AND (
p
ꢀNITROBENZYLOXY)METHYL GROUPS IN SYNTHESIS
257
98–99%. After the synthesis was completed, the oligoꢀ
nucleotides bound to the support were treated with
1ꢀM potassium iodide in acetonitrile (5 h at room
temperature) to deblock phosphate residues and then
with concentrated ammonia to cleave oligonucleꢀ
otides from the support and to remove protecting
(a)
(b)
1
2
3
1
2
3
4
XC
XC
groups from heterocyclic bases. pꢀNitrobenzyloxymꢀ
ethyl groups were removed from 2'ꢀOH oligonucleꢀ
otide functions by the treatment with TBAF in THF.
Oligonucleotides were then isolated by gel filtration
on columns with Sephadex Gꢀ25. Homogeneity of
the products were examined by electrophoresis in
polyacrylamide gel (figure a) and reverseꢀphase chroꢀ
matography. Oligonucleotides with the same
sequences obtained by the phosphoroamidite method
using commercial monomers were used as control
compounds. Our results showed the reasonable effiꢀ
ciency of using the (4ꢀNBOM)ꢀprotecting group for
the protection of 2'ꢀhydroxyls during the oligoribonuꢀ
cleotide synthesis by the phosphotriester method.
ВРВ
ВРВ
Electrophoregrams in 15% denatured PAAG: (a) is oligonuꢀ
cleotide , prepared with the use of 2'ꢀ ꢀ(4ꢀNBOM) proꢀ
tecting group before ( ) its removal, and the
same deprotected oligonucleotide prepared by the phosꢀ
phoroamidite method ( ); (b) is oligonucleotides 1, 2
and 5'ꢀCGAUCUCAUCACCUCUCCAU (3, 4) prepared
with the use of 2'ꢀ ꢀМОМ protecting group before (1, 3
U
О
15
1
) and after (2
3
U
(
)
15
О
)
and, after (2, 4), its removal with 1ꢀM LiI in a acetonitrile–
water mixture (20 : 1) in the presence of 0.01ꢀM HCl. The
photograph was taken in reflected UV light at 254 nm
(XC, xylene cyanol FF; BPB, bromophenol blue).
It should be noted that attempts to remove the
2'ꢀОꢀМОМ group from the synthesized oligoribonuꢀ
cleotides with acidic reagents, e.g., 30% aqueous triꢀ
fluoroacetic acid [15, 18] or by the recently proposed
ACKNOWLEDGMENTS
The authors are grateful to M.S. Smirnova (Sheꢀ
method based on the treatment with ZnBr2 in the myakin–Ovchinnikov Institute of Bioorganic Chemꢀ
istry, Russian Academy of Sciences) for participation
at different stages of the project.
presence of mercaptan [19], led to a significant degꢀ
radation of the oligomer chain. We found that the
treatment of MOMꢀcontaining nucleosides with
1ꢀM LiI in a acetonitrile–water mixture (20 : 1) in
the presence of 0.01ꢀM HCl for 2–3 h resulted in an
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