2-C-Methyluridine modified hammerhead ribozyme against the estrogen receptor

Article abstract of DOI:10.1016/j.bmcl.2010.03.060

A new synthesis of 2′-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2′-hydroxyl group. Comparison to previous protecting strategies and analysis of stability under 5′-DMTr removing conditions are discussed. The synthetic incorporation of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estrogen receptor α protein (ER-α), and transfection experiments in MCF-7 cell line are also presented.

Full text of DOI:10.1016/j.bmcl.2010.03.060

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2806–2808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-C-Methyluridine modified hammerhead ribozyme against the
estrogen receptor
Rodrigo Pontiggia a, Osvaldo Pontiggia b, Marina Simian b, Javier M. Montserrat a,c, Joachim W. Engels d,
Adolfo M. Iribarren a,e,
*
a INGEBI (CONICET),Vuelta de Obligado 2490-(1428), Buenos Aires, Argentina
b Instituto de Oncología A. H. Roffo, UBA, Av. San Martín 5481, CABA, Argentina
c Instituto de Ciencias, Universidad Nacional de Gral. Sarmiento, J. M. Gutierrez 1150, Los Polvorines (B1613GSX), Prov. de Bs. As., Argentina
d Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany
e Laboratorio de Biotransformaciones, Universidad Nacional de Quilmes, Roque Saenz Peña 352 (1876) Bernal, Prov. de Bs. As., Argentina
a r t i c l e i n f o
a b s t r a c t
A new synthesis of 20-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to
the improvement of the protection of the tertiary 20-hydroxyl group. Comparison to previous protecting
strategies and analysis of stability under 50-DMTr removing conditions are discussed. The synthetic incor-
poration of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estro-
Article history:
Received 12 February 2010
Revised 9 March 2010
Accepted 11 March 2010
Available online 17 March 2010
gen receptor
a
protein (ER-
a), and transfection experiments in MCF-7 cell line are also presented.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
20-C-methyluridine
Modified ribozyme
Estrogen receptor alpha
The development of synthetic oligoribonucleotides with non
conventional activities, like catalytic ribozymes,1 aptamers2 and
more recently siRNA,3 found applications in different fields such
as therapeutics, biosensors and molecular biology. The susceptibil-
ity of RNA to the attack of nucleases limited its utilization, espe-
cially in medicine. A way to overcome this restriction is the use
of chemical modifications, which must fulfill certain requisites.4
Another important feature is related to RNA structure, in this sense
sugar puckering of modified nucleosides can play an important
role, like in the case of locked nucleic acids (LNA).5
The direct modification of the ribose moiety of a natural ribonu-
cleoside can be also accomplished by two different routes, both
involving as a common synthon, the 20-ketonucleoside. In one case,
the 20-keto group is attacked by a methyl organometallic, yielding
mixtures of the ribo and arabino epimeres. The second option con-
sists in a Wittig reaction of the keto derivative followed by a ster-
eoselective dihydroxylation.7
In order to introduce 2-C-methyl nucleosides in an oligonucleo-
tide, proper phosphoramidite building blocks must be synthesized.
This includes an adequate protection of the 20-hydroxyl. In previ-
ous publications we used tetrahydropyranyl (THP) protection of
the tertiary alcohol,6 but unfortunately this group is not stable en-
ough to the conditions used during 50-dimethoxytrityl (DMT)
deprotection throughout oligonucleotide solid phase synthesis.13
An interesting model for testing this modification is the hammer-
head ribozyme.14,15 This molecule catalyses the sequence specific
hydrolysis of a phosphodiester linkage of RNA targets containing
the UX motif (X = C, A or U). Taking into account these antecedents
we decided to explore the preparation of 20-C-methyl phosphorami-
dite by alkaline degradation of fructose, using an improved lactone
reduction procedure and a more acid-stable protective group at
the tertiary 20-position. The modified phosphoramidite was intro-
duced in the catalytic core of a hammerhead ribozyme directed
(20R)-20-C-methylribonucleosides are attractive candidates to be
used in modified RNA structures, because they showed increased
nuclease resistance, when incorporated to RNA sequences;6 and
can adopt C-30endo (RNA-like) conformation with anti orientation
of the base.7 An important attribute of 20-C-methylribonucleosides
is that they posses the 20-hydroxyl. This functional group could be
essential to mimic RNA polar interactions and hydrogen bonding
of complex RNA tertiary structures. 20-C-methyl nucleosides have
been synthesized using two main strategies: (i) construction of the
modified sugar moiety and posterior glycosidation reaction, and
(ii) modification of the ribose portion of a natural ribonucleoside. Re-
lated to the first approach, glucose,8 fructose9 or ribose10 have been
employed as starting materials making use of regioselective depro-
tection at position 211 or of alkaline sugar degradation.12
against the ER-a
16 mRNA, which expression was measured by wes-
tern blot in cancer MCF-7 cells.
In an attempt to improve 20-C-methyl uridine preparation, by the
construction of the 2-modified sugar moiety first, 2-C-methylribon-
* Corresponding author. Tel.: +54 11 4783 2871; fax: +54 11 4786 8578.
E-mail address: airibarren@unq.edu.ar (A.M. Iribarren).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.060

R. Pontiggia et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2806–2808
2807
Figure 1. Synthesis of 20-C-methylphosphoramidite (14) from fructose.
o-
c
-lactone (1, Fig. 1) was prepared, starting from fructose and using
yield. Then, the 50-position was protected using DMT-Cl to give
13 in 72% yield. Finally, the phosphoramidite 14 (Fig. 1) was pre-
pared, by reaction of 13 with b-cyanoethoxy-N,N-aminochloro-
phosphine in a mixture of THF, diisopropylethylamine and CH2Cl2
under reflux, in 83% yield.
an alkaline degradation with calcium hydroxide. In our experience
the key step of this procedure is the exhaustive elimination of
remaining calcium ions by ion exchange chromatography (see
Supplementary data) and a percolation on silica gel, previous to
crystallization. In spite the low yield of this step (11%), the low cost
starting materials (food grade fructose, calcium hydroxide and
water) justifies the procedure. Positions 2- and 3- were then
protected using 2,2-dimethoxypropane to yield 2,3-di-O-isopropyl-
In order to test the enhanced acid stability of the 20-protection,
a comparative acid hydrolysis of 20-O-tetrahydropyranyl-20-C-
methyluridine (15) and compound 12 was performed using 2.5%
dichloroacetic acid in CH2Cl2.7 The remaining protected nucleoside
vs reaction time is depicted in Figure 2. As can be seen, (2-chloro-
ethoxy)ethyl protection is more stable (half life ca. 50 min) than
THP stable (half life ca. 7 min).
As a proof of concept and taking into account previous re-
sults15,6,14, we decided to synthesize a modified hammerhead ribo-
zyme carrying 20-C-methyluridine in position 7- of the catalytic
iden-2-C-methylribo-c-lactone (2, Fig. 1). This product was then
benzylated in 85% yield, to give 3 (Fig. 1). This compound was
reduced using 1.7 equivalents of DIBAL-H at ꢀ70 °C, obtaining 5-
O-benzyl-2,3-O-isopropyliden-2-C-methyl-
a,b-D
-ribose (4, Fig. 1),
practically as an equimolar anomeric mixture, in 84% yield.
Subsequently, 2,3-protection was removed using an acid resin
(DOWEX-50AG). Compound 5 was then perbenzoylated in 69%
core. In order to test its applicability we selected the ER-
a mRNA
yield, to obtain 5-O-benzyl-1,2,3-tri-O-benzoyl-2-C-methyl-
a
,b-
D
-
for the experiments in cell culture, motivated by its critical role
in the proliferation of certain estrogen-dependent mammary
breast tumors.17
ribofuranose (6, Fig. 1). This intermediate was stereoselectively
glycosylated under Vörbruggen conditions10 with uracil to give
50-O-benzyl-20,30-O-dibenzoyl-20-C-methyluridine (7, Fig. 1) in 70%
yield. The nucleoside was hydrogenated to remove the 50-protec-
tion, using Pd(OH)2/C as catalyst, practically in quantitative yield
to afford 8 (Fig. 1), which was debenzoylated by NH3(g) in ethanol,
to give 20-C-methyluridine (9, Fig. 1) in 75% yield.
For this purpose, a modified ribozyme designed to cleave the
956 nt of the mRNA of ER-
a was prepared. In order to increase
the ribozyme lifetime, the sequence was modified using 20-O-
methylnucleosides (blue capital letters captions, Fig. 3), two phos-
phorothiates at 30 and 50-ends (blue lower case, Fig. 3), the (20R)-20-
C-methyluridine (red capital letters caption, Fig. 3) and keeping
natural ribonucleotides (black captions, Fig. 3) at positions where
The next step consisted in the preparation of the corresponding
phosphoramidite. For this purpose, 30 and 50 positions were regio-
selectively acetylated. This alternative was preferred, because tra-
ditional Marckiewicz protection of positions 30 and 50, generates a
steric hindrance that hampers the access to the 20-tertiary alco-
hol.13 In this way 30,50-di-O-acetyl-20-C-methyluridine (10, Fig. 1)
was prepared from 9 in 90% yield. Then, different protective
groups, orthogonal to 50-DMT protection, were tested. Classical
tert-butyl-dimethylsilyl (TBDM) ether was attempted obtaining
negative results using either the chloride or the triflate sililating
agents. Then, (triisopropylsilyl)oxymethyl chloride (TOM-Cl),17
was tested. TOM-Cl is known to have less steric hindrance. In order
to introduce this protective group different bases (DBU, BDDDP,
BuLi) and reaction conditions (different solvents and tempera-
tures) were unsuccessfully assessed. Finally, 2-chloroethoxyethyl
ether was tested, carrying out the reaction in the absence of sol-
vent using an excess of 33 equiv of 2-chloroethylvinyl ether and
pyridonium p-toluensulfonate as catalyst. In this way product 11
(Fig. 1) was obtained in 74% yield. The next step consisted in the
removal of the acetyl groups under basic conditions to give 20-O-
(1-(2-choroethoxy)ethyl)-20-C-methyluridine (12, Fig. 1) in 83%
Figure 2. Relative acid stability of (12) and (15).

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R. Pontiggia et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2806–2808
Figure 3. Hammerhead ribozyme sequence and western blot results.
20-hydroxyl is important for activity (Ract, Fig. 3). An inactive ribo-
zyme (Rinact, Fig. 3) was prepared by deletion of a G from the cata-
lytic core (Fig. 3). This oligonucleotide has no catalytic activity but
maintains its antisense function.
These sequences were used to transfect MCF-7 cells using cat-
ionic liposomes (Oligofectamine, Sigma), incubating the cells for
Supplementary data
Supplementary data (detailed description of the experimental
procedures and NMR assignments) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2010.03.060.
24 and 36 h. ER-a protein expression was verified at these times
References and notes
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This work was financially supported by ANPCyT (PICT
05:38348) and CONICET (PIP 5462). R.P. thanks Boehringer Ingel-
heim Fonds for a fellowship. J.M.M. and A.M.I. are CONICET
members.