ties of the modified U6 were evaluated in human serum. In
all these media, the four U6 19-22 exhibited a high resistance
against enzymatic phosphodiester hydrolysis compared to the
parent hexuridylate degraded within 30 min. The stabilities
of 19 and 21 with acetalesters were greater than those of 20
and 22 with thioacetalesters considering the same ester
group: acetyl or pivaloyl. Furthermore, the two U6 bearing
pivaloyl esters 21 and 22 were more stable against nuclease
hydrolysis than the two U6 with acetyl esters 19 and 20. In
all these media, the less stable RNA was the hexauridylate
20 bearing acetylthioester groups. So the 2′-modifications
enhance the lifetime of the RNA by protection against
nuclease hydrolysis.
that was enhanced when the salt ionic strength was 1 M NaCl
(entry 2). Among the four 2′-O-protected U12 tested, only
the uridylates bearing the acetyloxymethyl or pivaloyloxy-
methyl groups were able to form a stable duplex with
C2A12C2 RNA.
To be effective for gene silencing, the RNA duplex must
retain conformationally RNA-like A-type helical character-
istics. We try to explain the differences in duplex stability
between the different RNA analogues studying the sugar
puckering modes of the new modified ribonucleosides in D2O
1
by H NMR. The percentage of the C3′-endo form (% N)
was determined for each ribonucleoside and compared with
this of the uridine (% N 52).18 It was found that the % N
values of the 2′-O-acetyloxymethyl (% N 53) and 2′-O-
acetylthiomethyl (% N 53) uridine were similar to that of
uridine but in the 2′-O-pivaloyloxymethyl (% N 47) or
pivaloylthiomethyl (% N 40) uridine, the C3′-endo form was
decreased to a degree of 5% and 12% respectively. Sugar
puckering modes of the ribonucleosides are not sufficient to
explain the thermal stability of the modified duplexes since
PivOM-U12 25 formed the more stable duplex and AcSM-
U12 24 the less stable one. As the substitution of the 2′-O-
acyloxymethyl for 2′-acylthiomethyl groups had an adverse
effect on the binding affinity, the steric hindrance of the
sulfur atom larger than the oxygen atom may play an
essential role in the destabilization of the RNA duplex.
In conclusion, we have synthesized novel 2′-O-modified
oligouridylates with four different biolabile 2′-protections.
Within the context of using RNAs in vivo, the 2′-O-acyl-
(Ac or Piv)oxymethyl uridylates successfully fulfill the
criteria of nuclease resistance, demasking upon esterase
activation in intracellular medium and good affinity for RNA
target to form a stable dsRNA. A potentiel advantage of our
biolabile protecting groups approach is that the 2′-modifica-
tions are removed into the cells to release the functional RNA
molecule. These properties make this acetalester modification
a promising candidate for further evaluation for RNAi and
such efforts are in progress since we are currently preparing
the 2′-protected ribonucleotides containing the nucleobases
A, C, and G for RNA synthesis.
Finally, since our strategy involves an intracellular car-
boxyesterase activation, we studied the fate of the modified
U6 in total cell extract from CEM-SS cells used as mimic
for the intracellular medium. Their metabolization was
monitored by MALDI-TOF MS. The data confirmed that
the acetalester groups in 2′ provide a relative RNA stability
against nuclease hydrolysis inside the cells compared to the
parent RNA, completely degraded in 30 min. Moreover, this
study showed that the 2′-O-protecting groups were efficiently
demasked by cellular carboxyesterases (Table 2). We found
that the pivaloyl groups slowed the demasking process
compared to the acetyl groups. In conclusion of these stability
studies, we established that the acetylthiomethyl group was
not a good candidate for biolabile 2′-protection of RNA
because of its low stability in human serum. Indeed, a relative
stability in extracellular medium associated with a rapid
intracellular demasking are key factors for compounds
designed to selectively release inside the cells the parent
RNA through enzymatic activation.17
The next step was to examine the ability of these RNA
analogues to efficiently hybridize to their complementary
RNA sequence keeping in mind that dsRNA are the active
molecules in several biological phenomena, especially in the
RNAi mechanism. The four dodecauridylates 23-26 were
hybridized to RNA target C2A12C2 and the corresponding
Tm values for the melting of the duplex were determined by
standard UV/melting-curve techniques (Table 1). The melting
curves of U12 24 and 26 bearing thioacetalesters (AcSM and
PivSM) showed that no duplex transition could be detected
in 0.1 M NaCl (entry 1) and PivSM-U12 26 only formed a
duplex in 1 M NaCl (entry 2) with a very low stability (Tm
< 12 °C). In contrast, in the case of U12 with acetalester 23
and 25, the stability of the duplex with RNA target was much
higher and PivOM-U12 25 formed a more stable duplex (Tm
22 °C at 0.1 M NaCl) than the parent duplex (Tm 16 °C at
0.1 M NaCl). Although the Tm value (12.7 °C) of the duplex
AcOM 23-U12/RNA target was lower than the Tm of the
parent duplex, the melting curve indicates a duplex formation
Acknowledgment. The authors thank Dr. Isabelle Lefe-
bvre for the gift of the CEM-SS cell extract and for advice
on metabolism studies in various biological media.
Supporting Information Available: Experimental pro-
cedures, spectral data for compounds, synthesis of oligo-
nucleotides, enzymatic studies, and hybridization experi-
ments. This material is available free of charge via the
OL0616182
(17) Pe´rigaud, C.; Gosselin, G.; Imbach, J.-L. Curr. Top. Med. Chem.
1997, 2, 15-29.
(18) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973, 95, 2333-
2344.
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Org. Lett., Vol. 8, No. 17, 2006