Use of 2-(tert-butyldiphenylsilyloxymethyl) benzoyl as N-protecting group for the synthesis of prooligonucleotides.

Article abstract of DOI:10.1016/S0960-894X(01)00515-7

A short TCCT Me-SATE prooligonucleotide was successfully synthesized using 2-(tert-butyldiphenyloxymethyl) benzoyl protecting group, after its removal by means of trimethylsilyl chloride and water.

Full text of DOI:10.1016/S0960-894X(01)00515-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2813–2816
Use of 2-(tert-Butyldiphenylsilyloxymethyl) Benzoyl as
N-Protecting Group for the Synthesis of Prooligonucleotides
Typhaine Guerlavais, Albert Meyer, Jean-Louis Imbach and Francois Morvan*
´ ` ´
Laboratoire de Chimie Organique Biomoleculaire de Synthese, UMR 5625 CNRS-UM II, Universite de Montpellier II,
Place E. Bataillon, 34095 Montpellier Cedex 5, France
Received 5April 2001; revised 11 June 2001; accepted 25July 2001
Abstract—A short TCCT Me-SATE prooligonucleotide was successfully synthesized using 2-(tert-butyldiphenyloxymethyl) benzoyl
protecting group, after its removal by means of trimethylsilyl chloride and water. # 2001 Elsevier Science Ltd. All rights reserved.
Antisense oligonucleotides (oligos) are designed to
interact with mRNA targets to downregulate protein
expression. However, these polyanionic compounds
pass through the hydrophobic lipid cellular membrane
with only poor efficiency. To overcome that limitation
we applied a prodrug strategy to the oligos with the
synthesis of prooligonucleotides (prooligos).1 We have
shown that polythymidine prooligos are rapidly and
efficiently taken up by HeLa cells.2 Now, to evaluate the
efficiency of the prooligonucleotide approach to inhibit
gene expression it is necessary to synthesize prooligos
bearing the three other nucleobases (i.e., A, C, and G).
Since prooligos are highly sensitive to ammonia treat-
ment, we cannot use the standard acyl protecting groups
(i.e., benzoyl and isobutyryl) for the protection of the
exocyclic amine of the nucleosides. Several protecting
groups were evaluated for this purpose in our labora-
tory.3 Using photocleavable 2,20-bis(2-nitrophenyl)
ethoxycarbonyl protection, we were able to synthesize
the first prooligonucleotides constituted with the four
nucleobases.4 However, a photolabile protecting group
presents some drawbacks as we must photolyze at low
concentration to avoid any saturation because of the
absorption of the nucleobases. Hence the longer the
oligo, the harder the photolysis.
synthesis of prooligonucleotides. This acyl protecting
group was introduced previously for the synthesis of
RNAs which are sensitive to long ammonia treatment.5
The SiOMB group was removed by treatment with
TBAF which leads to the hydrolysis of the silyl ether
and then the free hydroxyl via an intramolecular
nucleophilic attack yields the deprotected nucleobase
and the phthalide. Such a treatment could not be used
with prooligos but silyl groups could be removed under
a wide type of mild conditions.
To evaluate the interest of this protection for the
synthesis of prooligos, we first introduced the SiOMB
on deoxycytidine, converted it as a phosphoramidite
building block, and then we synthesized a short Me-
SATE TCCT prooligo on a photolabile solid support.6
The deprotection was performed by means of a solu-
tion of trimethylsilyl chloride (TMSCl)/water in aceto-
nitrile.
The synthesis of the Me-SATE TCCT prooligo requires
the preparation of Me-SATE phosphoramidite building
blocks of thymidine7 and deoxycytidine. This latter was
synthesized as follows. 4-N-[2-(tert-Butyldiphenylsilyl-
oxymethyl)]benzoyl deoxycytidine was synthesized
according to the transient di-O-30,50-trimethylsilyl pro-
tection (Fig. 1).8 The deoxycytidine hydrochloride, dis-
solved in dry pyridine, was treated with 1 equiv of
triethylamine to recover the free amine. The 30- and 50-
hydroxyls were transitorily protected with trimethylsilyl
groups and the N-4 amine was treated with the 2-(tert-
butyldiphenylsilyloxymethyl) benzoyl chloride (1.1
equiv)9 to yield the fully protected deoxycytidine 1. The
We present here the use of 2-(tert-butyldiphenylsilyloxy-
methyl) benzoyl (SiOMB) as a protecting group for the
*Corresponding author. Tel.: +33-467-144-961; fax: +33-467-042-
029; e-mail: morvan@univ-montp2.fr
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00515-7

2814
T. Guerlavais et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2813–2816
Figure 1. Synthesis of deoxycytidine Me-SATE phosphoramidite bearing the SiOMB protecting group. (i) Et3N (1 equiv), dry pyridine; (ii) tri-
methylsilyl chloride (10 equiv); (iii) SiOMBCl (2.2 equiv); (iv) MeOH, H2O; (v) DmtrCl (1.2 equiv), dry pyridine; (vi) N,N,N0N0-tetraisopropyl-S-
acetyl-2-thioethyl phosphorodiamidite (1.2 equiv), diisopropylammonium tetrazolide (0.5equiv), dry methylene chloride.
trimethylsilyl groups were removed by treatment with
methanol/water to give 2 (overall yield 42%). The N-4-
SiOMB deoxycytidine was 50-dimethoxytritylated and
then converted into the phosphoramidite derivative by
reaction with N,N,N0N0-tetraisopropyl-S-acetyl-2-thio-
ethyl phosphorodiamidite activated with diisopropyl-
ammonium tetrazolide (0.5equiv) in freshly distilled
methylene chloride (4: 50%, 31P NMR 148.9 and 149.2
ppm).
amount of prooligo having lost one Me-SATE and we
believe that phenomenon mainly occurred during the
synthesis and not during the treatment with TMSCl/
water.
Finally, the prooligo was released from the solid
support by UV irradiation.7 The crude mixture was
analyzed by HPLC on a C18 reverse-phase column
(Fig. 3). The HPLC profile showed two main peaks
centered at 32.4 and 39.4 min corresponding to the
TpCpCpTp with three Me-SATE and four Me-SATE
groups, respectively.
The TCCT Me-SATE prooligo was synthesized on a
photolabile solid support according to the already pub-
lished procedure7 (average coupling 97% calculated
from the trityl assay).
Since the phosphate group is chiral diastereoisomers
are visible as multiple peaks for each prooligo. To
verify that point, we withdrew one drop of solution
from each peak (32.4, 38.6, 39.4 and 40.2 min)
during the HPLC purification and analyzed by MS
The removal of the SiOMB groups was performed
directly on solid support by treatment with a solution of
trimethylsilyl chloride/water/acetonitrile (5 mL/2.5 mL/
50 mL).10 This treatment leads to the in situ generation
of a small amount of HCl that hydrolyzes the tert-
butyldiphenylsilyl group and then the free hydroxyl
attacks the carbonyl to give the unprotected nucleobase.
The deprotection was directly monitored on the support
by MALDI-TOF mass spectrometry.11 Thus, after 30,
90 and 120 min, few beads were withdrawn, mixed with
matrix (2,4,6-trihydroxyacetophenone, THAP), spotted
on the target to cocrystallize and then analyzed. Spectra
showed that after 30 min only a small amount of pro-
oligo still bearing one SiOMB group was present (Fig.
2A). After 90 min, the prooligo was fully deprotected to
afford the Me-SATE TpCpCpTp prooligo still bearing
the Me-SATE groups on the phosphates (Fig. 2B). A
small peak corresponding to a loss of one Me-SATE
group (1510.20 Da) was also detected but extended
reaction time did not give much more release of Me-
SATE groups (Fig. 2C). We always observe a small
MALDI-TOF. The first peak had
a
mass of
1510.26 corresponding to that of TCCT prooligo
bearing 3 Me-SATE groups (calcd 1510.25, negative
mode). The three other peaks displayed the same
mass spectrum (1612.79, 1612.63 and 1612.53, respec-
tively) corresponding to the fully Me-SATE prooligo.
Finally, the pure prooligo was characterized by MS
MALDI-TOF (MS calcd 1612.40, found 1612.64,
negative ion).
Using SiOMB protecting group, we were able to syn-
thesize a short prooligo constituted with deoxycytidine.
This protection will soon be applied to the two other
nucleobases (i.e., adenine and guanine). Thus, we
believe that using tert-butyldiphenyl-silyloxymethyl
benzoyl group we could synthesize prooligos, con-
stituted with the four nucleobases, targeted against
specific genes.

T. Guerlavais et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2813–2816
2815
Figure 2. (Top) structure of Me-SATE TCCT prooligo; (bottom) monitoring of the deprotection by MALDI-TOF analysis directly on the solid
support bearing a photolabile linker. Treatment with TMSCl/H2O/acetonitrile: (A) 30 min; (B) 90 min; (C) 120 min.

2816
T. Guerlavais et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2813–2816
Figure 3. (Left) crude HPLC profile of the Me-SATE TCCT prooligo after deprotection; (right) MALDI-TOF spectra of Me-SATE TCCT prooligo
(negative mode) after purification by HPLC.
Acknowledgements
4. Alvarez, K.; Vasseur, J. J.; Beltran, T.; Imbach, J. L. J.
Org. Chem. 1999, 64, 6319.
5. Dreef-Tromp, C. M.; Van Dam, E. M. A.; Van den Elst,
H.; Van den Boogaart, J. E.; Van der Marel, G. A.; Van
Boom, J. H. Rec. Trav. Chim. Pays-Bas 1991, 110, 378.
6. Dell’Aquila, C.; Imbach, J. L.; Rayner, B. Tetrahedron
Lett. 1997, 38, 5289.
This work was supported by grant from ‘Association
pour la Recherche contre le Cancer’ (ARC).
7. Tosquellas, G.; Alvarez, K.; Dell’Aquila, C.; Morvan, F.;
Vasseur, J. J.; Imbach, J. L.; Rayner, B. Nucleic Acids Res.
1998, 26, 2069.
References and Notes
1. Morvan, F.; Vasseur, J.-J.; Vives, E.; Rayner, B.; Imbach,
J.-L. The Oligonucleotide Prodrug Approach: The Pro-oligo-
nucleotides. In Pharmaceutical Aspects of Oligonucleotides;
Couvreur, P., Malvy, C., Eds.; Taylor & Francis: London, 2000;
Chapter 3, p 79.
8. Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc.
1982, 104, 1316.
9. Dreef-Tromp, C. M.; Van Dam, E. M. A.; Van den Elst,
H.; Van der Marel, G. A.; Van Boom, J. H. Nucleic Acids Res.
1990, 18, 6491.
2. Vives, E.; Dell’Aquila, C.; Bologna, J. C.; Morvan, F.;
Rayner, B.; Imbach, J. L. Nucleic Acids Res. 1999, 27,
4071.
3. Alvarez, K.; Tworkowski, I.; Vasseur, J. J.; Imbach, J. L.;
Rayner, B. Nucleosides Nucleotides 1998, 17, 365.
10. Grieco, P. A.; Markworth, C. J. Tetrahedron Lett. 1999,
40, 665.
11. Meyer, A.; Spinelli, N.; Bres, J. C.; Dell’Aquilla, C.;
Morvan, F.; Lefebvre, I.; Rayner, B.; Imbach, J. L.; Vasseur,
J. J. Nucleosides, Nucleotides and Nucleic Acid 2001, 20, 963.