Silyl protecting groups for oligonucleotide synthesis removed by a ZnBr2 treatment

Article abstract of DOI:10.1081/NCN-200060342

An oligonucleotide protected with N-(trimethylsilyloxycarbonyl) (Teoc) and P-(trimethylsilylethanol) (Tse) groups was synthesized and deprotected by a single ZnBr₂ treatment. Finally it was released from the solid support by cleavage of a disul

Full text of DOI:10.1081/NCN-200060342

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SILYL PROTECTING GROUPS FOR OLIGONUCLEOTIDE
SYNTHESIS REMOVED BY A ZnBr2 TREATMENT
Fernando Ferreira a & François Morvan a
a Laboratoire de Chimie Organique Biomoléculaire de Synthèse , UMR 5625 CNRS—UM II,
Université de Montpellier II , Montpellier Cedex 5, France
Published online: 15 Nov 2011.
To cite this article: Fernando Ferreira & François Morvan (2005) SILYL PROTECTING GROUPS FOR OLIGONUCLEOTIDE
SYNTHESIS REMOVED BY A ZnBr2 TREATMENT, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 1009-1013, DOI: 10.1081/
NCN-200060342
To link to this article: http://dx.doi.org/10.1081/NCN-200060342
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5--7):1009–1013, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060342
SILYL PROTECTING GROUPS FOR OLIGONUCLEOTIDE SYNTHESIS
REMOVED BY A ZnBr2 TREATMENT
5
Fernando Ferreira and Fran¸cois Morvan
Laboratoire de Chimie Organique
Biomole´culaire de Synth`ese, UMR 5625 CNRS-----UM II, Universite´ de Montpellier II,
Montpellier Cedex 5, France
5
An oligonucleotide protected with N-(trimethylsilyloxycarbonyl) (Teoc) and P-(trimethylsilylethanol)
(Tse) groups was synthesized and deprotected by a single ZnBr2 treatment. Finally it was released from
the solid support by cleavage of a disulfide linkage with TCEP. The oligonucleotide was obtained
without any basic treatment.
INTRODUCTION
Protecting groups for the amino functions of nucleobase have been extensively
developed. Protecting groups able to be removed under mild or neutral conditions
are of great interest for the synthesis of base-sensitive oligonucleotides.
Here we present a strategy to synthesize oligonucleotides without any basic
treatment by using the trimethylsilylethoxycarbonyl (Teoc) group for the protection
of nucleobases and of the trimethylsilylethyl (Tse) group[1,2] for the protection of the
internucleotidic linkages.
RESULTS AND DISCUSSION
Deoxycytidine was efficiently protected using a transient 3’ and 5’-OH
protection with the trimethylsilyl group and then with 4-nitrophenyl-2-(trimethylsilyl)
ethylcarbonate[3] in presence of DMAP as catalyst. After 16 h, a 20 min treatment
with ammonia yielded the expected N4-Teoc-dC 3 (80%), (Scheme 1). Finally, it was
5’-dimethoxytritylated 10a (85%).
For the protection of deoxyadenosine and deoxyguanosine, the more reactive
2-(trimethylsilyl)ethyl-carbonochloridate (TeocCl)[4] must be used. Introduction of
Address correspondence to Fran¸cois Morvan, Laboratoire de Chimie Organique Biomol´eculaire de
Synth`ese, UMR 5625 CNRS—UM II, Universit´e de Montpellier II, Place E. Bataillon, 34095 Montpellier Cedex 5,
France; Fax: +33-467-042-029; E-mail: morvan@univ-montp2.fr
1009
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F. Ferreira and F. Morvan
SCHEME 1 Synthesis of 5’-O-dimethoxytrityl-N-Teoc nucleosides.
the Teoc group was performed starting from 3’,5’-di-O-acetyl dA and dG.[5] The N-6
of adenine was protected by reaction with TeocCl in presence of N-methyl
imidazole in dichloromethane for 16 h (95%) (Scheme 1). Then a 10-min treatment
with 0.2 N NaOH in THF/MeOH/H2O (25:15:10,v/v/v) led to the expected N6-
Teoc-dA. After work-up, the crude product was directly dimethoxytritylated 10b
(80%). The O-6 of guanine was protected with Tse group in two steps. First 2,4,6-
triisopropylbenzenesulfonyl chloride in presence of DMAP and TEA reacted on
the O-6, then the trimethylsilylethanol in presence of DABCO displaced this leaving
group to yield the O6-Tse-dG 8 (60%). Alternatively it was synthesized by a
Mitsonobu reaction using triphenylphosphine, diethyl azodicarboxylate and
trimethylsilylethanol in dioxane with 48% yield. The Teoc group was introduced
on N-2 by treatment with TeocCl in presence of tert-butyl magnesium chloride in
THF for 16 h (70%). Acetyl groups were removed by a solution of 0.2 N NaOH in
THF/MeOH/H2O for 10 min. After work up, the crude product was directly
dimethoxytritylated 10c (75%).
The conditions of removal of Teoc group were studied at the nucleoside level.
Treatment with various fluoride reagents (HF.pyridine, Et3N.3HF, TBAF-AcOH,
and TBAF), in THF, led to no or incomplete deprotection. In contrast, treatment
with ZnBr2 and ZnCl2 gave rapid deprotection. The full deprotection was faster
with ZnBr2 (30 to 60 min) than with ZnCl2 (about 2 h). The order of silyl remov-
al was dC > dA > dG. Note that Tse group on O-6 of dG was removed by the same
way. As a DNA synthesis cycle involves an acidic treatment, we studied the stability
of N6-Bz-dA and 3’,5’-di-O-Ac-N6-Teoc-dA in acidic conditions. While the former

F. Ferreira and F. Morvan
1011
SCHEME 2 Synthesis of Tse phosphoramidites derivatives.
was fully depurinated in 80% acetic acid at 20°C within 1 h the latter was fully stable
up to 2 h. Furthermore 3’,5’-di-O-Ac-N6-Teoc-dA was found stable in 3% TCA or
2.5% DCA in CH2Cl2. Hence, the standard detritylation solution could be used
on synthesizer.
Then, the 5’-O-Dmtr-N-protected nucleosides 10a--d were converted into Tse
phosphoramidite derivatives (Scheme 2) in the presence of diisopropyl ammonium
tetrazolide as catalyst in CH2Cl2 using Tse bis-N,N’-diisopropyl phosphine:[1] 90%
for dC 11a; 80% for dA 11b; 85% for dG 11c, and 90% for dT 11d.
An oligonucleotide GCATTAGCATpOCH3 was synthesized from the Tse
phosphoramidites on a solid support with a disulfide linkage. The phosphoramidite
building blocks were used at a standard 0.1 M concentration in dry acetonitrile with
a 120-s coupling step. Since acetic anhydride usually used for the capping step
could also react on the exocyclic amino function especially of adenine, we capped
with di-tert-butyl diethyl phosphoramidite (0.05M in CH3CN) for 10 s. Oxidation
was performed with a 0.067% 2-butanoneperoxide solution in CH2Cl2 for 60 s[6] and
detritylation with standard 3% TCA solution for 60 s. In order, to be released from
the solid support without ammonia treatment we used a solid support with a
disulfide linkage[7] that can be cleaved by tris-2-carboxyethylphosphine (TCEP).
SCHEME 3 Deprotection of oligo GCATTAGCATpOCH3 by ZnBr2 and release from solid support by TCEP.

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F. Ferreira and F. Morvan
FIGURE 1 HPLC profiles of the crude and pure oligonucleotide and MALDI-TOF MS.
After elongation, the CPG-supported oligonucleotide was treated overnight
with a saturated solution of ZnBr2 in nitromethane/isopropanol (1:1, v/v) (Scheme 3).
Then the beads were thoroughly washed with water and with a 0.1M EDTA
solution to scavenge the Zn2+ cations. Finally, the oligo was cleaved from the solid
support by treatment with TCEP in triethylammonium acetate buffer pH 7 with 80%
acetonitrile for 2 h. This treatment led to a 3’-phosphotriester with a thio-ethyl group
that rearranged spontaneously to a 3’-phosphodiester after elimination of episulfide.
As that elimination is very slow with a diester due to the negative charge, we started
the synthesis using a methoxyphosphoramidite to obtain after ZnBr2 treatment a
3’-methyl triester linkage that rearranged the expected 3’-methyl phosphodiesters.
The crude HPLC showed a peak (Figure 1, left) with other peaks at higher reten-
tion times corresponding to the short of mers 5’-di-tert-butylphosphotriester as
determined by MALDI-TOF MS. After purification, the pure decamer was
obtained (Figure 1 middle) and characterized by MALDI-TOF MS (negative mode
m/z for C99H126N37O61P10 calc. 3120.07, found 3121.34) (Figure 1, right).
CONCLUSION
Using silyl protecting groups on the nucleobases (Teoc) and on the phosphate
(Tse), we synthesized an oligonucleotide and deprotected it by a single treatment
with a ZnBr2 solution. This strategy could be applied to the synthesis of base-
sensitive oligonucleotides.
REFERENCES
1. Wada, T.; Sekine, M. 2-(Trimethylsilyl)ethyl as a phosphate group in oligonucleotide synthesis. Tetrahedron
Lett. 1994, 35, 757–760.
2. Guerlavais Dagland, T.; Meyer, A.; Imbach, J.L.; Morvan, F. Fluoride-labile protecting groups for the synthesis
of base-sensitive methyl-SATE oligonucleotide prodrugs. Eur. J. Org. Chem. 2003, 2327–2335.
3. Dhanak, D.; Reese, C.B. Studies in the protection of pyrrole and indole derivatives. J. Chem. Soc., Perkin
Trans. 1986, 1, 2181–2186.
4. Shute, R.E.; Rich, D.H. Synthesis and evaluation of novel activated mixed carbonate reagents for the
introduction of the 2-(trimethylsilyl)ethoxycarbonyl(Teoc)-protecting group. Synthesis 1987, 346–349.
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enzymatisch abspaltbaren schutzgruppen versehenen nukleosiden und nukleosid-derivaten in der synthese von
oligonukleotiden. In EP 0 649 855 A1, 1994.

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1013
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efficient reagent for the oxidation of nucleoside phosphites into phosphates under nonbasic anhydrous
conditions. Org. Lett. 2001, 3, 815–818.
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Oligonucleotides and Analogues. A Practical Approach; Oxford University Press: Oxford, 1991; 283–308.