Preparation of 6-thioguanosine phosphoramidite for oligoribonucleotide synthesis

Article abstract of DOI:10.1081/NCN-120022847

6-Thioguanosine phosphoramidite was prepared, using 2,4-dinitrophenyl as protection group for thio-function, and its stability towards conditions of RNA synthesis was investigated. The results show that the monomer was stable under the conditions of RNA synthesis and suitable for incorporation of thioguanine into oligoribonucleotides.

Full text of DOI:10.1081/NCN-120022847

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Preparation of 6-Thioguanosine Phosphoramidite for
Oligoribonucleotide Synthesis
Yang Wang a , Eric Lattmann a & Qinguo Zheng a b
a School of Life and Health Sciences , Aston University , Birmingham, England
b School of Life and Health Sciences , Aston University , Aston Triangle, Birmingham,
England , B4 7ET
Published online: 31 Aug 2006.
To cite this article: Yang Wang , Eric Lattmann & Qinguo Zheng (2003) Preparation of 6-Thioguanosine Phosphoramidite for
Oligoribonucleotide Synthesis, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1247-1249, DOI: 10.1081/NCN-120022847
To link to this article: http://dx.doi.org/10.1081/NCN-120022847
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1247–1249, 2003
Preparation of 6-Thioguanosine Phosphoramidite for
Oligoribonucleotide Synthesis
*
Yang Wang, Eric Lattmann, and Qinguo Zheng
School of Life and Health Sciences, Aston University,
Birmingham, England
ABSTRACT
6-Thioguanosine phosphoramidite was prepared, using 2,4-dinitrophenyl as pro-
tection group for thio-function, and its stability towards conditions of RNA
synthesis was investigated. The results show that the monomer was stable under
the conditions of RNA synthesis and suitable for incorporation of thioguanine
into oligoribonucleotides.
Key Words: 6-Thioguanosine; RNA Synthesis; Modified RNA; Modified base.
Oligonucleotides containing thio-substituted base are useful for various pur-
poses in different areas, such as molecular biology and cancer study.[1,2] In concert
with the goal of developing methods for chemical synthesis of oligonucleotides con-
taining thiobases, we[3–5] and others[6,7] have established methods by which various
thiobases are successfully incorporated into DNA and RNA. Here we describe
synthesis of 6-thioguanosine phosphoramidite suitable for synthesis of oligoribonu-
cleotides containing thioguanine.
*Correspondence: Qinguo Zheng, School of Life and Health Sciences, Aston University,
Aston Triangle, Birmingham, B4 7ET England; Fax: þ44 (0) 121 359 0733.
1247
DOI: 10.1081/NCN-120022847
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1248
Wang, Lattmann, and Zheng
Scheme 1. i, dinitrofluorobenzen=Et3N; ii, (CH3)3SiCl; iii, phenylacetyl chloride; iv, 30%
NH3H2O; V, MMTCl/pyridine; vi, t-butyldimetylsily chloride=AgNO3=pyridine; vii,
i-Pr2NP(Cl)OCH2CH2CN=i-Pr2NEt.
The synthetic route for 6-thioguanosine phosphoramidite monomer (6)
is shown in Scheme 1. Commercially available 6-thioguanosine (1) was reacted
with 2,4-dinitrofluorobenzene in the presence of triethylamine to give com-
pound 2 with isolated yields of 70–80%. The reaction was highly selective because
6-thio function is much more nucleophilic than 20-, 30- and 50- hydroxyl groups
and 2-amino group. Phenoxylacetyl group was chosen to protect 2-amino group
of the nucleoside because it can be removed under mild conditions. It was intro-
duced into the 2-amino group using transient protection scheme, in which 3
hydroxyl groups were first protected temporally with silyl groups that were
removed by treatment of 30% aqueous ammonia after phenoxyacetyl group had
been introduced. N-acylated compound 3 was tritylated with monomethoxyltrityl
chloride (MMT-Cl). Although there are three free hydroxyl groups, due to its
bulky structure, the MMT group was dominantly reacted with 50-OH group (1ꢀ
OH group) in regioselective fashion. Using standard methods, compound 4 was
treated with t-butyldimethylsilyl chroride, then phosphitilating reagent to give
the desired phosphoramidite monomer (6).
The stability of 6-(2,4-dinitrophenyl)thioguanine monomer (6) to the reagents
used in oligonucleotide synthesis was studied before the monomer was used for
RNA synthesis. Compound 3 was dissolved in 3% dichloroacetic acid=dichloro-
ethane (deblocking reagent); and compound 5 in acetic anhydride=lutidine=THF
(1 : 1 : 8) (capping reagent A), 4.4% N-methylimidazole in THF (capping reagent
B), and 0.1 M iodine in THF=pyridine=water (40 : 9 : 1) (oxidation reagent). By moni-
toring changes in these solutions, it was shown that compound 5 was stable towards

6-Thioguanosine Phosphoramidite
1249
the reagents used in oligomer assembly for at least 24 h at room temperature, and
compound 3 was stable towards 3% dichloroacetic acid in dichloroethane at room
temperature for at least 3 h. It was also found that the 2,4-dinitrophenyl group pro-
tecting the 6-thioketo function can be removed completely by 1 M mercaptoethanol
in CH3CN in the presence of triethylamine within 30 min. These results shown that
monomer 6 would be stable during the synthesis, the protecting group easily remo-
vable after synthesis. We are currently investigating its incorporation into oligomers
and use of the thio function as a platform for further post-synthetic modification of
the oligonucleotides.
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