Solid-phase synthesis of oligodeoxynucleotides containing 3′-S-phosphorothiolate linkages

Article abstract of DOI:10.1081/NCN-100002553

For the first time a fully automated procedure has been developed for the incorporation of a 3′-S-phosphorothiolate linkage into DNA, using phosphorothioamidite monomers. Coupling yields with either of the activators 5-ethylthiotetrazole or 4,5-dicyanoimi

Full text of DOI:10.1081/NCN-100002553

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SOLID-PHASE SYNTHESIS OF
OLIGODEOXYNUCLEOTIDES CONTAINING 3′-S-
PHOSPHOROTHIOLATE LINKAGES
Kevin J. Fettes a , Ian O'Neil a , Stanley M. Roberts a & Richard Cosstick b
a Department of Chemistry , University of Liverpool , Crown Street, Liverpool, L69 7ZD,
United Kingdom
b Department of Chemistry , University of Liverpool , Crown Street, Liverpool, L69 7ZD,
United Kingdom
Published online: 07 Feb 2007.
To cite this article: Kevin J. Fettes , Ian O'Neil , Stanley M. Roberts & Richard Cosstick (2001) SOLID-PHASE SYNTHESIS OF
OLIGODEOXYNUCLEOTIDES CONTAINING 3′-S-PHOSPHOROTHIOLATE LINKAGES, Nucleosides, Nucleotides and Nucleic Acids,
20:4-7, 1351-1354
To link to this article: http://dx.doi.org/10.1081/NCN-100002553
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1351–1354 (2001)
SOLID-PHASE SYNTHESIS OF
OLIGODEOXYNUCLEOTIDES CONTAINING
3ꢀ-S-PHOSPHOROTHIOLATE LINKAGES
Kevin J. Fettes, Ian O’Neil, Stanley M. Roberts,
and Richard Cosstick∗
Department of Chemistry, University of Liverpool,
Crown Street, Liverpool L69 7ZD, United Kingdom
ABSTRACT
For the first time a fully automated procedure has been developed for the in-
corporation of a 3ꢀ-S-phosphorothiolate linkage into DNA, using phosphoroth-
ioamidite monomers. Coupling yields with either of the activators 5-ethylthio-
tetrazole or 4,5-dicyanoimidazole were in the range of 80–90%. Coupling yields
were equally good when performed on either a 0.2 or 1 µmole reaction column,
thus facilitating large scale synthesis.
In recent years DNA and RNA analogues have been extensively investigated
as potential chemotherapeutic agents (1) and have become essential tools for prob-
ing structural and mechanistic aspects of nucleic acid biochemistry (2). The 3ꢀ-S-
phosphorothiolate linkage (Fig. 1), in which the 3ꢀ-oxygen is replaced by sulfur, has
attracted increasing attention and been constructed using a variety of approaches
including phosphoramidite chemistry (3–6) and methods based on a Michaelis-
Arbusovreaction(7–10)VeryrecentlyNMRstudiesonthephosphorothiolatedimer
TspT have shown that 3ꢀ-sulfur substitution results in a sugar conformation that is
predominately 3ꢀ-C-endo making these analogues interesting as potential antisense
agents (11).
∗Corresponding author. E-mail: rcosstic@liverpool.ac.uk
1351
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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FETTES ET AL.
Figure 1.
Despite the increasingly wide spread use of oligonucleotides containing a sin-
gle 3ꢀ-S-phosphorothiolate linkage, there is no report of a fully automated synthesis
protocol for incorporation of these analogues. We now wish to report signifi-
cant developments in the synthesis of oligodeoxynucleotides containing a 3ꢀ-S-
phosphorothiolate linkage including: a shortened procedure for the synthesis of
phosphorothioamidite derived from 3ꢀ-thiothymidine (4) and a fully automated
synthesis protocol for incorporation of this thioamidite based on commercially
available activators and reagents.
Synthesis of 3ꢀ-thiothiothymidine. The previously reported synthesis of
2ꢀ-deoxy-3ꢀ-thiothymidine (3,4) (3) started from the xylo–configured nucleoside
involving displacement of the sulfonate ester with sodium thiobenzoate. We now
report a more immediate route to 2ꢀ-deoxy-3ꢀ-thiothymidine through direct ring-
opening of a 2,3ꢀ-anhydrothymidine with thiobenzoate. Synthesis started from
DMT-protected thymidine; this is in contrast to the originally reported work on
3ꢀ-thiothymidine that used the more robust MMT group (4). Thus, DMT-protected
2,3ꢀ-anhydrothymidine (1) was converted through to the corresponding 3ꢀ-thiothy-
midine (3) as shown in Figure 2. The thionucleoside was converted to its phospho-
rothioamidite, (4) as previously described (4).
Oligonucleotide synthesis. In formerly described procedures for the syn-
thesis of oligonucleotides containing 3ꢀ-S-phosphorothiolate linkages the key
phosphorothioamidite coupling step has been performed by removing the reac-
tion column from the synthesizer and manually introducing the thioamidite and
activator [5-(p-nitrophenyl)tetrazole] by syringe (6,12). This manual coupling pro-
cedure made the rigorous exclusion of moisture difficult and the result was a time
consuming and intricate operation that also gave inconsistent yields. Thus, there
was an obvious requirement for a fully automated and relatively simple procedure
that would make these analogues more accessible to the nucleic acid community.
Original studies had shown that phosphorothioamidites are much less reac-
tive than the standard amidites, presumably due to stabilization of the protonated
phosphorus tautomer by sulfur, and that 5-(p-nitrophenyl)tetrazole was a supe-
rior to tetrazole for coupling phosphorothioamidites (4). Unfortunately however,

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SOLID-PHASE SYNTHESIS OF OLIGODEOXYNUCLEOTIDES
1353
Figure 2. Reagents and Conditions i) CsBz, DMF, 110◦C, 82%; ii) NaOH, EtOH, H2O, 83%;
iii) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, 1-methylimidazole, diisopropylethyla-
mine, CH2Cl2, 87%.
5-(p-nitrophenyl)tetrazole is poorly soluble in acetonitrile and not ideally suited
to automated synthesis. The activators 5-ethylthiotetrazole (13) (SET) and 4,5-
dicyanoimidazole(14)(DCI)havebeenfoundtobemorepotentactivators, although
DCI is preferred due its reduced acidity.
To assess these commercially available activators and optimize the synthesis
cycle two test sequences (5ꢀ-TTT TTsT TTT T and 5ꢀ-CCT AAA TTsT GCC, where
ꢀ
=
Ts 3 -thiothymidine) which contain a single phosphorothiolate residue were syn-
thesized. When coupling times, activator and thioamidite concentrations were used
at standard values coupling yields were disappointing. Under these conditions tetra-
zole failed to catalyze any incorporation of the thioamidite, as determined by the
trityl release assay; whereas DCI and SET gave coupling yields that were detectable,
but less than 10%. Using the activators at a concentration of 1M and with extended
coupling times, good yields were obtained for the introduction of the thioamidite.
The activators SET and DCI were essentially equally effective both giving
coupling yields of 80–90% with a 15 min coupling. Coupling yields were also
equally good when performed on either a 0.2 or 1 µmole reaction column.
Notable in the synthesis protocol is the use of the standard iodine oxidation
solution. There was some apprehension with regard to the use of this oxidant as
–
aqueous iodine solutions have been shown to cleave the P S bond in a phospho-
rothiolate diester (4). However, the trityl release assay (not shown) reveals that the
coupling yields are consistently maintained subsequent to the introduction of the
phosphorothiolate linkage and suggests that the iodine oxidant is not detrimental
to the phosphorothiolate triester to any significant extent.
Oligonucleotides were deprotected using standard conditions, isolated by
reverse-phase HPLC and characterised by electrospray mass spectrometry (15).

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FETTES ET AL.
Experimental. All oligonucleotides were synthesised on an ExpediteTM
8909 DNA synthesiser using standard protocols for the phosphoramidite-based
coupling approach. The standard amidites were used at the recommended concen-
tration (50 mg/mL), whilst the thymidine-3ꢀ-thioamidite was used at 100 mg/mL.
Optimal coupling conditions were achieved using one of the activators SET or DCI
at 1M concentration with coupling time of 15 min.
ACKNOWLEDGMENTS
We would like to thank the EPSRC for generous support to KJF and are
grateful to the EPSRC service at Swansea for obtaining mass spectra.
REFERENCES
1. Crooke, S.T.; Lebleu, B. Antisense Research and Applications. CRC Press, Boca
Raton, Fl, USA, 1993.
2. Verma, S.F.; Eckstein, F. Annu. Rev. Biochem., 1998, 67, 99.
3. Cosstick, R.; Vyle, J.S. Tetrahedron Lett., 1989, 30, 4693.
4. Cosstick, R.; Vyle, J.S. Nucleic Acids Res., 1990, 18, 829.
5. Sun, S.G.; Piccirilli, J.A. Nucleosdes Nucleotides, 1997, 16, 1543.
6. Sun, S.G.; Yoshida, A.; Piccirilli, J.A. RNA–A Publication of the RNA Society, 1997,
3, 1352.
7. Vyle, J.S.; Li X.; Cosstick, R. Tetrahedron Lett., 1992, 33, 3017.
8. Li, X.; Scott, G.K.; Baxter, A.D.; Taylor, R.J.; Vyle, J.S.; Cosstick, R. J. Chem. Soc.
Perkin Trans. 1, 1994, 2123.
9. Liu, X.; Reese, C.B. Tetrahedron Lett., 1996, 37, 925.
10. Weinstein, L.B.; Earnshaw, D.J.; Cosstick, R.; Cech, T.R. J. Am. Chem. Soc., 1996,
118, 10341.
11. Beevers, A.P.G.; Witch, E.; Jones, B.C.N.M.; Cosstick, R.; Arnold, J.R.P.; Fisher,
J. Magn. Reson. Chem., 1999, 37, 814.
12. Vyle, J.S.; Kemp, D.; Cosstick, R.; Connolly, B.A. Biochemistry, 1992, 31, 3012.
13. Wincott, F.; Di Renzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.; Workman, C.; Sweedler,
D.; Gonzalez, C.; Scaringe, S; Usman, N. Nucleic Acids Res., 1995, 23, 2677.
14. Vargesse, C.; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.; Kropp, E.; Paterson, K.;
Pieken, W. Nucleic Acids Res., 1998, 26, 1046.
15.
Sequence
Calculated mass Measured mass
5ꢀ-TTT TTsT TTT T
2996.0
3596.4
2995.9 0.3
3597.1 0.7
5ꢀ-CCT AAA TTsT GCC

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