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Fig. 2. HPLC trace (254 nm) of: (A) crude PS-hexamer 17, (B) purified 17.
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Fig. 3. 31P NMR (80.7 MHz, CH3CN and H3PO4 as internal standard)
HO
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G ApsCpsGpsTpsTOH).
ps
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identical (HPLC, MS) with the same hexamer prepared via
standard solid-phase synthesis.
In conclusion, the sequential use of phenylacetyl disulf-
ide (PADS, 11) and 3H-1,2-benzodithiol-3-one (9) is an
efficient procedure for the one-pot sulfurization of phos-
phite and H-phosphonate intermediates. Implementation
of this sulfurization procedure in
a solution-phase
approach for the synthesis of phosphorothioate oligo-
nucleotides allows the replacement of chromatographic
purifications of protected phosphorothioate intermediates
by simple extractions. This solution-phase approach was
demonstrated by the synthesis of hexameric oligothioate
17 which was obtained in both good yield and purity on
a 0.5 mmol scale, with no apparent formation of side
products caused by oxidation.
25. Rao, M. V.; Macfarlane, K. Tetrahedron Lett. 1994, 35, 6741.
26. The detected oxidation by-products represent the corresponding
phosphates of either 3 or 4.
27. It is reported that TEDT (10) is able to convert H-phosphonates into
H-phosphonothioates under basic conditions (0.5 M TEDT (10) in
carbon disulfide/triethylamine 9:1 (v/v) for 10 min).22
28. Attempts to oxidize H-phosphonate 4 into the extractable phosphate
diester by the addition of iodine was accompanied by oxidation
by-products of 5 due to desulfurization of the corresponding phos-
phorothioate diester linkage, which is generated from the phosphoro-
thioate triester due to some premature loss of the 2-cyanoethyl
phosphate protective group.
Acknowledgements
This work was supported by Diosynth BV (Organon
NV), The Netherlands and the Netherlands Organisation
for Scientific Research (NWO).
29. The overall reaction rate of the two-step sulfurization protocol was
negatively affected in terms of yield and purity by the simultaneous
addition of PADS (11) and 9 or the reversal of the order of addition,
that is, first 9 and then 11.
30. TCEP was sulfurized by both PADS (11) and reagent 9.
31. Bauer, S.; Kirschning, C. J.; Hacker, H.; Redecke, V.; Hausmann, S.;
Akira, S.; Hermann, W.; Lipford, G. B. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 9237.
Supplementary data
Experimental procedures, spectroscopic and chromato-
graphic data for all new compounds. Supplementary data
associated with this article can be found, in the online
32. The HPLC-purification was performed at 60 °C with triethylammo-
nium acetate to suppress peak broadening due to P-diastereomers.
33. The yield was estimated by A260 absorption units using e =
58200 L molꢀ1 cmꢀ1
34. Application of
.
a
hexafluoroisopropanol/triethylamine buffer in
MeOH/H2O suppressed peak broadening due to P-diastereomers.
35. Fountain, K. J.; Gilar, M.; Gebler, J. C. Rapid Commun. Mass
Spectrom. 2003, 17, 646.
References and notes
1. Crook, S. T. Antisense Drug Technology: Principles, Strategies and
Applications, New York, 2001.