1.6 In 1989, Eckstein and Ludwig described a new and
efficient procedure for the synthesis of 1 employing trico-
ordinated phosphorus compounds as phosphitylating re-
agents.7 Condensation of appropriately protected nucleosides
and 2-chloro-4H-1,3,2-benzodioxaphosphorine resulted in an
intermediate, which without isolation, reacted first with bis-
(tri-n-butylammonium) pyrophosphate followed by an ad-
dition of elemental sulfur, providing, after deprotection, the
desired R-thiotriphosphates 1 with 60-75% yield. The
Ludwig-Eckstein method was also applied for the synthesis
of some R-thiotriphosphates on a solid support.8
The successful synthesis of various biophosphates using
oxathiaphospholane methodology (OTP)9 prompted us to
examine its use for the synthesis of nucleoside 5′-O-(R-
thiotriphosphates) (1). Early results10 on the opening of the
2-thio-1,3,2-dithiaphospholane ring system with inorganic
pyrophosphate leading to nucleoside 5′-O-R-dithiotriphos-
phate provided evidence that ring-opening condensation with
pyrophosphate is feasible, albeit the yield of desired product
was rather discouraging. Results of our recent studies on the
synthesis of nucleoside 5′-O-R-thiotriphosphates via an OTP
approach are presented in this communication.
First attempts to obtain 1 in reaction of 3′-O-acetylthy-
midine 5′-O-(2-thio-1,3,2-oxathiaphospholane) (2a, mixture
of diastereoisomers, dr ca. 1:1) with tris(tetra-n-butylammo-
nium) hydrogen pyrophosphate (3) in the presence of an
equimolar amount of DBU9a,11 failed. The total consumption
of 2a was observed after 30 min. Analysis of 31P NMR spec-
tra12 of the reaction mixture revealed the presence of two
groups of signals at ca. 67 and 56 ppm that suggested the
formation of “dimeric” product 4 as a result of ring-opening
nucleophilic substitution in 2a with water11 (Scheme 1).
monium hydroxide, and the resulting solution was lyophi-
lized.14 Prepared in this way, pyrophosphate 3 exists,
however, as a hydrate.15 Removal of water by the method
described by Poulter14 (crystallization from ethyl acetate or
coevaporation from acetonitrile solutions) appeared to be
insufficient to obtain 3 dry enough to produce 3′-O-
acetylthymidine 5′-O-(R-thiotriphosphate) (5a) in its reaction
with 2a. Sufficiently dry 3 was prepared by pretreatment of
acetonitrile solution of 3 with 3 Å molecular sieves before
its reaction with a diastereomeric mixture (ca. 1:1) of 2a. A
10% molar excess of DBU was used, and after 2 h, the
resonance signal from 2a vanished (31P NMR assay),
providing 5a with 58% NMR yield. Compound 5a was
isolated from the reaction mixture by DEAE-Sephadex
chromatography in 32% yield (as calculated on the basis of
starting 2a), and its structure was proved by 31P NMR and
FAB-MS. Applicability of this procedure for the synthesis
of 1 was examined using 5′-O-(2-thio-1,3,2-oxathiaphos-
pholanes) of all eight common deoxyribo- and ribonucleo-
sides 2a-h (Scheme 2). Starting from suitably protected
nucleosides16 in a reaction with 2-chloro-1,3,2-oxathia-
phospholane9c,17 in the presence of elemental sulfur in a
pyridine solution, we obtained compounds 2a-h with
excellent yields. Oxathiaphospholanes 2a-h (mixture of
diastereomers, ca. 1:1) reacted with a dry solution of 3 (as
described above) in the presence of DBU (10% molar
excess), providing protected nucleoside 5′-O-R-thiotriphos-
phates 5a-h in 48-78% yield, as estimated by 31P NMR
(Table 1). No stereoselectivity was observed in these
reactions since both diastereomers of 5a-h were formed in
an equimolar ratio. Deprotection reactions were performed
by the use of concentrated ammonia solution, and conditions
(9) (a) Stec, W. J.; Grajkowski, A.; Koziolkiewicz, M.; Uznanski, B.
Nucleic Acid Res. 1991, 19, 5883-5888. (b) Stec, W. J.; Wilk, A. Angew.
Chem., Int. Ed. Engl. 1994, 33, 709-722. (c) Stec, W. J.; Grajkowski, A.;
Kobylanska, A.; Karwowski, B.; Koziolkiewicz, M.; Misiura, K.; Okruszek,
A.; Wilk, A.; Guga, P.; Boczkowska, M. J. Am. Chem. Soc. 1995, 117,
12019-12029. (d) Misiura, K.; Pietrasiak, D.; Stec, W. J. J. Chem. Soc.,
Chem. Commun. 1995, 613-614. (e) Stec, W. J.; Karwowski, B.; Bocz-
kowska, M.; Guga, P.; Koziolkiewicz, M.; Sochacki, M.; Wieczorek, M.
W.; Blaszczyk, J. J. Am. Chem. Soc. 1998, 120, 7156-7167. (f) Guga, P.;
Doman˜ski, K.; Stec, W. J. Angew. Chem., Int. Ed. 2001, 40, 610-613. (g)
Guga, P.; Okruszek, A.; Stec, W. J. Top. Curr. Chem. 2002, 220, 170-
200. (h) Olesiak, M.; Krajewska, D.; Wasilewska, E.; Korczyn˜ski, D.;
Baraniak, J.; Okruszek, A.; Stec, W. J. Synlett 2002, 967-971. (i) Baraniak,
J.; Kaczmarek, R.; Korczynski, D.; Wasilewska, E. J. Org.Chem. 2002,
49, 7267-7274. (j) Zmudzka, K.; Nawrot, B.; Chojnacki, T.; Stec, W. J.
Org. Lett. 2004, 6, 1385-1387.
Scheme 1. Ring-Opening Condensation of
3′-O-Acetylthymidine 5′-O-(2-Thio-1,3,2-oxathiaphospholane)
with Pyrophosphate
(10) Okruszek, A.; Olesiak, M.; Balzarini, J. J. Med. Chem. 1994, 37,
3850-3854.
(11) Misiura, K.; Szymanowicz, D.; Olesiak, M.; Stec, W. J. Tetrahedron
Lett. 2004, 45, 4301-4305.
(12) See Supporting Information.
(13) It was emphasized only recently that during the synthesis of
morpholine nucleoside triphosphates via the Ludwig-Eckstein method,
traces of water were also deleterious for reliable synthesis of triphosphates
(see: Abramova, T. V.; Bakharev, P. A.; Vasilyeva, Silnikov, V. N.
Tetrahedron Lett. 2004, 45, 4361-4364.
Since the above reaction was performed under strictly
anhydrous conditions13 (oven-dried glass equipment, argon
atmosphere, acetonitrile with less than 20 ppm of water),
the only source of water could come from a solution of
pyrophosphate 3. Reagent 3 was obtained from potassium
pyrophosphate by cation exchange on Dowex 50WX8 and
neutralization of pyrophosphoric acid with tetra-n-butylam-
(14) Dixit, V. M.; Laskovics, F. M.; Noall, W. I.; Poulter, C. D. J. Org.
Chem. 1981, 46, 1967-1969.
(15) (a) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-4779. (b)
Wu, W.; Freel Meyers, C. L.; Borch R. F. Org. Lett. 2004, 6, 2257-2260.
(16) Jones, R. A. In Oligonucleotide Synthesis; Gait, M. J., Ed.; IRL
Press: Oxford, 1984; pp 23-34.
(17) Guga, P.; Stec, W. J. In Current Protocols in Nucleic Acid
Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A.,
Eds.; John Wiley & Sons: Hoboken, NJ, 2003; pp 4.17.1-4.17.28.
(7) Ludwig, J.; Eckstein F. J. Org. Chem. 1989, 54, 631-635.
(8) Gaur, R. K.; Sproat, B. S.; Krupp, G. Tetrahedron Lett. 1992, 33,
3301-3304.
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