Synthesis and chemical and enzymatic reactivity of thymidine 3′-O- and 5′-O-phosphorofluoridothioates

Article abstract of DOI:10.1039/a706636h

5′-O- or 3′-O-Protected thymidine 3′-O- or 5′-O-(2-thiono-1,3,2-oxathiaphospholanes) react with triethylammonium fluoride in a presence of DBU and furnish, after deprotection, thymidine 3′-O- and 5′-O-phosphorofluoridothioates; the latter undergoes stereo

Full text of DOI:10.1039/a706636h

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Synthesis and chemical and enzymatic reactivity of thymidine 3A-O- and
5A-O-phosphorofluoridothioates
Konrad Misiura, Daria Szymanowicz and Wojciech J. Stec*†
Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry,
Sienkiewicza 112, 90-363 Lo´dz´, Poland
5A-O- or 3A-O-Protected thymidine 3A-O- or 5A-O-(2-thiono-
1,3,2-oxathiaphospholanes) react with triethylammonium
fluoride in a presence of DBU and furnish, after deprotec-
tion, thymidine 3A-O- and 5A-O-phosphorofluoridothioates;
the latter undergoes stereoselective hydrolysis by snake
venom phosphodiesterase.
published earlier results on the stereochemistry of nucleophilic
substitution at phosphorus by fluoride ion.7,8 Also, thymidine
5A-O-phosphorofluoridothioate 4 has been synthesized accord-
ing to the reaction sequence presented in the Scheme 2.
Phosphitylation of 3A-O-methoxyacetylthymidine with N,N-
diisopropylamino-1,3,2-oxathiaphospholane2 in a presence of
1H-tetrazole, followed by sulfurization yielded oxathiaphos-
pholane 5. Reaction of 5 with triethylammonium fluoride–DBU
furnished intermediate 3A-O-methoxyacetylthymidine 5A-O-
phosphorofluoridothioate 6 which subsequently was depro-
tected with a concentrated solution of ammonia providing, after
purification on Sephadex A-25, the final product 4** in 75%
yield. The phosphorofluoridothioate monoesters 3 and 4 were
hydrolytically stable even under basic conditions (conc. ammo-
nia, room temp., 1 h), as proven by 31P NMR assay. Similarly,
the resistance of 3 and 4 towards methanolysis, attempted in the
presence of triethylamine or pyridine, has been observed.
Attempts at internucleotide bond formation in reaction of 2 with
3A-O-acetylthymidine in the presence of strong bases such as
ButOK, DBU and 2-tert-butylimino-2-diethylamino-1,3-di-
methyl-1,3,2-diazaphosphinane (BEMP) have failed. Reactions
Within the course of our studies on the development of
oxathiaphospholane methodology for phosphorylation,1 phos-
phorothioylation2 and phosphorodithioylation3 of biomolecules
we have found that the endocyclic P–S bond of a 2-thio-
substituted 1,3,2-oxathiaphospholane ring attached to nucleo-
side moiety can be easily broken in the presence of DBU by
fluoride ions leading, after spontaneous episulfide elimination,
to
3A-O-methoxyacetyl
nucleoside
5A-O-phosphoro-
fluoridothioates or 5A-O-dimethoxytrilyl nucleoside 3A-O-phos-
phorofluoridothioates.4,5 Thus, 5A-O-dimethoxytritylthymidine
3A-O-(2-thiono-1,3,2-oxathiaphospholane) 1 (ratio of diaster-
eomers ca. 1:1), after treatment with triethylammonium
fluoride‡ in a presence of DBU gives 5A-O-dimethoxy-
tritylthymidine 3A-O-phosphorofluoridothioate 2§ in 78% yield
(Scheme 1). The dimethoxytrityl protecting group was removed
by the treatment of 2 with 80% AcOH. The final product,
thymidine 3A-O-phosphorofluoridothioate 3,¶ was purified by
anion-exchange chromatography on Sephadex A-25 using
triethylammonium hydrogen carbonate buffer (pH 7.5, 0.02–0.5
m) as eluent. Pure 3 has also been obtained from 1 in 68% yield
in a one-pot synthesis. Reaction of 1 with triethylammmonium
fluoride in a presence of DBU is, unlike the 1,3,2-ox-
athiaphospholane ring-opening condensation with alcohols2 or
amines,6 a non-stereospecific process. Starting from a mixture
of partially separated diastereomers of 1 [ratio 73:27, dP
(CD3CN), 105.78 and 105.83] diastereomers of 2 were obtained
in a ratio of 54:46 [dF (CD3CN), 230.06 and 229.96]. The
epimerisation at phosphorus was not unexpected in the light of
were performed in DMF and their progress was followed by 31
P
NMR spectroscopy. Under these conditions, even after 18 h,
formation of dithymidylyl (3A,5A)phosphorothioate (TPST) was
not observed. Instead, both 3 and 4 underwent intramolecular
cyclization in the presence of an excess of ButOK (five-fold
molar excess) leading to thymidine cyclic (3A,5A)phosphor-
othioate (cTMPS). Similarly, as previously observed9 for
ButOK-catalyzed cyclization of nucleoside 5A-O-p-nitrophenyl
phosphorothioates,
in
the
reactions
of
phospho-
rofluoridothioates 3 and 4 [SP]-cTMPS10 was also formed
preferentially (ratio of [SP]-: [RP]-cTMPS ca. 2:1). Yields of
Sephadex-purified cTMPS obtained from 3 and 4 were 57 and
38%, respectively. The lack of formation of TPST in ButOK
assisted condensation of 2 with 3A-O-acetylthymidine was rather
unexpected in the light of the results of von Tigerstrom and
Smith11 on effective formation of TPT and other medium-sized
oligothymidylates in the reaction of protected thymidine 3A-O-
phosphorofluoridate with 3A-O-acetylthymidine.
T
T
DMTO
DMTO
O
O
i
S
O
O
P
O
P
F
T
HO
T
P
O
O
S
O
O
S–
i
S
S
O
1
2
OCOCH2OMe
OCOCH2OMe
ii
ii
5
T
HO
O
S–
P
S–
P
F
O
T
T
F
O
O
O
iii
O
O
O
O
P
S–
OH
OCOCH2OMe
6
F
DMT = 4,4′-dimethoxytrityl
4
3
Scheme 2 Reagents and conditions: i, N,N-diisopropylamino-1,3,2-oxa-
thiaphospholane, 1H-tetrazole, 2 h, then elemental sulfur, 18 h; ii, Et3NHF,
DBU, CH2Cl2 MeCN, 15 min; iii, conc. NH4OH, EtOH, 2.5 h
Scheme 1 Reagents and conditions: i, Et3NHF, DBU, MeCN, 15 min; ii,
80% AcOH, 1 h
Chem. Commun., 1998
515

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HO
T
HO
T
idate. Adenosine 5A-O-phosphorofluoridate was found as the
product of sv PDE-assisted hydrolysis of the same substrate.
Besides the hydrolytic instability of the P–F bond of dinucleo-
side (3A,5A)phosphorofluoridates in buffered aqueous media18
yielding appropriate phosphates, even if thymidine 3A-O-
phosphorofluoridate or adenosine 5A-O-phosphorofluoridate
were the respective products of PDE-catalyzed hydrolyses, they
would necessarily undergo further enzymatic degradation to
phosphomonoesters.
O
O
O
S–
O
O
O
P
P
T
T
O
–S
O
O
O
OH
OH
Studies presented here were financially supported by the
State Committee of Scientific Research (KBN), grant no 4
PO5F 023 10.
sv PDE
(RP)-TPS
T
(SP)-TPST
O
P
–O
–S
O
T
T
HO
O
O
Notes and References
+
† E-mail: wjstec@bio.cbmm.lodz.pl
‡ Triethylammonium fluoride (1 m solution in THF) was obtained by
mixing triethylamine tris(hydrofluoride) (1 equiv.) with triethylamine (2
equiv.).
OH
or
OH
O
F –
§ Compound 2 consists of a 1:1 mixture of diastereomers, dP(CD3CN, 81
F
S–
O
F
MHz) 54.52 (1JP–F 1043 Hz), 54.58 (1JP–F 1046 Hz); m/z (2FAB) 641.4
sv PDE
P
P
(M+ 21).
·
T
T
O
O
–S
¶ Compound 3 consists of a mixture of diastereomers (ratio 48:52),
dP(D2O, 81 MHz) 53.72, (1JP–F 1053 Hz), 53.74 (1JP–F 1055 Hz); dF(D2O,
188 Mz) 231.4 (1JP–F 1043 Hz), 231.2 (1JP–F 1046 Mz); m/z (2FAB)
O
O
339.1 (M+ 21).
·
OH
(RP)-4 (SLOW)
OH
∑ Compound 5 was obtained as a mixture of diastereomers, dP(CDCl3, 81
(SP)-4 (FAST)
MHz) 106.64, 106.82 (ratio 1 :1); m/z (+FAB) 453.2 (M+ +1).
·
**Compound 4 consists of mixture of diastereomers (ratio 59:41), dP(D2O,
Fig. 1 Tentative assignment of absolute configuration of diastereomers of 4
81 Mz) 54.17, 54.20 (1JP–F 1053 Hz); dF(D2O, 188 Mz) 235.6, 235.7
(1JP–F 1053 Hz); m/z (2FAB) 339.2 (M+ 21).
·
In the light of earlier results on an enzymatic cleavage of
thymidine 5A-O- or 3A-O-phosphorofluoridate assisted by snake
venom (sv PDE)12,13 and spleen12 phosphodiesterases (spleen
PDE) it was tempting to study the thio analogues 4 and 3 as
substrates for these enzymes. From the pioneering work of
Eckstein14 and Benkovic15 demonstrating the stereoselectivity
of sv PDE towards P-chiral diesters of phosphorothioic acid it
was of interest to check if this enzyme can discriminate between
the diastereomers of 3 or 4.
5A-O-Phosphorofluoridothioate 4 was incubated with sv
PDE†† and the progress of the enzymatic digestion was
analyzed by RP-HPLC. It was found that sv PDE, if added to a
diastereomeric mixture of 4, causes the stereoselective hydroly-
sis of the P–F bond of slow-eluted 4 leaving the fast-eluted
diastereomer intact. Also, in the case of diastereomeric mixture
of 3†† only slow-eluted 3 underwent hydrolysis in a presence of
sv PDE, albeit the reaction proceeded much slower than that
observed for slow-eluted 4. Interestingly, under analogous
conditions, the rate of hydrolysis of slow-4 by sv PDE was
similar to that obtained during digestion of dithymidylyl
(3A,5A)phosphate (TPT). In the presence of spleen PDE both
diastereomers of 3 were hydrolyzed while both diastereomers of
4 were resistant to this enzyme.** It was also found that 3 and
4 have no inhibitory activity§§ towards either phosphodiester-
ase. Results on the use of 4 for inhibition of thymidylate
synthase will be published separately.16
†† The reaction mixture consists of 0.1 mm 4 or 3, 100 mm Tris–HCl pH 8.0,
20 mm MgCl2, and sv PDE (0.01 U ml21); 37 °C; incubation time: 0.5 h for
4 and 16 h for 3.
‡‡ The reaction mixtures consist of 0.1 mm 3 (or 4), 50 mm acetate buffer
pH 5.0, and spleen PDE (0.15 U ml21), 37 °C; incubation time: 1 h for 3 and
16 h for 4.
§§ Enzymatic digestions were performed under conditions mentioned
above. TPT and 3 (or 4) were used at equimolar concentrations (0.1 mm).
1 W. J. Stec, B. Karwowski, P. Guga, M. Koziolkiewicz, M. Boczkowska,
M. W. Wieczorek and J. Blaszczyk, J. Am. Chem. Soc., submitted for
publication.
2 W. J. Stec, A. Grajkowski, M. Koziolkiewicz and B. Uznanski, Nucleic
Acids Res., 1991, 19, 5883; W. J. Stec, A. Grajkowski, B. Karwowski,
A. Kobylanska, M. Koziolkiewicz, K. Misiura, A. Okruszek, A. Wilk,
P. Guga, M. Boczkowska, J. Am. Chem. Soc., 1995, 117, 12019.
3 A. Okruszek, M. Olesiak, D. Krajewska, W. J. Stec, J. Org. Chem.,
1997, 62, 2269; A. Okruszek, A. Sierzchala, M. Sochacki and W. J. Stec,
Tetrahedron Lett., 1992, 33, 7585; A. Okruszek, A. Sierzchala,
K. L. Fearon and W. J. Stec, J. Org. Chem., 1995, 60, 6998.
4 W. Dabkowski and I. Tworowska, Chem. Lett., 1995, 727.
5 M. Bollmark and J. Stawinski, Tetrahedron Lett., 1996, 37, 5739.
6 K. Misiura, M. Olesiak and W. J. Stec, unpublished results.
7 M. Mikolajczyk and M. Witczak, J. Chem. Soc., Perkin Trans. 1, 1977,
2213.
8 R. J. P. Corriu, J. P. Dutheil and G. F. Lanneau, J. Am. Chem. Soc., 1984,
106, 1060.
9 F. Eckstein, L. P. Simonson and M. P. Baer, Biochemistry, 1974, 13,
3806.
In conclusion, we have found that the 1,3,2-oxathiaphos-
pholane ring can be opened in the presence of DBU by fluoride
anion leading to the appropriate phosphorofluoridothioates. The
nucleoside 5A-O- or 3A-O-phosphorofluoridothioates obtained
can be used in studies of the mode of action of nucleolytic
enzymes. Comparative topological analysis of diastereomers of
TPST and 4 undergoing sv PDE- assisted hydrolysis allows the
tentative assignment of absolute configuration of the slow-
eluted 4 as RP (Fig. 1). Spleen and sv PDE-assisted hydrolysis of
P–F bonds in compounds 3 and 4 is in agreement with earlier
findings12,13 that these enzymes split nucleoside 3A-O- or 5A-O-
phosphorofluoridate, respectively, giving rise to the appropriate
nucleoside phosphates. From this perspective our data on
enzymatic hydrolyses of 3 and 4 disagree with the results of
Dabkowski et al.,17 who characterized thymidine 3A-O-phos-
phorofluoridate as the product of spleen PDE-assisted degrada-
tion of thymidin-3A-yl 2A-deoxyadenosin-5A-yl phosphorofluor-
10 W. S. Zielinski and W. J. Stec, J. Am. Chem. Soc., 1977, 99, 8365.
11 R. G. von Tigerstrom and M. Smith, Science, 1970, 167, 1266.
12 R. Wittmann, Chem.Ber., 1963, 96, 771.
13 Z. Kucerova and J. Skoda, Biochim. Biophys. Acta, 1971, 247, 194.
14 P. M. J. Burgers, F. Eckstein and D. H. Hunneman, J. Biol. Chem., 1979,
254, 7476.
15 F. R. Bryant and S. J. Benkovic, Biochemistry, 1979, 18, 2825.
16 B. Golos, K. Misiura, M. Olesiak, A. Okruszek, W. J. Stec and W. Rode,
Acta Biochim. Pol., 1998, in the press.
17 W. Dabkowski, F. Cramer and J. Michalski, J. Chem. Soc., Perkin
Trans. 1, 1992, 1447; W. Dabkowski, J. Michalski, J. Wasiak and
F. Cramer, ibid., 1994, 817.
18 K. Misiura, D. Pietrasiak and W. J. Stec, J. Chem. Soc., Chem.
Commun., 1995, 613; K. Misiura, D. Szymanowicz and W. J. Stec,
Collect. Czech. Chem. Commun., 1996, 61, S101; M. Bollmark and
J. Stawinski, Chem. Commun., 1997, 991.
Received in Glasgow, UK, 12th September 1997; 7/06636H
516
Chem. Commun., 1998