SCHEME 4. Activation of Prochiral Thymidine
Methylphosphonate (11)
separately as benzene solutions over activated molecular sieves 4
Å for 3 days. To a solution of triazine in benzene (15 mL), cooled
in an water-ice bath, was added dropwise a solution of morpholine.
The precipitated salt was washed with petroleum ether, dried, and
used without further purification. Yield: 71%.
1: 1H NMR (CDCl3) δ 3.01 (s, 5H), 3.72 (t, 4H), 3.83 (t, 4H),
3.95 (s, 6H). Anal. (C10H17N4O3Cl): C, 43.15; H, 6.18; Cl, 6.34;
N, 21.05. Calcd: C, 43.39; H, 6.19; Cl, 12.81; N, 20.24.
Synthesis of 5′-O-DMT-thymidine 3′-O-Methylphosphono-
thioate (5). Into a solution of 1,2,4-triazole (0.345 g, 5 mmol, 2.5
equiv) and triethylamine (0.85 mL, 6 mmol, 3 equiv) in dry THF
(10 mL), cooled in ice bath, was added methyldichlorophosphine
(2.2 mmol, 1.1 equiv) with vigorous stirring, and the reaction
mixture was stirred for 20 min at this temperature. The immediate
formation of white precipitate was observed. 5′-O-DMT-thymidine
(1.09 g, 2 mmol) dissolved in THF (10 mL) was added to this
mixture dropwise at 0 °C, and the ice bath was removed. Stirring
was continued for 30 min. After this time, sulfur was added, and
the reaction mixture was allowed to sit overnight. Triethylamine
and water (1:4 v/v) were added to the reaction mixture until the
solution became clear, and stirring was continued for an additional
0.5 h. After this time, the reaction mixture was diluted with NaHCO3
(0.1 M) and extracted three times with chloroform. The combined
organic fractions were additionally washed with NaHCO3 and water,
dried with MgSO4, and concentrated. The obtained product (as a
foam) was dissolved in a small amount of CH2Cl2 and precipitated
to hexane or petroleum ether. The product (mixture of diastereomers
in ca. 1:1 ratio, 1.18 g) was separated by short path column
chromatography on silica gel (40 g) using a mixture of chloroform
and ethanol (19:1, v/v) containing 1% of triethylamine as an eluent.
The RP- and SP-5 were separated by the column chromatography
on fine silica gel (50 g) using a mixture of chloroform and ethanol
(39:1, v/v) containing up to 5% of Et3N as eluent.25
of class of active esters that can be used for efficient synthesis
of exclusively one diastereoisomer (either RP or SP) of meth-
ylphosphonothioates 5.
An analogous approach eventually leading to diastereomers
of 5′-O-DMT-thymidine 3′-O-(4,6-dimethoxy-1,3,5-triazin-2-
yl methylphosphonate) (10), prepared from the P-prochiral 11,
was appealing in the context of the expected diastereoselective
activation and possible separation of formed diastereomers 10
(Scheme 4). We found, however, that instead of the expected
ester 10 (31P NMR δ 29.09, 29.12 ppm, diastereomeric ratio
ca. 1:1, observed about 5%), the corresponding dithymidyl
(3′,3′)-pyrophosphonate (12) [31P NMR δ 24.85, 24.98, 25.1
ppm, 1:2:1 ratio, 85%)] was observed as the foremost product.
Pyrophosphonate 12 was, most probably, produced in reaction
of the active ester 10 with methylphosphonic acid 11. Different
conditions tested for this reaction did not improve the yield of
the required 10, observed only transiently and at low concentra-
tions during the course of the reaction.23
B ) Thy: 31PNMR (CDCl3) δ (FAST-RP-5) 77.18; (SLOW-SP-
5) 77.46 ppm.
In conclusion, we established that N-methyl-N-4,6-dimethoxy-
1,3,5-triazinylmorpholinium salts 1 or 2 can be applied as
activators of nucleoside methylphosphonates 11 and their
thiocongeners 5.24 In the case of methylphosphonotioates 5, the
reaction is both chemoselective and stereospecific leading to
stable active esters 7, converted in base-catalyzed reactions with
O-nucleophiles, including water and alcohols into the corre-
sponding esters (or acids) with inversion of configuration.
Therefore, esters 7 can constitute convenient tools in chirotech-
nology for exchange of the P-chirality in these methylphospho-
nothioates 5, but at this stage, they are not competitive
monomers for synthesis of modified oligonucleotides.
FAST-RP-5 (B ) Thy) (0.44 g, 30% yield): TLC Rf 0.50
(chloroform-ethanol (19:1, v/v) containing 5% of Et3N); 1H NMR
(CDCl3) δ 1.26 (t, J 7.3 Hz, 9H), 1.34 (d, J 1.2 Hz, 3H), 1.70 (d,
J 14.3 Hz, 3H), 2.40 (m, 1H), 2.94 (m, 1H), 3.05 (q, J 7.3 Hz,
6H), 3.40 (dd, J 10.4, 2.3 Hz, 1H), 3.54 (dd, J 10.4, 2.3 Hz, 1H),
3.78 (s, 6H), 4.36 (m, 1H), 5.38 (m, 1H), 6.47 (dd, J 8.5, 5.7 Hz,
1H), 6.82 (m, 4H), 7.33 (m, 9H), 7.62 (d, J 1.2 Hz, 1H), 8.26 (br
s, 1H), 12.44 (br s, 1H); MS-FAB (M - H)- 637.4 (calcd 637.603).
SLOW-SP-5 (B ) Thy) (0.41 g, 28% yield): TLC Rf 0.42
(chloroform-ethanol (19:1, v/v) with 5% of Et3N); 1H NMR
(CDCl3) δ 1.24 (t, J 7.3 Hz, 9H), 1.39 (d, J 1.1 Hz, 3H), 1.60 (d,
J 14.4 Hz, 3H), 2.38 (m, 1H), 2.71 (m, 1H), 2.95 (q, J 7.3 Hz,
6H), 3.43 (d, J 2.7 Hz, 2H), 3.78 (s, 6H), 4.23 (m, 1H), 5.35 (m,
1H), 6.42 (dd, J 8.2, 5.8 Hz, 1H), 6.82 (m, 4H), 7.32 (m, 9H), 7.62
(d, J 1.1 Hz, 1H); MS-FAB (M - H)- 637.4 (calcd 637.603).
B ) AdeBz: 31P NMR (CDCl3) δ (FAST-RP-5) 77.52; (SLOW-
SP-5) 77.59 ppm.
B ) CytBz: 31P NMR (CDCl3) δ (FAST-RP-5) 77.02; (SLOW-
SP-5) 77.24 ppm.
B ) Guaibu 31P NMR (CDCl3) δ (FAST-RP-5) 76.98; (SLOW-
:
SP-5) 77.04 ppm.
Experimental Section
Reactions were carried out under positive pressure of dry argon.
Solvents and reagents were purified according to standard laboratory
techniques and distilled directly into reaction vessels. Column
chromatography and TLC analyses were performed on silica gel
(240-400 mesh) and silica gel HP TLC precoated F254 plates,
respectively. NMR spectra were recorded at 500.13 MHz (1H) and
202.46 MHz (31P). Chemical shifts (δ) are reported relative to TMS
(1H) and 80% H3PO4 (31P) as external standards.
Synthesis of 5′-O-DMT-thymidine 3′-O-(4,6-Dimethoxy-1,3,5-
triazin-2-yl) Methylphosphonothioate (7). Substrate SLOW-RP-5
(B ) Thy, 31P NMR 77.27) and N-methyl-N-[4,6-dimethoxy-1,3,5-
triazin-2-yl)morpholinium chloride] (1) (2 equiv) were stirred at rt
for 0.5 h in dry MeCN. After the reaction was complete (TLC
control), the reaction mixture was concentrated and applied to a
silica gel column. Product SLOW-SP-7 (31P NMR 95.68 ppm) was
eluted with 2% methanol in CHCl3 in 88% yield. Analogously,
Synthesis of N-Methyl-N-[4,6-dimethoxy-1,3,5-triazin-2-yl)-
morpholinium chloride] (1). 2-Chloro-4,6-dimethoxy-1,3,5-triazine
(4.4 g, 25 mmol) and N-morpholine (2.75 mL, 25 mmol) were dried
(23) The attempts to obtain the active ester 10 in a reaction of the stable
ester 7 with tBuOOH or SeO2 under mild anhydrous conditions have failed.
(24) Our observations suggest that methyl phosphonoselenoates are, in
contrast, activated in a nonselective way, and both Se-esters and O-esters
are formed.
(25) Misiura, K; Stec, W. J. Synlett 2004, 2143-2146.
8586 J. Org. Chem., Vol. 72, No. 22, 2007