concentrated ammonium hydroxide, occurs in less than 3 h
at ∼90 °C, affording the corresponding dithymidylyl mono-
phosphate 18 in essentially quantitative yields.15
Scheme 3a
While the cleavage of phosphate protecting groups from
10-17 proceeds cleanly, deprotection of the parent thio-
phosphate protecting groups from 11, 13, and 15 under
identical conditions produces dithymidylyl phosphorothioate
19 along with 10% to 20% of the native phosphodiester 18.
Such an extent of desulfurization precludes the use of these
thiophosphate protecting groups in routine oligonucleoside
phosphorothioate syntheses. However, heating the dinucleo-
side thiophosphate triesters 10, 12, 14, 16, and 17 under
conditions identical to those used for the related phospho-
triesters results in complete removal of the thiophosphate
protecting groups without significant desulfurization (<0.5%)
of 19.16 These findings raise interesting questions about the
deprotection mechanism of these thiophosphate protecting
groups.
Preliminary mechanistic studies using 12 as a model
strongly suggest that the presence of water is required for
rapid phosphate/thiophosphate deprotection;17 a tentative
mechanism involving cyclodeesterification3 of the dinucleo-
side phosphotriester is proposed in Scheme 3.18 Whether the
proposed phosphate/thiophosphate deprotection mechanism
applies to the dinucleoside phosphotriesters 10, 11, and 13-
17 remains to be determined and will be addressed at a later
date.
a Conditions: (i) 0.1 M triethylammonium acetate pH 7.0, 90
°C, 3 h.
1 was selected as a model and used in the solid-phase
synthesis of an octadecanucleotide. The synthesis of 1 begins
with the preparation of the phosphordiamidite 20. Thus,
addition of anhydrous N,N-diisopropylamine to a solution
of freshly distilled phosphorus trichloride in dry benzene
gives bis(N,N-diisopropylamino)chlorophosphine, which is
then immediately reacted with 2-(N-formyl-N-methyl)-
aminoethan-1-ol. 31P NMR analysis of the reaction mixture
indicates that the formation of the phosphordiamidite 20 (δP
118.0 and 118.7 ppm) is nearly complete (∼96%) after 2 h
at 25 °C. After workup, the phosphinylating reagent is
purified from hydrolysis side products by silica gel chro-
matography and is isolated as an oil in 73% yield.
Since the groups used for phosphate/thiophosphate protec-
tion of 10, 12, 14, 16, and 17 are easily removed under
neutral conditions, the deoxyribonucleoside phosphoramidite
(12) The sulfurization reaction is effected by 3H-1,2-benzodithiol-3-one
1,1-dioxide, see: (a) Beaucage, S. L.; Iyer, R. P.; Egan, W.; Regan, J. B.
Ann. New York Acad. Sci. 1990, 616, 483-485. (b) Iyer, R. P.; Phillips, L.
R.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Org. Chem. 1990, 55, 4693-
4699. (c) Regan, J. B.; Phillips, L. R.; Beaucage, S. L. Org. Prep. Proc.
Int. 1992, 24, 488-492.
(13) Boal, J. H.; Wilk, A.; Harindranath, N.; Max, E. E.; Kempe, T.;
Beaucage, S. L. Nucl. Acids Res. 1996, 24, 3115-3117.
(14) Selected RP-HPLC chromatograms recorded before and after
phosphotriester deprotection are provided in the Supporting Information.
(15) Under these conditions, phosphate deprotection of the dinucleoside
phosphotriesters 13 and 14 is complete within 16 and 4 h, respectively.
(16) It should also be noted that heating 17 for an extended period of
time (>12 h) in 0.1 M triethylammonium acetate pH 7.0 at 90 °C will
result in significant desulfurization of 19 (∼10%). This problem can
essentially be eliminated by selecting 1X phosphate buffered saline (PBS)
pH 7.4 as a buffer formulation for thermolytic thiophosphate deprotection.
(17) Heating the dinucleoside phosphotriester 12 for 3 h at ∼90 °C in
MeCN containing 50 ppm water affords only ∼15% phosphate deprotection.
Complete phosphate deprotection is accomplished within 3 h (t1/2 ∼ 20
min) in 0.1 M triethylammonium acetate pH 7.0 and is consistent with a
pseudo-first-order kinetic pathway. The enhanced rate of deprotection does
not however result from water attacking the phosphate function and releasing
2-(N-formyl-N-methyl)aminoethanol. This argument is supported by the fact
that hydrolysis of triethyl phosphate in 0.1 M triethylammonium acetate
pH 7.0 for 8 h at ∼90 °C gives only ∼1% diethyl phosphate, thereby
exhibiting a phosphate deprotection rate considerably slower (t1/2 ∼ 600 h)
than that of 12. Data are shown in the Supporting Information.
Phosphinylation of 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-
thymidine with 20 is performed essentially as reported by
Barone et al.19 and affords the deoxyribonucleoside phos-
phoramidite 1. The crude phosphoramidite is purified by
silica gel chromatography and isolated as a white foam in
92% yield.20 The pure deoxyribonucleoside phosphoramidite
1 is then used in the solid-phase synthesis of an oligonucle-
otide (18-mer) according to standard protocols.21 The 5′-
deprotected oligonucleoside phosphotriester is released from
(18) Removal of thiophosphate protecting groups that are structurally
related to 11 or 13 has also been reported by others, see: (a) Iyer, R. P.;
Yu, D.; Devlin, T.; Ho, N.-H.; Agrawal, S. J. Org. Chem. 1995, 60, 5388-
5389. (b) Iyer, R. P.; Guo, M. J.; Yu, D.; Agrawal, S. Tetrahedron Lett.
1998, 39, 2491-2494. (c) Wang, J.-C.; Just, G. Tetrahedron Lett. 1997,
38, 3797-3800. (d) Wang, J.-C.; Just, G. J. Org. Chem. 1999, 64, 8090-
8097. (e) Guzaev, A. P.; Manoharan, M. J. Am. Chem. Soc. 2001, 123,
783-793. Unfortunately, deprotection of 11 or 13 in the presence or absence
of concentrated ammonium hydroxide leads to desulfurization of the
dinucleoside phosphorothioate 19 in unacceptable levels.
(19) Barone, A. D.; Tang, J.-T.; Caruthers, M. H. Nucl. Acids Res. 1984,
12, 4051-4061.
(20) The detailed preparation of 20 and 1 along with characterization
data are provided in the Supporting Information.
Org. Lett., Vol. 3, No. 9, 2001
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