Stereocontrolled synthesis of dinucleoside phosphorothioates using a fluorous tag

Article abstract of DOI:10.1016/j.jfluchem.2013.03.013

Dinucleoside phosphorothioates were synthesized in a stereocontrolled manner (diastereoselectivity > 99:1) by using thymidine derivatives bearing a simple perfluoroalkyl tag (C₆F₁₃ or C₈F ₁₇) at the 3′-end and diastereopure nucleoside 3′-O-oxazaphospholidine monomers. Fluorous-tagged intermediates in the synthesis were easily purified by a simple fluorous solid-phase extraction process.

Full text of DOI:10.1016/j.jfluchem.2013.03.013

Journal of Fluorine Chemistry 150 (2013) 85–91
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Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Short communication
Stereocontrolled synthesis of dinucleoside phosphorothioates using a
fluorous tag
*
Natsuhisa Oka, Ryosuke Murakami, Tomoaki Kondo, Takeshi Wada
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Building 702, 5-1-5 Kashiwanoha,
Kashiwa, Chiba 277-8562, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Dinucleoside phosphorothioates were synthesized in
a
stereocontrolled manner (dia-
Received 23 February 2013
Received in revised form 15 March 2013
Accepted 15 March 2013
Available online 26 March 2013
stereoselectivity > 99:1) by using thymidine derivatives bearing a simple perfluoroalkyl tag (C6F13 or
C8F17) at the 30-end and diastereopure nucleoside 30-O-oxazaphospholidine monomers. Fluorous-tagged
intermediates in the synthesis were easily purified by a simple fluorous solid-phase extraction process.
ß 2013 Elsevier B.V. All rights reserved.
Keywords:
Stereocontrolled synthesis
Phosphorothioate
Fluorous
Oxazaphospholidine
1. Introduction
To overcome this problem, we turned our attention to fluorous
chemistry [12]. In contrast to the solid-phase synthesis, the
Chemically modified oligonucleotides have found a wide range
of applications, such as therapeutics, diagnostics, and nanotech-
nology [1–4]. In particular, their therapeutic applications have
received renewed interest due to some newly discovered gene
silencing processes, such as RNA interference, in which relatively
short oligonucleotides regulate gene expression in a sequence-
specific manner [5–7]. Many kinds of chemically modified
oligonucleotides have been developed for these applications and
the methods for their synthesis have also been continuously
studied [1]. We have also developed a method to synthesize
backbone-modified oligonucleotide analogs [8], such as phosphor-
othioates [9] and boranophosphates [10], in a stereocontrolled
manner by using diastereopure nucleoside 30-O-oxazaphospholi-
dine derivatives [11] as monomer units, because the properties of
these oligonucleotide analogs as drugs can be significantly affected
by the configuration of phosphorus atoms [8f–i,9c,d].
fluorous chemistry consists of reactions in homogeneous media
with stoichiometric amounts of reagents and facile purifications
of the synthetic intermediates by liquid–liquid [12b] or solid–
liquid extraction [12d] procedures utilizing the affinity of fluorous
tags or supports [12b], which are attached to the intermediates, to
fluorous solvents or silica gel. Fluorous chemistry has already
been applied to the synthesis of oligopeptides and oligosacchar-
ides [13,14], whereas its applications to the synthesis of
oligonucleotides are scarce. To date, the only application is a
fluorous-tagging of oligonucleotides, which are synthesized on
insoluble solid-supports, while syntheses of oligonucleotides on
fluorous supports have not been reported [15]. Under these
circumstances, we have started to explore the applicability of the
fluorous chemistry to a large-scale synthesis of stereoregulated
oligonucleoside phosphorothioates. In this paper, we describe the
preliminary results of this study.
One of the problems of these methods including ours is how to
scale up the synthesis. Because these methods use reactions
between oligonucleotides on insoluble solid-supports and reagents
in solution-phase, large excess amounts of reagents are generally
required to complete the reactions, and thus direct application of
these methods to a large scale synthesis would be problematic.
2. Results and discussion
First, we attempted to tag the 30-end of 50-O-(4,40-dimethox-
ytrityl)thymidine [50-O-(DMTr)thymidine] 1 with an acyl chlo-
ride bearing a C8F17 group (2) (Scheme 1). However, although
the fluorous-tagged thymidine derivative
3 was generated
smoothly, the product 3 turned out to be sparingly soluble in
common organic solvents. Because the poor solubility of 3 in
organic solvents would hamper the following synthesis of
* Corresponding author. Tel.: +81 4 7136 3611.
E-mail address: wada@k.u-tokyo.ac.jp (T. Wada).
0022-1139/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2013.03.013

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N. Oka et al. / Journal of Fluorine Chemistry 150 (2013) 85–91
Scheme 1. Synthesis of 30-fluorous-tagged thymidine derivative 3. Th = thymin-1-yl.
Scheme 2. 30-Fluorous-tagging of thymidine derivative via succinate linker.
oligonucleotides, we inserted a succinate linker between the
thymidine derivative and a fluorous tag so that the resultant
fluorous-tagged thymidine would be sufficiently soluble in
organic solvents. As shown in Scheme 2, a 50-O-(DMTr)thymi-
dine derivative bearing a succinate linker at the 30-position (4)
was condensed with commercially available 3-(perfluoroalk-
yl)propanols in the presence of 1,3-dimethyl-2-(3-nitro-1,2,
4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium
hexafluorophosphate (MNTP) [16] as a condensing agent. Three
kinds of 3-(perfluoroalkyl)propanols (perfluoroalkyl = C8F17
,
C6F13, C4F9) were employed as fluorous tags. The resultant
fluorous-tagged thymidine derivatives 5a–c were soluble in
common organic solvents, such as dichloromethane, and were
purified by a regular silica gel column chromatography.
Next, we investigated the affinity of 5a–c to fluorous silica gel
by using a regular fluorous solid-phase extraction (FSPE) process
Scheme 3. Stereocontrolled synthesis of dinucleoside phosphorothioates 15t,a,c,g using fluorous tags. BPRO = protected nucleobase; Cyac = N4-acetylcytosin-1-yl; Addmf = N6-
(dimethylamino)methyleneadenin-9-yl; Guce,pac = O6-cyanoethyl-N2-phenoxyacetylguanin-9-yl.

N. Oka et al. / Journal of Fluorine Chemistry 150 (2013) 85–91
87
Fig. 1. 31P NMR spectra of crude 13t (left) and 13t purified by FSPE (right).
Fig. 2. 31P NMR spectra of crude 14t,a,c,g (left) and of those purified by FSPE (right).

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N. Oka et al. / Journal of Fluorine Chemistry 150 (2013) 85–91
[12c,d], because the fluorine contents of 5a–c are relatively low
(19–29 wt%) due to the large non-fluorous 4,40-DMTr group and
they might be useful as model compounds of the corresponding
fluorous-tagged di- or oligonucleoside phosphorothioates. When
5a,b were loaded onto a short fluorous silica gel column and
washed with a non-fluorous solvent (methanol–H2O (8:2, v/v)),
they were sufficiently retained in the column. In contrast, 5c
bearing a C4F9 group was mostly eluted from the column,
indicating that the C4F9 tag was not useful for this method.
Therefore, the former two compounds were employed for further
study on the fluorous synthesis of stereoregulated dinucleoside
phosphorothioates by the oxazaphospholidine method. Com-
pounds 5a,b were treated with 3 vol% dichloroacetic acid (DCA)
to give the fluorous-tagged thymidine derivatives having a free 50-
OH group (6a,b) (Scheme 2).
phosphite intermediate 9t. Subsequent N-trifluoroacetylation
with N-(trifluoroacetyl)imidazole, P-sulfurization with N,N0-
dimethylthiuram disulfide (DTD) [17] and 50-detritylation with
DCA in the presence of Et3SiH as a trityl cation scavenger [18] gave
the fluorous-tagged dithymidine phosphorothioate triester in-
termediate 13t. The whole process from 6a to 13t was carried out
without purification of the intermediates. The intermediate 13t
was then purified by FSPE. A 31P NMR analysis showed that the
same FSPE procedure as attempted with 5a,b completely removed
the residues of the oxazaphospholidine monomer and any other
phosphorus-containing byproducts (Fig. 1). The intermediate 13t
was not observed in the non-fluorous eluent and the recovery of
13t was virtually quantitative.
The thymidine derivative bearing a C6F13 tag 6b was also
used for the synthesis of dinucleoside phosphorothioates
(Scheme 3). The same procedure as for 6a was employed; the
only difference between 6a and 6b was the solubility in MeCN;
6a can be dissolved in MeCN only at a low concentration
(0.056 M), whereas 6b can be dissolved at a higher concentra-
tion (e.g., 0.1 M). The one-pot procedure gave the four kinds of
dinucleoside phosphorothioate triester intermediates 14t,a,c,g,
Next, we investigated the stereocontrolled synthesis of
dinucleoside phosphorothioates using the fluorous tags (Scheme 3).
First, the thymidine derivative tagged with a C8F17 group (6a)
was reacted with a thymidine 30-O-oxazaphospholidine deriva-
tive (7t) [8d] in the presence of N-(cyanomethyl)pyrrolidinium
triflate (CMPT, 8) [8b] in dry MeCN to give the dinucleoside
Fig. 3. RP-HPLC profiles of crude dinucleoside phosphorothioates 15t,a,c,g.

N. Oka et al. / Journal of Fluorine Chemistry 150 (2013) 85–91
89
which were then purified by FSPE. As in the case of 6a, all of
4.3. Compound 5a (Rf = (CH2)3C8F17)
these intermediates were well-purified by FSPE (Fig. 2). The
purified 14t,a,c,g obtained from 6b were then treated with
concentrated ammonia at an elevated temperature to remove
the base protecting groups, the chiral auxiliary at the
phosphorothioate linkage and the fluorous tag. The resultant
products were washed with CHCl3 to remove any hydrophobic
materials and analyzed by RP-HPLC with authentic samples
synthesized by the method we previously reported [8d] (Fig. 3).
The analysis showed that the desired (Sp)-dinucleoside phos-
phorothioates were obtained in good to excellent yields. The
diastereopurity of the dinucleoside phosphorothioates was as
high as that obtained by the previously reported synthesis on
solid-support (>99:1).
0.517 g (0.47 mmol, 94%) of 5a was obtained as a colorless foam
from 0.375 g (0.50 mmol) of 4. 1H NMR (300 MHz, CDCl3)
9.51 (s,
d
1H, 3-H of thymine), 7.62 (s, 1H, 6-H of thymine), 7.41–7.24 (m, 9H,
3
meta to OCH3, ArH of DMTr), 6.84 (d, JHH = 8.1 Hz, 4H, ortho to
3
OCH3), 6.47 (t, JHH = 7.1 Hz, 1H, 10-H), 5.49 (s, 1H, 30-H), 4.18 (t,
3JHH = 6.5 Hz, 2H, OCH2), 4.15 (s, 1H, 40-H), 3.79 (s, 6H, OCH3 of
DMTr), 3.48 (s, 2H, 50-H), 2.66 (s, 4H, CH2 of succinate), 2.48–2.44
(m, 2H, 20-H), 2.27–2.10 (m, 2H, CF2CH2), 2.01–1.94 (m, 2H,
CF2CH2CH2), 1.37 (s, 3H, 5-CH3). 13C NMR (75 MHz, CDCl3)
d 172.2,
172.0, 164.2, 159.0, 151.0, 144.4, 135.6, 135.5, 135.3, 130.3, 130.3,
128.4, 128.3, 127.4, 122–104 (indistinguishable cluster of peaks
due to CFx groups), 113.5, 112.0, 87.4, 84.5, 84.2, 76.2, 64.0, 63.6,
55.4, 38.1, 29.2, 28.9, 28.0 (t, 2JCF = 22.5 Hz), 20.1, 11.8. ESI-HRMS:
m/z calcd for C46H42F17N2O10 [(M+H)+] 1105.2563, found
+
3. Conclusion
1105.2550.
In conclusion, diastereopure dinucleoside phosphorothioates
4.4. Compound 5b (Rf = (CH2)3C6F13
)
were synthesized in
a stereocontrolled manner by using
thymidine derivatives bearing a simple perfluoroalkyl tag at
the 30-end and the diastereopure nucleoside 30-O-oxazapho-
spholidine monomers. The thymidine derivatives having a C6F13
or a C8F17 group attached to the 30-OH via a succinate linker
have sufficient solubility in organic solvents and affinity to
fluorous silica gel so that they can be used for syntheses in
homogeneous media and the intermediates of the synthesis can
be purified by a simple FSPE procedure. Further studies on the
applicability of this method to the synthesis of oligodeoxyr-
ibonucleoside phosphorothioates are in progress.
0.486 g (0.48 mmol, 81%) of 5b was obtained as a colorless foam
from 0.449 g (0.60 mmol) of 4. 1H NMR (300 MHz, CDCl3)
9.39 (s,
d
1H, 3-H of thymine), 7.62 (s, 1H, 6-H of thymine), 7.40–7.25 (m, 9H,
3
meta to OCH3, ArH of DMTr), 6.84 (d, JHH = 7.8 Hz, 4H, ortho to
3
OCH3), 6.47 (t, JHH = 7.1 Hz, 1H, 10-H), 5.49 (s, 1H, 30-H), 4.19 (t,
3JHH = 6.5 Hz, 2H, OCH2), 4.15 (s, 1H, 40-H), 3.79 (s, 6H, OCH3 of
DMTr), 3.48 (s, 2H, 50-H), 2.67 (s, 4H, CH2 of succinate), 2.49–2.44
(m, 2H, 20-H), 2.27–2.10 (m, 2H, CF2CH2), 2.02–1.91 (m, 2H,
CF2CH2CH2), 1.36 (s, 3H, 5-CH3). 13C NMR (75 MHz, CDCl3)
d 172.3,
172.1, 164.4, 159.0, 151.1, 144.4, 135.6, 135.5, 135.3, 130.3, 128.4,
128.3, 127.4, 122–106 (indistinguishable cluster of peaks due to
CFx groups), 113.5, 112.0, 87.4, 84.5, 84.2, 76.2, 64.0, 63.6, 55.4,
4. Experimental
2
38.0, 29.2, 28.9, 28.0 (t, JCF = 22.4 Hz), 20.1, 11.8. ESI-HRMS: m/z
4.1. General
calcd for C44H42F13N2O10+ [(M+Na)+] 1027.2446, found 1027.2436.
All NMR spectra were recorded on a Varian Mercury 300. 1H
NMR spectra were obtained at 300 MHz with tetramethylsilane
4.5. Compound 5c (Rf = (CH2)3C4F9)
(TMS) (
d
0.0) as an internal standard in CDCl3. 13C NMR spectra
0.202 g (0.22 mmol, 75%) of 5c was obtained as a colorless foam
from 0.224 g (0.30 mmol) of 4. 1H NMR (300 MHz, CDCl3)
d 9.56 (s,
were obtained at 75 MHz with CDCl3 as an internal standard (
d
77.0) in CDCl3 or with pyridine-d5 as an internal standard (
d
1H, 3-H of thymine), 7.62 (s, 1H, 6-H of thymine), 7.40–7.24 (m, 9H,
meta to OCH3, ArH of DMTr), 6.84 (d, JHH = 7.8 Hz, 4H, ortho to
3
123.5) in pyridine-d5. 31P NMR spectra were obtained at
121.5 MHz with 85% H3PO4
(
d
0.0) as an external standard in
OCH3), 6.47 (t, 3JHH = 7.2 Hz, 1H, 10-H), 5.49 (s, 1H, 30-H), 4.20–4.14
(m, 3H, 40-H, OCH2), 3.79 (s, 6H, OCH3 of DMTr), 3.47 (s, 2H, 50-H),
2.66 (s, 4H, CH2 of succinate), 2.48–2.44 (m, 2H, 20-H), 2.27–2.09
(m, 2H, CF2CH2), 2.01–1.92 (m, 2H, CF2CH2CH2), 1.36 (s, 3H, 5-CH3).
CDCl3. ESI mass spectra were recorded on an Applied Biosystems
QSTAR. Dry organic solvents were prepared by appropriate
procedures. The other organic solvents were reagent grade and
used as received. Silica gel column chromatography was carried
13C NMR (75 MHz, CDCl3)
d 172.0, 171.7, 163.7, 158.7, 150.5, 144.1,
out using Kanto silica gel 60N (spherical, neutral, 63–210
m
mm
m).
135.3, 135.2, 135.0, 130.1, 128.1, 128.0, 127.2, 122–106 (indistin-
guishable cluster of peaks due to CFx groups), 113.2, 111.7, 87.2,
84.2, 83.9, 76.0, 63.7, 63.3, 55.2, 37.8, 29.0, 28.7, 27.6 (t,
FSPE was carried out by using FluoroFlash1 Silica Gel, 40
(Fluorous Technologies, Inc.).
2JCF = 22.4 Hz), 19.8, 11.5. ESI-HRMS: m/z calcd for C42H42F9N2O10
+
4.2. General procedure for synthesis of 5a–c
[(M+H)+] 905.2690, found 905.2671.
Compound 4 (0.375 g, 0.50 mmol) was dried by repeated
coevaporations with dry pyridine and dissolved in dry dichlor-
omethane (5.0 mL) under Ar. Dry pyridine (0.12 mL, 1.5 mmol),
4.6. Compound 6a (Rf = (CH2)3C8F17)
6 vol% DCA solution in dichloromethane (25 mL) was added to a
stirred solution of compound 5a (0.517 g, 0.47 mmol) in dichlor-
omethane (25 mL) at rt. The mixture was then stirred for 20 min at
rt and washed with saturated NaHCO3 aqueous solutions
(3 ꢀ 30 mL). The aqueous layers were combined and back-
extracted with dichloromethane (2 ꢀ 20 mL). The organic layers
were combined, dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography [2.2 cm id, 15 g of silica gel, dichlor-
omethane–methanol (100:0 then 100:3, v/v)] to afford 6a (0.358 g,
3-(perfluoroalkyl)propanol (perfluoroalkyl = C8F17
,
C6F13 or
C4F9) (0.55 mmol), and MNTP (0.537 g, 1.2 mmol) were added,
and the mixture was stirred for 12 h at rt. The mixture was then
diluted with CHCl3 (30 mL) and washed with saturated NaHCO3
aqueous solutions (3 ꢀ 20 mL). The aqueous layers were
combined and back-extracted with CHCl3 (2 ꢀ 10 mL). The
organic layers were combined, dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography [2.7 cm id, 27 g of silica
gel, dichloromethane–methanol–pyridine (100:0:0.5 then
100:1:0.5, v/v/v)] to afford 5a, b or c.
0.45 mmol, 95%) as a colorless solid. 1H NMR (300 MHz, CDCl3)
8.13 (s, 1H, 3-H of thymine), 7.49 (s, 1H, 6-H of thymine), 6.23
d

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N. Oka et al. / Journal of Fluorine Chemistry 150 (2013) 85–91
3
3
(t, JHH = 7.1 Hz, 1H, 10-H), 5.39 (s, 1H, 30-H), 4.20 (t, JHH = 6.0 Hz,
2H, OCH2), 4.11 (s, 1H, 40-H), 3.93 (s, 2H, 50-H), 2.67 (s, 4H, CH2 of
succinate), 2.45–2.40 (m, 2H, 20-H), 2.22–2.11 (m, 2H, CF2CH2),
2.00–1.97 (m, 2H, CF2CH2CH2), 1.93 (s, 3H, 5-CH3). 13C NMR
30-O-oxazaphospholidine derivative 7t (75.5 mg, 0.10 mmol),
which was dried in vacuo overnight, and a 0.3 M solution of
CMPT 8 (0.5 mL), which was dried over MS3A overnight, were
successively added, and the mixture was stirred for 20 min at rt
(75 MHz, pyridine-d5)
d
172.5, 172.3, 164.8, 151.9, 136.1, 120–106
under Ar. N-(Trifluoroacetyl)imidazole (17.0
mL, 0.15 mmol) and i-
(indistinguishable cluster of peaks due to CFx groups), 110.9, 86.0,
84.9, 76.6, 63.4, 62.4, 37.8, 29.5, 29.2, 27.7 (t, 2JCF = 21.9 Hz), 20.2,
12.7. ESI-HRMS: m/z calcd for C25H24F17N2O8+ [(M+H)+] 803.1256,
found 803.1209.
Pr2NEt (44.0 L, 0.25 mmol) were added and the mixture was
m
stirred for 30 min at rt. Then DTD (31.9 mg, 0.15 mmol) was added
and the mixture was stirred for 50 min at rt. The mixture was then
concentrated under reduced pressure, and the residue was
dissolved in dichloromethane (5.0 mL). A 6 vol% DCA solution in
dichloromethane (5.0 mL) and Et3SiH (1.2 mL, 7.5 mmol) were
added and the mixture was stirred for 20 min at rt. The mixture
was washed with saturated NaHCO3 aqueous solutions
(3 ꢀ 10 mL). The aqueous layers were combined and back-
extracted with dichloromethane (2 ꢀ 10 mL). The organic layers
were combined, dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by FSPE [1.6 cm
column id, 4.8 g of fluorous silica gel, methanol–H2O 16 mL (80:20,
v/v) then THF 25 mL] to give 14t, a, c or g as a colorless foam, which
was analyzed by 31P NMR (Fig. 2). The 14t, a, c or g thus obtained
was dissolved in ethanol (8.0 mL) and a 25% NH3 aqueous solution
(40 mL) was added. The mixture was put in a sealed flask and
heated at 55 8C for 12 h while stirring. The mixture was then cooled
to rt, concentrated under reduced pressure to ca. 20 mL The
mixture was diluted with a 0.1 M ammonium acetate buffer (pH
7.0) (20 mL) and washed with CHCl3 (4 ꢀ 20 mL). The aqueous
layer was then concentrated under reduced pressure. The residue
was repeatedly lyophilized from distilled H2O to remove ammo-
nium acetate to give crude the dinucleoside phosphorothioate
(15t, a, c or g), which was analyzed by RP-HPLC [Senshu Pak
PEGASIL ODS, 4 ꢀ 150 mm, linear gradient of 0–20% MeCN
(60 min) in 0.1 M triethylammonium acetate buffer (pH 7.0),
50 8C, 0.5 mL/min]. Authentic samples of 15t,a,c,g were synthe-
sized via the solid-phase synthesis using the nucleoside 30-O-
oxazaphospholidines 7t,a,c,g as monomer units [8d].
4.7. Compound 6b (Rf = (CH2)3C6F13
)
6 vol% DCA solution in dichloromethane (25 mL) was added to a
stirred solution of compound 5b (0.486 g, 0.48 mmol) in dichlor-
omethane (25 mL) at rt. The mixture was then stirred for 20 min at
rt and washed with saturated NaHCO3 aqueous solutions
(3 ꢀ 50 mL). The aqueous layers were combined and back-
extracted with dichloromethane (30 mL). The organic layers were
combined, dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography [2.2 cm id, 17 g of silica gel, dichloromethane–
methanol (100:0 then 100:2.5, v/v)] to afford 6b (0.325 g,
0.46 mmol, 95%) as a colorless solid. 1H NMR (300 MHz, CDCl3)
d
9.82 (s, 1H, 3-H of thymine), 7.61 (s, 1H, 6-H of thymine), 6.28 (t,
3JHH = 7.1 Hz, 1H, 10-H), 5.40 (s, 1H, 30-H), 4.20 (t, 3JHH = 6.0 Hz, 2H,
OCH2), 4.11 (s, 1H, 40-H), 3.91 (s, 2H, 50-H), 3.36 (s, 1H, OH), 2.68 (s,
4H, CH2 of succinate), 2.48–2.40 (m, 2H, 20-H), 2.26–2.11 (m, 2H,
CF2CH2), 2.03–1.93 (m, 2H, CF2CH2CH2), 1.91 (s, 3H, 5-CH3). 13C
NMR (75 MHz, CDCl3)
d 172.4, 172.3, 164.5, 151.0, 136.8, 124–104
(indistinguishable cluster of peaks due to CFx groups), 111.5, 86.1,
85.3, 75.5, 63.6, 62.6, 37.4, 29.1, 28.9, 27.9 (t, 2JCF = 22.5 Hz), 20.1,
12.7. ESI-HRMS: m/z calcd for C23H24F13N2O8+ [(M+H)+] 703.1320,
found 703.1308.
4.8. Stereocontrolled synthesis of dithymidine phosphorothioate
triester intermediate 13t
The thymidine 30-O-oxazaphospholidine derivative 7t (75.5 mg,
0.10 mmol) was dried by coevaporation with dry MeCN (3 mL)
under Ar and dissolved in a 0.3 M solution of CMPT 8 (0.5 mL),
which was dried over MS3A overnight. The resultant solution was
Acknowledgments
We thank Professor Kazuhiko Saigo (Kochi University of
Technology) for helpful suggestions and Ms. Sakie Hirochi for
her skilled technical assistance. This research was supported by
grants from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) Japan.
added to the compound 6a (40.1 mg, 50
mmol), which was dried by
coevaporation with dry MeCN prior to use, and the resultant
mixture was stirred for 20 min at rt under Ar. N-(Trifluoroacety-
l)imidazole (17.0
mL, 0.15 mmol) and i-Pr2NEt (44.0 mL,
0.25 mmol) were added and the mixture was stirred for 30 min
at rt. Then DTD (31.9 mg, 0.15 mmol) was added and the mixture
was stirred for 50 min at rt. The mixture was then concentrated
under reduced pressure, and the residue was dissolved in
dichloromethane (5.0 mL). A 6 vol% DCA solution in dichloro-
methane (5.0 mL) and Et3SiH (1.2 mL, 7.5 mmol) were added and
the mixture was stirred for 20 min at rt. The mixture was washed
with saturated NaHCO3 aqueous solutions (3 ꢀ 10 mL). The
aqueous layers were combined and back-extracted with dichlor-
omethane (2 ꢀ 10 mL). The organic layers were combined, dried
over Na2SO4, filtered and concentrated under reduced pressure.
The residue was purified by FSPE [1.6 cm column id, 4.8 g of
fluorous silica gel, methanol–H2O 16 mL (80:20, v/v) then THF
25 mL] to give 13t (73.3 mg, colorless foam), which was analyzed
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