Hydroxyalkylated phosphoramidate, phosphoramidothioate and phosphorodiamidothioate derivatives as thiophosphate protecting groups in the development of thermolytic DNA prodrugs

Article abstract of DOI:10.1039/b9nj00692c

The hydroxyalkylated phosphoramidate 4a, phosphoramidothioates 4b, 4e-j, and phosphorodiamidothioates 4c and 4d have been identified as a new class of heat-sensitive thiophosphate protecting groups in the development of thermolytic immunomodulatory DNA prodrugs. These alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives 6a-j, which are then used in the preparation of the thermosensitive dinucleoside phosphorothioates 7a-j. The negatively charged thiophosphate protecting groups of 7a-b and 7e-j presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially neutral conditions. The thiophosphate protecting groups of 7e and 7f show relatively rapid deprotection kinetics at 37 °C (t_1/2 = 20 and 42 h, respectively) and are therefore suitable for the protection of phosphodiester functions flanking the CpG motifs of immunomodulatory DNA sequences, whereas the thiophosphate protecting groups of 7g-j with thermolytic deprotection half-lives in the range of 94-265 h at 37 °C are more appropriate for the thiophosphate protection of CpG motifs. Furthermore, the thermostability of the group protecting the thiophosphate function of 7a (t _1/2 = 82 min at 90 °C) should offer adequate protection of the 5'- and/or 3'-terminal phosphodiester functions of DNA prodrugs against ubiquitous extracellular and intracellular exonucleases.

Full text of DOI:10.1039/b9nj00692c

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Hydroxyalkylated phosphoramidate, phosphoramidothioate and
phosphorodiamidothioate derivatives as thiophosphate protecting
groups in the development of thermolytic DNA prodrugswz
a
a
b
a
´
Andrzej Grajkowski, Jacek Cieslak, Alexei Gapeev and Serge L. Beaucage*
Received (in Montpellier, France) 19th November 2009, Accepted 6th January 2010
First published as an Advance Article on the web 23rd February 2010
DOI: 10.1039/b9nj00692c
The hydroxyalkylated phosphoramidate 4a, phosphoramidothioates 4b, 4e–j, and
phosphorodiamidothioates 4c and 4d have been identified as a new class of heat-sensitive
thiophosphate protecting groups in the development of thermolytic immunomodulatory DNA
prodrugs. These alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives
6a–j, which are then used in the preparation of the thermosensitive dinucleoside
phosphorothioates 7a–j. The negatively charged thiophosphate protecting groups of 7a–b and 7e–j
presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially
neutral conditions. The thiophosphate protecting groups of 7e and 7f show relatively rapid
1
deprotection kinetics at 37 1C (t = 20 and 42 h, respectively) and are therefore suitable for the
protection of phosphodiester functions flanking the CpG motifs of immunomodulatory DNA
2
sequences, whereas the thiophosphate protecting groups of 7g–j with thermolytic deprotection
half-lives in the range of 94–265 h at 37 1C are more appropriate for the thiophosphate protection
of CpG motifs. Furthermore, the thermostability of the group protecting the thiophosphate
0
1
2
function of 7a (t = 82 min at 90 1C) should offer adequate protection of the 5 - and/or
30-terminal phosphodiester functions of DNA prodrugs against ubiquitous extracellular and
intracellular exonucleases.
consists of masking the phosphodiester functions of DNA
Introduction
sequences with thermosensitive phosphate/thiophosphate
protecting groups. Such a modification eliminates the require-
ment for esterases activity; only an aqueous environment at
elevated temperature (430 1C) is necessary to thermolytically
convert oligonucleoside phosphotriesters to bioactive oligo-
nucleoside phosphodiesters. This approach to the preparation
of DNA prodrugs allows the selection of phosphate/
thiophosphate protecting groups with differential thermo-
sensitivity properties in order to optimize cellular uptake of
the prodrugs through better control of their lipophilic and
hydrophilic attributes.
The polyanionic nature of DNA or RNA sequences is known
to negatively affect the cellular uptake of these biomolecules,
thereby limiting their abilities to modulate the expression of
therapeutically relevant proteins through either an antisense or
an RNA interference pathway. An additional complication
stemming from negatively charged DNA or RNA sequences,
is their vulnerability to ubiquitous extracellular and intra-
cellular nucleases that are present in biological environments.
In order to mitigate these shortcomings, the concept of
oligonucleotide prodrugs has been proposed1 and has evolved
over the years as a viable approach to facilitate cellular uptake
of DNA sequences while imparting these with increased
stability to extracellular and intracellular nucleases. The
prodrug approach consists of converting the negatively
charged phosphodiester groups of DNA sequences to neutral
acylated or S-acylated phosphotriester functions,2–4 which
upon cellular entry, are hydrolyzed by intracellular esterases
to their bioactive phosphodiester state. A modification of the
original prodrug approach has been proposed by us5 and
When applied in the context of immunomodulatory single-
stranded DNA oligonucleoside phosphorothioates containing
unmethylated CpG motifs (type K CpG ODNs),6 oligonucleoside
phosphorothioates with thermolytic 2-(N-formyl-N-methyl)-
aminoethyl (FMA) thiophosphate protection induced an
immunostimulatory response in mice similar to that generated
from the administration of a conventional CpG ODN.5
These findings prompted us to identify thermosensitive thio-
phosphate protecting groups, which by virtue of the range of
their deprotection rates at 37 1C, should generate bioactive
CpG ODNs in a time-dependent manner and produce an
immunostimulatory response in animal models lasting longer
than that obtained with conventional CpG ODNs.
a Division of Therapeutic Proteins, Center for Drug Evaluation and
Research, Food and Drug Administration, Bethesda, Maryland,
USA. E-mail: serge.beaucage@fda.hhs.gov; Fax: +1 301-480-3256;
Tel: +1 301-827-5162
b Department of Chemistry and Biochemistry, University of Maryland,
Baltimore County, Baltimore, Maryland, USA
We recently investigated thermolytic thiophosphate protecting
groups derived from 2-(N-formyl-N-methyl)aminoethanol
and 4-(methylthio)butan-1-ol;7 the majority of these groups
revealed deprotection rates that were either faster or
w This article is dedicated to Professor Wojciech J. Stec on the
occasion of his 70th birthday.
z This article is part of a themed issue on Biophosphates.
ꢀc
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considerably slower than that of the FMA group at 37 1C
after removal of the 4,4-dimethoxytrityl (DMTr) group under
1
2
(t = 72 h). The thermolytic groups with relatively faster
acidic conditions and purification by chromatography on silica
gel, the hydroxyalkylated phosphoramidate 4a or the
phosphoramidothioate 4b in yields of 80% and 84%, respec-
tively. The hydroxyalkylated phosphoramidothioates 4e and
4h were similarly prepared from the protected alcohols 2 and
3, respectively, and were isolated in similar yields after
silica gel chromatography. The replacement of 2-cyanoethyl
(N,N-diisopropyl)phosphoramidochloridite with either 2-cyano-
ethyl (N,N-diethyl)phosphoramidochloridite12 or 2-cyanoethyl
(N-morpholinyl)phosphoramidochloridite13 under the condi-
tions used for the synthesis of 4b afforded the hydroxy-
alkylated phosphoramidothioates, 4f, 4g, 4i and 4j, the yields
of which were in the range of 79–83%. The hydroxyalkylated
phosphorodiamidothioate derivatives 4c and 4d were obtained
from the reaction of alcohol 1 with either bis(N,N-diisopropyl-
amino)chlorophosphine or bis(N,N-diethylamino)chlorophosphine
under the conditions employed for the preparation of 4b
(Scheme 1). The hydroxyalkylated phosphorodiamidothioate
derivatives 4c and 4d were isolated after purification by
chromatography on silica gel in yields comparable to those
of 4a and 4b. The identity of the alcohols 4a–j was confirmed
deprotection kinetics may be used for the thiophosphate
protection of immunomodulatory CpG DNA motifs and/or
that of their flanking DNA sequences, whereas those with
exceedingly slow deprotection rates offer DNA prodrugs with
adequate protection of terminal phosphodiester functions
against exonucleases that may be found in biological systems.7
In order to better assess the biological consequences of
sustained CpG ODN immunostimulation in animal models,
it would be desirable to identify thiophosphate protecting
groups with thermolytic deprotection half-lives in the range
of 100–200 h at 37 1C. On the basis of our earlier work on the
application of
a 3-hydroxypropyl phosphoramidothioate
function to the thermosensitive release of DNA sequences
from controlled-pore glass under near neutral conditions,8
we are now reporting the results of our investigations regarding
the thermolytic properties of hydroxyalkylated phosphoramidate,
phosphoramidothioate and phosphorodiamidothioate deriva-
tives as thiophosphate protecting groups, as well as their
potential application to the development of thermosensitive
immunomodulatory CpG ODN prodrugs.
1
by H, 13C and 31P NMR spectroscopy.
The deoxyribonucleoside phosphoramidites 6a–j (Scheme 2)
were prepared from the reaction of 50-O-(4,40-dimethoxytrityl)-
30-O-bis(N,N-diisopropylamino)phosphinyl-20-deoxythymidine
(5)8 with equimolar amounts of any of the alcohols 4a–j in
dry MeCN and solid 1H-tetrazole, which was added to the
solution in small portions over a period of 1 h at ambient
temperature. The crude phosphoramidites 6a–j were purified
by chromatography on silica gel that was equilibrated in
benzene containing Et3N (10% v/v) to prevent premature
activation of 6a–j during purification. This solution was
also used to elute 6a–j from the chromatography column.
Complete removal of residual Et3N from purified phosphor-
amidites by lyophilization from dry benzene under high
vacuum is critically important for optimal coupling of 6a–j
in the solid-phase synthesis of the dinucleoside phosphorothioate
triesters 7a–j (Scheme 3). The identity of phosphoramidites
6a–j was determined by 31P NMR spectroscopy and by
high resolution mass spectrometry, which also confirmed the
identity of 4a–j.
Results and discussion
The synthesis of hydroxyalkylated phosphoramidate, phosphor-
amidothioate and phosphorodiamidothioate derivatives (4a–j)
began with the preparation the protected alcohols 1–3
(Scheme 1) according to published literature procedures.9–11
The reaction of 1 with 2-cyanoethyl (N,N-diisopropyl)-
phosphoramidochloridite (1.1 molar equiv.) and N,N-diiso-
propylethylamine (5 molar equiv.) in CH2Cl2 was followed by
oxidation of the resulting phosphoramidite intermediate with
either tert-butyl hydroperoxide or elemental sulfur to give,
The synthesis of 7a–j was performed by the reaction of
any of the phosphoramidites 6a–j with the 50-hydroxy function
of deoxythymidine that is covalently attached to long
chain alkylamine controlled-pore glass (CPG) through a
30-O-hemisuccinate linker (TsuccCPG, Scheme 3). This
reaction proceeded smoothly in the presence of 1H-tetrazole
in dry MeCN to produce a phosphite triester intermediate,
Scheme 1
Scheme 2
ꢀc
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Scheme 3
which was oxidized to the corresponding dinucleoside
phosphorothioate derivative by treatment with 3H-1,2-benzo-
dithiol-3-one-1,1-dioxide in MeCN. Removal of the 50-DMTr
protecting group under acidic conditions was followed
by the cleavage of the 2-cyanoethyl group from the
phosphoramidate/phosphoramidothioate function and release
of 7a–j from CPG upon exposure to pressurized MeNH2 gas
(Scheme 3). Each of the dinucleoside phosphorothioate
triesters 7a–j was purified by reversed-phase (RP) HPLC
prior to its thermolytic conversion to the dinucleoside
phosphorothioate diester TpsT in phosphate-buffered saline
(PBS, pH 7.4) at 37 1C or 90 1C.
Table 1 Thermolytic deprotection rates of the dinucleoside
phosphorothioate triester derivatives 7a–j to TpsT
7a–j - TpsT
1
2
1
2
Triester derivative
t (90 1C) [min]
t (37 1C) [h]
7a
7b
7c
7d
7e
7f
7g
7h
7i
60
B1b
20
72
5
10
13
30
36
49
NDa
2.5
NAc
NA
20
42
94
135
245
265
The thermolytic conversion of dinucleoside phosphotriester
models to TpsT rather than to TpT is preferred for assessing
the deprotection rates of thermosensitive protecting groups
because S-alkylation of the internucleoside thiophosphate
function, if occurring during the thermolytic deprotection
reaction, can be easily monitored by RP-HPLC. S-Alkylation
of the internucleoside phosphorothioate diester by deprotection
side products typically results in its hydrolytic desulfurization
and formation of TpT along with other minor degradation
products. Since the structural integrity of phosphorothioate
diester groups is critically important for safeguarding the
immunostimulatory properties of type K6 and type D6,14
CpG ODNs by providing resistance to the nucleolytic activities
of extracellular and intracellular nucleases, S-alkylation and
desulfurization of phosphorothioate diester functions must be
avoided.
7j
a
b
Not determined. The deprotection rate is too rapid to be accurately
c
determined. Not applicable.
from 7b was too rapid under the same thermolytic conditions
1
2
(t = B1 min at 90 1C or 2.5 h at 37 1C) to be of practical use
in the development of CpG ODN produgs.
As shown in Scheme 4, the thermolytic deprotection of 7b
followed a cyclodeesterification pathway.5,7,15 31P NMR
analysis of the deprotection reaction conducted at 37 1C in
0.1 M triethylammonium acetate (TEAA, pH 7) revealed the
formation of TpsT (dP 54 ppm) and that of 8 (dP 48 ppm) as a
The thermolytic thiophosphate deprotection of RP-HPLC
purified 7a in PBS (pH 7.4) to give TpsT occurred with a
half-life of 60 min at 90 1C (Table 1). The deprotection rate of
the 2-[O-(N,N-diisopropylphosphoramido)]ethyl group would
definitely be too slow at 37 1C for thiophosphate protection of
the CpG motifs and adjacent flanking sequences of CpG ODN
prodrugs. This group may nonetheless be useful in the
protection of terminal 50- and/or 30-thiophosphate functions
of oligonucleotide prodrugs. Conversely, the cleavage of
the 2-[O-(N,N-diisopropylphosphoramidothioyl)]ethyl group
Scheme 4
ꢀc
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the immunostimulatory function of CpG ODN prodrugs
in vivo. Further investigations are therefore necessary to
characterize these side products and assess their immuno-
stimulatory properties, if any, in animal models.
An expansion of the five-membered cyclic transition state
operating in the thermal thiophosphate deprotection of 7b to a
six-membered cyclic transition state decreased, as expected,
the thermal thiophosphate deprotection of 7e, which occurred
with a half-life of 5 min at 90 1C or 20 h at 37 1C (Table 1).
Because of the relatively rapid thermolytic cleavage of the
negatively charged 3-[O-(N,N-diisopropylphosphoramido-
thioyl)]prop-1-yl group from 7e, this functional group may
find application in the thiophosphate protection of the DNA
sequences flanking the CpG motif of CpG ODN prodrugs and
provide increased aqueous solubility to lipophilic oligo-
nucleotide prodrugs.7 As discussed above, replacement of the
3-[O-(N,N-diisopropylphosphoramidothioyl)]prop-1-yl group
in 7e with a 3-[O-(N,N-diethylphosphoramidothioyl)]prop-1-yl
group gave 7f, which underwent thermolytic thiophosphate
Fig. 1 31P NMR analysis of the thermolytic conversion of RP-HPLC
purified 7b to TpsT and 8 in 0.1 M TEAA (pH 7). (A) Spectrum of 7b
after being heated at 37 1C for 2.5 h. (B) Spectrum of the reference
standard TpsT in 0.1 M TEAA (pH 7). (C) Spectrum of 7b after being
heated at 37 1C for 20 h. (D) Spectrum of synthetic 8 in 0.1 M TEAA
(pH 7).
1
deprotection at a slower rate (t = 10 min at 90 1C or 42 h at
2
37 1C) than 7e under identical conditions (Table 1). These
findings are consistent with the thermal thiophosphate depro-
cyclodeesterification side product (Fig. 1). The identity of 8
was corroborated by its chemical synthesis, which was carried
out by the oxidation of 2-(N,N-diisopropylamino)-1,3,2-
oxathiaphospholane16 with tert-butyl hydroperoxide, and
through spiking experiments (data not shown). It is therefore
likely that the thermolytic thiophosphate deprotection of 7e–7j
proceeds generally through a cyclodeesterification process
with the concomitant formation of cyclic phosphoramidothioate
side products.
1
tection rate of 7g, which was found slower (t = 13 min at
2
90 1C or 94 h at 37 1C) than that of 7f. These results suggest
that the inductive effect of the oxygen atom in the morpholine
ring of 7g had negatively affected the deprotection rate of the
3-[O-(N-morpholinophosphonothioyl)]prop-1-yl group and
thus further support the significance of electronic effects in
the thermolytic cyclodeesterification of these thiophosphate
protecting groups. The negatively charged 3-[O-(N,N-diethyl-
phosphoramidothioyl)]prop-1-yl and 3-[O-(N-morpholino-
phosphonothioyl)]prop-1-yl groups can be used for
thiophosphate protection of the CpG motif of CpG ODN
prodrugs.
In an effort to slow down the thiophosphate deprotection of
7b, the 2-[O-bis(N,N-diisopropylphosphorodiamidothioyl)]-
ethyl group for thiophosphate protection was then investigated.
The uncharged bis(N,N-diisopropylphosphorodiamidothioyl)
function of 7c is significantly less nucleophilic than the
negatively charged N,N-diisopropylphosphoramidothioyl
functionality of 7b and should therefore slow down thio-
phosphate deprotection rates. Indeed, the thiophosphate
deprotection of 7c proceeded with a half-life of 20 min at
90 1C (Table 1). RP-HPLC analysis of the deprotection
reaction showed a clean conversion of 7c to TpsT at 90 1C,
but not at 37 1C; in addition to TpsT, new unidentified peaks
with longer retention times were also produced. Replacement
of the bis(N,N-diisopropylphosphorodiamidothioyl) function
in 7c with a bis(N,N-diethylphosphorodiamidothioyl) function
gave 7d. The thermolytic thiophosphate deprotection kinetics
of 7d should provide information on the steric effect of the
diisopropyl groups on the rates of deprotection. Surprisingly,
An expansion of the six-membered cyclic transition state
taking place in the thermal thiophosphate deprotection of 7e
was necessary to decrease its deprotection rate. Thus, replace-
ment of the negatively charged 3-[O-(N,N-diisopropyl-
phosphoramidothioyl)]prop-1-yl group in 7e with the negatively
charged 4-[O-(N,N-diisopropylphosphoramidothioyl)]but-1-yl
group led to 7h, which under thermolytic conditions produced
TpsT with a half-life of 30 min at 90 1C or 135 h at 37 1C
(Table 1). When the 4-[O-(N,N-diethylphosphoramidothioyl)]-
but-1-yl or 4-[O-(N-morpholinophosphonothioyl)]but-1-yl
group was used for thiophosphate protection of 7i or 7j,
respectively, the thermolytic cleavage of these protecting
groups to TpsT proceeded with respective half-lives of
36 min at 90 1C and 245 h at 37 1C or 49 min at 90 1C and
265 h at 37 1C (Table 1). These groups are also suitable for
thiophosphate protection of the CpG motif of CpG ODN
prodrugs.
1
the rate of thermal deprotection of 7d was slower (t = 72 min)
2
than that of 7c by a factor greater than 3 at 90 1C (Table 1).
These findings suggest that electronic rather than steric factors
were predominant in the transition state of the thiophosphate
deprotection reaction. RP-HPLC analysis of the thermal
thiophosphate deprotection of 7d also indicated that TpsT
was exclusively formed at 90 1C only. On the basis of these
results, the use of bis(N,N-dialkylphosphorodiamidothioyl)-
ethyl groups as thiophosphate protecting groups for CpG
ODN prodrugs must be avoided; the formation of unexpected
side products at 37 1C may interfere with the interpretation of
Conclusion
In this report, many of the thiophosphate protecting groups
listed in Table 1 have shown thermolytic deprotection half-
lives in the range of 94 h to 265 h at 37 1C. These deprotection
rates are complementary to those of thiophosphate protecting
groups identified earlier,7 the thermolytic deprotection half-lives
ꢀc
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of which were in the range of 6 h to 40 h at 37 1C.
Altogether, these heat-sensitive groups are likely to protect
adequately the thiophosphate functions of CpG motifs and
those of adjacent DNA sequences in immunomodulatory CpG
ODN prodrugs. The judicious selection of uncharged and
negatively charged phosphate/thiophosphate protecting
groups, in proper ratios, should provide aqueous solubility
to lipophilic oligonucleotide prodrugs and enhance their
pharmacokinetics and pharmacodynamic properties in vivo.
Phosphate/thiophosphate protecting groups with deprotection
half-lives exceeding 300 h7 at 37 1C should be useful for the
protection of terminal 50- and 30-phosphodiester functions of
CpG ODN prodrugs and increase the resistance of these
biomolecules to pervasive nucleases. Furthermore, in the
context of immunomostimulatory CpG ODN prodrugs, a
library of CpG motifs modified with thermolytic phosphate/
thiophosphate groups is being prepared to evaluate the
correlation between extended immunostimulation and resis-
tance to viral and/or bacterial infections in animal models.
As discussed earlier,7 thermosensitive groups have also been
applied to the protection of the 50-hydroxyl17 and exocyclic
amino18 functions of deoxyribonucleosides, thereby supporting
the potentially general application of these groups as alcohols
and amine protecting groups. Interestingly, the incorporation
of thermolabile phosphate protecting groups into DNA
oligonucleotide primers19 prevents the premature extension
of these primers at the initial set up stages of the polymerase
chain reaction. A thermal activation step induces the cleavage
of the heat-sensitive phosphate protecting groups and
generates the corresponding unmodified DNA primers, which
then enable a clean amplification of the desired DNA target
sequences. These useful applications highlight the importance
and versatility of thermolytic groups in the development of
DNA prodrugs and DNA diagnostics.
obtained on a Bruker Daltonics Apex III FT-ICR mass
spectrometer. Electrospray ionization in positive ion mode
was used to generate [M + H]+ and [M + Na]+ ions out of
0.1 mg mLꢁ1 test sample solutions in 2-propanol–water
(1:1 v/v). Spectra were externally calibrated using a 1 mg mLꢁ1
solution of CsI, which yielded a series of peaks in the mass
range used for analysis (200–2000 m/z).
Procedures
General procedure for the preparation of hydroxyalkylated
phosphoramidate, phosphoramidothioate and phosphorodiamido-
thioate derivatives (4a–j). To a stirred solution of any of the
alcohols 1–3 (5 mmol) and dry N,N-diisopropylethylamine
(25 mmol) in anhydrous CH2Cl2 (20 mL) was added, under an
inert gas atmosphere, the appropriate phosphoramido-
chloridite (5.5 mmol). After 2 h at 25 1C, the reaction was
complete (TLC); a 5.5 M solution of tert-butyl hydroperoxide
(10 mmol) in decane or elemental sulfur (10 mmol) was added
to the reaction mixture. The tert-butyl hydroperoxide
oxidation reaction was allowed to proceed for 30 min, whereas
the sulfurization reaction was left stirring for 16 h at 25 1C.
The reaction mixture was then diluted to a final volume of
50 mL with CH2Cl2 and was washed with water (3 ꢂ 25 mL);
the organic extracts were collected, dried over anhydrous
Na2SO4, and evaporated to dryness under reduced pressure.
The material left was dissolved in 80% AcOH (30 mL); the
solution was stirred for 3 h at 25 1C and was then evaporated
to an oil under reduced pressure. The crude product was
purified by chromatography on silica gel (B30 g) using a
gradient of CH3OH (0 - 5%) in CHCl3. The alcohol deriva-
tives 4a–j were isolated in yields ranging from 79% to 86%.
O-(1-Hydroxyethyl)-O-(2-cyanoethyl)-N,N-diisopropyl phos-
phoramidate (4a). Yield: 1.10
g (80%). dH(300 MHz;
DMSO-d6; Me4Si) 4.83 (1H, br s, OH), 4.02 (2H, m,
CH2CH2OH), 3.95–3.77 (2H, m, CH2CH2OH), 3.57 (2H, t,
J = 5.8 Hz, CH2CH2CN), 3.44 (1H, hept, J = 6.8 Hz,
(CH3)2CHN), 3.37 (1H, hept, J = 6.8 Hz, (CH3)2CHN),
2.87 (2H, t, J = 5.8 Hz, CH2CH2CN), 1.16 (12H, d, J =
6.8 Hz, (CH3)2CHN); dC(75 MHz; DMSO-d6) 118.4
Experimental
Methods and materials
Common chemicals and solvents in addition to
bis(N,N-diethylamino)chlorophosphine, bis(N,N-diisopropyl-
amino)chlorophosphine and 2-cyanoethyl (N,N-diisopropyl)-
phosphoramidochloridite were purchased from commercial
suppliers and were used as received. 2-[(4,40-Dimethoxytrityl)-
oxy]ethan-1-ol (1),9 3-[(4,40-dimethoxytrityl)oxy]propan-1-ol
(2),10 4-[(4,40-dimethoxytrityl)oxy]butan-1-ol (3),11 2-cyano-
ethyl (N,N-diethyl)phosphoramidochloridite,12 2-cyanoethyl
(N-morpholinyl)phosphoramidochloridite,13 2-(N,N-diisopropyl-
amino)-1,3,2-oxathiaphospholane16 and 50-O-(4,40-dimethoxy-
trityl)-30-O-bis(N,N-diisopropylamino)phosphinyl-20-deoxy-
thymidine (5)8 were prepared as described in the literature.
Silica gel 60 (EMD, 0.040–0.063 mm) was used for chromato-
graphic purifications. All NMR experiments were carried out
using a Bruker Avance DRX 300 spectrometer operating
at fields of 300.13 MHz for 1H, 75.47 MHz for 13C, and
121.5 MHz for 31P. 1H, proton-decoupled 13C, and proton-
decoupled 31P NMR spectra were recorded in deuterated
solvent as indicated. High resolution mass spectra used to
determine the elemental composition of compounds 6a–j were
2
(CH2CH2CN), 66.7 (d, JCꢁP = 5.7 Hz, CH2CH2OH), 60.1
3
2
(d, JCꢁP = 6.9 Hz, CH2CH2OH), 60.0 (d, JCꢁP = 4.6 Hz,
2
CH2CH2CN), 45.2 (d, JCꢁP = 4.6 Hz, (CH3)2CHN), 22.1
3
3
(d, JCꢁP = 14.9 Hz, (CH3)2CHN), 19.1 (d, JCꢁP = 8.0 Hz,
CH2CH2CN); dP(121 MHz, C6D6; extern. H3PO4/D2O) 9.1.
O-(1-Hydroxyethyl)-O-(2-cyanoethyl)-N,N-diisopropyl phos-
phoramidothioate (4b). Yield: 1.25 g (84%). dH(300 MHz;
DMSO-d6; Me4Si) 4.83 (1H, br s, OH), 4.12–3.99 (2H, m,
CH2CH2OH), 3.94 (1H, m, CH2CH2OH), 3.84 (1H, m,
CH2CH2OH), 3.75 (1H, hept, J = 6.8 Hz, (CH3)2CHN),
3.68 (1H, hept, J = 6.8 Hz, (CH3)2CHN), 3.59 (2H, t, J =
5.2 Hz, CH2CH2CN), 2.89 (2H, t, J = 5.8 Hz, CH2CH2CN),
1.22 (12H, d, J = 6.8 Hz, (CH3)2CHN); dC(75 MHz; DMSO-d6)
2
118.4 (CH2CH2CN), 67.7 (d, JCꢁP = 5.7 Hz, CH2CH2OH),
2
3
60.9 (d, JCꢁP = 4.6 Hz, CH2CH2CN), 59.9 (d, JCꢁP
=
10.3 Hz, CH2CH2OH), 46.4 (d, 2JCꢁP = 4.6 Hz, (CH3)2CHN),
3
22.1 (CH3)2CH2CN), 18.8 (d, JCꢁP = 9.2 Hz, CH2CH2CN);
dP(121 MHz, C6D6; extern. H3PO4/D2O) 72.4.
ꢀc
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884 | New J. Chem., 2010, 34, 880–887

View Online
O-(1-Hydroxyethyl)-bis(N,N-diisopropyl) phosphorodiamido-
thioate (4c). Yield: 1.40 g (86%). dH(300 MHz; DMSO-d6;
Me4Si) 4.77 (1H, br s, OH), 3.91 (1H, t, J = 5.7 Hz,
CH2CH2OH), 3.88 (1H, t, J = 5.7 Hz, CH2CH2OH), 3.64
(1H, t, J = 6.0 Hz, CH2CH2CN), 3.17 (4H, m, CH2CH2N),
2.90 (1H, t, J = 6.0 Hz, CH2CH2CN), 1.76 (2H, quint, J = 6.3 Hz,
CH2CH2CH2OH); dC(75 MHz; DMSO-d6) 118.4
(CH2CH2CN), 66.1 (d, JCꢁP = 5.7 Hz, CH2CH2CH2OH),
63.9 (d, 3JCꢁP = 4.6 Hz, CH2CH2N), 61.2 (d, 2JCꢁP = 4.6 Hz,
CH2CH2CN), 56.9 (CH2CH2CH2OH), 44.8 (CH2CH2N), 32.7
2
(2H, m, CH2CH2OH), 3.61 (2H, hept,
J = 6.8 Hz,
(CH3)2CHN), 3.57 (2H, hept, J = 6.8 Hz, (CH3)2CHN),
1.25 (12H, d, J = 6.8 Hz, (CH3)2CHN), 1.21 (12H, d, J =
3
3
(d, JCꢁP = 8.0 Hz, CH2CH2CH2OH), 18.7 (d, J = 9.2 Hz,
CH2CH2CN); dP(121 MHz, C6D6; extern. H3PO4/D2O) 74.4.
6.8 Hz, (CH3)2CHN); dC(75 MHz; DMSO-d6) 65.1 (d, 2JCꢁP
=
3
4.6 Hz, CH2CH2OH), 59.9 (d, JCꢁP
= 10.3 Hz,
3
CH2CH2OH), 45.5 (d, JCꢁP = 5.7 Hz, (CH3)2CHN), 23.0
(CH3)2CHN), 21.2 (CH3)2CHN); dP(121 MHz, C6D6; extern.
H3PO4/D2O) 71.8.
O-(1-Hydroxybutyl)-O-(2-cyanoethyl)-N,N-diisopropyl phos-
phoramidothioate (4h). Yield: 1.35 g (83%). dH(300 MHz;
DMSO-d6; Me4Si) 4.44 (1H, t, J = 5.2 Hz, OH), 4.10–3.83
(4H, m, CH2CH2CH2CH2OH), 3.72 (1H, hept, J = 6.8 Hz,
(CH3)2CHN), 3.69 (1H, hept, J = 6.8 Hz, (CH3)2CHN), 3.42
(1H, t, J = 6.3 Hz, CH2CH2CN), 3.40 (1H, t, J = 6.3 Hz,
CH2CH2CN), 2.90 (2H, m, CH2CH2CN), 1.65 (2H, m,
CH2CH2CH2CH2OH), 1.48 (2H, m, CH2CH2CH2CH2OH),
1.22 (12H, d, J = 6.8, (CH3)2CHN); dC(75 MHz; DMSO-d6)
O-(1-Hydroxyethyl)-bis(N,N-diethyl) phosphorodiamido-
thioate (4d). Yield: 1.15 g (85%). dH(300 MHz; DMSO-d6)
4.74 (1H, br s, OH), 3.82 (1H, t, J = 5.4 Hz, CH2CH2OH),
3.79 (1H, t, J = 5.4 Hz, CH2CH2OH), 3.57 (2H, t, J = 5.4 Hz,
CH2CH2OH), 3.12–2.94 (8H, m, CH3CH2N), 1.04 (12H, t,
J = 7.1 Hz, CH3CH2N); dC(75 MHz; DMSO-d6) 65.8
118.4 (CH2CH2CN), 66.1 (d, 2JCꢁP
=
5.7 Hz,
2
3
CH2CH2CH2CH2OH), 60.9 (d, 2JCꢁP = 4.6 Hz, CH2CH2CN),
(d, JCꢁP = 4.6 Hz, CH2CH2OH), 60.0 (d, JCꢁP = 10.3
Hz, CH2CH2OH), 39.4 (d, 2JCꢁP = 5.7 Hz, CH3CH2N), 39.2
2
60.1 (CH2CH2CH2CH2OH), 46.4 (d, JCꢁP = 5.7 Hz,
2
3
3
(d, JCꢁP = 5.7 Hz, CH3CH2N), 13.7 (d, JCꢁP = 4.6 Hz,
(CH3)2CHN), 28.7 (CH2CH2CH2CH2OH), 26.2 (d, JCꢁP
=
3
CH3C2HN), 13.6 (d, JCꢁP
= 4.6 Hz, CH3CH2N);
9.2 Hz, CH2CH2CH2CH2OH), 22.1 (CH3)2CHN), 18.8
(d, 3JC–P = 9.2 Hz, CH2CH2CN); dP(121 MHz, C6D6; extern.
H3PO4/D2O) 72.0.
dP(121 MHz, C6D6; extern. H3PO4/D2O) 79.0.
O-(1-Hydroxypropyl)-O-(2-cyanoethyl)-N,N-diisopropyl phos-
phoramidothioate (4e). Yield: 1.3 g (84%). dH(300 MHz;
DMSO-d6; Me4Si) 4.06–3.90 (4H, m, CH2CH2CH2OH), 3.71
(1H, hept, J = 6.8 Hz, (CH3)2CHN), 3.69 (1H, hept, J =
6.8 Hz, (CH3)2CHN), 3.49 (2H, t, J = 6.3 Hz, CH2CH2CN),
2.88 (2H, t, J = 6.3 Hz, CH2CH2CN), 1.76 (2H, quint, J = 6.3 Hz,
CH2CH2CH2OH), 1.22 (12H, d, J = 6.8 Hz, (CH3)2CHN);
O-(1-Hydroxybutyl)-O-(2-cyanoethyl)-N,N-diethyl phosphor-
amidothioate (4i). Yield: 1.20
g (81%). dH(300 MHz;
DMSO-d6; Me4Si) 4.42 (1H, t, J = 5.2 Hz, OH), 4.08–3.79
(4H, m, CH2CH2CH2CH2OH), 3.42 (1H, t, J = 6.3 Hz,
CH2CH2CN), 3.40 (1H, t, J = 6.3 Hz, CH2CH2CN), 3.18
(2H, q, J = 7.1 Hz, CH3CH2N), 3.13 (2H, q, J = 7.1 Hz,
CH3CH2N), 2.88 (2H, t, J = 5.7 Hz, CH2CH2CN), 1.65 (2H, m,
CH2CH2CH2CH2OH), 1.47 (2H, m, CH2CH2CH2CH2OH),
1.06 (6H, t, J = 7.1 Hz, CH3CH2N); dC(75 MHz; DMSO-d6)
dC(75 MHz; DMSO-d6) 118.4 (CH2CH2CN), 63.5 (d, 2JCꢁP
=
2
5.7 Hz, CH2CH2CH2OH), 60.9 (d, JCꢁP
= 4.6 Hz,
2
CH2CH2CN), 57.0 (CH2CH2CH2OH), 46.4 (d, JCꢁP
5.7 Hz, (CH3)2CHN), 32.8 (d, JCꢁP
CH2CH2CH2OH), 22.1 (CH3)2CHN), 18.8 (d, JCꢁP
=
3
=
8.0 Hz,
118.3 (CH2CH2CN), 66.1 (d, 2JCꢁP
=
5.7 Hz,
3
=
CH2CH2CH2CH2OH), 60.8 (d, 2JCꢁP
=
4.6 Hz,
2
9.2 Hz, CH2CH2CN); dP(121 MHz, C6D6; extern. H3PO4/
D2O) 72.0.
CH2CH2CN), 60.1 (CH2CH2CH2CH2OH), 39.6 (d, JCꢁP
4.6 Hz, CH3CH2N), 28.6 (CH2CH2CH2CH2OH), 26.2
=
3
3
(d, JCꢁP = 9.2 Hz, CH2CH2CH2CH2OH), 18.7 (d, JCꢁP
=
O-(1-Hydroxypropyl)-O-(2-cyanoethyl)-N,N-diethyl phos-
phoramidothioate (4f). Yield: 1.15 g (81%). dH(300 MHz;
DMSO-d6; Me4Si) 4.08–3.85 (4H, m, CH2CH2CH2OH), 3.48
(2H, t, J = 6.0 Hz, CH2CH2CN), 3.17 (2H, q, J = 7.1 Hz,
CH3CH2N), 3.13 (2H, q, J = 7.1 Hz, CH3CH2N), 2.89 (2H, t,
J = 6.0 Hz, CH2CH2CN), 1.75 (2H, quint, J = 6.3 Hz,
9.2 Hz, CH2CH2CN), 14.0 (CH3CH2N); dP(121 MHz, C6D6;
extern. H3PO4/D2O) 76.0.
O-(1-Hydroxybutyl)-O-(2-cyanoethyl)-N-morpholino phos-
phonothioate (4j). Yield: 1.25 g (80%). dH(300 MHz; DMSO-d6;
Me4Si) 4.43 (1H, t, J = 5.2 Hz, OH), 4.07 (1H, t, J = 5.7 Hz,
CH2CH2CH2OH), 1.06 (6H, t, J = 7.1 Hz, CH3CH2N);
2
CH2CH2CH2CH2OH), 4.04 (1H, t,
J
=
5.7 Hz,
dC(75 MHz; DMSO-d6) 118.4 (CH2CH2CN), 63.5 (d, JCꢁP
=
2
CH2CH2CH2CH2OH), 3.93 (2H, m, CH2CH2CH2CH2OH),
3.55 (4H, t, J = 4.9 Hz, CH2CH2N), 3.42 (1H, t, J = 6.3 Hz,
OCH2CH2CN), 3.40 (1H, t, J = 6.3 Hz, CH2CH2CN), 3.17
(4H, m, CH2CH2N), 2.90 (2H, t, J = 6.3 Hz, CH2CH2CN),
1.65 (2H, m, CH2CH2CH2CH2OH), 1.47 (2H, m,
CH2CH2CH2CH2OH); dC(75 MHz; DMSO-d6) 118.3
4.6 Hz, CH2CH2CH2OH), 60.8 (d, JCꢁP
= 4.6 Hz,
3
CH2CH2CN), 57.0 (CH2CH2CH2OH), 39.6 (d, JCꢁP
=
4.6 Hz, CH3CH2N), 32.7 (d, 2JCꢁP = 8.0 Hz, CH2CH2CH2OH),
2
18.7 (d, JCꢁP = 9.2 Hz, CH2CH2CN), 14.0 (CH3CH2N);
dP(121 MHz, C6D6; extern. H3PO4/D2O) 76.0.
3
O-(1-Hydroxypropyl)-O-(2-cyanoethyl)-N-morpholino phos-
phonothioate (4g). Yield: 1.15 g (79%). dH(300 MHz; DMSO-d6;
Me4Si) 4.52 (1H, t, J = 5.2 Hz, OH), 4.07 (1H, t, J = 5.8 Hz,
CH2CH2CH2OH), 4.04 (1H, t, J = 5.8 Hz, CH2CH2CH2OH),
4.03–3.95 (2H, m, CH2CH2CH2OH), 3.55 (4H, t, J = 4.9 Hz,
CH2CH2N), 3.49 (1H, t, J = 6.0 Hz, CH2CH2CN), 3.47
(CH2CH2CN), 66.6 (d, JCꢁP = 4.6 Hz, CH2CH2N), 66.1
2
2
(d, JCꢁP =5.7 Hz, CH2CH2CH2OH), 61.2 (d, JCꢁP
4.6 Hz, CH2CH2CN), 60.1 (CH2CH2CH2CH2OH), 44.8
=
3
(CH2CH2N), 28.5 (CH2CH2CH2CH2OH), 26.2 (d, JCꢁP
8.0 Hz, CH2CH2CH2OH), 18.7 (d, JCꢁP
=
3
= 9.2 Hz,
CH2CH2CN); dP(121 MHz, C6D6; extern. H3PO4/D2O) 74.4.
ꢀc
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New J. Chem., 2010, 34, 880–887 | 885

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+ESI-MS: Calcd for C47H65N5O10P2S [M + H]+ 954.4006,
General procedure for the synthesis of the deoxyribonucleo-
side phosphoramidites 6a–j. 50-O-(4,40-Dimethoxytrityl)-30-O-
bis(N,N-diisopropylamino)phosphinyl-20-deoxythymidine (5,
1 mmol, Scheme 2) and any of the alcohols 4a–j (1 mmol)
were placed in a flame-dried round-bottom flask to which was
added, by syringe, anhydrous MeCN (10 mL) under an inert
gas atmosphere. Solid 1H-tetrazole was added to the solution
by portions (4 ꢂ 0.25 mmol) over a period of 1 h at 25 1C. The
reaction mixture was left stirring for an additional 2 h and was
then concentrated to a syrup under reduced pressure. The
crude phosphoramidite was purified by chromatography on
silica gel (25 g), which was equilibrated in C6H6–Et3N (9 : 1 v/v).
The equilibration solvent was also used as the eluent and
found 954.3991.
50-O-(4,40-Dimethoxytrityl)-30-O-[3-[(2-cyanoethyl)-N-
morpholinophosphonothioyl]prop-1-oxy]-(N,N-diisopropylamino)-
phosphinyl-20-deoxythymidine (6g). Yield: 794 mg (82%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.8, 146.7, 146.5,
146.4, 73.5. +ESI-MS: Calcd for C47H63N5O11P2S [M + H]+
968.3793, found 968.3807.
50-O-(4,40-Dimethoxytrityl)-30-O-[4-[(2-cyanoethyl)-N,N-
diisopropylphosphoramidothioyl]but-1-oxy]-(N,N-diisopropyl-
amino)phosphinyl-20-deoxythymidine (6h). Yield: 797 mg
(80%). dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.3,
146.0, 145.9, 70.3. +ESI-MS: Calcd for C50H71N5O10P2S
[M + H]+ 996.4470, found 996.4448.
fractions (10 mL) containing the product were identified by 31
P
NMR spectroscopy. These fractions were pooled together and
evaporated to dryness under reduced pressure. The purified
phosphoramidite was dissolved in dry benzene (5 mL); the
resulting solution was frozen in a dry ice–acetone bath and
was then lyophilized under high vacuum to give the phosphor-
amidite 6a–j as a white powder. Phosphoramidites were
isolated in yields ranging from 80% to 85%.
50-O-(4,40-Dimethoxytrityl)-30-O-[4-[(2-cyanoethyl)-N,N-
diethylphosphoramidothioyl]but-1-oxy]-(N,N-diisopropylamino)-
phosphinyl-20-deoxythymidine (6i). Yield: 784 mg (81%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.3, 146.0, 145.9,
74.7. +ESI-MS: Calcd for C48H67N5O10P2S [M + H]+
968.4157, found 968.4160.
50-O-(4,40-Dimethoxytrityl)-30-O-[2-[(2-cyanoethyl)-N,N-
diisopropylphosphoramidyl]ethoxy]-(N,N-diisopropylamino)-
phosphinyl-20-deoxythymidine (6a). Yield: 790 mg (83%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 147.8, 147.4, 7.4.
+ESI-MS: Calcd for C48H67N5O11P2 [M + Na]+ 974.4205,
found 974.4201.
50-O-(4,40-Dimethoxytrityl)-30-O-[4-[(2-cyanoethyl)-N-
morpholinophosphonothioyl]but-1-oxy]-(N,N-diisopropylamino)-
phosphinyl-20-deoxythymidine (6j). Yield: 805 mg (82%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.1, 145.9, 73.0.
+ESI-MS: Calcd for C48H65N5O11P2S [M + Na]+ 1004.3769,
found 1004.3740.
50-O-(4,40-Dimethoxytrityl)-30-O-[2-[(2-cyanoethyl)-N,N-
diisopropylphosphoramidothioyl]ethoxy]-(N,N-diisopropylamino)-
phosphinyl-20-deoxythymidine (6b). Yield: 794 mg (82%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 147.7, 147.4, 147.2,
71.4, 71.3, 71.2, 71.1. +ESI-MS: Calcd for C48H67N5O10P2S
[M + H]+ 968.4157, found 968.4163.
General procedure for the manual solid-phase synthesis of
the thermosensitive dinucleoside phosphorothioate triester
derivatives 7a–j. A solution of 3% TCA in CH2Cl2 (3 mL)
was slowly pushed by syringe through a commercial DNA
synthesis column packed with 50-DMTrTsuccCPG (1 mmol)
until complete disappearance of any orange color (B2 min).
Excess acid was washed away from TsuccCPG (Scheme 3)
with CH2Cl2 (10 mL) and then with dry MeCN (10 mL). A
premixed solution of any of the 50-DMTrT-30-phosphor-
amidites 6a–j (30 mmol) and 0.45 M 1H-tetrazole in MeCN
(0.3 mL) was then added by syringe to TsuccCPG; the
suspension was manually agitated for 5 min prior to expelling
the excess reagents from the synthesis column with MeCN
(2 ꢂ 10 mL). The CPG support was treated with 0.05 M 3H-
1,2-benzodithiol-3-one-1,1-dioxide20 in MeCN (1 mL) for 2 min.
The excess oxidant was washed off the synthesis column with
MeCN (2 ꢂ 10 mL). Removal of the terminal 50-DMTr group
was effected by agitating the CPG support immersed in a
solution of 3% TCA in CH2Cl2 (2 mL) over a period of
2 min. After carefully washing the support with MeCN
(2 ꢂ 10 mL), the dinucleoside thiophosphate triester 7a–j
was released from the CPG support upon exposure (30 min)
to pressurized (B2.5 bar) methylamine gas.21 Crude 7a–j was
purified by RP-HPLC prior to determining its thermolytic
thiophosphate deprotection kinetics (vide infra).
50-O-(4,40-Dimethoxytrityl)-30-O-[2-[bis(N,N-diisopropyl)-
phosphorodiamidothioyl]ethoxy]-(N,N-diisopropylamino)phos-
phinyl-20-deoxythymidine (6c). Yield: 848 mg (85%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.9, 146.8, 70.7.
+ESI-MS: Calcd for C51H77N5O9P2S [M + Na]+ 1020.4809,
found 1020.4772.
50-O-(4,40-Dimethoxytrityl)-30-O-[2-[bis(N,N-diethyl)phos-
phorodiamidothioyl]ethoxy]-(N,N-diisopropylamino)phosphinyl-
20-deoxythymidine (6d). Yield: 801 mg (85%). dP(121 MHz,
C6D6; extern. H3PO4/D2O) 147.2, 146.9, 78.3, 78.1.
+ESI-MS: Calcd for C47H69N5O9P2S [M + Na]+ 964.4183,
found 964.4182.
50-O-(4,40-Dimethoxytrityl)-30-O-[3-[(2-cyanoethyl)-N,N-
diisopropylphosphoramidothioyl]prop-1-oxy]-(N,N-diisopropyl-
amino)phosphinyl-20-deoxythymidine (6e). Yield: 786 mg (80%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.3, 146.2, 146.1,
145.9, 70.3, 70.2. +ESI-MS: Calcd for C49H69N5O10P2S
[M + Na]+ 1004.4133, found 1004.4108.
50-O-(4,40-Dimethoxytrityl)-30-O-[3-[(2-cyanoethyl)-N,N-
diethylphosphoramidothioyl]prop-1-oxy]-(N,N-diisopropylamino)-
phosphinyl-20-deoxythymidine (6f). Yield: 782 mg (82%).
dP(121 MHz, C6D6; extern. H3PO4/D2O) 146.7, 146.5, 75.2.
Thermolytic deprotection of the dinucleoside phosphorothioate
triesters 7a–j. RP-HPLC-purified 7a–j (B200 nmol) were
dissolved in PBS (1X, pH 7.4, 500 mL) and were heated to
the desired temperature (37 1C or 90 1C). Aliquots (50 mL)
ꢀc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010
886 | New J. Chem., 2010, 34, 880–887

View Online
were taken out at predetermined time points for analysis
by RP-HPLC. The analyses were performed using a 5 mm
Supelcosil LC-18S column (4.6 mm ꢂ 25 cm) under the
following conditions: starting from 0.1 M triethylammonium
acetate (pH 7.0), a linear gradient of 1% MeCN/min was
pumped at a flow rate of 1 mL/min for 40 min. Thermolytic
thiophosphate deprotection rates of 7a–j are listed in Table 1.
7 C. Ausı
S. L. Beaucage, Tetrahedron, 2010, 66, 68.
8 A. Grajkowski, J. Cieslak, J. S. Kauffman, R. J. Duff, S. Norris,
´
n, J. S. Kauffman, R. J. Duff, S. Shivaprasad and
´
D. I. Freedberg and S. L Beaucage, Bioconjugate Chem., 2008, 19,
1696.
9 M. Koizumi, R. Koga, H. Hotoda, K. Momota, T. Ohmine,
H. Furukawa, T. Agatsuma, T. Nishigaki, K. Abe, T. Kosaka,
S. Tsutsumi, J. Sone, M. Kaneko, S. Kimura and K. Shimada,
Bioorg. Med. Chem., 1997, 5, 2235; F. Ferreira, A. Meyer,
J.-J. Vasseur and F. Morvan, J. Org. Chem., 2005, 70, 9198.
10 W. Bannwarth, A. Dorn, P. Iaiza and X. Pannekouke, Helv. Chim.
Acta, 1994, 77, 182.
11 M. M. Greenberg, T. J. Matray, J. D. Kahl, D. J. Yoo and
D. L. McMinn, J. Org. Chem., 1998, 63, 4062; Y.-C. Chang,
J. Herath, T. H.-H. Wang and C. S. Chow, Bioorg. Med. Chem.,
2008, 16, 2676.
2-(N,N-Diisopropylamino)-2-oxo-1,3,2-oxathiaphospholane (8).
To a stirred solution of 2-(N,N-diisopropylamino)-1,3,2-
oxathiaphospholane16 (0.2 g, 1.0 mmol) in CH2Cl2 (5 mL)
was added 5.5 M tert-butyl hydroperoxide in decane (0.36 mL,
2.0 mmol). The oxidation reaction was allowed to proceed for
30 min at 25 1C. The reaction mixture was concentrated to an
oil under reduced pressure. The crude product was purified by
chromatography on silica gel (B10 g) using a gradient of
CH3OH (0 - 3%) in CHCl3, affording 8 as a white crystalline
material in a yield of 85% (0.19 g, 0.85 mmol).
12 M. H. Lyttle, P. B. Wright, N. D. Sinha, J. D. Bain and
A. R. Chamberlin, J. Org. Chem., 1991, 56, 4608.
13 N. D. Sinha, J. Biernat, J. McManus and H. Koster, Nucleic Acids
¨
Res., 1984, 12, 4539.
14 M. Puig, A. Grajkowski, M. Boczkowska, C. Ausın, S. L. Beaucage
´
and D. Verthelyi, Nucleic Acids Res., 2006, 34, 6488.
15 A. Wilk, M. K Chmielewski, A. Grajkowski, L. R. Phillips and
S. L. Beaucage, J. Org. Chem., 2002, 67, 6430; J. Cies
S. L. Beaucage, J. Org. Chem., 2003, 68, 10123; J. Cies
A. Grajkowski, V. Livengood and S. L. Beaucage, J. Org. Chem.,
2004, 69, 2509; A. Grajkowski, C. Ausın, J. S. Kauffman,
´
lak and
´
lak,
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