An efficient oxidizing reagent for the synthesis of mixed backbone oligonucleotides via the H-phosphonate approach

Article abstract of DOI:10.1016/S0968-0896(02)00615-6

The mixture of carbon tetrachloride, N-methyl morpholine (NMM), pyridine and water in acetonitrile has been exploited for the oxidation of dinucleoside H-phosphonate diesters to the corresponding phosphates. The system is found to be inert to the phosphoramidate (P-N) and the phosphorothioate (P-S) linkages and has successfully been applied to the solid phase synthesis of mixed-backbone oligonucleotides (MBOs).

Full text of DOI:10.1016/S0968-0896(02)00615-6

Bioorganic & Medicinal Chemistry 11 (2003) 1419–1431
An Efficient Oxidizing Reagent for the Synthesis of Mixed
Backbone Oligonucleotides via the H-Phosphonate Approach
Nikhil U. Mohe, Kamlesh J. Padiya and Manikrao M. Salunkhe*
Department of Chemistry, The Institute of Science, 15-Madam Cama Road, Mumbai-400 032, India
Received 26 August2002; revised 13 November 2002; accep te d 14 November 2002
Abstract—The mixture of carbon tetrachloride, N-methyl morpholine (NMM), pyridine and water in acetonitrile has been exploi-
ted for the oxidation of dinucleoside H-phosphonate diesters to the corresponding phosphates. The system is found to be inert to
the phosphoramidate (P-N) and the phosphorothioate (P-S) linkages and has successfully been applied to the solid phase synthesis
of mixed-backbone oligonucleotides (MBOs).
#
2003 Elsevier Science Ltd. All rights reserved.
Introduction
issue. The oxidation procedure with the 0.1 M I in Py/
2
H O/THF (5/5/90) provides inconsistent oxidation of
2
3
The synthesis of oligonucleotides by the phosphor-
the internucleotide H-phosphonate linkages. Results
1
2
amidite and the H-phosphonate methodologies has
acclerated the development of oligonucleotides as
potential therapeutics and diagnostic agents and for the
appplications in the field of molecular biology. A sig-
nificant potential for success to control the natural cel-
lular functions, is the use of the synthetic
oligonucleotides with modified backbones that are
complementary to the gene or mRNA responsible for
those functions. Some of the important examples of the
phosphate modifications being investigated are the
phoshorothioates, phosphoramidates and phospho-
tiesters. These modifications in the oligonucleotide can
lead to enhanced cell membrane permeability, increased
resistance to exo and/or endonuclease activity and
increased stability of oligonucleotide/targeted sequence
duplex. The nucleoside H-phosphonates provide a con-
venient route to these types of modifications with the
advantage that a common intermediate would be
required to obtain any of the various modifications.
have been improved by using a two step oxidation,
beginning with iodine solution containing a weak base
like N-methyl imidazole followed by a solution con-
3
taining a strong base like triethyl amine. Additionally,
iodine solution containing water looses the ability to
oxidize support bound oligo H-phosphonate after a
couple of days. Hence mixing the fresh iodine solution
prior to oxidation is advised. An alternative method
for oxidation is a solution of 2% iodine in Py/H O
(98:2,v/v). However this oxidation strategy by aqueous
iodine in pyridine is problematic because the aqueous
condition brings about partial hydrolysis of the inter-
3
2
4
3
5
nucleotidic H-phosphonate linkage. de Vroom etal.
has developed an alternative method of oxidation using
tert-butyl hydroperoxide (TBHP) in the presence of
N,O-bis(trimethylsilyl) acetamide (BSA). In this case
BSA, not only activates the nucleoside H-phosphonate
but also acts as a scavenger of all traces of water in the
oxidation reaction. However the excess use of BSA,
converts TBHP to the less reactive trimethylsilyl tert-
butyl hydroperoxide, and consequently causes retarding
of the oxidation reaction.
The synthetic cycle for the H-phosphonate chemistry is
relatively simple as compared to the phosphoramidite
approach since the oxidation step is carried out at the
end of the synthesis, before the final detritylation. The
efficiency of the oxidation is therefore a highly critical
As the oligonucleotide-based applications continue to
evolve, synthetic methodologies need to be refined to
meet the needs. The use of phosphorothioate oligo-
deoxynucleotides (PS-oligos), has resulted in the first
antisense drug Fomivirsen (vitravene). However, these
PS-oligos are not optimal antisense molecules and their
6
*Corresponding author. Tel./fax: +91-22-2816750; e-mail: mmsalunkhe
@
hotmail.com
0
968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00615-6

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N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
drawbacks have been revealed in the development pro-
7
phosphates both in the solution phase and the solid
phase synthesis of oligonucleotides.
cess of the drugs. Therefore at the current stage of
technology, the mixed backbone oligonucleotides
(MBOs) are considered as the best choice as the second
8
generation antisense compounds.
Results and Discussion
As part of our interest in the field of oligonucleotide
9
In order to explore the role of CCl /H O/NMM/Py/
4
CH CN as an oxidizing reagentin ht e H-phosphona et
3
2
synthesis we have optimized CC1 /H O/NMM/Py/
4
2
1
0
CH CN oxidizing mixture to the H-phosphonate
3
chemistry, preliminary experiments were carried out in the
solution phase. The unoxidized dithymidyl H-phos-
phonate diester 1 (0.01 mmol) (d=12.1 ppm) was subjected
t o oxidat ion wit h C C/ Hl O/NMM/Py/CH CN (2.5/1.0/
chemistry and in turn to the synthesis of MBOs con-
taining the phosphorothioate and phosphoramidate link-
ages. The system is efficient and fast in converting
nucleoside H-phosphonate diesters to the corresponding
4
2
3
1.0/6.0/1.0, v/v) (Scheme 1). The addition of CCl /H O/
4
2
Figure 1. 31P NMR spectra of the dithymidyl phosphate, 2 synthesized by using: (a) CCl
4 2 3
/H O/NMM/Py/CH CN (2.5/1.0/1.0/6.0/1.0, v/v), (b) with
2 2 2
0.035 M I in Py/THF/H O (1/300/33, v/v), (c) 0.2 M iodine in Py/H O (98:2, v/v).

N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
1421
NMM/Py/CH CN to the unoxidized dithymidyl
3
(3.0 ppm), and the other a side product (d=ꢀ9.8 ppm)
H-phosphonate diester resulted in almost immediate
(
(Fig. 1(b)).
<5 min) and quantitative conversion to dithymidyl
phosphate 2 (d=2.9 ppm), with no detectable side pro-
However, when 0.2 M I2 in Py/H O (98:2,v/v) was
2
3
1
ducts as judged by P NMR spectroscopy (Fig. 1(a)). A
similar experimentwas performed w iht 0.035 M I in
Py/THF/H O (1/300/33, v/v). The oxidation reaction in
added, the oxidation was immediate (<5 min) and
quantitative, with no detectable side products (Fig. 1C).
These results indicate that the same combination of
2
2
this case did not lead to complete formation of 2. Two
products were formed, one of them was the phosphate
0.035 M I2 in Py/THF/H O (1/300/33, v/v) which is
2
conventionally used in the phosphoramidite approach,
Figure 2. RP-HPLC (31P NMR is shown in the inset) of d(AA), 3 synthesized by: (a) I
method (negative control), (c) CCl /H O/NMM/Py/CH CN method.
2
2
in Py//H O method (positive control), (b) no oxidation
4
2
3

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N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
cannot be used as an oxidant in the H-phosphonate
chemistry which demands 0.2 M iodine in Py/H O (98:2,
standard protocols for the H-phosphonate chemistry.
The oxidation was effected by the CCl /H O/NMM/Py/
CH CN (2.5/0.2/1.0/6.0/9.3, v/v) system for 45 min. In
3
2
4
2
1
1
v/v). In contrast, the CCl /H O/NMM/Py/CH CN (2.5/
4
2
3
1
.0/1.0/6.0/1.0, v/v) mixture can be used as a versatile
order to check the oxidizing efficiency of the system,
control experiments were performed. One control
experiment (positive control) involved the oxidation of
the unoxidized d(ApHA) dimer supported on lcaa CPG-
oxidizing reagent for the solution phase oxidation of
nucleoside phosphite triester to the corresponding
phosphotriester via the phosphoramidite approach as
well as for the conversion of the nucleoside H-phos-
phonate diesters to corresponding phosphodiesters via
the H-phosphonate approach.
1
0
˚
500 A with the conventionally used 0.2 M I in Py/H O
2
2
(98:2, v/v) for 30 min. In the second control experiment
(negative control), the d(Ap A) dimer supported on
H
˚
lcaa CPG-500 A was notoxidized atall. A fet r per-
In order to check the suitability of the developed CCl4/
H O/NMM/Py/CH CN mixture on solid phase, the
bz
dimer d(AA) (3) was synthesized by coupling of dA
forming the standard deprotection under basic condi-
tions, the crude deprotection mixtures of the three
experiments were analyzed by RP-HPLC and P NMR
spectroscopy and were compared with each other. The
2
3
31
bz
˚
H-phosphonate with dA lcaa CPG 500 A using the
4 2 3
Figure 3. RP-HPLC profile (ESI-MS shown in the inset) of d(GTTAAGACTTTTAC), 4 synthesized by CCl /H O/NMM/Py/CH CN method. ESI-
MS of 4 MWfound: 4234.8 MWcalcd: 4237.
4 2 3 2 2
Figure 4. Solution phase stability of d(TpsT), 5 in: (a) CCl /H O/NMM/Py/CH CN system and (b) I in Py/H O system.

N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
1423
4 2 3 2 2
Figure 5. Solution phase stability of d(TpNT) 6, in: (a) CCl /H O/NMM/Py/CH CN system and (b) I in Py/H O system.
RP-HPLC analysis of the 3 synthesized by I in Py/H O
2
linkages in the backbone, the dimer d(TpsT) (5) (1.0
A260 units) (d=57.0 and 57.4 ppm) was stirred for 16 h
with CCl /H O/NMM/Py/CH CN (2.5/1.0/1.0/6.0/1.0,
2
showed the oxidized dimer 3, as the major peak at
3
1
retention time 7.76 min, its P NMR showed signal at
d=0.93 ppm arising due to the phosphodiester linkages
4
2
3
v/v) mixture at ambient temperature. A similar experi-
ment was carried out under identical scale, conditions
and protocols using 0.2 M I in Py/H O (98:2, v/v). In
(Fig. 2A). The negative control showed two peaks one
at retention time 5.95 min (adenosine) and the other at
retention time 6.48 min arising due to the formation of
H-phosphonate, with <3% formation of 3 (retention
2
2
order to judge the potential occurrence of desulfuriza-
3
1
tion by these oxidizing reagents, the P NMR analysis
31
3
1
time 7.79 min), while its P NMR showed two signals
at d=6.9 and 8.3 ppm (Fig. 2B). The RP-HPLC analy-
sis of the crude deprotection mixture by CCl /H O/
of the reaction mixtures were carried out. The
NMR of the crude reaction mixture using CCl /H O/
P
4
2
NMM/Py/CH CN reagent( Fig. 4(a)) indicated ca. 6%
3
4
2
NMM/Py/CH CN method, showed the major peak at
3
desulfurization d=0.9 ppm). However, the crude
retention time 7.76 min (expected product 3), with no
evidence for the formation of the H-phosphonate aris-
ing due to the cleavage of the unreacted H-phosphonate
diester linkages under basic conditions used to release
the oligonucleotide from the solid support (Fig. 2(c)).
reaction mixture using I in Py/H O (98:2, v/v) method
2
2
indicated complete desufurization of 5, to give d(TpoT)
as the only detectable product (d=0.96 ppm) (Fig. 4(b))
as judged by the P NMR analysis. The desulfurization
3
1
process by I in Py/H O has been reported previously in
2
2
3
1
13
Also, P NMR of its crude deprotection mixture indi-
cated only one signal at d=0.9 ppm (shown in the inset
of Fig. 2C). These experiments clearly indicate that the
conversion of oligonucleotide H-phosphonate diesters
to the corresponding phosphates results in quantitative
conversion in case of the optimized CCl /H O/NMM/
literature.
Similarly in order to check the compatibility of the
CCl /H O/NMM/Py/C CN system towards the phos-
4
2
3
phoramidate linkages in the backbone, the dimer
d(TpNT)(6) (1.0 A260 units)=12.08 and 12.00 ppm) was
stirred individually for 16 h with the CCl /H O/NMM/
4
2
Py/CH CN system. In case of I in Py/H O system ca.
2
3
2
4
2
2
% formation of H-phosphonate (retention time 6.4
Py/CH CN (2.5/1.0/1.0/6.0/1.0, v/v) and 0.2 M I in
3 2
min) was detected. It has been reported that the oxida-
tion of H-phosphonate with the I in Py/H O system is
Py/H O) (98:2, v/v) oxidizing reagents, as described
2
3
1
previously. The P NMR analysis of the crude reaction
mixtures were then carried out. The dimer 6 stirred with
the CCl /H O/NMM/Py/CH CN did notshow any
2
2
slow and it is preferable to do it in an automated mode
using a three reagent combination in a DNA synthesi-
4
2
3
1
2
zer.
cleavage of the phosphoramidate linkages as judged by
the phosphorus NMR (Fig. 5(a)). However, the phos-
phoramidate linkages were completely cleaved by I in
The oxidizing system has sucessfully been applied to the
synthesis of 14 mer oligo d(GTTAAGACTTTTAC) (4).
The RP-HPLC profile of the crude deprotection mixture
of 4 is shown in Figure 3. All the fractions were char-
acterized simultaneously by ESI-MS analysis (ESI-MS
of the major fraction, 4 is shown in the inset of Fig. 3).
2
Py/H O reagent to give the phosphodiester signal at
2
d=0.97 ppm which was the only detectable product
(Fig. 5(b)). These solution phase analysis clearly indi-
cate that the CCl /H O/NMM/Py/CH CN oxidation
4
2
3
method is far superior than the conventionally used I2
in Py/H O in retaining the phosphorothioate and
2
In order to assess the chemical stability of the CCl /H O/
4
NMM/Py/CH CN system towards the phosphorothioate
3
the phosphoramidate linkages in the oligonucleotide
backbone.
2

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N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
4 2 3 2 2
Figure 6. RP-HPLC profile of d(TpoTpsT), 7 synthesized by: (a) CCl /H O/NMM/Py/CH CN method and (b) I in Py//H O method.

N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
1425
4 2 3 2 2
Figure 7. RP-HPLC profile of d(TpoTPNT), 7 synthesized by: (a) CCl /H O/NMM/Py/CH CN method and (b) I in Py//H O method.

1426
N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
Figure 8. RP-HPLC profile (ESI-MS shown in the inset) of d(TpoTpsTpsTpsTpsTpsTpsT), 9 synthesized by CCl
ESI-MS of 9 MWcalcd: 2459.95, MWfound: 2466.
4 2 3
/H O/NMM/Py/CH CN method.
Scheme 1. Solution phase oxidation of dithymidyl H-phosphonate
diester 1 with CCl /H O/NMM/Py/CH CN system.
4 2 3
Scheme 2.
Encouraged by these positive results on the dimers 5
and 6 we decided to investigate the use of the CCl4/
H O/NMM/Py/CH CN method to the synthesis of trimers
The trimer 8 sufferd ca. 7% loss of the phosphor-
amidate linkages, while the major fraction was eluted at
retention time 10.2 and 10.4 min (Fig. 7(b)).
2
3
d(TpoTpsT) (7) and d(TpoPNT) (8)[where PN corresponds
to P-NH-(CH ) N(CH ) linkages] on solid phase using
2
3
3 2
the standard protocols for the H-phosphonate chem-
istry (Scheme 2). After the standard deprotection step,
the crude deprotection mixtures were analysed by
0
dual advantage of high nuclease resistance and
enhanced binding affinity have been reported. The
Presently, a number of 2 -modifications that offer the
1
4
RP-HPLC. The RP-HPLC of
7
synthesized by
CCl /H O/NMM/Py/CH CN (2.5/0.2/1.0/6.0/9.3, v/v)
0
0
15
2 -O-(2-methoxyethyl) (2 -O-MOE), with the phos-
phodiester (PO) backbone provides sufficient nuclease
resistance, at approximately the same level as the
4
2
3
method showed ca. 2% desulfurization (retention time
.11 min), while the expected product 7 was obtained as
9
0
the major peak at retention time 9.8 and 10.35 min (two
peaks are due to the diasteriomers resulting from the
chiral phosphorus centre) (Fig. 6(a)). The RP-HPLC of
2 -O-deoxyphosphorothioate modification. A typical
0
approach entails the use of the 2 -O-alkylated phospho-
diester to replace part of the PS linkers in the antisense
compounds, leading to a MBO with reduced phosphoro-
thioate content. The preliminary experiments have
shown that these MBOs exhibit more acceptable safety
7
synthesised by 0.2 M I in Py/H O (98:2, v/v) method
2 2
however resulted in nearly complete loss of sulfur to
give the desulfurized trimer, d(TpoTpoT) as the major
peak at retention time 9.15 min. The expected product 7
obtained was ca. 4% (Fig. 6(b)).
1
6
profiles than those of the PS oligos. As a consequence
of which, the antisense oligos containing PS and PO
linkages in the backbone are presently undergoing
1
7
Similarly the trimer 8 was synthesized by using CCl4/
H O/NMM/Py/CH CN (2.5/0.2/1.0/6.0/9.3, v/v) and
human clinical trials. Taking into account the superior
attributes of the developed CCl /H O/NMM/Py/
CH CN (2.5/0.2/1.0/6.0/9.3, v/v) oxidation method and
2
3
4
2
0
.2 M I in Py/H O (98:2, v/v) method on solid phase.
2
2
3
The RP-HPLC analysis of the crude deprotection mix-
tures were then carried out. The CCl /H O/NMM/Py/
CH CN method, although was stable towards the
the importance of the MBOs containing both phos-
phorothioate and phosphodiester linkages, we have
extended our developed methodology to the synthesis of
d(TpoTpsTpsTpsTpsTpsTpsT) (9). The expected pro-
duct was obtained at retention time 31.0 min (Fig. 8),
which was simultanesouly characterized by ESI–MS
(shown in the inset of Fig. 8). The LC-MS profile of 9
indicated that the overall desulfurization was less than
4% (desulfurized 8 mer oligo retention time 30.35 min,
containing 2 phosphodiester and 5 phosporothioate
linkages in the backbone).
4
2
3
phosphoramidate linkages in the solution phase, in the
solid phase cleaved ca. 2% of the phosphoramidate lin-
kages (retention time 9.0 min), while 8 was obtained as
major peak (retention time 10.2 and 10.4 min) as the
mixture of diasteriomers (Fig. 7(a)). As one could
expect from the solution phase experiments, 8 surpris-
ingly did not undergo extensive loss of the phosphor-
amidate linkage on solid phase by I in Py/H O method.
2
2

N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
1427
Conclusion
diode Array Detector). All these RP-HPLC fractions
were simultaneously characterized by electrospray
ionization mass spectrometry (ESI-MS) using a Hew-
lett-Packard 59987A electrospray quadrupole mass
analyzer using negative polarity.
In the present study we have developed CCl /H O/
4
NMM/Py/CH CN mixture as a mild and efficient oxi-
3
2
dizing reagent, that can quantitatively convert nucleo-
side H-phosphonate diesters to the corresponding
phosphates both in the solution phase as well as in
the solid phase synthesis of oligonucleotides. The data
presented suggests that this oxidation method can
serve as an advantageous replacement for the con-
ventionally used aqueous iodine in pyridine oxidation
method, particularly in the synthesis of Mixed Backbone
Oligonucleotides.
Solution phase synthesis of dithymidyl H-phosphonate
diester (1)
To a well stirred solution of dT-H-phosphonate TEA
0
salt(0.1 mmol) and 3 -O-acetyl thymidine (0.11 mmol)
dissolved in 1.0 mL of anhydrous pyridine, was added
pivaloyl chloride (0.1 mmol). The reaction mixture was
stirred at ambient temperature for 15 min. The reaction
mixture was monitored by TLC (ethyl acetate/pet
ether=8:2). After completion of the reaction, the reac-
tion mixture was concentrated and was subjected to
column chromatography to get the product 1 (isolated
yield 85%).
Experimental
Materials
˚
All the nucleoside H-phosphonates, lcaa CPG-500 A
3
1
were purchased from Glen Res. (Virginia). Acetonitrile
(
P NMR (CD CN) 1, d=12.1 ppm.
3
HPLC grade, Spectrochem India Ltd.) was distilled
˚
and stored over activated 3 A molecular sieves (Aldrich
Chemical Co.). Pyridine (E. Merck, India) was dried by
Solution phase synthesis of dithymidyl phosphonate (2)
using CCl /H O/NMM/Py/CH CN oxidation method
˚
refulxing over KOH and was stored over activated 3 A
4
2
3
molecular sieves. Pivaloyl chloride (AR grade, Spectro-
chem India Ltd.) was used as received. The solid phase
oligonucleotide syntheses were performed manually in a
The dithymidyl H-phosphonate diester 1 (10 mg, ca.
0.01 mmol) d=12.1 ppm) was dissolved in CD CN (200
mL) and was transferred into a NMR sample tube. The
3
2
plug at the inlet.
.5 mL Hamilton gas tight syringe with a glass wool
8
2.0 mL of CCl /H O/NMM/Py/CH CN (2.5/1.0/1.0/
6.0/1.0, v/v) mixture was added at ambient temperature
4
2
3
1
3
1
to the above solution. The reaction was monitored by
P
NMR. After 5 min, quantitative formation of 2 (d=2.9
ppm) was observed as the only detectable product.
Methods
31P NMR analysis: the 31
P NMR was performed on a
Bruker AMX 500 instrument. The experiments were
Solution phase synthesis of 2 using 0.035 M I in Py/
2
THF/H O (1/300/33, v/v) oxidation method
2
carried outin an NMR sample tu be (5 mm ꢁ180 mm) at
ꢂ
31
using 85% H PO as external reference. In each
2
5 C with proton decoupling at 202 Mz for P NMR
The solution of 1 (10 mg, ca. 0.01mmol) in CD CN (200
3
mL) was transferred into a NMR sample tube. To this
3
4
measurement, data accumulation was carried out 64
times with a pulse width of 10 ms, an acquisition time of
solution was added 2.0 mL of 0.035 M I in Py/THF/
H O (1/300/33, v/v) at ambient temperature, the pro-
gress of the reaction was monitored by P NMR spec-
troscopy. After 5 min formation of 2 (d=3.0 ppm)
along with the formation of another product at d=ꢀ9.8
ppm was detected.
2
2
3
1
0
.196 s, and a pulse delay of 3.0 s. The spectrum was
acquired with 16384 data points for the spectral width
of 41666 Hz.
RP-HPLC techniques: the RP-HPLC analysis of oligo-
nucleotides 3 along with the positive and negative con-
trol experiments were performed on a Licrosphere RP-
Solution phase synthesis of 2 using 0.2 M I in Py/H O
2
2
(98:2, v/v) oxidation method
1
8 (e) 5 mm, (125ꢁ4 mm) column using a Merck Hitachi
LC pump L-7100 which was connected to a diode array
detector (model L-7455). The system used was A:
As previously described, to the solution of 1 (10 mg, ca.
0.01 mmol) dissolved in CD CN (200 mL) was added 2.0
3
0
TEAA (pH=7.0) using a gradientA-B in 20 min, flow
.1 M TEAA, pH=7.0, B: 30% CH CN in 0.1 M
mL of 0.2 M I in Py/H O (98:2, v/v). After 5 min
2
quantitative formation of 2 (d=2.5 ppm) was observed
as the only detectable product.
3
2
ꢀ
1
rate: 1.5 mL min . RP-HPLC analysis of oligonu-
cleotides 7 and 8 were performed on a HP spherisorb
ODS-2, 5 mm, (125ꢁ4 mm) column using a Varian
Prostar LC pump to which was attached a Dynamax
absobance detector (model UV-D11). The solvent sys-
tem and the conditions were same as described above.
The RP-HPLC analysis of the oligonucleotides 4 and 9
Synthesis of oligodeoxynucletides (ODNs)
Solid phase synthesis of d(AA) (3) using CCl /H O/
4
2
NMM/Py/CH CN oxidation method (protocol 1).
3
TM
The DMT-Abz loaded 1caa CPG 500 A (22 mg, 1
mmole, loading 44.3 mmol/g) was poured into a well
dried 2.5 mL Hamilton gas tight syringe with a glass wool
˚
were performed on a Waters YMC-Pack
AQ
ODS-
S-5 column (2.0ꢁ250 mm) using a Waters
HPLC system (600 E system controller, 996 Photo-
TM

1428
N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
plug at the inlet. The support was initially washed with
CH CN (3ꢁ0.5 mL). The DMT protecting group was
Solid phase synthesis of d(GTTAAGACTTTTAC) (4)
using CCl /H O/NMM/Py/CH CN oxidation method
3
4
2
3
removed by washing with dichloroacetic acid (DCA)/
CH Cl (3/100, v/v, 1.0 mL). Additional washes with
The DMT-Cbz loaded 1caa CPG 500 A (21 mg, 1
mmole, loading 46.9 (mmol/g) was poured into a well
dried 2.5 mL Hamilton gas tight syringe with a glass
wool plug at the inlet. The support was initially washed
˚
2
2
DCA/CH Cl (8ꢁ0.5 mL) were performed and all the
2
2
orange effluents were collected for subsequent spectro-
scopy (448 nm) and calculation of the coupling effi-
ciency. The supportwas washed successively wi ht Py/
CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry CH CN (4ꢁ0.5
with CH CN (3ꢁ0.5 mL). The DMT protecting group
3
was removed by washing with dichloroacetic acid
(DCA)/CH Cl (3/100, v/v, 1.0 mL). Additional washes
3
3
bz
mL). The dA -H-phosphonate solution (10 mg, ca.
2
2
0
.012 mmol) in dry CH CN (0.25 mL) and dry pyridine
3
with DCA/CH Cl (8ꢁ0.5 mL) were performed and all
2
2
(
0.25 mL) containing pivaloyl chloride (8 mL, ca. 0.075
mmole) was drawn into the syringe and was agitated for
min. The coupling agents were ejected from the syr-
inge, and the support was washed with dry CH CN
the orange effluents were collected for subsequent spec-
troscopy (448 nm) and calculation of the coupling effi-
ciency. The support was then washed with Py/CH CN
2
3
(1/4, v/v, 1ꢁ0.5 mL) and dry CH CN (4ꢁ0.5 mL). The
3
3
bz
(3ꢁ0.5 mL). The internucleoside H-phosphonate link-
ages were then oxidized with 2.0 mL of CCl /H O/
dA -H-phosphonate solution (10 mg, ca. 0.012 mmol)
in dry CH CN (0.25 mL) and dry pyridine (0.25 mL)
4
2
3
NMM/Py/CH CN (2.5/0.2/1.0/6.0/9.3, v/v) for 45 min.
3
Washings were performed with Py/CH CN (1/4, v/v,
3
containing pivaloyl chloride (8 L, ca. 0.075 mmole) was
drawn into the syringe and was agitated for 2 min. The
coupling agents were ejected from the syringe, and the
supportwas washed wi th dry CH 3CN (3ꢁ0.5 mL). The
terminal DMT protecting group was removed by wash-
ing with DCA/CH Cl (3/100, v/v, 1.0 mL) and addi-
3
ꢁ1.5 mL) and dry CH CN (3ꢁ1.5 mL). The terminal
3
DMT protecting group was removed by washing with
dichloroacetic acid (DCA)/CH Cl (3/100, v/v, 1.0 mL)
2
2
and additional washes with DCA/CH Cl (8ꢁ0.5 mL)
2
2
2
2
followed by subsequentwashings wi th Py/CH CN (1/4,
3
tional washes with DCA/CH Cl (8ꢁ0.5 mL) followed
2
2
v/v, 1ꢁ0.5 mL) and dry CH CN (4ꢁ0.5 mL). The sup-
by subsequentwashings w hi t Py/CH
CN (1/4, v/v,
3
3
port bound oligonucleotide was then completely depro-
tected using concentrated NH OH, 55 C, 16 h.
1ꢁ0.5 mL) and dry CH CN (4ꢁ0.5 mL). This cycle was
3
ꢂ
repeated by coupling of the appropriate nucleosidic
unit, until the desired length of the oligonucleotide
was achieved. The oxidation step was then carried
4
RP-HPLC of 3 (retention time 7.76 min).
outw hi t 2.0 mL of CCl
/H O/NMM/Py/CH CN
2 3
4
3
1
P NMR (D O) 3, d=0.9 ppm.
(2.5/0.2/1.0/6.0/9.3, v/v) for 45 min at the end of the
synthesis and before final detritylation. After the final
detritylation and washings steps, the oligonucleotide
2
The negative control experiment was performed exactly
identical to protocol 1. The only difference was that the
oxidation step was omitted. The support bound oligo-
was completely deprotected using concentrated
ꢂ
NH OH, 55 C, 24 h.
4
nucleotide was then completely deprotected using con-
ꢂ
centrated NH OH, 55 C, 16 h.
4
RP-HPLC of 4 (retention time 27.8 min).
ESI-MS of 4, Mwfound: 4234.8 Mw calcd: 4237.
RP-HPLC of 3 (retention time 7.79 min), adenosine
(retention time 5.95 min), dA-H-phosphonate (retention
time 6.4 min).
Solution phase stability of d(TpsT) (5) in CCl /H O/
4
2
NMM/Py/CH CN oxidizing system
3
31
P NMR (D O) dA-H-phosphonate, d=6.9 and 8.3 ppm.
2
˚
The DMT-T loaded 1caa CPG 500 A (20 mg, 1 mmol,
loading 48.3 m mol/g) was poured into a well dried 2.5
mL Hamilton gas tight syringe with a glass wool plug at
Solid phase synthesis of 3 using 0.2 M I in Py/H O
2
2
(
98:2, v/v) oxidation method (positive control)
the inlet. The support was initially washed with CH CN
3
bz
The dimer was synthesized by coupling dA -H-phos-
bz
(3ꢁ0.5 mL). The DMT protecting group was removed
phonate with the support bound dA loaded 1caa CPG
5
by washing with DCA/CH Cl (3/100, v/v, 1.0 mL).
2
2
˚
00 A as described in protocol 1. At the end of the
Additional washes with DCA/CH Cl (8ꢁ0.5 mL) were
2
2
synthesis, before the final detritylation, the internucleo-
side H-phosphonate linkages were oxidized with 2.0 mL
of 0.2 M solution in Py/H O (98:2, v/v) for 30 min. The
performed and all the orange effluents were collected for
subsequent spectroscopy (448 nm) and calculation of
the coupling efficiency. The support was washed suc-
2
support was then washed with CH CN/Py (4/1, v/v,
3
cessively with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry
3
6
removal of the DMT group, the support bound oligo-
ꢁ1.5 mL) and with CH CN (3ꢁ1.5 mL). After the
CH CN (4ꢁ0.5 mL). The dT-H-phosphonate solution
3
3
(10 mg, ca. 0.015 mmol) in dry CH CN (0.25 mL) and
3
nucleotide was then completely deprotected using con-
ꢂ
dry pyridine (0.25 mL) containing pivaloyl chloride
(8 mL, ca. 0.075 mmol) was drawn into the syringe and
was agitated for 2 min. The coupling agents were
ejected from the syringe, and the support was washed
centrated NH OH, 55 C, 16 h.
4
RP-HPLC of 3 (retention time 7.76 min), dA-H phos-
phonate (retention time 6.4 min).
with dry CH CN (3ꢁ0.5 mL). The internucleotidic
3
H-phosphonate linkages were sulfurized by using 2.0
mL mixture of 10% S in CS /Py/TEA (35/35/1) for
3
1
P NMR (D O) 3, d=0.9 ppm.
2
8
2

N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
1429
3
h. Washings were performed with Py/CH CN (1/4, v/
3
purified by RP-HPLC. The purified oligo 5 (1.0 A260
units) was then stirred in a Wheaton glass vial for 16 h,
at ambient temperature with 1.0 mL of CCl /H O/
v, 6ꢁ1.5 mL) and dry CH CN (3ꢁ1.5 mL). The term-
3
inal DMT protecting group was removed by washing
with DCA/CH Cl (3/100, v/v, 1.0 mL) and additional
4
2
NMM/Py/CH CN (2.5/1.0/1.0/6.0/1.0, v/v) mixture.
3
2
2
3
1
washes with DCA/CH Cl (8ꢁ0.5 mL) followed by
The P NMR analysis was carried outaf et r lipholiza-
tion of the reaction mixture.
2
2
subsequentwashings wi ht Py/CH CN (1/4, v/v, 1ꢁ0.5
3
mL) and dry CH CN (4ꢁ0.5 mL). After performing
3
ꢂ
31
the deprotection with concentrated NH OH, 5 C, 16
4
P NMR (D O) 6, d=12.08 ppm and 12.02 ppm.
2
h, the oligo was purified by RP-HPLC. The purified
oligo 5 (1.0 A260 units) was then stirred in a Whea-
ton glass vial for 16 h at ambient temperature with
Solution phase stability of 6 in 0.2 M I2 in Py/H O
2
(98:2, v/v) oxidizing system
1
.0 mL of CCl /H O/NMM/Py/CH CN (2.5/1.0/1.0/
4 2 3
6
.0/1.0, v/v) mixture. The reaction mixture was
3
As described above, the purified oligo 6 (1.0 A260 units)
was stirred with 1.0 mL of 0.2 M I in Py/H O (98:2,
1
lipholyzed and was then analyzed by
spectroscopy.
P NMR
2
2
v/v) mixture in a Wheaton glass vial for 16 h at ambient
temperature. The lipholyzed reaction mixture was sub-
3
1
31
P NMR (D O) 5, d=57.4 and 57.0 ppm; desulfurized
jected to P NMR analysis.
2
dimer d(TpoT) d=0.9 ppm.
3
1
P NMR (D O) desulfurized dimer d(TpoT) d=0.97
2
ppm.
Solution phase stability of 5 in 0.2 M I2 in Py/H O
2
(
98:2, v/v) oxidizing system
Solid phase synthesis of d(TpoTpsT) (7) using CCl4/
H O/NMM/Py/CH CN oxidation method
As described above the purified oligo 5 (1.0 A260 units)
was stirred in a Wheaton glass vial for 16 h at ambient
temperature with 1.0 mL of 0.2 M I in Py/H O (98:2,
v/v) mixture. After lipholyzation the P NMR analysis
was performed.
2
3
˚
The DMT-T loaded 1caa CPG 500 A (20 mg, 1 mmole,
loading 48.3 (mmol/g) was poured into a well dried 2.5
mL Hamilton gas tight syringe with a glass wool plug at
2
3
2
1
the inlet. The support was initially washed with CH CN
3
3
1
P NMR (D O) desulfurized dimer d(TpoT)=0.9
2
(3ꢁ 0.5 mL). The DMT protecting group was removed
ppm
by washing with DCA/CH Cl (3/100, v/v, 1.0 mL).
2
2
Additional washes with DCA/CH Cl (8ꢁ0.5 mL) were
2
2
performed and all the orange effluents were collected for
subsequent spectroscopy (448 nm) and calculation of
the coupling efficiency. The support was washed suc-
Solution phase stability of d(TPNT) (6) in CCl /H O/
2
NMM/Py/CH CN oxidizing system
3
4
˚
The DMT-T loaded 1caa CPG 500 A (20 mg, 1 mmol,
loading 48.3 (mmol/g) was poured into a well dried 2.5
mL Hamilton gas tight syringe with a glass wool plug at
cessively with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry
3
CH CN (4ꢁ0.5 mL). The dT-H-phosphonate solution
3
(10 mg, ca. 0.015 mmol) in dry CH CN (0.25 mL) and
3
the inlet. The support was initially washed with CH CN
3
dry pyridine (0.25 mL) containing pivaloyl chloride (8
mL, ca. 0.075 mmol) was drawn into the syringe and was
agitated for 2 min. The coupling agents were ejected
from the syringe, and the support was washed with dry
(
by washing with DCA/CH Cl (3/100, v/v, 1.0 mL).
3ꢁ0.5 mL). The DMT protecting group was removed
2
2
Additional washes with DCA/CH Cl (8ꢁ0.5 mL) were
2
2
performed and all the orange effluents were collected for
subsequent spectroscopy (448 nm) and calculation of
the coupling efficiency. The support was washed suc-
CH CN (3ꢁ0.5 mL). The internucleotidic H-phospho-
3
nate linkages were sulfurized by using 2.0 mL mixture
of 10% S in CS2/Py/TEA (35/35/1) for 3 h. Washings
8
cessively with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry
were performed with Py/CH CN (1/4, v/v, 6ꢁ1.5 mL)
3
3
CH CN (4ꢁ0.5 mL). The dT-H-phosphonate solution
and dry CH CN (3ꢁ1.5 mL). The terminal DMT pro-
3
3
(10 mg, ca. 0.015 mmol) in dry CH CN (0.25 mL) and
dry pyridine (0.25 mL) containing pivaloyl chloride
tecting group was removed by washing with DCA/
CH Cl (3/100, v/v, 1.0 mL) and additional washes
3
2
2
(
8 mL, ca. 0.075 mmol) was drawn into the syringe and
with DCA/CH Cl (8ꢁ0.5 mL), followed by sub-
2
2
was agitated for 2 min. The coupling agents were ejec-
ted from the syringe, and the support was washed with
sequentwashings w hi t Py/CH
mL) and dry CH CN (4ꢁ0.5 mL). The nextcoupling
CN (1/4, v/v, 1ꢁ0.5
3
3
dry CH CN (3ꢁ0.5 mL). The dithymidyl H-phospho-
step was then effected by drawing the dT-H-phospho-
nate solution (10 mg, ca. 0.015 mmol) in dry CH CN
3
nate diester linkages were converted to the phosphor-
amidate linkages by using 2.0 mL mixture of 15%
N,N-dimethyl-1,3- propane diamine in CCl for 1 h.
3
(0.25 mL) and dry pyridine (0.25 mL) containing
pivaloyl chloride (8 mL, ca. 0.075 mmol) into the syr-
inge and agitating the mixture for 2 min. The coupling
agents were ejected from the syringe, and the support
4
Washings were performed with Py/CH CN (1/4, v/v,
3
3
DMT protecting group was removed by washing with
DCA/CH Cl (3/100, v/v, 1.0 mL) and additional
ꢁ1.5 mL) and dry CH CN (3ꢁ1.5 mL). The terminal
3
was washed with dry CH CN (3ꢁ0.5 mL). The inter-
3
nucleoside H-phosphonate linkages were then oxidized
with 2.0 mL of CCl /H O/NMM/Py/CH CN (2.5/0.2/
1.0/6.0/9.3, v/v) for 45 min. Washings were performed
2
2
washes with DCA/CH Cl (8ꢁ0.5 mL) followed by
2
2
4
2
3
washings with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry
3
CH CN (4ꢁ0.5 mL). After performing the deprotection
with Py/CH CN (1/4, v/v, 3ꢁ1.5 mL) and dry
3
3
ꢂ
with concentrated NH OH, 55 C, 16 h, the oligo was
4
CH CN (3ꢁ1.5 mL). The terminal DMT protecting
3

1430
N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
group was removed by washing with DCA/CH Cl
2
0.015 mmol) in dry CH CN (0.25 mL) and dry pyridine
3
2
(
3/100, v/v, 1.0 mL) and additional washes with DCA/
(0.25 mL) containing pivaloyl chloride (8 mL, ca. 0.075
mmol) into the syringe and agitating the mixture for 2
min. The coupling agents were ejected from the syringe,
CH Cl (8ꢁ0.5 mL) followed by subsequentwashings
2
2
with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry CH CN
3
3
(
then completely deprotected using concentrated
4ꢁ0.5 mL). The supportbound oligonucleoi td e was
and the support was washed with dry CH CN (3ꢁ0.5
3
mL). The internucleoside H-phosphonate linkages were
then oxidized with 2.0 mL of CCl /H O/NMM/Py/
CH CN (2.5/0.2/1.0/6.0/9.3, v/v) for 45 min. Washings
ꢂ
NH OH, 55 C, 16 h.
4
4
2
3
RP-HPLC of 7 (retention time 9.8 and 10.3 min);
desulfurized trimer d(TpoTpoT) retention time 9.1 min.
were performed with Py/CH CN (1/4, v/v, 3ꢁ1.5 mL)
3
and dry CH CN (3ꢁ1.5 mL). The terminal DMT pro-
3
tecting group was removed by washing with DCA/
CH Cl (3/100, v/v, 1.0 mL) and additional washes with
2
2
Solid phase synthesis of 7 using 0.2 M I in Py/H O
2
2
DCA/CH Cl (8ꢁ0.5 mL) followed by subsequent
2
2
(
98:2, v/v) oxidation method
washings with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry
3
The synthesis of 7 was carried out identically as for the
synthesis described above by the CCl /H O/NMM/Py/
CH CN (4ꢁ0.5 mL). The supportbound oligonucleo-
3
tide was then completely deprotected using con-
centrated NH OH, 55 C, 16 h.
4
2
ꢂ
CH CN oxidation method. The only difference was that
3
4
the oxidation reaction was carried out at the end of the
synthesis, before final detritylation by 2.0 mL of 0.2 M
I in Py/H O (98:2, v/v) method for 30 min instead of
RP-HPLC of 8 (retention time 10.2 and 10.4 min);
desulfurized trimer d(TpoTpoT) (retention time 9.08
min).
2
2
CCl /H O/NMM/Py/CH CN method. The support was
3
4
2
then washed with CH CN/Py (4/1, v/v, 6ꢁ1.5 mL) and
3
with CH CN (3ꢁ1.5 mL). After the removal of the
3
Solid phase synthesis of 8 using 0.2 M I in Py/H O
2
2
DMT group, the support bound oligonucleotide was
then completely deprotected using concentrated
(98:2, v/v) oxidation method
ꢂ
NH OH, 55 C, 16 h.
4
The synthesis of 8 was carried out identically as for the
synthesis described above by the CCl /H O/NMM/Py/
4
2
RP-HPLC of 7 (retention time 9.9 and 10.3 min),
desulfurized trimer d(TpoTpoT) (retention time 9.1
min).
CH CN oxidation method. The only difference was that
3
the oxidation reaction was carried out at the end of the
synthesis, before final detritylation by 2.0 mL of 0.2 M
I in Py/H O (98:2, v/v) method for 30 min instead of
2
2
CCl /H O/NMM/Py/CH CN method. The support was
4
2
3
Solid phase synthesis of d(TpoTPNT) (8) using CCl4/
H O/NMM/Py/CH CN oxidation method
then washed with CH CN/Py (4/1, v/v, 6ꢁ1.5 mL) and
3
2
3
with CH CN (3ꢁ1.5 mL). After the removal of the
3
˚
The DMT-T loaded 1caa CPG 500 A (20 mg, 1 mmol,
loading 48.3 mmol/g) was poured into a well dried 2.5
mL Hamilton gas tight syringe with a glass wool plug at
DMT group, the support bound oligonucleotide was
then completely deprotected using concentrated
ꢂ
NH OH, 55 C, 16 h.
4
the inlet. The support was initially washed with CH CN
3
(
by washing with DCA/CH Cl (3/100, v/v, 1.0 mL).
3ꢁ0.5 mL). The DMT protecting group was removed
RP-HPLC of 8 (retention time 10.2 and 10.4 min),
desulfurized trimer d(TpoTpoT) (retention time 9.04
min).
2
2
Additional washes with DCA/CH Cl (8ꢁ0.5 mL) were
2
2
performed and all the orange effluents were collected for
subsequent spectroscopy (448 nm) and calculation of
the coupling efficiency. The support was washed suc-
Synthesis of d(TpoTpsTpsTpsTpsTpsTpsT) (9) using
CCl /H O/NMM/Py/CH CN oxidation method
4
2
3
cessively with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry
3
˚
CH CN (4ꢁ0.5 mL). The dT-H-phosphonate solution
The DMT-T loaded 1caa CPG 500 A (20 mg, 1 mmol,
loading 48.3 mmol/g) was poured into a well dried 2.5
mL Hamilton gas tight syringe with a glass wool plug at
3
(
10 mg, ca. 0.015 mmol) in dry CH CN (0.25 mL) and
3
dry pyridine (0.25 mL) containing pivaloyl chloride
8 mL, ca. 0.075 mmol) was drawn into the syringe and
(
the inlet. The support was initially washed with CH CN
3
was agitated for 2 min. The coupling agents were ejected
from the syringe, and the support was washed with dry
(3ꢁ0.5 mL). The DMT protecting group was removed
by washing with DCA/CH Cl (3/100, v/v, 1.0 mL).
2
2
CH CN (3ꢁ0.5 mL). The dithymidyl H-phosphonate
Additional washes with DCA/CH Cl (8ꢁ0.5 mL) were
3
2
2
diester linkages were converted to the phosphoramidate
linkages by using 2.0 mL mixture of 15% N,N-dimethyl-
performed and all the orange effluents were collected
for subsequent spectroscopy (448 nm) and calculation
of the coupling efficiency. The support was washed
1
,3-propane diamine in CCl for 1 h. Washings were
4
performed with Py/CH CN (1/4, v/v, 3ꢁ1.5 mL) and
successively with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and
3
3
dry CH CN (3ꢁ1.5 mL). The terminal DMT protecting
dry CH CN (4ꢁ0.5 mL). The dT-H-phosphonate solu-
3
3
group was removed by washing with DCA/CH Cl
2
tion (10 mg, ca. 0.015 mmol) in dry CH CN (0.25 mL)
3
2
(
3/100, v/v, 1.0 mL) and additional washes with DCA/
and dry pyridine (0.25 mL) containing pivaloyl chlo-
ride (8 mL, ca. 0.075 mmol) was drawn into the syringe
and was agitated for 2 min. The coupling agents were
ejected from the syringe, and the support was washed
CH Cl (8ꢁ0.5 mL) followed by subsequentwashings
2
2
with Py/CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry CH CN
3
3
(
drawing the dT-H-phosphonate solution (10 mg, ca.
4ꢁ0.5 mL).The next coupling step was then effected by
with dry CH CN (3ꢁ0.5 mL). This cycle was repeated
3

N. U. Mohe et al. / Bioorg. Med. Chem. 11 (2003) 1419–1431
1431
until the oligonucleotide, d(TPHTPHTPHTPHTPHTPHT)
where PH corresponds to the H-phosphonate diester
2. (a) Garegg, P. J.; Regberg, T.; Stawanski, J.; Stromberg, R.
Chem. Scr. 1985, 25, 280. (b) Froehler, B. C.; Matteucci, M. D.
Tetrahedron Lett. 1986, 27, 469.
(
linkages in the backbone) was achieved. The inter-
nucleotidic H-phosphonate linkages were sulfurized by
using 2.0 mL mixture of 10% S8 in CS /PyTEA (35/35/
3
. Froehler, B. C.; Ng, P. G.; Matteucci, M. D. Nucl. Acids
Res. 1986, 14, 5399.
. Garegg, P. J.; Regberg, T.; Stawanski, J.; Stromberg, R.
Tetrahedron Lett. 1986, 27, 4051.
. de Vroom, E.; Dreef, C. E.; van der Marcel, G. A.; van
2
4
1
) for 3 h. Washings were performed with Py/CH CN
3
(
1/4, v/v, 6ꢁ1.5 mL) and dry CH CN (3ꢁ1.5 mL). The
3
5
terminal DMT protecting group was removed by wash-
ing with dichloroacetic acid (DCA)/CH Cl (3/100, v/v,
Boom, J. H. Recl. Trav. Chim. Pays-Bas 1988, 107, 592.
6. Searchrist, L. Bioworld Today 1998, 9, 1.
2
2
1
.0 mL) and additional washes with DCA/CH Cl
2
7. (a) Bennett, C. F. Biochem. Pharmacol. 1998, 55, 9. (b)
Juliano, R. L.; Alahari, S.; Yoo, H.; Cho, M. Pharm. Res.
1999, 16, 494. (c) Akhtar, S.; Agrawal, S. Trends Pharmacol.
Sci. 1997, 18, 12.
2
(
8ꢁ0.5 mL) followed by subsequentwashings wi ht Py/
CH CN (1/4, v/v, 1ꢁ0.5 mL) and dry CH CN (4ꢁ0.5
3 3
mL). The lastdT nucleosidic unitwas added by drawing
the dT-H-phosphonate solution (10 mg, ca. 0.015
mmol) in dry CH CN (0.25 mL) and dry pyridine (0.25
8
. (a) Agrawal, S. Trends Biotechnol. 1996, 14, 376. (b) Altmann,
K. H.; Dean, N. M.; Fabbro, D.; Freier, S. M.; Geiger, T.;
Haner, R.; Husken, D.; Martin, P.; Monia, B. P.; Muller, M.;
3
¨
¨
¨
mL) containing pivaloyl chloride (8 mL, ca. 0.075 mmol)
into the syringe and agitating the mixture for 2 min. The
coupling agents were ejected from the syringe, and the
supportwas washed wi th dry CH 3CN (3ꢁ0.5 mL). The
internucleoside H-phosphonate linkage was then oxi-
dized with 2.0 mL of CCl /H O/NMM/Py/CH CN
Natt, F.; Nicklin, P.; Phillips, J.; Pieles, U; Sasmor, H.; Moser,
H. E. Chimia 1996, 50, 168.
9
. (a) Padiya, K. J.; Salunkhe, M. M. Bioorg. Med. Chem.
2000, 8, 337. (b) Padiya, K. J.; Mohe, N. U.; Salunkhe, M. M.
Syn. Comm. 2002, 32 (6), 917.
10. Padiya, K. J.; Salunkhe, M. M. J. Chem. Res. (S) 1998,
804.
4
2
3
(
2.5/0.2/1.0/6.0/9.3, v/v) for 45 min. Washings were
1
1
4 2 3
1. The CCl /H O/NMM/Py/CH CN with the ratio 2.5/0.2/
.0/6.0/9.3, v/v was the optimized system for the solid phase
performed with Py/CH CN (1/4, v/v, 3ꢁ1.5 mL) and dry
3
CH CN (3ꢁ1.5 mL). The terminal DMT protecting group
3
oxidation. A contact time of 10 min with this optimized sys-
tem results in ca. 80% oxidized product, 3 as judged by RP-
HPLC analysis. However, quantitative oxidation takes 45 min.
was removed by washing with DCA/CH Cl (3/100, v/v,
2
2
1
.0 mL) and additional washes with DCA/CH C (8ꢁ0.5
2
12
mL) followed by subsequentwashings wi th Py/CH CN (1/
3
1
2. Froehler, B. C. Oligonucleotide synthesis; H-phosphonate
4
, v/v, 1ꢁ0.5 mL) and dry CH CN (4ꢁ0.5 mL). The sup-
3
Approach. In Protocols for Oligonucleotides and Analogues;
Agrawal, S., Ed.; Humana Press: New Jersey, p 63.
13. (a) Connolly, B.; Potter, B.; Eckstein, F.; Pingoud, A.;
Grotjahn, L. Biochemistry 1984, 23, 3443. (b) Eckstein, F.
Nucleosides Nucleotides 1985, 4 (1-2), 77.
portbound oligonucleo itd e was ht en comple et ly depro-
tected using concentrated NH OH, 55 C, 16 h.
ꢂ
4
RP-HPLC of 9 (retention time 31.0 min).
1
4. (a) Manoharan, M.; Kawasaki, A. M.; Prakash, T. P.;
Fraser, A. S.; Prhave, M.; Inamati, G. B.; Casper, M. D.;
Cook, P. D. Nucleosides Nucleotides 1999, 18, 1737. (b)
Kawasaki, A. M.; Casper, M. D.; Prakash, T. P.; Manalili, S.;
Sasmor, H.; Manoharan, M. Tetrahedron Lett. 1999, 40, 661.
ESI-MS spectrum of 9, Mwcalc: 2459.95 Mwfound: 2466.
Acknowledgements
(
c) Prakash, T. P.; Kawasaki, A. M.; Vasquez, G.; Fraser,
A. S.; Casper, M. D.; Cook, P. D.; Manoharan, M. Nucleo-
sides Nucleotides 1999, 18, 1381. (d) Review: Manoharan, M.
Biochim. Biophys. Acta 1999, 1489, 117.
15. Martin, P. Helv. Chim. Acta 1995, 78, 486.
16. Zhou, W.; Agrawal, S. Bioorg. Med. Chem. 1998, 8, 3269.
We thank the D.S.T., New Delhi for the financial assis-
tance. We thank Dr. Yogesh Sanghvi, Isis Pharmaceu-
ticals, Carlsbad, CA, USA for the LC/MS analysis of
our samples.
1
7. (a) Sanghvi, Y. S.; Andrade, M.; Deshmukh, R. R.;
Holmberg, L.; Scozzari, A. N.; Cole, D. L. In Manual of
Antisense Methodology, Ch. 1; Hartmann, G., Endres, S. Eds.;
Kluwer Academic Publishers: London, p 1. (b) Agrawal, S.
Biochimica et. Biophysica Acta 1999, 53.
18. Tanaka, T.; Letsinger, R. L. Nucleic Acids Res. 1982, 10,
3249.
References and Notes
1
1
. (a) Matteucci, M. D.; Caruthers, M. H. J. Am. Chem. Soc.
981, 103, 3185. (b) Beaucage, S. L.; Caruthers, M. H. Tetra-
hedron Lett. 1981, 22, 1859.