New approach to the synthesis of oligodeoxyribonucleotides modified with phosphorothioates of predetermined sense of P-chirality

Article abstract of DOI:10.1016/j.tetlet.2005.07.158

Appropriately protected, diastereomerically pure dinucleoside phosphorothioates, obtained by the stereocontrolled oxathiaphospholane method, were S-alkylated with 2-nitrobenzyl bromide and then converted into their 3′-O-phosphoramidites. The corresponding S-protected dinucleotide building blocks were successfully used for the synthesis of oligonucleotides containing P-stereodefined phosphorothioate bonds at preselected positions.

Full text of DOI:10.1016/j.tetlet.2005.07.158

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 6641–6644
New approach to the synthesis of
oligodeoxyribonucleotides modified with phosphorothioates
of predetermined sense of P-chirality
*
´ ´
Barbara Nawrot, Beata Re˛bowska, Katarzyna Cieslinska and Wojciech J. Stec
Centre of Molecular and Macromolecular Studies of Polish Academy of Sciences, Department of Bioorganic Chemistry,
Sienkiewicza 112, 90-363 Ło´dz´, Poland
Received 17 June 2005; revised 20 July 2005; accepted 29 July 2005
Available online 15 August 2005
Abstract—Appropriately protected, diastereomerically pure dinucleoside phosphorothioates, obtained by the stereocontrolled
oxathiaphospholane method, were S-alkylated with 2-nitrobenzyl bromide and then converted into their 30-O-phosphoramidites.
The corresponding S-protected dinucleotide building blocks were successfully used for the synthesis of oligonucleotides containing
P-stereodefined phosphorothioate bonds at preselected positions.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Oligonucleotides containing, at selected position(s),
internucleotide phosphorothioate linkages of a predeter-
mined sense of P-chirality (PS-oligos) are attractive as
probes in mechanistic studies of various enzymes, partic-
ularly endo- and exonucleases.1 They are accessible via
the phosphoramidite approach2 where at selected steps
the routine oxidation of intermediary phosphites with
iodine–water is replaced by sulfurization.3 However,
the resulting PS-oligos are a mixture of diastereoisomers
that have to be separated, preferably by RP-HPLC.4 In
numerous cases such an attempted separation is not
effective.5 Pure diastereoisomers of PS-oligos modified
with a single phosphorothioate moiety are accessible
via preparation of dinucleoside phosphorothioates con-
taining an O-protected phosphorothioate function, their
separation at the dinucleotide stage, 30-O-activation
with phosphoramidite and subsequent incorporation
into the oligonucleotide chain at a selected position.6
Appropriate protection of the phosphorothioate func-
tion is necessary since unprotected phosphorothioate
diesters are converted effectively into phosphates under
treatment with iodine–water, which is unavoidable dur-
ing chain elongation. Attention has to be paid to proper
selection of the phosphorothioate-protecting group
which, after completion of the synthesis of the desired
oligonucleotide, has to be removed without loss of sulfur
and with known stereochemistry.6,7
Recently, we demonstrated that dinucleoside-(30,50)-O-
p-nitrophenyl phosphorothioates can be used as build-
ing blocks due to adventitious separation of the diaste-
reomers (this is not always the case for dinucleoside
(30,50)-phosphorothioate O-alkyl triesters) and clean ste-
reoinvertive removal of the p-nitrophenyl protection by
means of syn-4-nitrobenzaldoxime/1,1,3,3-tetramethyl-
guanidine.8
Here, we present an alternative approach based upon
the stereocontrolled synthesis of dinucleoside phos-
phorothioates using the oxathiaphospholane method
(OTP) developed in our laboratory.9 Suitably protected
30-O-(2-thio-1,3,2-oxathiaphospholane) nucleosides
1
were afforded from the reaction of the 30-OH group of
50-DMT-O-protected nucleosides with N,N-diisoprop-
ylamino-1,3,2-oxathiaphospholane, followed by sul-
furization and chromatographic separation into pure P-
diastereoisomers.10 The yields from the DBU-promoted
ring opening condensations with 50-OH-nucleosides
were excellent and the stereospecificity was above
99.5%. Therefore, the corresponding P-chiral dinucleo-
side phosphorothioates 3 of high diastereoisomeric
purity are now available. Diastereomerically pure
Keywords: P-Chiral dinucleoside phosphorothioate; Oligonucleotide
phosphorothioate; PS-DNA; S-Protection; Oligonucleotide synthesis.
*
Corresponding author. Tel.: +48 42 681 6970; fax: +48 42 681
5483; e-mail: bnawrot@bio.cbmm.lodz.pl
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.07.158

6642
B. Nawrot et al. / Tetrahedron Letters 46 (2005) 6641–6644
B1
B1
B1
_
DMTO
O
DMTO
O
DMTO
aq. ammonia/dioxane,
1/1, v/v
25-30 min
O
B2
O
HO
DBU
_
O
O
O
O
+
S
S
O
O
S
*
*
*
P
P
P
B2
B2
O
O
S
O
O
OiPA
3
1
2
4
OiPA
OH
B1
B1
DMTO
O
DMTO
O
NO2
B2
NO2
B2
(2-NO2)-PhCH2-Br
phosphitylation
CH2
O
CH2
O
S
O
S
*
*
P
P
O
O
O
O
O
5
6
OH
O
P
B1 = Thy, GuaDpc,iBu, GuaiBu
B2 = Thy, CytBz, AdoBz
O
NC
N
Scheme 1. Synthesis of S-protected P-stereodefined dinucleoside units.
phosphorothioate dimers are S-alkylated and converted
into building blocks via 30-O-deprotection followed by
activation with phosphoramidite. The obvious advan-
tage of the present method results from the fact that
only four oxathiaphospholane monomers 1 have to be
separated into their RP and SP diastereoisomers in order
to access all 16 diastereomerically pure common dinu-
cleotides 3. As depicted in Scheme 1, diastereomerically
pure compounds 1 were condensed with appropriately
protected nucleosides 2 providing the dinucleoside
phosphorothioates 3. As established earlier, the slow-
migrating diastereoisomers of 1 furnished the dinucleo-
tides 3 of RP configuration (Scheme 1).11
led to removal of the diphenylcarbamoyl (Dpc) protect-
ing group from O-6 of the guanine nucleobase. Before
the conversion of dimers 4 into building blocks 6, the
sulfur atom of the internucleotide phosphorothioate
function was alkylated with 2-nitrobenzyl bromide,13
which was selected among several tested chloro- and
nitrobenzyl halides, as the most suitable S-protecting
agent. The 2-nitrobenzyl protecting group was found
to be stable during oligonucleotide synthesis and could
be efficiently and stereospecifically removed by means
of thiophenolate anion7 before aqueous ammonia-as-
sisted cleavage of the oligonucleotide from the solid sup-
port. The alkylation reaction was optimized for the
diastereomeric mixture of dithymidine phosphorothio-
ate 4 (B1 = B2 = Thy). Physical data and yields of 5
are given in Table 1. Routine phosphitylation of 5 with
Removal of the iso-propoxyacetyl protecting group12
from the 30-OH position of 3 was performed using
28% aqueous ammonia/dioxane mixture (1/1, v/v) at
room temperature in a time-controlled reaction, moni-
tored by TLC. Typically, the reaction was terminated
after 25–30 min by removal of ammonia under slightly
reduced pressure. Dimers 4 were obtained in preparative
yields >80%. Extended reaction times (up to 120 min)
2-cyanoethyl
bis-(N,N-diisopropyl)phosphoramidite/
1-H-tetrazole in acetonitrile,2 followed by silica gel
chromatographic purification, afforded the desired
products 6, 80–90% yields, which were ready to use as
building blocks for standard phosphoramidite synthesis
of oligonucleotides.
Table 1. Spectral and chromatographic characteristics of S-protected dinucleoside phosphorothioates 5 (Scheme 1)
No.
P-conf.
B1
B2
MALDI-TOF
31P NMR (d, ppm)
Yield 4 ! 6 (%)
Rf (CHCl3/MeOH, 9/1)a
5
Mix
RP
SP
Thy
Thy
999.4b [M+H]+
1380.3 (1379)
1380.3 (1379)
1111.9 (1111)
1111.7 (1111)
1182.8 (1182)
1182.8 (1182)
29.00; 29.18
26.59
26.96
27.83
27.96
91
66
57
84
82
75
79
0.31
0.76
0.71
0.30
0.33
0.52
0.45
5a
5b
5c
5d
5a0
5b0
Guadpc, i-Bu
Guadpc, i-Bu
Thy
CytBz
CytBz
AdoBz
AdoBz
CytBz
CytBz
RP
SP
Thy
RP
SP
Guai-Bu
Guai-Bu
27.29
27.65
a Thin layer chromatography, silica gel 60 F254 (Merck).
b FAB MS.

B. Nawrot et al. / Tetrahedron Letters 46 (2005) 6641–6644
Table 2. Sequences and characteristics of oligonucleotide phosphorothioates 7–15
6643
PS-ODN
No.
Sequence 50 ! 30
P-conf.
HPLCa
DMT-ON
tR (min)b
MALDI-TOF
DMT-OFF
HPLCa
DMT-OFF
tR (min)b
Yield
(A260)
Calcd
Exp.
7
GACTTPSTCGAT
Mix
RP
SP
20.42
19.40
21.01
19.51
21.62
19.91
21.13
19.54
19.39
3032
9586
9586
9586
9586
9586
9586
9586
9586
3031
9591
9592
9583
9580
9591
9589
9577
9585
19.61
21.14
20.97
20.85
20.70
16.21
14.79
14.83
14.79
15.0
11.3
27.0
14.3
12.0
7.2
10.0
7.0
15.3
8
AGGGTTGAGGCTAGpsCTACAACGATCATCTGT
AGGGTTGAGGCTAGpsCTACAACGATCATCTGT
AGGGTTGAGGpsCTAGCTACAACGATCATCTGT
AGGGTTGAGGpsCTAGCTACAACGATCATCTGT
AGGGTTGAGGCTpsAGCTACAACGATCATCTGT
AGGGTTGAGGCTpsAGCTACAACGATCATCTGT
AGGGTTGAGGCTAGCTpsACAACGATCATCTGT
AGGGTTGAGGCTAGCTpsACAACGATCATCTGT
9
10
11
12
13
14
15
RP
SP
RP
SP
RP
SP
a Hypersil BDS C-18 column, 4 · 250 mm, eluting buffers—A: 1 M triethylammonium bicarbonate, pH 7.5; B: 40% acetonitrile/60% A; DMT-ON
gradient—B: 15% ! 100% in 0 ! 20 min; B: 100% in 20 ! 35 min; DMT-OFF gradient—B: 0% ! 50% in 0 ! 20 min; 50% ! 100% in
20 ! 24 min; 100% in 24 ! 32 min; 100% ! 0% in 32–36 min.
b Purity of oligomers was >98%.
S-Protected P-chiral phosphorothioate dinucleosides
were successfully introduced into the DNA chain of
the model octamer 7 and eight 31-mers (8–15). These
later constructs containing the 15-nt consensus sequence
of a catalytic domain of deoxyribozyme 10–2314 and
substrate recognition arms complementary to the
selected region (from 1801 to 1817) of the human aspar-
tyl protease Asp2 (BACE) mRNA15 were designed for
mechanistic studies on the mode of action of the deoxy-
ribozyme 10–23.
gel electrophoresis (20% polyacrylamide/7 M urea) (data
not shown) was >98%. The structure of the oligomers
was confirmed by MALDI-TOF mass spectrometry
(Table 2). The absolute configuration at the phosphorus
atoms in the phosphorothioate internucleotide bonds of
oligonucleotides 8–15 was confirmed enzymatically using
snake venom phosphodiesterase (PDE I) and 30-endo-
nuclease P1 (nP1) (see Supplementary data).19,20
The methodology presented in this letters demonstrates
that S-protected diastereomerically pure dinucleoside
phosphorothioates 5 can be prepared effectively from
nucleoside oxathiaphospholanes 1. These, after S-pro-
tection and 30-OH activation, are introduced into the
DNA chain by solid-phase phosphoramidite methodol-
ogy. This approach permits the preparation of PS-
deoxyribozymes with a predetermined sense of chirality
at the phosphorus atom of the internucleotide phos-
phorothioate function at each selected internucleotide
position of the catalytic core. Since some of the catalytic
nucleic acids facilitate magnesium-assisted hydrolysis of
phosphodiester bonds in complementary RNA, their
activity is expected to be modulated by thio-substitu-
tions in selected positions of the catalytic core. In subse-
quent studies, the stereochemical aspects of reactions
catalyzed by chimeric 10–23 DNAzymes containing a
single PS linkage of RP- and SP-configuration will be
evaluated. In addition, future studies will be targeted
at the preparation of diastereomerically pure di-ribo
nucleoside phosphorothioates and their incorporation
into the RNA chain. Various sources report that such
P-chiral oligoribonucleotides containing phosphoro-
thioate functions are indispensable tools for evaluation
of the stereochemical outcome of RNA-binding21 and
RNA-cleaving proteins.19
The syntheses of PS-modified DNAzymes were per-
formed on 1 lmol scale according to the routine proto-
col with the coupling time for the modified units
extended to 120 s. The coupling yields for the incorpora-
tion of the building blocks 6 were in the range of 90–
99% (DMT cation assay), depending on the incorpo-
rated dimer. After the syntheses were complete, the
oligonucleotides were treated with 1 M piperidine/
acetonitrile solution to remove the 2-cyanoethyl protect-
ing groups from the phosphate functions. This deprotec-
tion protocol was designed to avoid P-Sꢀ center
modification with the acrylonitrile released during phos-
phate deprotection.16,17 Subsequently, the polymer-
bound oligonucleotides were treated for a short time
with a mixture of dioxane/triethylamine/thiophenol (1/
2/2, v/v/v). HPLC analysis confirming that treatment
of the oligomers with thiophenolate for 5 min resulted
in effective removal of the S-protecting group from the
phosphorothioate function.
It should be pointed out that in the case of GuaDpc
derivatives, this procedure did not result in substitution
of the O-diphenylcarbamoyl moiety with a S-Ph residue,
though such a transformation was observed when the
guanosine protected with Dpc was exposed to thiophe-
nol for 2–3 h.18
Acknowledgements
The oligomers with deprotected internucleotide bonds
were further treated with 28% aqueous ammonia,
followed by RP-HPLC (DMT-ON) purification, acidic
removal of the DMT group, and a final RP-HPLC puri-
fication (DMT-OFF).4 The purity of the oligomers,
determined by reverse phase HPLC and polyacrylamide
This work is dedicated to Professor Jan Michalski on
the occasion of his 85th birthday. Financial support
from the State Committee for Scientific Research in
Poland (Project PBZ-KBN-059/T09/09) is gratefully
acknowledged.

6644
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available in electronic version: detailed synthetic proce-
dures for 1–6; spectral data of oxathiaphospholanes 1
and dimers 3 and 4, chromatographic profiles and
MALDI-TOF mass spectra of oligomer 14 and HPLC
analysis of the products of enzymatic digestion of 12
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