The synthesis of dithymidine boranophosphate by the oxathiaphospholane approach

Article abstract of DOI:10.1081/NCN-100107195

The application of the oxathiaphospholane approach for the synthesis of dithymidine boranphospate was evaluated. It was shown, that although the nucleoside-3′-O-oxathiaphospholane-borane complexes 2 or 6 could not be chromatographically separated into dia

Full text of DOI:10.1081/NCN-100107195

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THE SYNTHESIS OF DITHYMIDINE BORANOPHOSPHATE
BY THE OXATHIAPHOSPHOLANE APPROACH
Andrzej Okruszek a , Agnieszka Sierzchała a , Katarzyna Żmudzka a & Wojciech J. Stec a
a Department of Bioorganic Chemistry , Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences , Sienkiewicza 112, Łódź, 90-363, Poland
Published online: 19 Aug 2006.
To cite this article: Andrzej Okruszek , Agnieszka Sierzchała , Katarzyna Żmudzka & Wojciech J. Stec (2001) THE SYNTHESIS
OF DITHYMIDINE BORANOPHOSPHATE BY THE OXATHIAPHOSPHOLANE APPROACH, Nucleosides, Nucleotides and Nucleic Acids,
20:10-11, 1843-1849, DOI: 10.1081/NCN-100107195
To link to this article: http://dx.doi.org/10.1081/NCN-100107195
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(10&11), 1843–1849 (2001)
THE SYNTHESIS OF DITHYMIDINE
BORANOPHOSPHATE BY THE
OXATHIAPHOSPHOLANE APPROACH
_
Andrzej Okruszek,* Agnieszka Sierzchała, Katarzyna Zmudzka, and
Wojciech J. Stec
Department of Bioorganic Chemistry, Centre of Molecular and
Macromolecular Studies, Polish Academy of Sciences,
Sienkiewicza 112, 90-363 Lodz, Poland
ABSTRACT
The application of the oxathiaphospholane approach for the synthesis of
dithymidine boranphospate was evaluated. It was shown, that although
the nucleoside-30-O-oxathiaphospholane-borane complexes 2 or 6 could
not be chromatographically separated into diastereomerically pure spe-
cies due to their apparent instability to moisture, they can be successfully
applied to the non-stereocontrolled formation of internucleotide bor-
anophosphate bond by reaction with 50-OH-nucleoside in the presence of
DBU. Attempts to apply the related dithiaphospholane approach for the
preparation of dithymidine boranophosphorothioate were unsuccessful.
INTRODUCTION
Boranophosphates, containing negatively charged borane moiety
(BHÀ3 ) in place of one of the non-bridging oxygen atoms, constitute a new
backbone-modified class of oligonucleotides1. The boranophosphate group is
isoelectronic to the natural phosphodiester group and isosteric to the me-
thylphosphonate modification. Introduction of a boranophosphate moiety
*Corresponding author. Fax: þ4842681 54 83; E-mail: okruszek@bio.cbmm.lodz.pl
1843
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1844
OKRUSZEK ET AL.
retains the ability of oligonucleotides to form reasonably stable complexes
with the complementary DNA or RNA2–4 and to activate RNase H4,5, and
leads to significant increase in resistance of oligonucleotides towards nu-
cleolytic degradation2,3 thus making them promising candidates for ther-
apeutic applications including antisense=antigene approach or boron neutron
capture therapy (BNCT)6.
Although the first chemical synthesis of oligo(thymidine boranopho-
sphate)s was performed by phosphoramidite approach1 it soon became ob-
vious that much better results could be obtained by H-phosphonate
methodology with simultaneous boronation of pre-synthesized oligo(thymi-
dine-H-phosphonate) after its trimethylsilylation3,4,7. These syntheses pro-
vide more or less random mixtures of diastereomers due to the chirality of
each internucleotide boranophosphate centre. Full stereocontrol was only
observed during enzymatic primer template-directed synthesis of bor-
anophosphate oligonucleotides from diastereomerically pure Rp-nucleoside
a-boranotriphosphates, which were found to be good substrates for a
number of DNA polymerases2,8. For SP dithymidine boranophosphate a
stereoselective synthesis was reported by Jin and Just9,10 using (S)-3-hydroxy-
4-(2-indolyl)butyronitrile as a chiral auxiliary. These authors were also able
to separate into individual diastereomers appropriately protected dithymidyl-
b-cyanoethylphosphite-borane complexes and transform them into diaster-
eomerically pure dithymidine boranophosphates.
RESULTS AND DISCUSSION
Since the oxathiaphospholane approach proved to be a method of
choice for a stereocontrolled synthesis of oligo(nucleoside phosphor-
othioate)s11–13, it was of interest to check its applicability to the synthesis of
oligo(nucleoside boranophosphate)s. Thus, freshly prepared 3-N-anisoyl-50-
O-dimethoxytritylthymidine-30-O-1,3,2-oxathiaphospholane (1)14 (0.1 mmol)
was reacted with 6 equivalents of borane-diisopropylethylamine (DIPEA)
complex (10.5 mL, Aldrich) in THF (5 mL, freshly distilled over CaH2). The
reaction was followed by 31P NMR (Bruker AC 200, 85% H3PO4 as a
standard) and after 2h at rt a complete disappearance of signals corre-
sponding to the substrate was observed with the formation of a broad signal
at d 164.4 ppm (half-line width 1.9 ppm) which was assigned to
oxathiaphospholane-borane complex 2 (Scheme 1)15.
The attempts to isolate complex 2 by column chromatography in order
to separate P-chiral diastereomers were unsuccessful. The complex 2 ap-
peared to be quite reactive and decomposed readily in the presence of
moisture. Therefore, no separation of the crude product 2 was attempted.
Thus, the THF solution of 2 prepared from 1 mmol of 1 and 6 mmol of BH3-
DIPEA was reacted with 1 mmol of 3-N-anisoyl-30-O-acetylthymidine in the

DITHYMIDINE BORANOPHOSPHATE
1845
Scheme 1.
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1 mmol). After 2h at rt
the 31P NMR control (CD3CN added), showed complete disappearance of
the signal of 2 and the appearance of a new broad signal at d 93.7 ppm which
was assigned to the formation of protected dithymidine boranophosphate 316
(see Scheme 1). The compound 3 was isolated by silica gel column chroma-
tography in 97% yield and was characterized by 31P NMR (CD3CN, d 91.6,
93.5 ppm, broad dublet)16, 11B NMR (CD3CN, d À 36.6 ppm, broad signal,
half-line width 2.2 ppm, BF3xEt2O as a standard)16, and by FAB MS (m=z
1155.9; calcd MW 1155.94). A sample of 3 was deprotected at the 30-position
with 30% aq. ammonia (48 h, rt), and purified by RP HPLC (ODS Hypersil
5 m column, linear gradient of CH3CN in 0.1 M TEAB, 1.5%=min). The
substance collected at RT 32.7 min (main peak) was identified by FAB MS as
50-O-DMT-dithymidine boranophosphate 4 (m=z 845.6, calcd MW 845.62).
Although compound 4 was supposed to be obtained as a mixture of dia-
stereomers due to phosphorus chirality, only a single peak was observed by
HPLC under the conditions employed.
The same reaction was performed under the conditions of solid-phase
synthesis, using LCA CPG support loaded with 50-O-dimethoxy-
tritylthymidine via a sarcosinylated17 linker. Thus, the support (1 mmole) was
placed in a DNA synthesis column (Applied Biosystems) attached to a syr-
inge, and was treated with 3% trichloroacetic acid in dichloromethane in
order to remove the 50-DMT group. After washing (CH2Cl2, CH3CN) and
drying (argon flush) the THF solution of 218, premixed with DBU, was ap-
plied to the column and reacted with the support bound nucleoside for a
given period of time (see Table 1). Then, the column was subjected to
washing (THF, CH3CN) and drying (argon), and the product was cleaved
from the support by overnight treatment with 30% aq. ammonia. The so-
lution was evaporated and analyzed by RP HPLC (conditions as above). The
yield of 50-O-DMT-dithymidine boranophosphate 4 under the conditions of
solid phase synthesis was calculated from integrated chromatograms taking
into account the intensity of the peak of 4 (RT 32.7 min) and the peak of
unreacted thymidine cleaved from the support (RT 8.7 min). The results of
syntheses performed under various experimental conditions are listed in
Table 1.

1846
OKRUSZEK ET AL.
Table 1. The Synthesis of Dithymidine Boranophosphate on the Solid Support (1 mmol)
Oxathiaphospholane
Complex 2
(Molar Equivalents)
DBU
(Molar Equivalents)
Reaction Time
(min)
Yield of 4
(%)
6
6
5
18
5
1212
24
55
24
30
30
50
100
200
30
5
5
10
5
5
5
10
58
68
68
58
65
58
35a
30
30
50
30
30
30
a With CH3CN instead of THF as a solvent.
It was found, that the best yield of 4 (68%) was obtained when THF
was used as a solvent, with a 30-fold molar excess of both 2 and DBU with
respect to support bound nucleoside, and the reaction time restricted to
5 min. Further increase in the concentration of the reagents or prolongation
of the reaction time did not lead to any improvement of the yield of con-
densation. A change of the solvent to acetonitrile resulted in a substantial
decrease of the yield of 4.
These experiments proved that the internucleotide boranophosphate
bond is formed under the conditions of oxathiaphospholane synthesis.
Since acidic deprotection of the 50-hydroxyl function in 4 would inevitably
lead to the degradation of boranophosphate by its reaction with di-
methoxytrityl cation4, further experiments aimed at the preparation of the
fully deprotected dithymidine boranophosphate were performed with the
substrate protected at the 50-hydroxyl with a t-butyldimethylsilyl (TBDMS)
group. Thus, the 50-O-TBDMS-thymidine-30-O-l,3,2-oxathiaphospholane
(5)19 was reacted in THF with 10 molar equivalents of borane-DIPEA
complex. After 2h at rt the 31P NMR inspection (CD3CN added) showed
the formation of oxathiaphospholane-borane complex 6 as a broad signal
at d 164.0 ppm (Scheme 2). The complex 6 appeared to be too unstable to
be purified by flash column chromatography, so the THF solution pre-
pared as above was used for further experiments without purification.
Thus, the THF solution of 6, prepared from 1 mmol of 5 and 10 mmol of
BH3-DIPEA, was reacted with 0.9 mmol of 3-N-anisoyl-30-O-acet-
ylthymidine in the presence of DBU (3 mmol). The formation of protected
dinucleoside boranophosphate 7 was evidenced by 31P NMR analysis
(CD3CN added, broad signal at d 93.2ppm) 16. The compound 7
was purified by silica gel column chromatography (CH2Cl2-MeOH as an

DITHYMIDINE BORANOPHOSPHATE
1847
Scheme 2.
eluent) in 60% yield, and was characterized by 31P NMR (CD3CN, d
91.2ppm, broad signal) 16, and by FAB MS (m=z 833.5; calcd MW 833.70).
A sample of 7 was incubated with 1 M HFxEt3N (THF, 48 h, rt) followed
by treatment with 30% aq. ammonia (48 h, rt). The fully deprotected di-
thymidine boranophosphate 8 was isolated by RP HPLC (Econosil 5 m
column, linear gradient of CH3CN in 0.1 M TEAB, 1.5%=min) at RT
10.2min. The unsymmetrical appearance of the HPLC peak of
8
(shoulder) was related to the fact that boranophosphate 8 was supposed to
be formed as a mixture of diastereomers. However no separation of the
peaks related to diastereomers was achieved under the conditions em-
ployed. The structure of compound 8 isolated as above was confirmed by
FAB MS (m=z 543.2; calcd MW 543.25).
The above results on the application of the oxathiaphospholane ap-
proach to the preparation of dinucleoside boranophosphate prompted us to
check the possibility of the synthesis of dinucleoside boranopho-
sphorothioate20 by the dithiaphospholane approach21. Thus, 50-O-TBDMS-
thymidine-30-O-l,3,2-dithiaphospholane (9)22 was reacted with 10 molar
equivalents of borane-DIPEA complex in THF solution. After 2h at rt the
31P NMR inspection (CD3CN added) showed the presence of the signal of 9
and the appearance of a broad signal at d 173 ppm which could be assigned to
the corresponding dithiaphospholane-borane complex (10). The integration
of the spectrum showed the ratio 9:10 to be ca 1:1. The attempts to shift the
equilibrium towards the formation of a higher proportion of 10 by pro-
longation of the time of experiment or by increasing of the excess of borane-
DIPEA complex failed. Similarly, the isolation of pure 10 by flash column
chromatography was not possible. In this situation it was attempted to react
the crude mixture prepared as above with 3-N-anisoyl-30-O-acetylthymidine
in the presence of DBU (10 molar equivalents). Although in the obtained
mixture the 31P NMR analysis showed the presence of some amounts (ca
30%) of the broad signal at d 166 ppm which could be attributed to the
presence of corresponding dithymidine boranophosphorothioate20, isolation
attempts by column chromatography were not successful, and further at-
tempts of dinucleoside boranophosphorothioates by this approach were
abandoned.

1848
OKRUSZEK ET AL.
CONCLUSIONS
The results presented in this paper indicate that the formation of in-
ternucleotide boranophosphate bond can be accomplished under the condi-
tions of oxathiahospholane synthesis both in solution and on the solid
support, although the yields for the solid support synthesis are far from being
satisfactory. Unfortunately, due to the apparent instability of oxathiapho-
spholane-borane complexes 2 and 6, their chromatographic separation into
diastereomerically pure species was not possible, so we could not pursue the
matter of the stereospecificity of ring-opening condensation. An attempt to
apply the dithiaphospholane approach for the preparation of dithymidine
boranophosphorothioate was not successful.
ACKNOWLEDGMENTS
The research presented in this paper was financially assisted by the State
Committee for Scientific Research (KBN), Grant no. 4.P05F.006.17.
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14. 1 Was Prepared From 3-N-Anisoyl-50-O-DMT-Thymidine and 2-N,N-Diisopro-
pylamino-1,3,2-Oxathiaphospholane11 in CH2Cl2 in the Presence of Tetrazole

DITHYMIDINE BORANOPHOSPHATE
1849
and was Isolated by Flash Column Chromatography (Silica Gel, Benzene-
CHCl3 as Eluent) in 60% Yield. 31P NMR (CD3CN) d 171.05, 172.90 ppm.
The 3-N-Anisoyl Protection of Thymine was Employed to Prevent the Reduc-
tive Degradation of the Base by the Borane Complex4.
15. Similar Results were Obtained when THF-BH3 or Me2S-BH3 Complexes were
used Instead of DIPEA-BH3 for the Reaction with 1.
16. For D2O Solutions of Unprotected Dithymidine Boranophosphates Jin and
Just9 Reported Following NMR Chemical Shift Data: 31P NMR, d 93.51,
93.76 ppm (Broad Signals, for Sp and Rp Diastereomers, Respectively); 11B
NMR, d À 41.64, 41.29 ppm (Broad Signals, for Sp and Rp Diastereomers, Re-
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in a Separated Flask from a Given Amount of 1 (see Table 1) and 6 Molar
Equivalents of BH3-DIPEA (2h at rt, total volume 140 mL). Before Applying
to the Column the Solution of 2 was Treated with DBU (Amounts are Given
in Table 1). In this Series of Experiments the Thymidine Derivative Without 3-
N-anisoyl Protection was used.
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1,3,2-oxathiaphospholane11 in CH2Cl2 in the Presence of Tetrazole and was
Isolated by Flash Column Chromatography (Silica Gel, CHCl3-MeOH as Elu-
ent) in 78% Yield. 31P NMR (C6D6) d 172.20, 173.64 ppm.
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22. 9 was Prepared from 50-O-TBDMS-thymidine and 2-N,N-diisopropylamino-
1,3,2-dithiaphospholane21 in CH2Cl2 in the Presence of Tetrazole, and was Iso-
lated by Flash Column Chromatography (Silica Gel, CHCl3-MeOH as Eluent)
in 93% Yield. 31P NMR (CD3CN) d 150.95 ppm.
Received December 27, 2000
Accepted July 2, 2001