Nucleoside H-boranophosphonates: A new class of boron-containing nucleotide analogues

Article abstract of DOI:10.1039/b901045a

A study on the synthesis of nucleoside H-boranophosphonates, a new class of nucleotide analogues having a P→BH₃ and a P-H group, via condensation of the corresponding nucleosides with H-boranophosphonate derivatives is described. The Royal Soci

Full text of DOI:10.1039/b901045a

Showcasing research from Professor Takeshi Wada’s
laboratory, The University of Tokyo, Japan.
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Title: Nucleoside H-boranophosphonates: a new class of boron-
Chemical Communications
www.rsc.org/chemcomm
Number 18 | 7 May 2009 | Pages 2413–2592
containing nucleotide analogues
A new class of boron-containing nucleotide analogues, nucleoside
H-boranophosphonates, have been developed. These compounds
ĺ
have a P–H and a P BH3 group, both of which can be used
for further modifications. The unique chemical properties of
H-boranophosphonates will open up access to a wide variety of as
yet unknown phosphate-modified nucleotide analogues.
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Nucleoside H-boranophosphonates: a new class of boron-containing
nucleotide analoguesw
Renpei Higashida, Natsuhisa Oka, Toshihide Kawanaka and Takeshi Wada*
Received (in Cambridge, UK) 16th January 2009, Accepted 6th February 2009
First published as an Advance Article on the web 19th February 2009
DOI: 10.1039/b901045a
A study on the synthesis of nucleoside H-boranophosphonates, a
new class of nucleotide analogues having a P-BH3 and a P–H
group, via condensation of the corresponding nucleosides with
H-boranophosphonate derivatives is described.
Chemical modifications of natural nucleotides and oligo-
nucleotides by replacing the non-bridging oxygen atoms of
their phosphate groups with other elements or substituents
(e.g., sulfur atom) have been widely used, especially for the
development of nucleic acid-based drug candidates.1,2 Nucleo-
side boranophosphates, in which a non-bridging oxygen atom
of the phosphate group is replaced with a BH3 group, are one
of the most promising candidates owing to their significant
stability to nucleases and high lipophilicity, which may save
the need for an elaborate delivery system.3 Their low cyto-
toxicity has also been suggested.4 In addition, oligonucleoside
Fig. 1 Strategy for synthesis of various P-modified oligonucleotide
analogues 2 and 3 by substitution of P–H and P–B bonds of oligo-
nucleoside H-boranophosphonates 1, respectively. B = nucleobase.
boranophosphates have shown promising RNA interference
activity,5 and may also be useful as target-specific 10B carriers
for boron-neutron capture therapy (BNCT).6
difficult to apply these methods to the synthesis of more functiona-
lized molecules, such as nucleotide analogues.
These studies on the nucleoside boranophosphates have
generated interest in other nucleotide analogues containing a
P-BH3 group. However, only a limited number of such
analogues are available7,8 with the methods developed for
the synthesis of nucleoside boranophosphates.9–11
We anticipated that an H-boranophosphonate derivative
having at least one free P–Oꢀ function would undergo
condensation reactions with the hydroxy group of nucleosides
to give the nucleoside H-boranophosphonates under mild
reaction conditions. Based on this idea, we synthesized inorganic
H-boranophosphonate as a monopyridinium salt (Scheme 1, 7).
Phosphinic acid 4 was treated with N,O-bis(trimethylsilyl)-
benzamide15 to afford bis(trimethylsilyl) phosphonite 5, which
was then boronated by treatment with BH3ꢁTHF. Montchamp
et al. have also synthesized bis(trialkylsilyl) H-borano-
phosphonates and alkyl trialkylsilyl H-boranophosphoates
via the corresponding phosphonite derivatives, though the
bis(TMS) derivative 5 was not used for further applications
due to its instability. In contrast, we used the silylation as a
transient protection and the intermediate 6 was desilylated by
treatment with MeOH and pyridine to synthesize 7. The
To expand the availability of the boron-containing nucleotide
analogues, we designed a novel nucleotide analogue having a
P-BH3 and a P–H group (nucleoside H-boranophosphonate 1,
Fig. 1) with the expectation that it would work as a versatile
precursor of a variety of boron-containing P-modified nucleotide
analogues via the activation of the P–H function by bases or
transition metals followed by reactions with electrophiles.12 The
P-BH3 moiety may also work as a modifiable site via deboro-
nation and subsequent reaction with electrophiles, which would
expand the availability of P-modified nucleotide analogues.12a,13
To the best of our knowledge, there are only two reports on
the synthesis of H-boranophosphonate derivatives.12c,14 Cento-
fanti reported the synthesis of dimethyl H-boranophosphonate
from dimethyl phosphonite decades ago.14 In addition, the
synthesis of silyl H-boranophosphonate derivatives and their
applications as precursors of alkylboranophosphonates were
reported by Montchamp et al. very recently.12c However, it is
Department of Medical Genome Sciences, Graduate School of
Frontier Sciences, The University of Tokyo, Bioscience Building 702,
5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
E-mail: wada@k.u-tokyo.ac.jp; Fax: +81 4 7136 3612;
Tel: +81 4 7136 3612
w Electronic supplementary information (ESI) available: Experimental
details and NMR spectra. See DOI: 10.1039/b901045a
Scheme 1 Synthesis of pyridinium H-boranophosphonate 7.
ꢂc
This journal is The Royal Society of Chemistry 2009
2466 | Chem. Commun., 2009, 2466–2468

Table 1 Synthesis of nucleoside 50-H-boranophosphonates 9a–e by 50-boranophosphonylation of nucleosides 8a–ea
Yield of
9a–e (%)
Entry
8
BPRO
R2
R3
Reagents and conditions
1
2
3
4
5
6
7
a
a
a
b
c
Thbz
Thbz
Thbz
Th
Th
Th
H
H
H
H
H
H
OPac
DMTr
DMTr
DMTr
DMTr
Bz
7 (1.5 equiv.), Piv-Cl (1.5 equiv.), iPr2NEt (3 equiv.), NT (1.5 equiv.), MeCN, rt, 3 h
7 (2 equiv.), Bop-Cl (2 equiv.), iPr2NEt (4 equiv.), NT (2 equiv.), MeCN, rt, 2 h
7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 2 h
7 (1.6 equiv.), Bop-Cl (1.6 equiv.), Py, rt, 1 h
7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 30 min
39
43
95
95
92
90
98
d
e
Pac
Pac
7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 40 min
7 (2 equiv.), Bop-Cl (2 equiv.), Py, rt, 2 h
Ur
a
TEAB = triethylammonium bicarbonate; BPRO = (protected) nucleobase; Thbz = N3-benzoylthymin-1-yl; DMTr = 4,40-dimethoxytrityl;
Piv-Cl = pivaloyl chloride; NT = 3-nitro-1H-1,2,4-triazole; Bop-Cl = bis(2-oxo-3-oxazolidinyl)phosphinic chloride; Pac = phenoxyacetyl.
resultant pyridinium H-boranophosphonate 7 was stable to air
oxidation and water, and can be stored for at least several
months at ꢀ30 1C without decomposition.
Table 2 Deprotection of nucleoside 50-H-boranophosphonates 9b,d,e
Pyridinium H-boranophosphonate 7 was applied to the
synthesis of nucleoside H-boranophosphonate derivatives.
Firstly, nucleoside 50-H-boranophosphonates were synthe-
sized by condensation of 7 with appropriately protected
nucleosides having a 50-hydroxy group (8a–e, Table 1). When
the reaction was carried out under conditions similar to those
used for the boranophosphorylation of nucleosides,11a
the isolated yields of the desired thymidine 50-H-borano-
phosphonate 9a were low mainly due to the formation of a
50,50-dinucleoside H-boranophosphonate diester (entries 1 and 2).
In sharp contrast, condensations promoted by Bop-Cl in
pyridine afforded the desired nucleoside 50-H-borano-
phosphonates 9a–e in excellent yields without observable
side-reactions (entries 3–7).
Deprotection
conditions
Yield
10 of 10 (%)
Entry
1
B
9
b
R2
H
R3
a
Th
DMTr 80% AcOH,
rt, 30 min
a
a
b
—
71
68
2
3
Th
Ur
d
e
H
Pac
sat. NH3–MeOH,
rt, 50 min
sat. NH3–MeOH,
rt, 3 h
OPac Pac
a
Decomposition of H-boranophosphonate moiety was observed.
Next, we attempted to synthesize fully-deprotected nucleo-
side 50-H-boranophosphonates, which would be interesting as
a new class of boron-containing nucleoside monophosphate
analogues. Firstly, the detritylation of 30-O-DMTr-thymidine
50-H-boranophosphonate 9b was attempted by treatment with
an 80% AcOH aqueous solution. However, decomposition of
the H-boranophosphonate moiety (ca. 70%) was observed
after 30 min (Table 2, entry 1). It is probably due to the
reaction of the liberated DMTr+ with the BH3 group of the
H-boranophosphonate moiety as observed in the case of
boranophosphates.10b,11a,16 In contrast, the H-boranophosphonate
monoesters were stable under conventional ammonolysis
conditions, and the nucleoside 50-H-boranophosphonates
10a,b were isolated in good yields (entries 2 and 3).
low yields of 12 mainly due to the generation of a 30,30-dithymidine
H-boranophosphonate derivative (entries 1 and 2).
The 50-O-protected nucleoside 30-H-boranophosphonates are
potentially useful as monomer units to synthesize oligo-
nucleotide analogues having an H-boranophosphonate diester
backbone. As we mentioned above, such oligonucleotide
analogues would work as precursors of a variety of back-
bone-modified oligonucleotide analogues through the P–H or
P–B modifications. To investigate the potential of the H-borano-
phosphonates,
a dithymidine H-boranophosphonate was
synthesized from the 50-O-DMTr-nucleoside 30-H-borano-
phosphonate 12a. The 30-H-boranophosphonate 12a was
allowed to condense with 30-O-DMTr-N3-benzoyl-thymidine
8a in MeCN in the presence of Bop-Cl and 2,2,6,6-tetramethyl-
piperidine (Scheme 2, reaction i). The reaction proceeded
smoothly at rt. Although a partial decomposition during silica
gel column chromatography was observed, the dithymidine
H-boranophosphonate diester 13a was isolated in a modest yield.
To explore the potential of the nucleoside H-borano-
phosphonates, we carried out the following two experiments.
Firstly, a 30-O-benzoyl-dithymidine H-boranophosphonate 13b
The reaction conditions optimized for the 50-borano-
phosphonylation were also applicable to the synthesis of
nucleoside 30-H-boranophosphonates. The condensation of
50-O-DMTr-nucleosides 11a,b with
7 in pyridine in
the presence of Bop-Cl afforded the desired nucleoside
30-H-boranophosphonates 12a,b in excellent yields (Table 3,
entries 3 and 4), whereas the reactions promoted by Piv-Cl or
Bop-Cl in MeCN in the presence of iPr2NEt and NT resulted in
ꢂc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 2466–2468 | 2467

boranophosphorothioate 15 (dP 162.5, 161.0)8a,b within 3 h at
rt, and 15 was isolated in good yield (Scheme 2, reaction iii).
In conclusion, nucleoside H-boranophosphonates were syn-
thesized via condensation of the corresponding nucleosides and
H-boranophosphonate derivatives. The mild reaction conditions
and high efficiency of this method are attractive for further
applications to the synthesis of a broad spectrum of H-borano-
phosphonate derivatives. In addition, conversion of the P–H
group of a dinucleoside H-boranophosphonate into a P–S– group
demonstrated that the H-boranophosphonate derivatives are
potential precursors of a variety of boron-containing oligo-
nucleotide analogues. Further studies on the synthesis of nucleoside
H-boranophosphonates and their applications are in progress.
We thank Professor Kazuhiko Saigo (University of Tokyo)
for helpful suggestions. This research was supported by a
Grant-in-Aid for Young Scientists (B) (No. 20750127) from
MEXT Japan (N.O.).
Table 3 Synthesis of nucleoside 30-H-boranophosphonates 12a,b by
30-boranophosphonylation of nucleosides 11a,b
Yield of
12 (%)
Entry 11 BPRO Reagents and conditions
1
a
Thbz
7 (1.2 equiv.), Piv-Cl (1.5 equiv.),
iPr2NEt (3 equiv.), NT (1.5 equiv.),
MeCN, rt, 4 h
7 (1.2 equiv.), Bop-Cl (3 equiv.),
iPr2NEt (6 equiv.), NT (3 equiv.),
MeCN, rt, 1.5 h
7 (1.2 equiv.), Bop-Cl (1.2 equiv.),
Py, rt, 1 h
7 (2 equiv.), Bop-Cl (2 equiv.),
Py, rt, 3 h
41
2
a
Thbz
50
3
4
a
b
Thbz
Th
95
82
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Scheme 2 (i) Synthesis of dithymidine H-boranophosphonates 13a,b,
(ii) 50-O-detritylation of 13b and (iii) conversion of 13a into dithymi-
dine boranophosphorothioate 15 by P–H modification. Reagents and
conditions: (i) N3-benzoyl-30-O-R3-thymidine (0.75 equiv.), Bop-Cl
(2.5 equiv.), 2,2,6,6-tetramethylpiperidine (6 equiv.), MeCN, rt, 1 h;
(ii) 3% dichloroacetic acid in CH2Cl2–Et3SiH (3 : 1, v/v), rt, 1 min;
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was synthesized as a crude product in a similar manner and treated
with 3% dichloroacetic acid in CDCl3–Et3SiH (1 : 1, v/v)11b,16
for 50-O-detritylation. In contrast to the detritylation of the
H-boranophosphonate monoester 9b by treatment with 80%
AcOH (Table 2, entry 1), which caused the decomposition of the
H-boranophosphonate moiety, the 50-O-DMTr group of 13b was
quantitatively removed without decomposition of the product
(Scheme 2, reaction ii). It indicates that a solid-phase synthesis
of oligonucleoside H-boranophosphonates via condensation and
50-O-detritylation is feasible.
Secondly, the possibility of the P–H modification of the
H-boranophosphonate diester linkage was investigated by
treating the dithymidine H-boranophosphonate 13a with S8 in
the presence of Et3N under anhydrous conditions. A 31P NMR
analysis of the reaction showed that 13a (dP 135.1, 133.7) was
quantitatively converted into the corresponding dithymidine
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ꢂc
This journal is The Royal Society of Chemistry 2009
2468 | Chem. Commun., 2009, 2466–2468