Synthesis of dinucleoside phosphates and their analogs by the boranophosphotriester method using azido-based protecting groups

Article abstract of DOI:10.1016/j.tetlet.2007.01.064

Oligodeoxyribonucleotides and their backbone-modified analogs were synthesized in good yields by the boranophosphotriester method in solution. The oligodeoxyriobonucleoside boranophosphates, fully protected with 2-(azidomethyl)benzoyl groups, were convert

Full text of DOI:10.1016/j.tetlet.2007.01.064

Tetrahedron Letters 48 (2007) 1973–1976
Synthesis of dinucleoside phosphates and their analogs by the
boranophosphotriester method using azido-based protecting groups
Toshihide Kawanaka, Mamoru Shimizu and Takeshi Wada*
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
Received 8 December 2006; revised 10 January 2007; accepted 15 January 2007
Available online 18 January 2007
Abstract—Oligodeoxyribonucleotides and their backbone-modified analogs were synthesized in good yields by the boranophospho-
triester method in solution. The oligodeoxyriobonucleoside boranophosphates, fully protected with 2-(azidomethyl)benzoyl groups,
were converted to the various backbone-modified DNA analogs via the corresponding H-phosphonate intermediates. A new effi-
cient protecting group for the O6-position of 20-deoxyguanosine, 4-[(2-azidomethyl)benzoyloxy]benzyl (AZBn) group, was also
developed. The AZBn group was found to be quickly removed by treatment with MePPh2 in dioxane–2-mercaptoethanol–H2O.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Recently, a number of backbone-modified DNA ana-
logs have been synthesized for anticancer and antiviral
drugs such as antisense DNAs. H-Phosphonate DNAs
have been used as versatile intermediates for the synthe-
sis of DNA and a wide variety of backbone-modified
DNA analogs because the phosphorous atom in H-
phosphonate diesters can be modified by various chemi-
cal reactions.1,2 However, the H-phosphonate DNA is
chemically unstable, and therefore, only short oligomers
can be synthesized in good yields by the current method.
regarded as a protecting group for the H-phosphonate
diester linkage. Based on this concept, we have devel-
oped a new method for the synthesis of DNA and its
analogs: First, the boranophosphate DNA is synthe-
sized on a solid support, then it is converted to the
H-phosphonate DNA, and finally, it is converted to
DNA and backbone-modified DNA analogs.
Base-sensitive DNA analogs such as phosphoramidate
DNA4,5 and methyl phosphate DNA6,7 cannot be
synthesized by the use of the conventional base-labile
protecting groups for nucleobases. Therefore, we
employed a 2-(azidomethyl)benzoyl (AZMB), which
can be removed under neutral and reductive conditions,
as a protecting group for nucleobases (Scheme 2).8 In
the boranophosphotriester method, which has been
developed for the synthesis of boranophosphate DNA
by our group, both the guanine and thymine bases react
with the boranophosphorylating reagent.9 Therefore,
the N3-position of thymidine was protected with the
AZMB group. In addition, the O6-position of 20-deoxy-
guanosine has to be protected, and it was necessary to
Recently, we have established a new reaction for the
transformation of boranophosphate diesters into the
corresponding H-phosphonate diesters in the presence
of trityl cation under acidic conditions (Scheme 1).3
From another point of view, the borane group is
BzO
T
BzO
T
O
P
O
P
trityl cation,
acid
BH3
O
O
O
O
H
O
Et3NH O
T
T
O
O
OBz
Scheme 1. Novel transformation reaction.
OBz
O
O
O
O
R
R
N
HN
N
H
reduction
H
+
Keywords: Backbone-modified DNA analog; H-Phosphonate DNA;
Boranophosphotriester method; Protecting group.
N3
AZMB
N3
NH2
R NH2
*
Corresponding author. Tel./fax: +81 4 7136 3612; e-mail: wada@
k.u-tokyo.ac.jp
Scheme 2. AZMB group.
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.01.064

1974
T. Kawanaka et al. / Tetrahedron Letters 48 (2007) 1973–1976
N3
OAZBn
O
N
MePPh2
(4 equiv)
N
N
N
N
NH
O
TBSO
N
TBSO
N
N
N
H
Pa
Pa
O
O
O
AZBn
dioxane-H2O
(4:1, v/v),
rt, 10 min
65%
H
OTBS
OTBS
N3
NH2
3
1
reduction
O
O
O
O
O
R
O
R
Scheme 5. Removal of the AZBn group.
O
(4:1, v/v),8 which are the same conditions as those for
the removal of the AZMB group (Scheme 5).
+
R O
HN
O
Although the AZBn group was quickly removed, the
side reaction was observed and the O6-deprotected 20-
deoxyguanosine derivative 1 was isolated in a moderate
yield. TLC analysis of the reaction mixture suggested
that the side reaction occurred. It was assumed that
the quinone methide, which was formed during the
deprotection reaction, was added to the guanine
base.13,14 In order to suppress this side reaction, depro-
tection reaction was carried out in the presence of
2-mercaptoethanol, which was an efficient scavenger
for the quinone methide.15,16 Consequently, the AZBn
group could be quantitatively removed without any side
reactions (Scheme 6).
Scheme 3. AZBn group.
develop a novel protecting group, which can be removed
under neutral conditions in the present sturdy. A 4-[(2-
azidomethyl)benzoyloxy]benzyl (AZBn) group was
designed as a protecting group for the O6-position.
The AZBn group can be removed under essentially the
same conditions as those for the AZMB group. The
AZMB moiety in the AZBn group is removed sub-
sequent to the reduction of the azido function, then
the deprotection reaction proceeds with the generation
of quinone methide (Scheme 3).
In order to apply the novel method to the synthesis of
various kinds of backbone-modified DNA analogs,
nucleobases and hydroxy functions were protected with
the AZMB and AZBn groups. Dinucleoside borano-
phosphates were synthesized by the boranophosphotri-
ester method.9,17,18 The dinucleoside boranophosphate
derivatives 4 were allowed to react with a DMTr cation,
which was generated by the reaction of dimethoxytrityl
methyl ether (DMTrOMe) with dichloroacetic acid
(DCA) in CH2Cl2 (Table 1). Then, the excess trityl cat-
ion was reduced with triethylsilane, and the products
were analyzed by 31P NMR spectroscopy after the aque-
ous work-up of the reaction mixture.
In this Letter, we wish to describe a novel method for
the synthesis of DNA and its analogs via the borano-
phosphate DNA, and a novel protecting group for the
O6-position of the 20-deoxyguanosine.
First, the introduction and removal reactions of the
AZBn group were attempted using a model compound.
The AZBn group was introduced at the O6-position of
the N2-phenylacetyl-20-deoxyguanosine derivative 1 via
a C6-ammonium intermediate with 4-[(2-azidomethyl)-
benzoyloxy]benzyl alcohol (AZBnOH)10 by a slightly
modified literature procedure (Scheme 4).11,12 Next,
the removal reaction of the AZBn group in 20-deoxy-
guanosine derivative 3 was carried out by treatment
with methyldiphenylphosphine in 1,4-dioxane–H2O
In all the cases, the 31P NMR analysis suggested that
dinucleoside H-phosphonates 5 were obtained in almost
quantitative yields. In the next stage, dinucleoside phos-
phates 6 were synthesized from boranophosphates 4 via
H-phosphonates 5 as a control reaction for the synthesis
of various backbone-modified DNA analogs. Because
the H-phosphonate diester is chemically unstable, inter-
mediates 5 were purified by simple extraction. After the
aqueous work up of the reaction mixtures subsequent to
the transformation reactions of 4 to 5, the treatment of 5
with I2 in pyridine–H2O (98:2, v/v)19 gave dinucleoside
phosphates 6 in good yields.20
O
N
OTPS
N
N
N
N
N
NH
i
TBSO
TBSO
N
H
N
N
Pa
Pa
O
O
H
OTBS
OTBS
1
2
OAZBn
N
N
ii, iii
TBSO
N
O
N
N
Pa
H
OTBS
OAZBn
N
O
N
3
N
N
N
N
NH
N Pa
Pa = phenylacethyl
TBS = -butyldimethylsilyl
TPS = 2,3,4-triisopropylbenzenesulfonyl
i
TBSO
TBSO
t
N
N Pa
O
O
H
H
OTBS
OTBS
1
Scheme 4. Introduction of the AZBn group into the O6 of 20-
deoxyguanosine. Reagents and conditions: (i) TPSCl (2 equiv), Et3N
(4 equiv), DMAP (0.05 equiv)/CH2Cl2, rt, 1 h, 85%, (ii) N-methylpyrr-
olidine (10 equiv)/CH2Cl2, 0 ꢁC, 10 min, (iii) AZBnOH (5 equiv), DBU
(1.5 equiv)/CH2Cl2, 0 ꢁC, 1.5 h, two steps 77%.
3
Scheme 6. Removal of the AZBn group in the presence of 2-
mercaptoethanol. Reagents and conditions: (i) MePPh2/dioxane–
H2O–2-mercaptoethanol (3:1:1, v/v/v), rt, 20 min, quant.

T. Kawanaka et al. / Tetrahedron Letters 48 (2007) 1973–1976
Table 1. Synthesis of dinucleoside phosphates from dinucleoside boranophosphates
1975
B1
B1
AZMBO
B1
HO
T
AZMBO
AZMBO
O
P
O
P
O
P
O
P
i, ii
iv
iii
BH3
O
O
O
O
O
O
O
O
O
O
H
O
B3
Et3NH O
B2
Et3NH O
B2
B2
Et3NH O
O
O
O
O
OH
OAZMB
OAZMB
OAZMB
6
7
5
4
B1 =
N
3-(2-azidomethyl)benzoylthymin-1-yl
B3 = A, C, G, T
Entry
B2
Yield of 5a (%)
Isolated yield of 6 (%)
Isolated yield of 7 (%)
1
2
3
N6-(2-Azidomethyl)benzoyladenin-9-yl
N4-(2-Azidomethyl)benzoylcytosin-1-yl
N2-(2-Azidomethyl)benzoyl-O6-4-
Quant
95
Quant
Quant
78
Quant
33
45
41
[(2-azidomethyl)benzoyloxy]benzylguanin-9-yl
4
N3-(2-Azidomethyl)benzoylthymin-1-yl
Quant
Quant
34
Reagents and conditions: (i) DMTrOMe (5 equiv), 3% DCA/CH2Cl2, 0 ꢁC, 1 min, (ii) Et3SiH (excess), ext. with satd NaHCO3 aq, (iii) I2 (3 equiv)/
pyridine–H2O (98:2, v/v), rt, 10 min, (iv) MePPh2 (8 equiv) in dioxane–H2O (4:1, v/v), rt, 20 min (B3 = A, C, T) or in dioxane–2-mercaptoethanol–
H2O (3:1:1, v/v), rt, 20 min (B3 = G).
a Yields of 5 were estimated by 31P NMR.
Table 2. Synthesis of backbone-modified DNA analogs
Finally, the removal of the AZMB and AZBn groups
was attempted. In the case of 6 (B2 = N2-2-(azido-
AZMBO
B1
AZMBO
iii
B1
methyl)benzoyl-O6-4-[(2-azidomethyl)benzoyloxy]benz-
ylguanin-9-yl), the reaction was carried out in the
presence of 2-mercaptoethanol. After purification by
the reverse-phase silica gel column chromatography,
we obtained fully deprotected dinucleoside phosphates
7. It was difficult to remove phosphine oxide from the
reaction mixture and the desired products were lost dur-
ing the purification to some extent. Therefore, the iso-
lated yields of 7 were moderate in all the cases.
However, the TLC and 31P NMR analyses of the reac-
tion indicated that the deprotection reaction proceeded
quantitatively.
O
P
O
P
i, ii
BH3
O
O
O
X
O
O
Et3NH O
B2
B2
O
O
OAZMB
OAZMB
8
4
HO
T
O
iv
O
X
O
O
P
T
O
OH
9
B1 = B2
=
N
3-(2-azidomethyl)benzoylthymin-1-yl
Upon using the transformation reaction described
above, various kinds of backbone-modified nucleic acid
analogs were synthesized from dithymidine boranophos-
phate 4 (Table 2). In a similar manner to the synthesis of
phosphates, H-phosphonate intermediates were not iso-
lated in these cases.
Entry
X
Isolated yield
of 8 (%)
Isolated yield
of 9 (%)
1
2
3
4
Sꢀ
90
32
33
50
48
N(CH2CH2)2O
OMe
NH2
96a
72
78
Reagents and conditions: (i) DMTrOMe (5 equiv), 3% DCA/CH2Cl2,
0 ꢁC, 1 min, (ii) Et3SiH (excess), ext. with satd NaHCO3 aq, (iii)
transformation reaction, (iv) MePPh2 (8 equiv)/dioxane–H2O (4:1,
v/v), rt, 20 min.
The dithymidine phosphorothioate21 (entry 1) and
phosphoromorpholidate4,22 (entry 2) were obtained
from the corresponding boranophosphate in the excel-
lent yields. Dithymidine methyl phosphate4,15 (entry 3)
and phosphoramidate4,23 (entry 4) were synthesized in
good yields. In the case of the synthesis of the phos-
phoromorpholidate, the reaction time was longer than
those for other cases, and therefore, the AZMB groups
at the N3-position of thymine base were removed during
the reaction (entry 2).
a Thymine bases were not protected.
In conclusion, we have developed a novel method for
the synthesis of oligodeoxyribonucleotides and their
analogs by the boranophosphotriester approach utiliz-
ing azido-based protecting groups. A new protecting
group for the O6-position of 20-deoxyguanosine, which
can be removed under neutral and reductive conditions
was developed and successfully applied to the synthesis
of dinucleoside phosphate derivatives. The transforma-
tion reaction conditions for the boranophosphate diest-
ers to the H-phosphonate diesters were optimized. Upon
applying these reactions, dinucleoside phosphates and
their analogs were synthesized from the dinucleoside
boranophosphates. Therefore, the present method will
be useful for the synthesis of nucleic acids and various
Finally, the AZMB groups were removed from the hy-
droxy functions and the nucleobases in 8 by treatment
with methyldiphenylphosphine in 1,4-dioxane–H2O. In
all the cases, quantitative deprotection reactions of 8
were ascertained by the TLC monitoring and 31P
NMR spectroscopy after the extraction. Consequently,
the fully deprotected TPT analogs 9, including the
base-labile analogs, were obtained.

1976
T. Kawanaka et al. / Tetrahedron Letters 48 (2007) 1973–1976
14. Pande, P.; Shearer, J.; Yang, J.; Greenberg, W. A.; Rokita,
S. E. J. Am. Chem. Soc. 1999, 121, 6773.
15. Toteva, M. M.; Moran, M.; Amyes, T. L.; Richard, J. P. J.
Am. Chem. Soc. 2003, 125, 8814.
kinds of base-labile backbone-modified nucleic acid ana-
logs. The solid-phase synthesis of nucleic acid deriva-
tives by the present method is now in progress.
16. Murata, A.; Wada, T. Tetrahedron Lett. 2006, 47,
2147.
Acknowledgement
17. Wada, T.; Shimizu, M.; Oka, N.; Saigo, K. Tetrahedron
Lett. 2002, 43, 4137.
18. Shimizu, M.; Saigo, K.; Wada, T. J. Org. Chem. 2006, 71,
4262.
We are indebted to Professor K. Saigo, for the helpful
suggestions.
19. Garegg, P. J.; Lindh, I.; Regberg, T.; Stawinski, J.;
Stro¨mberg, R. Tetrahedron Lett. 1986, 27, 4051.
20. A general procedure for the synthesis of dinucleoside
phosphates and their analogs; dinucleoside boranophos-
phate 4 (64.0 mg, 50.0 lmol) was added to a solution of
3% DCA in CH2Cl2 (5 mL), and the solution was stirred
at 0 ꢁC. The solution of DMTrOMe (84.0 mg, 250 lmol)
and 3% DCA in CH2Cl2 (5 mL) was added at 0 ꢁC and
the solution was stirred for 1 min. Then triethylsilane
(5 mL) was added to the reaction mixture and the
mixture was stirred at rt for 10 min. The solution was
diluted with CH2Cl2 (5 mL) and washed with satd
NaHCO3 aq (3 · 30 mL). The aqueous layer was back-
extracted with CH2Cl2 (3 · 30 mL) and the organic layer
was dried over Na2SO4, filtered, and concentrated to
dryness under reduced pressure. The residue and I2
(38 mg, 150 lmol) were dissolved in pyridine–H2O (98:2,
v/v, 0.5 mL) and the solution was stirred at rt for 10 min.
Satd Na2S2O3 aq (10 mL) was added, and the solution
was diluted with CHCl3 (10 mL). The solution was
washed with satd Na2S2O3 aq (4 · 10 mL) and the
aqueous layer was back-extracted with CHCl3
(3 · 10 mL). The organic layer was washed with 0.1 M
triethylammonium hydrogencarbonate buffer (pH 7.0,
3 · 40 mL) and the aqueous layer was back-extracted
with CHCl3 (3 · 100 mL).The organic layer was dried
over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The crude product was purified by
silica gel chromatography using a gradient of MeOH (0–
3%) in CH2Cl2 with 0.5% triethylamine as an eluent. The
fractions containing dinucleoside phosphate 6 were com-
bined and concentrated to dryness. Excess triethylamine
was removed by repeated coevaporation with toluene and
concentrated to dryness under reduced pressure to give 6
as a colorless foam.
References and notes
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10. To a suspension of 4-hydroxybenzyl alcohol (0.621 g,
5.00 mmol) in ethyl acetate (8.3 mL) at 0 ꢁC under Ar was
added triethylamine (2.1 mL, 15 mmol) followed by a
solution of 2-(azidomethyl)benzoyl chloride8 (0.978 g,
5.00 mmol) in ethyl acetate (4 mL). The mixture was
stirred at 0 ꢁC for 5 h and the precipitate was filtered off.
The filtrate was evaporated to dryness under reduced
pressure. The crude product was purified by silica gel
chromatography using a gradient of ethyl acetate (10–
40%) in hexane as an eluent. The fractions were combined
and concentrated to dryness under reduced pressure to
give AZBnOH (0.944 g, 71%) as a yellow oil.
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¨
´
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23. The dithymidine H-phosphonate 520 was allowed to react
with 2% I2 and satd NH3 in pyridine at rt. The reaction
mixture was stirred for 1 min.