Solid-phase synthesis of backbone-modified DNA analogs by the boranophosphotriester method using new protecting groups for nucleobases

Article abstract of DOI:10.1016/j.bmcl.2008.05.053

Backbone-modified DNA analogs were synthesized in good yields by the boranophosphotriester method on a solid support. The oligodeoxyribonucleoside boranophosphates, protected with 2-(azidomethyl)benzoyl groups for nucleobases, were converted into DNA and its backbone-modified analogs via the corresponding H-phosphonate intermediates. A new protecting group for the O⁶ position of 2′-deoxyguanosine, 4-azidobenzyl (ABn) group, was also developed. The ABn group can be quickly removed by treatment with MePPh₂ and H₂O in the presence of 2-mercaptoethanol.

Full text of DOI:10.1016/j.bmcl.2008.05.053

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3783–3786
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Solid-phase synthesis of backbone-modified DNA analogs by the
boranophosphotriester method using new protecting groups for nucleobases
*
Toshihide Kawanaka, Mamoru Shimizu, Noriko Shintani, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Backbone-modified DNA analogs were synthesized in good yields by the boranophosphotriester method
on a solid support. The oligodeoxyribonucleoside boranophosphates, protected with 2-(azidomethyl)
benzoyl groups for nucleobases, were converted into DNA and its backbone-modified analogs via the cor-
responding H-phosphonate intermediates. A new protecting group for the O6 position of 20-deoxyguano-
sine, 4-azidobenzyl (ABn) group, was also developed. The ABn group can be quickly removed by
treatment with MePPh2 and H2O in the presence of 2-mercaptoethanol.
Received 1 February 2008
Revised 25 April 2008
Accepted 9 May 2008
Available online 17 May 2008
Keywords:
Solid-phase synthesis
DNA analogs
Ó 2008 Elsevier Ltd. All rights reserved.
Boranophosphate DNA
H-Phosphonate DNA
Protecting group
Base-sensitive DNA analogs, such as O-alkyl phosphate DNA,
alkylphosphonate DNA, and phosphoramidate DNA, have been re-
garded and synthesized as potentially useful therapeutic oligonu-
cleotides.1,2 Chemical synthesis of oligonucleotides and their
analogs is generally carried out on a solid support with base-labile
nucleobase-protecting groups.3 However, base-sensitive oligonu-
cleotide analogs are decomposed under the conditions prescribed
for the removal of the protecting groups. Recently, we have re-
ported a new strategy for the synthesis of backbone-modified
DNA analogs by the boranophosphotriester method by the use of
azido-based protecting groups which can be removed under mild
and neutral conditions in solution (Scheme 1).4
R1O
B1
R1O
B1
HO
B3
O
P
O
P
O
P
trityl cation
acid
BH3
O
O
X
O
O
O
O
H
O
B4
O
B2
O
B2
O
O
O
OR1
R1 = (2-azidomethyl)benzoyl (AZMB)
B1, B2 = azmb6A, azmb4C, azmb2azbn6G, azmb3T
OR1
OH
X = O-, S-, OMe, NH2
B3, B4 = A, C, G, T
Scheme 1. Synthesis of backbone-modified DNA analogs by the boranophospho-
triester method.
In this strategy, boranophosphate DNA is employed as a precur-
sor of H-phosphonate DNA. The boranophosphate DNA can be con-
verted into the corresponding H-phosphonate DNA, and is finally
transformed to the desired backbone-modified DNA analogs.
In the previous method, the exocyclic amino groups of nucleo-
bases and the imido function of thymine base are protected with
a 2-(azidomethyl)benzoyl (AZMB) group,5 and the lactam function
guanine residue. The 4-azidobenzyl group, which was reported
by Kusumoto and co-workers as a protecting group for the hydroxy
function, can be removed by oxidation with 2,3-dichloro-5,6-dic-
yanobenzoquinone (DDQ) or anodic oxidation after reduction to
4-aminobenzyl group with catalytic hydrogenation or oxidation
with DDQ after conversion to iminophosphorane (Scheme 2).6
The ABn group is expected to be removed under essentially the
same conditions as those for an AZMB group with the generation of
iminoquinone. In this letter, we describe the application of the
of guanine is protected with
a 4-[(2-azidomethyl)benzoyl-
oxy]benzyl (AZBn) group, which can be removed under neutral
and reductive conditions. Therefore, the method is suitable for
the synthesis of base-sensitive DNA analogs. However, the AZBn
group is bulky and unstable under basic conditions, hence the yield
of protection for the O6 position of guanine base was insufficient.4
Herein, we report a novel protecting group for the O6 position of
NH
N
3
NH
2
O
R
O
R
+
R OH
4-azidobenzyl (ABn)
* Corresponding author. Tel./fax: +81 4 7136 3612.
E-mail address: wada@k.u-tokyo.ac.jp (T. Wada).
Scheme 2. ABn group.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.053

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T. Kawanaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3783–3786
PyNTP, DMAN
boranophosphotriester method to the solid-phase synthesis of
backbone-modified DNA analogs by using a new protecting group
for the O6 position of guanine.
1
2
DMTrO
B
HO
B
O
P
O
3% DCA/CH2Cl2-Et3SiH
2,6-lutidine-Ac2O
BH
+
3
O
First, an ABn group was introduced to the O6 of N2-phenylace-
O
CEO
O HNEt
3
tyl-20-deoxyguanosine derivative
1 as a model compound.
6g
5
4-Azidobenzyl alcohol was allowed to react with the C6 quaternary
N-methylpyrrolidinium intermediate by the method reported in
the literature7 with some modifications to give 4 in 90% yield from
2 (Scheme 3). The O6-ABn group of 20-deoxyguanosine derivative 4
was deblocked under the same conditions as those for the removal
of AZMB group. Fortunately, the ABn group was smoothly removed
in 97% yield by the treatment with methyldiphenylphosphine in
1,4-dioxane–H2O (Scheme 4), whereas the side reaction occurred
in the case of AZBn group under the same conditions.4
In the next step, the ABn group was applied to the solid-phase
synthesis.8 The removal of the ABn group in a dinucleoside borano-
phosphate on a solid support was examined. 20-Deoxyguanosine
boranophosphate 6g, whose nucleobase was protected with the
AZMB and ABn groups, was used as a monomer unit for the bor-
anophosphotriester method.9–11 6g was condensed with a thymi-
dine derivative 5 anchored to the solid support in the presence of
PyNTP12 as a condensing reagent (Scheme 5). After capping and
decyanoethylation, the nucleobases were deprotected and the
dinucleoside boranophosphate was cleaved from the solid support.
The crude reaction mixture was analyzed by RP-HPLC. Under the
same deprotection conditions as those for in solution, some by-
products were observed by the HPLC analysis. The formation of
these by-products could be attributed to the addition reaction of
iminoquinone to the nucleobases or phosphate moiety (Fig. 1A).
In order to suppress the side-reaction to the phosphate moiety of
the boranophosphotriester 7gt, the cyanoethyl group was depro-
tected after the removal of the ABn group. As a result, the HPLC
analysis suggested that the by-products were slightly decreased
(Fig. 1B). Furthermore, in order to trap iminoquinone, 2-mercap-
toethanol was added, which was an effective scavenger for quinone
= CPG
1
AcO
B
HO
G
O
O
P
10% DBU
deprotection
conc NH3
BH
3
BH
O
P
3
O
2
CEO
O
B
H N O
4
O
T
O
O
O
OH
7gt
8gt
CE = 2-cyanothyl
DMAN = 1,8-bis(N,N'-dimethylamino)
naphthalene
B1
B2
=
N
2-(2-azidomethyl)benzoyl-O6
4-azidobenzylguanin-9-yl
3-(2-azidomethyl)benzoylthymin-1-yl
-
NO2
PF6
N
=
N
N P N
PyNTP =
N
3
Scheme 5. Solid-phase synthesis using the AZMB and ABn groups.
O
N
OTPS
N
N
N
N
N
NH
N Pa
i
iii
TBSO
TBSO
N
N Pa
O
O
H
H
OTBS
OTBS
2
1
OABn
N
N
N
N
N
N
TBSO
N
N
N
N Pa
iii
O
H
TBSO
N Pa
H
O
OTBS
4
OTBS
TBS = t-butyldimethylsilyl
TPS = 2,4,6-triisopropylbenzenesulfonyl
Pa = 2-phenylacetyl
3
Scheme 3. Introduction of ABn group into the O6 of 20-deoxyguanosine derivatives.
Reagents and conditions: (i) 2,4,6-triisopropylbenzenesulfonyl chloride (2 equiv),
Et3N (4 equiv), DMAP (0.05 equiv)/CH2Cl2, rt, 1 h, 85%, (ii) N-methylpyrrolidine
(10 equiv)/CH2Cl2, 0 °C, 10 min, (iii) ABnOH (5 equiv), DBU (1.5 equiv)/CH2Cl2, 0 °C,
1.5 h, two steps 90%.
O
N
OABn
N
N
N
N
N
NH
N Pa
H
Figure 1. Reverse-phase HPLC profiles of crude mixtures of d(GPBT), deprotected
with MePPh2 in dioxane–H2O (4:1, v/v), 1 h, after decyanoethylation (A), MePPh2 in
dioxane–H2O (4:1, v/v), 1 h, followed by decyanoethylation (B), MePPh2 in dioxane-
2-mercaptoethanol–H2O (3:1:1, v/v/v), 1 h, followed by decyanoethylation (C).
i
TBSO
TBSO
N
N Pa
H
O
O
OTBS
1
OTBS
4
methide.4,13,14 Consequently, the side reactions were almost sup-
pressed in the presence of 2-mercaptoethanol (Fig. 1C).
Scheme 4. Removal of ABn group. Reagents and conditions: (i) MePPh2 (4 equiv)/
1,4-dioxane–H2O (4:1, v/v), rt, 10 min, 97%.

T. Kawanaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3783–3786
3785
Table 1
Transformation of dinucleoside boranophosphate including four nucleobases
AcO
B1
AcO
B1
B3
O
P
O
P
HO
O
P
AcO
B1
HO
+
B2
O
BH3
O
O
O
O
H
oxidation
O
trityl reagent
O
O
O
O
B2
O
B2
BH3
O
CH2Cl2, rt,
10 min
O
O
T
O
O
O
O
P
OCE
O
O
OH
6
5
11
9
10
B3 = A, C, G, T
B2
=
N
3-(2-azidomethyl)benzoylthymin-1-yl
Entry
Compound
B1
Trityl reagent
Yield of 11b (%)
1
2
3
9ct
9tt
9at
9at
9at
9gt
N4-(2-azidomethyl)benzoylcytosin-1-yl
N3-(2-azidomethyl)benzoylthymin-1-yl
0.01 M TrBF4
0.01 M TrBF4
0.01 M TrBF4
0.01 M DMTrBF4
0.01 M DMTrBF4
0.01 M DMTrBF4
93
93
72
80
87
83
N6-bis(2-azidomethyl)benzoyladenin-9-yl
N6-bis(2-azidomethyl)benzoyladenin-9-yl
N6-bis(2-azidomethyl)benzoyladenin-9-yl
4
5a
6a
N2-(2-azidomethyl)benzoyl-O6-4-azidobenzylguanosin-9-yl
a
Transformation reaction was carried out after deprotection of nucleobases.
Estimated by RP-HPLC.
b
Next, backbone-modified DNA analogs were synthesized on the
9at, the N6-bis(2-azidomethyl)benzoyl-20-deoxyadenosine deriva-
tive 6a was used as a monomer unit to suppress the depurination
by the TrBF4. However, the dinucleoside phosphate 11at was ob-
tained in lower yield (entry 3). The result could be attributed to
the strong Lewis acidity of the unsubstituted Tr cation, which
may cause depurination. Based on this consideration, DMTrBF4,
which is a weaker Lewis acid than TrBF4, was used for the transfor-
mation of 9at. Accordingly, HPLC analysis showed that the yield of
solid support by the boranophosphotriester method. The nucleo-
side boranophosphodiesters 6 whose nucleobases were protected
with the AZMB and AZBn groups were used as monomer units.
The monomer units 6 were condensed with the 50-hydroxy
function of the thymidine derivative 5 (Table 1). After subsequent
capping of the 50-hydroxy group and removal of the cyanoethyl
group, the dinucleoside boranophosphate 9 was synthesized on
the solid support. The resulting dinucleoside boranophosphate 9
was allowed to react with trityl reagent.4,15 Since the H-phospho-
nate linkage was unstable under the deprotection conditions, 10
was oxidized to the dinucleoside phosphate by treatment with I2
in pyridine–H2O.16 After deprotection, the dinucleoside phosphate
was cleaved from the solid support, and the product was analyzed
by RP-HPLC.
The HPLC analysis showed nearly quantitative formation of the
desired dinucleoside phosphate 11ct, and this fact indicated that
the treatment of TrBF4 gave H-phosphonate 10ct (entry 1) in an
excellent yield. Next, we attempted to transform the dinucleoside
boranophosphates including other nucleobases to the correspond-
ing phosphates. On the basis of the HPLC analysis, the dithymidine
phosphate 11tt was synthesized in good yield under the same con-
ditions as those for the transformation of 9ct (entry 2). In the case
of the dinucleoside boranophosphate including 20-deoxyadenosine
Table 2
Chain elongation cycle, deprotection, and transformation for manual solid-phase
synthesis
Step
Manipulation
Reagents and solvents
Time
Chain elongation
1
2
3
4
5
Detritylation
Wash
Drying
Condensation
Wash
3% DCA in CH2Cl2–Et3SiH (1:1, v/v)
(i) CH2Cl2, (ii) CH3CN
15 s
10 min
20 min
6 (0.1 M), PyNTP (0.2 M) in DMAN/CH3CN
(i) CH3CN, (ii) CH2Cl2
Repeat steps 1–5
6
Detritylation
3% DCA in CH2Cl2–Et3SiH (1:1, v/v)
15 s
30 s
Deprotection and transformation
7
Capping
Ac2O-2,6-lutidine (1:9, v/v),
DMAP (10 mg/mL)
8
9
10
11
12
Decyanoethylation
Deprotection
Transformation
Oxidation
10% DBU in CH3CN
10 min
1 h
10 min
10 min
1 h
MePPh2 in 1,4-dioxane–H2O (4:1, v/v)
DMTrBF4 (0.01 M) in CH2Cl2
2% I2 in pyridine–H2O (98:2, v/v)
Concd NH3
Figure 2. Reverse-phase HPLC profiles of crude mixtures of d(CPCPT) (A and B), and
d(CPCPCPT), (C), transformed by 0.01 M DMTrBF4 in CH2Cl2 at rt, 10 min, (A), 0.01 M
DMTrBF4 in CH2Cl2, at 0 °C, 10 min (B and C).
Cleavage

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T. Kawanaka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3783–3786
coupling (Scheme 6).16,17 The treatment with methanol or
morpholine in CCl4 gave the methyl phosphate DNA and phosp-
horomorpholidate DNA in 70% and 88% yields, respectively
(Scheme 6).
AcO
C
HO
C
O
P
O
P
(i), (ii), (iii)
BH3
O
X
O
O
Et3NH O
O
T
O
T
O
O
In conclusion, we developed a novel protecting group, ABn
group, for the O6 of guanine which can be removed under mild
and neutral conditions. The ABn group was useful for the synthesis
of boranophosphate DNA as well as other DNA analogs. We also
established the synthesis of DNA and backbone-modified DNA ana-
logs by the boranophosphotriester method on the solid support.
The present method will be useful for the synthesis of various
backbone-modified oligodeoxyribonucleotide analogs via the H-
phosphonate intermediate. Solid-phase synthesis of longer oligo-
nucleotide analogs is now in progress.
O
OH
13ct
12ct
= CPG
(A) X = OMe : 70%a
: 88%a
(B) X = N
O
aEstimated by RP-HPLC.
Scheme 6. Synthesis of methylphosphate DNA and phosphoromorpholidate DNA.
Reagents and conditions: (i) DMTrBF4/CH2Cl2, 0 °C, 10 min, (ii) A—10% MeOH/N-
methylimidazloe-Et3N-CCl4 (5:5:90, v/v/v), rt, 1 h; B—10% morpholine/CCl4, rt, 1 h;
(iii) A—25 mM K2CO3/MeOH, rt, 5 h, B—concd NH3, rt, 1 h.
11at was improved (entry 4). Furthermore, the unprotected dinu-
cleoside boranophosphate, which was more stable than the N-acyl-
ated 20-deoxyadenosine derivatives under acidic conditions,17 was
used for the transformation reaction. After deprotection of the
nucleobases, the dimer was treated with DMTrBF4, and the dinu-
cleoside phosphate 11at was obtained in 87% yield (entry 5). The
dinucleoside boranophosphate including 20-deoxyguanosine 9gt
was transformed in 83% yield under the same conditions as those
for the transformation of 9at (entry 6).
In the next stage, d(CPCPT) and d(CPCPCPT) were synthesized on
the solid support. The trinucleoside boranophosphate, d(CPBCPBT),
was synthesized by the repeated detritylation and the condensa-
tion on the solid support (Table 2). The base-unprotected borano-
phosphate trimer was converted to the H-phosphonate
intermediate by treatment with the DMTrBF4 at room temperature,
and then it was oxidized to give d(CPCPT). The crude products were
analyzed by RP-HPLC (Fig. 2A). The HPLC profiles represented the
decomposition of the product in some degree. To decrease the deg-
radation, the transformation reaction was carried out at 0 °C (Fig.
2B). In this case, the desired trinucleoside phosphate was obtained
in excellent yield. The tetramer, d(CPCPCPT), was also synthesized
in good yield (Fig. 2C).
Acknowledgment
We are indebted to Professor K. Saigo, for the helpful
suggestions.
References and notes
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manually as described in literature 11.
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Then the method was applied to the solid-phase synthesis of
backbone-modified DNA analogs. The oxidation step (Table 2,
step 11) was replaced by the oxidative amination and oxidative
17. Remaud, G.; Zhou, X.-X.; Chattopadhyaya, J.; Oivanen, M.; Lönnberg, H.
Tetrahedron 1987, 43, 4453.