Stereocontrolled synthesis of oligodeoxyribonucleoside boranophosphates by an oxazaphospholidine approach using acid-labile N-protecting groups

Article abstract of DOI:10.1016/j.tetlet.2012.06.015

Oligodeoxyribonucleoside boranophosphates (PB-ODNs) were synthesized in a stereocontrolled manner via the corresponding H-phosphonates with fully deprotected nucleobases by using diastereopure 2′-deoxyribonucleoside 3′-O-oxazaphospholidine monomers bearin

Full text of DOI:10.1016/j.tetlet.2012.06.015

Tetrahedron Letters 53 (2012) 4361–4364
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Tetrahedron Letters
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Stereocontrolled synthesis of oligodeoxyribonucleoside boranophosphates
by an oxazaphospholidine approach using acid-labile N-protecting groups
⇑
Naoki Iwamoto, Natsuhisa Oka, 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:
Oligodeoxyribonucleoside boranophosphates (PB-ODNs) were synthesized in a stereocontrolled manner
via the corresponding H-phosphonates with fully deprotected nucleobases by using diastereopure 20-
deoxyribonucleoside 30-O-oxazaphospholidine monomers bearing acid-labile protecting groups on the
nucleobases. Using the resultant stereodefined PB-ODNs, we demonstrated that the thermal stability of
the duplexes of PB-ODNs with complementary oligonucleotides was dependent on the configuration of
their phosphorus atoms.
Received 25 April 2012
Revised 1 June 2012
Accepted 6 June 2012
Available online 13 June 2012
Keywords:
Stereocontrolled synthesis
Boranophosphate
Ó 2012 Elsevier Ltd. All rights reserved.
Oxazaphospholidine approach
H-Phosphonate
Oligonucleoside boranophosphates and their analogs have at-
tracted much attention lately as potential therapeutic oligonucleo-
tides.1 They are resistant to nucleases, adequately lipophilic, which
may facilitate transport across cell membranes, and have modest
to high affinity for complementary RNA sequences.1–3 Recent stud-
ies have shown that they serve as potent short interfering RNAs,3a,4
being comparable with or more effective than natural siRNAs.4 It
has also been demonstrated that effective cell transfection was
achieved with such P-boronated oligonucleotide analogs without
the aid of transfection agents.3 Furthermore, these types of oligo-
nucleotide analogs have been expected to work as target-specific
10B carriers for boron neutron capture therapy (BNCT).1
For these reasons, chemical synthesis of P-boronated oligonu-
cleotide analogs has become a significant subject of research. How-
ever, there are still two major problems to be solved: first,
nucleobases other than thymine bearing conventional acyl protect-
ing groups suffer from severe side reactions caused by boronating
agents, such as BH3ꢀTHF and BH3ꢀSMe2.5 Thymine also becomes
sensitive to such boronating agents after being silylated during
the silylation step for the conversion of oligonucleoside H-phos-
phonate intermediates into boranophosphates.6 Second, the phos-
phorus atoms of these P-boronated oligonucleotide analogs are
chiral and proper stereocontrol should have a positive impact on
their properties, such as the stability to nucleases and the affinity
for target RNA strands.7–10
base-unprotected nucleoside 30-H-phosphonate monomers,12
a
phosphoramidite method using base protecting groups unreactive
to the boronating agents (e.g., 4,40,400-trimethoxytrityl (TMTr)
groups),3,13 and a boranophosphotriester method using nucleoside
30-boranophosphate diester monomers.2,14 However, all of these
methods cannot control the stereochemistry at the phosphorus
atoms and the products are obtained as mixtures of P-diastereo-
mers. To date, there have been no reports for the stereoselective
chemical synthesis of P-boronated oligonucleotide analogs.15 The
enzymatic synthesis4,16 can incorporate only (Sp)-boranophos-
phate diester linkages into oligonucleotides due to the substrate
recognition specificity of the enzymes. Thus, a new synthetic strat-
egy to overcome both of these two problems is required.
Recently, we have developed a method for the stereocontrolled
synthesis of oligodeoxyribonucleoside H-phosphonates (PH-ODNs)
using diastereopure nucleoside 30-O-oxazaphospholidine mono-
mers,17 and all-(Rp)- and (Sp)-tetrathymidylate boranophosphates
have been synthesized by silylation and boronation of the resultant
stereodefined H-phosphonate intermediates.5,6,17,18 The side reac-
tions on silylated thymine bases have been suppressed by using
DMF as a solvent,17 but attempts to synthesize PB-ODNs contain-
ing four kinds of nucleobases using conventional acyl protections
resulted in failure due to significant side reactions on the acylated
nucleobases by boronation even in DMF (data not shown).
Given this result, we turned to the synthesis of stereodefined
PH-ODNs with unprotected nucleobases. As reported in the litera-
ture, unprotected nucleobases and the boronating agents can form
adducts, but regenerate the unmodified nucleobases during the
final deprotection of PB-ODNs.11 Since base protection is necessary
for the synthesis of the nucleoside 30-O-oxazaphospholidine
The first problem has been successfully overcome by several
methods developed recently: an H-phosphonate method11 using
⇑
Corresponding author. Tel./fax: +81 4 7136 3612.
E-mail address: wada@k.u-tokyo.ac.jp (T. Wada).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.06.015

4362
N. Iwamoto et al. / Tetrahedron Letters 53 (2012) 4361–4364
DMTrO
BPRO
O
N
O
P
H
−OTf
NC
+
N
O
Ph
Me
(Sp)- or (Rp)-1a−d
2
DMTrO
BPRO
O
1) BH3·SMe2, BSA
2) sat. NH3/CH3OH
TfO−
+
O
O
NH2
P
O
B
O
O
B
O
Ph
Me
HO
B
O
O
H3B
−O
O
O
3
P
O
O
B
H
O
P
O
B
O
H3B
−O
O
O
1% CF3COOH/
CH2Cl2−Et3SiH
P
B
O
O
4
O
stereodefined PB-ODNs
Scheme 1. Solid-phase synthesis of stereodefined PB-ODNs.
Table 1
Synthesis of oxazaphospholidine monomers 1a–d
DMTrO
BPRO
O
N
Cl
DMTrO
BPRO
O
P
P
O
Et3N
O
N
+
THF, −78 °C
O
Ph
OH
5a−d
then rt, 2 h
Me
Ph
Me
L- or D-6
BPROb
(Sp)- or (Rp)-1a−d
Entry
1
Yield (%)
trans:cis
1a
2
3
(Sp)-1a
(Sp)-1b
(Sp)-1c
(Sp)-1d
(Rp)-1a
(Rp)-1b
(Rp)-1c
(Rp)-1d
Th
74
80
80
76
83
88
80
71
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
Cytmt
Addmt
Gutse,dmt
Th
4
5a
6
Cytmt
Addmt
Gutse,dmt
7
8
a
Data taken from Ref. 17.
b
BPRO = protected nucleobase; Th = thymin-1-yl; Cytmt = N4-4,40,400-trimethoxytritylcytosin-1-yl; Addmt = N6-4,40-dimethoxytrityladenin-9-yl; Gutse,dmt = O6-trimethylsi-
lylethyl-N2-4,40-dimethoxytritylguanin-9-yl.
monomers, we employed acid-labile base protecting groups so
that the nucleobases would be deprotected during the 50-O-det-
ritylation. The method is outlined in Scheme 1. Diastereopure
nucleoside 30-O-oxazaphospholidine monomers (Sp)- or (Rp)-1a–
d were condensed with the 50-OH of a nucleoside on solid support
in the presence of N-(cyanomethyl)pyrrolidinium triflate (CMPT,
2).19 The resultant phosphite intermediates 3 were treated with
1% trifluoroacetic acid (TFA) in CH2Cl2–Et3SiH5a,14b for the 50-O-
detritylation and the conversion of the phosphite triester linkage
into an H-phosphonate diester stereospecifically.17 Acid-labile pro-
tecting groups on the nucleobases were removed simultaneously.
As a result, stereodefined PH-ODNs were synthesized on solid sup-
port with the nucleobases being fully deprotected (4). The desired
PB-ODNs were given by silylation and boronation of 4. We have
already confirmed that oxazaphospholidine monomers do not
form adducts with unprotected nucleobases.20
The synthesis of the nucleoside 30-O-oxazaphospholidine mono-
mers (Sp)- and (Rp)-1a–d is summarized in Table 1. 50-O-DMTr-
nucleosides 5a–d bearing acid-labile TMTr (N4-position of cyto-
sine), DMTr (N6-position of adenine and N2-position of guanine),
and trimethylsilylethyl (TSE)21 (O6-position of guanine) groups
were allowed to react with the 2-chloro-1,3,2-oxazaphospholidine

N. Iwamoto et al. / Tetrahedron Letters 53 (2012) 4361–4364
4363
Table 2
should be noted that the coupling reactions of the adenosine
monomers (1c) were conducted using a lower concentration of
CMPT (0.5 M) and a Lewis basic cosolvent 1-methyl-2-pyrrolidone
to suppress the detritylation of the N6-DMTr-adenine caused by
the acidity of CMPT, whereas the other monomers (1a, b, d) were
coupled using a higher concentration of CMPT (1.0 M) in CH3CN.
This should be the reason for the relatively low coupling yields
for 1c (entries 3, 7). The equally moderate yields for 1d (entries
4, 8) may be due to the steric hindrance of the bulky N2-DMTr
group. Although the coupling yields still remain to be improved,
it is noteworthy that the side reactions on silylated thymine as well
as the other protected nucleobases during the boronation step
were completely suppressed in either DMF or DMAc.
Synthesis of dinucleoside boranophosphates 7a–d
Entry
7
dNBTb
Yieldc (%)
Rp:Spc
1a
2
3
(Rp)-7a
(Rp)-7b
(Rp)-7c
(Rp)-7d
(Sp)-7a
(Sp)-7b
(Sp)-7c
(Sp)-7d
(Rp)-TBT
91
95
88
85
92
94
88
84
>99:1
>99:1
>99:1
>99:1
>1:99
>1:99
>1:99
>1:99
(Rp)-dCBT
(Rp)-dABT
(Rp)-dGBT
(Sp)-TBT
(Sp)-dCBT
(Sp)-dABT
(Sp)-dGBT
4
5a
6
7
8
a
b
c
Data taken from Ref. 17.
Subscript ‘B’ = boranophosphate diester linkage.
Determined by RP-HPLC.
Next, we synthesized stereodefined PB-ODN 4mers containing a
full set of nucleobases (Table 3, entries 1, 2) and dodecathymidy-
lates [(TB)11T] (entries 3, 4) by this method. All-(Rp)- and
all-(Sp)-(TB)11T (10, 11) were isolated by RP-HPLC for a thermal
denaturation study. Configurational assignment was conducted
by enzymatic digestion study using snake venom phosphodiester-
ase (svPDE) since it has been reported svPDE digests (Sp)-PB link-
ages stereospecifically over (Rp)-PB-linkages, though at a slower
rate compared to that for natural phosphodiester linkages at dimer
level.9a,d All-(Sp)-(TB)11T (11) was digested completely as expected,
whereas all-(Rp)-(TB)11T (10) was also partially hydrolyzed.22
Although it is still not clear whether the partial digestion was
due to incomplete stereocontrol of the synthesis or imperfect
stereospecificity of the enzyme,23 it should be mentioned that
the diastereoselectivity of the synthesis was >99% at least at the
dimer level as shown in Table 2.
Table 3
Synthesis of P-stereodefined PB-ODNs
Entry
PB-ODN
Yield (%)
1
2
3
4
all-(Rp)-d(CBABGBT) 8
all-(Sp)-d(CBABGBT) 9
all-(Rp)-(TB)11T 10
all-(Sp)-(TB)11T 11
73a
54a
13b
19b
a
Determined by RP-HPLC.
Isolated yield.
b
derivatives L- or D-6, which were synthesized from L- or D-proline,
respectively.17 Only the trans-isomers were generated stereoselec-
tively and isolated in good yields (Table 1).
Using these diastereopure monomers, we attempted to synthe-
size dinucleoside boranophosphates (Rp)- and (Sp)-7a–d on solid
support by the method shown in Scheme 1. The monomers
(Sp)- or (Rp)-1a–d were allowed to condense with the 50-OH of
thymidine attached to the support in the presence of CMPT 2 to af-
ford the corresponding dinucleoside phosphite intermediates. The
phosphite intermediates were then treated with 1% TFA in CH2Cl2–
Et3SiH (1:1, v/v) for the removal of the chiral auxiliary, base pro-
tecting groups and 50-O-DMTr group to give the base-unprotected
dinucleoside H-phosphonates. The resultant H-phosphonates were
converted into the corresponding boranophosphates by using
BH3ꢀSMe2 in DMF17 or N,N-dimethylacetamide (DMAc) in the pres-
ence of N,O-bis(trimethylsilyl)acetamide (BSA). After being re-
leased from the solid support by treatment with saturated NH3
in CH3OH, the crude products were analyzed by reversed-phase
HPLC (RP-HPLC). The analysis showed that diastereopure dinucle-
oside boranophosphates were obtained in modest to good yields
without significant side reactions on the nucleobases (Table 2). It
To evaluate the impact of the stereocontrol of PB-ODNs on their
duplex formation, a thermal denaturating study was carried out
with the duplexes of all-(Rp)-(TB)11T (10), all-(Sp)-(TB)11T (11),
stereo-random (TB)11T (12),24 and natural dodecathymidylate
[(TO)11T] (13) with the complementary dodecadeoxyadenylate
d[(AO)11A] (14) and dodecadenylate r[(AO)11A] (15) at low and high
ionic strength (Table 4). The data presented in Table 4 clearly show
that the configuration of phosphorus atoms of PB-ODNs signifi-
cantly affect the thermal stability of the duplexes. All-(Sp)-(TB)11T
(11) formed a duplex with d(AO)11A (14) with Tm values of 11.9
and 19.6 °C at low and high ionic strength, respectively (entries 3,
7, left), though the duplex was much less stable than that of natural
(TO)11T (13) (entries 1, 5). In sharp contrast, no detectable Tm values
were observed in the case of all-(Rp)-(TB)11T (10) (entries 2, 6, left).
Similarly, all-(Sp)-(TB)11T (11) formed a duplex with r(AO)11A (15)
(entries 3, 7, right), whereas all-(Rp)-(TB)11T (10) and stereo-
random (TB)11T (12) did not (entries 2, 4, 6, 8, right). The differences
in Tm values between the duplexes all-(Sp)-(TB)11T–r(AO)11
A
Table 4
Melting temperatures for duplexes formed with P-stereodefined or stereo-random PB-ODNs and natural DNA and RNA
Entry
ODN
d(AO)11A 14
r(AO)11A 15
Tm/mod.b (°C)
Tm (°C)
D
Tm (°C)
D
Tm/mod.b (°C)
a
a
Tm (°C)
D
Tm (°C)
D
0.1 M NaCl, NaH2PO4–Na2HPO4 buffer (pH 7.0)
1
2
3
4
(TO)11T 13
34.8
—
30.4
—
c
c
c
c
c
c
all-(Rp)-(TB)11T 10
all-(Sp)-(TB)11T 11
stereo-random (TB)11T 12
—
—
—
—
11.9
ꢁ22.9
ꢁ2.1
14.9
ꢁ15.5
ꢁ1.4
d
d
d
c
c
c
—
—
—
—
—
—
1 M NaCl, NaH2PO4–Na2HPO4 buffer (pH 7.0)
5
6
7
8
(TO)11T 13
46.1
—
38.2
—
c
c
c
c
c
c
all-(Rp)-(TB)11T 10
all-(Sp)-(TB)11T 11
stereo-random (TB)11T 12
—
—
—
—
19.6
ꢁ26.5
ꢁ2.4
17.5
ꢁ20.7
ꢁ1.9
d
d
d
c
c
c
—
—
—
—
—
—
a
b
c
Difference in Tm values between duplexes of PB-ODNs and natural counterparts.
Difference in Tm values per boranophosphate modification.
Tm value was not observed.
d
Not determined.

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N. Iwamoto et al. / Tetrahedron Letters 53 (2012) 4361–4364
(11–15) and (TO)11T–r(AO)11A (13–15) were smaller (
ꢁ1.4 °C and ꢁ1.9 °C at low and high ionic strength, respectively) than
those in the cases with d(AO)11A (14) (
Tm/mod. = ꢁ2.1 °C and
DTm/mod. =
References and notes
1. For
a comprehensive review on nucleoside boranophosphates and their
D
analogs, see: Li, P.; Sergueeva, Z. A.; Dobrikov, M.; Shaw, B. R. Chem. Rev.
2007, 107, 4746–4796.
ꢁ2.4 °C at low and high ionic strength, respectively), being in consis-
tent with the data we obtained with stereo-random PB-ODNs.2
Thus, the study showed that all-(Sp)-(TB)11T (11) formed du-
plexes with complementary oligo(deoxy)adenylates (14, 15),
though with much lower Tm values than those obtained with the
natural counterpart (TO)11T (13). In contrast, all-(Rp)-(TB)11T (10)
did not form duplexes under the same conditions. The data indi-
cate that the duplexes in which the BH3 groups are oriented ‘in-
ward’ are thermally more stable than those in which the BH3
groups are oriented ‘outward’. This stereo-dependence is generally
similar to that of oligodeoxyribonucleoside phosphorothioates (PS-
ODNs).25 One of the reasons for the lower thermal stability ob-
tained with the PB-ODNs is the sterically more demanding BH3
groups compared to the oxygen atoms of natural ODNs.1 However,
a more intensive physicochemical study using stereodefined PB-
ODNs containing four kinds of nucleobases would be necessary
for full clarification of the stereo-dependence in the duplex forma-
tion of PB-ODNs because it has been reported that duplexes con-
sisting of homothymidylates structurally differ in detail from
those consisting of two oligomers of mixed base-sequence.26
In conclusion, we developed a method to synthesize stereode-
fined PB-ODNs at the oligomer level for the first time and clarified
that the modifications of ODNs with (Sp)-boranophosphate link-
ages were better than those with (Rp)- and stereo-random coun-
terparts in terms of the duplex formation. Although the duplexes
of PB-ODNs used in this study were much less stable than those
of natural ODNs, it should be improved by using PB-ODNs contain-
ing four kinds of nucleobases as we demonstrated previously with
stereo-random PB-ODNs.2 Further studies on the synthesis of du-
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Acknowledgments
We thank Professor Kazuhiko Saigo (Kochi University of Technol-
ogy) for helpful suggestions. We also thank Dr. Mamoru Shimizu for
helpful discussions and the synthesis of stereo-random PB-ODNs.
This research was financially supported by grants from the Ministry
of Education, Culture, Sports, Science, and Technology (MEXT) Japan.
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
06.015.