A novel method for the synthesis of dinucleoside boranophosphates by a boranophosphotriester method

Article abstract of DOI:10.1021/jo0493875

2′-Deoxyribonucleoside-3′-boranophosphates (nucleotide monomers), including four kinds of nucleobases, were synthesized in good yields by the use of new boranophosphorylating reagents. We have explored various kinds of condensing reagents as well as nucle

Full text of DOI:10.1021/jo0493875

A Novel Meth od for th e Syn th esis of Din u cleosid e
Bor a n op h osp h a tes by a Bor a n op h osp h otr iester Meth od
Mamoru Shimizu, Takeshi Wada,* Natsuhisa Oka, and Kazuhiko Saigo
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo,
Bioscience Building 702, Kashiwa, Chiba 277-8562, J apan
wada@k.u-tokyo.ac.jp
Received April 13, 2004
2′-Deoxyribonucleoside-3′-boranophosphates (nucleotide monomers), including four kinds of nucleo-
bases, were synthesized in good yields by the use of new boranophosphorylating reagents. We have
explored various kinds of condensing reagents as well as nucleophilic catalysts for the borano-
phosphorylation reaction with nucleosides. In the synthesis of dinucleoside boranophosphates,
undesirable side reactions occurred at the O-4 of thymine and the O-6 of N2-phenylacetylguanine
bases. To avoid these side reactions, additional protecting groups, benzoyl (Bz) and diphenylcar-
bamoyl (Dpc) groups, were introduced to thymine and guanine bases, respectively. As a result, the
condensation reactions proceeded smoothly without any side reactions, and the dimers including
four kinds of nucleobases were obtained in excellent yields. In the deprotection of the 5′-DMTr
group, Et3SiH was found to be effective as a scavenger for the DMTr cation which caused a P-B
bond cleavage. After removal of the other protecting groups by the conventional procedure, four
kinds of dinucleoside boranophosphates were obtained in good yields.
In tr od u ction
boranophosphate with native dodecadeoxyadenylate is
lower than those of the corresponding native DNA/DNA
and phosphorothioate DNA/DNA duplexes.4 Neverthe-
less, the duplexes of boranophosphate DNAs including
cytosine and guanine bases are expected to have higher
Tm values.
A new class of nucleic acid analogues, oligodeoxyribo-
nucleotides bearing internucleotidic boranophosphate
linkages (boranophosphate DNAs), are regarded as po-
tentially useful antisense molecules.1 The DNA analogues
are isosteric with methylphosphonate DNAs and phos-
phorothioate DNAs, but the BH3 group is slightly larger
than the oxygen in native phosphate DNAs.2 Borano-
phosphate DNAs are also isoelectronic with phosphate
diesters, but imparts a considerable increase in hydro-
phobicity compared to native DNAs.2 In addition, a
duplex consisting of a boranophosphate DNA and its
complementary RNA is a good substrate for ribonuclease
H (RNase H) compared with the corresponding native
DNA/RNA and phosphorothioate DNA/RNA duplexes.3
Boranophosphate DNAs are, furthermore, stable and are
more resistant to nucleases than phosphorothioate DNAs.4
From another point of view, boranophosphate DNAs are
applicable to boron neutron capture therapy (BNCT)5 due
to the 10B atom property. Thus, boranophosphate DNAs
have many advantages as nucleic acid analogues. It has
been reported that the hybridization ability of borano-
phosphate DNAs are much poorer than that of native
DNAs; the Tm value of the duplex of dodecathymidylate
The methods previously reported for the synthesis of
boranophosphate DNAs are either enzymatic or chemical.
In an enzymatic approach for the synthesis of borano-
phosphate DNAs, 2′-deoxynucleoside 5′-(R-P-borano)-
triphosphates are employed as substrates and polymer-
ized by the use of T7 DNA polymerase.6 However, the
enzyme can recognize only one of the diasteromers (the
RP isomer), and the SP isomer cannot be incorporated into
a DNA strand. Hence, the enzymatic method yields only
boranophosphate oligonucleotides with the SP configu-
ration. Furthermore, the enzymatic method is obviously
not suitable for large-scale synthesis. On the other hand,
the chemical synthesis of boranophosphate oligonucle-
otides may be accomplished via the phosphoramidite or
H-phosphonate approach, followed by the boronation of
the P(III) intermediates.4,7-9 However, this chemical
approach requires the use of a borane reagent to give rise
to undesirable side reactions at the base moieties in the
boronation step; these base modifications are mainly
caused by the reduction of N-acyl protecting groups to
(1) Summers, J . S.; Shaw, B. R. Curr. Med. Chem. 2001, 8, 1147-
1155.
(2) (a) Li, H.; Huang, F.; Shaw, B. R. Bioorg. Med. Chem. 1997, 5,
787-795. (b) Summers, J . S.; Roe, D.; Boyle, P. D.; Colvin, M.; Shaw,
B. R. Inorg. Chem. 1998, 37, 4158-4159.
(3) Rait, V. K.; Shaw, B. R. Antisense Nucleic Acid Drug Dev. 1999,
9, 53-60.
(6) Li, H.; Porter, K.; Huang, F.; Shaw, B. R. Nucleic Acids Res. 1995,
21, 4495-4501.
(7) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J . Am. Chem. Soc. 1990,
112, 9000-9001.
(4) Sergueev, D. S.; Shaw, B. R. J . Am. Chem. Soc. 1998, 120, 9417-
(8) Zhang, J .; Terhorst, T.; Matteucci, M. D. Tetrahedron Lett. 1997,
38, 4957-4960.
(9) Higson, A. P.; Sierzchala, A.; Brummel, H.; Zhao, Z.; Caruthers,
M. H. Tetrahedron Lett. 1998, 39, 3899-3902.
9427.
(5) Barth, R. F.; Soloway, A. H.; Fairchild, R. G. Sci. Am. 1990, 263,
100-107.
10.1021/jo0493875 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/07/2004
J . Org. Chem. 2004, 69, 5261-5268
5261

Shimizu et al.
SCHEME 1
give the corresponding N-alkyl derivatives.10 Therefore,
the method is applicable only to thymine derivatives, in
which no acyl protecting group is required. This means
that oligonucleotide chains must be constructed without
base protection or with base protection by nonacyl-type
N-protecting groups in order to synthesize borano-
phophate DNAs including four kinds of nucleobases, A,
C, and G as well as T, by this method. However, the base-
unprotected method has some limitations, due to the
difficulty of the synthesis of long H-phosphonate DNAs
in acceptable yields,11 and due to the reduction of
unprotected thymine base moiety by a borane reagent
in the boronation step.9
TABLE 1. Syn th esis of Dim er s
entry
B1
yield of 6 (%)
1
2
3
4
N6-benzoyladenin-9-yl
N4-benzoylcytosin-1-yl
N2-phenylacetylguanin-9-yl
thymin-1-yl
59
94
47
92
These results strongly suggest that the development
of a new boranophosphorylating agent is very fascinating
for the effective synthesis of boranophosphate DNAs.
Imamoto et al. have reported a boranophosphorylation
reaction of nucleosides.12 In this method, tetramethyl
pyroboranophosphate is used as a boranophosphorylating
reagent. However, this reagent is less reactive for the
nucleophilic attack of an alcohol; the reaction has to be
carried out by the use of t-BuLi for the activation of the
hydroxy function at -78 °C in THF. Therefore, this
reaction is not applicable to solid-phase synthesis.
Recently, we have reported a new strategy for the
synthesis of boranophosphate DNAs (the boranophos-
photriester method).13 In this method, a subsequently
boronated phosphorylating reagent was used in order to
introduce a BH3 group into an internucleotidic phosphate
linkage; consequently, side reactions, caused by borane
reagents in the conventional methods for the synthesis
of boranophosphate DNAs, have been completely avoided.
In this paper, we report detailed study on the borano-
phosphotriester method involving the boranophosphoryl-
ation, internucleotidic bond formation, and deprotection
reactions.
SCHEME 2
sides (Scheme 1).13 Applying this boranophosphotriester
method, dithymidine boranophosphate was synthesized
in >90% yield by using N,N′-bis(2-oxo-3-oxazolidinyl)-
phosphonic chloride (Bop-Cl) as a condensing reagent in
the presence of 3-nitro-1,2,4-triazole (NT) and i-Pr2NEt
in THF.
Resu lts a n d Discu ssion
In a similar manner, we attempted to synthesize
dimers including other nucleobases, A, C, and G. How-
ever, in contrast to pyrimidine-pyrimidine dimers (dTpb-
dT and dCpbdT), purine-pyrimidine dimers (dApbdT and
dGpbdT) were obtained only in 40-60% yields (Table 1).
A precise monitoring of the reaction mixture by TLC
indicated that the monomer 4 reacted with the O4 of the
thymin-1-yl moiety and the O6 of the N2-phenyl-
acetylguanin-9-yl moiety in the nucleoside derivatives
Syn th esis of Dim er s. We have recently reported a
novel strategy for the boranophosphorylation of nucleo-
(10) Sergueeva, Z. A.; Sergueev, D. S.; Shaw, B. R. Nucleosides
Nucleotides 2000, 19, 275-282.
(11) Wada, T.; Sato, Y.; Honda, F.; Kawahara, S.; Sekine, M. J . Am.
Chem. Soc. 1997, 119, 12710-12721.
(12) Imamoto, T.; Nagato, E.; Wada, Y.; Masuda, H.; Yamaguchi,
K.; Uchimaru, T. J . Am. Chem. Soc. 1997, 119, 9925-9926.
(13) Wada, T.; Shimizu, M.; Oka, N.; Saigo, K. Tetrahedron Lett.
2002, 43, 4137-4140.
5262 J . Org. Chem., Vol. 69, No. 16, 2004

Novel Method for the Synthesis of BH3-ODNs
SCHEME 3
SCHEME 4
to give the corresponding boranophosphotriesters 7 and
8 (Scheme 2). In the formation of the dimers 6, the
boranophosphotriesters 7 and 8 with pyrimidine nucleo-
side derivatives would be reactive with the 5′-OH of the
nucleosides 5 to give the desired dimers, while those with
purine nucleoside derivatives may be less reactive due
to the bulky purine bases, resulting in low yields of the
dimers. Although similar side-reactions should take place
in the formation of the monomers, the side reactions did
not become a serious problem; since the boranophospho-
rylating reagent 2 is not bulky, even if a P-O bond was
formed at the base moieties, it would be readily hydro-
lyzed during the aqueous workup of the reaction mixture.
Syn th esis of New Bor a n op h osp h or yla tin g Re-
a gen ts. To prevent the side reactions described above,
additional protecting groups should be introduced at the
N3 of the thymin-1-yl moiety and the O6 of the N2-
phenylacetylguanin-9-yl moiety. For newly introduced
protecting groups, the following requirements should be
satisfied: (1) stable under acidic and basic conditions and
(2) deprotected under the same conditions prescribed for
the removal of the N-acyl protecting groups of A, C, and
G. To meet these criteria, we designed the heretofore
undescribed fully protected monomers, 9 and 10 (Scheme
3). In 9, a benzoyl (Bz) group was used to block the N3 of
thymine, whereas in 10 a diphenylcarbamoyl (Dpc) group
was used to additionally block the O6 of N2-pheny-
lacetylguanine. However, the Bz group in 9 was found
to be partially lost during demethylation with PhSH/Et3N
according to the literature method,16 yielding a mixture
of 11 and 12. Moreover, in agreement with a previous
report of displacement of the Dpc group during demethy-
lation with PhSH/Et3N,17 treatment of 10 with this
reagent led to the sulfide 13.
Therefore, we decided to change the phosphorus pro-
tecting group of the boranophosphorylating reagent from
a methyl group to one that could be removed without loss
of the nucleobase protecting groups. An attractive can-
didate for this purpose was the 2-(trimethylsilyl)ethyl
(TSE) group,18 which can be cleaved by a fluoride ion
under neutral conditions. Thus, we synthesized the new
TSE-protected boranophosphorylating reagent 17, ac-
cording to Scheme 4. In a similar manner, triethylam-
monium bis-2-(trimethylsilyl)ethyl boranophosphate (18)
was also synthesized.
P r ep a r a tion of F u lly P r otected Th ym id in e a n d
2′-Deoxygu a n osin e 3′-Bor a n op h osp h a te Mon om er s.
We at first attempted to synthesize thymidine deriva-
tives. When 5′-O-dimethoxytrityl-N3-benzoylthymidine
(1, B1 ) N3-benzoylthymin-1-yl) was condensed with 18
in the presence of Bop-Cl, NT, and i-Pr2NEt in THF, the
reaction was very slow, and the desired compound was
obtained only in 52% yield after 1 h. In this case, due to
the bulkiness of the TSE group, the nucleophilic catalyst,
NT, would not be able to attack at the phosphorus center
of tetrakis(2-(trimethylsilyl)ethyl) pyroboranophosphate,
which was considered to be the probable intermediate
in the boranophosphorylation reaction. To avoid the
formation of such a sterically crowded intermediate, 17
was used as the boranophosphorylating reagent instead
of 18. The reaction proceeded quickly, and 19 (B1 ) N3-
benzoylthymin-1-yl) was obtained in 91% yield after 30
min. In a similar manner, 1 (B1 ) O6-diphenylcarbamoyl-
N2-phenylacetylguanin-9-yl) was condensed with 17 to
give 19 (B1 ) O6-diphenylcarbamoyl-N2-phenylacetylgua-
nin-9-yl) in 85% yield (Scheme 5).
J . Org. Chem, Vol. 69, No. 16, 2004 5263

Shimizu et al.
TABLE 3. Syn th esis of F u lly Bese-P r otected Dim er s
SCHEME 5
entry
B2
yield of 6 (%)
1
N6-benzoyladenin-9-yl
N4-benzoylcytosin-1-yl
O6-diphenylcarbamoyl-
N2-phenylacetylguanin-9-yl
N3-benzoylthymin-1-yl
92
99
94
2a
3
4
quant
TABLE 2. Syn th esis of F u lly Ba se-P r otected Dim er s
a
The 3′-OH was protected with a TBDMS group, since 5 (B2
)
N4-benzoylcytosin-1-yl) protected with a Bz group was insoluble
in CH3CN.
Rem ova l of th e DMTr Gr ou p . It is well-known that
4,4′-dimethoxytrityl cation (DMTr+) causes the decom-
position of boranophosphate DNAs.9,21 Thus a DMTr+
scavenger is necessary to suppress this major side
reaction. We used 3 (B1 ) thymin-1-yl) as a prototypical
boranophosphate triester and tested two types of scav-
engers (Table 4). When the DMTr group was removed
with dichloroacetic acid (DCA), byproducts 22-24 were
observed (Table 4, entry 1). On the other hand, when Et3-
SiH was included as a scavenger the formation of
byproducts was decreased or entirely eliminated (Table
4, entries 2-4). Although byproducts were likewise not
observed when MeOH or H2O was used as the scavenger
(Table 4, entries 5-8), complete removal of the DMTr
group was much slower. Thus, Et3SiH appeared to be a
more effective scavenger than MeOH or H2O in this
deprotection step.
R em ova l of t h e Ot h er P r ot ect in g Gr ou p s. Once
the DMTr had been removed under the best conditions
described above (Table 4; entry 4), the other protecting
groups were cleaved by the conventional procedure using
aqueous ammonia.7 However, considerable degradation
of the dinucleoside boranophosphates 27 was observed
by an HPLC analysis. From this result, we concluded that
aqueous ammonia was unsuitable for the demethylation
of boranophosphotriesters. Accordingly, we remove the
methyl group with PhSH/Et3N prior to cleaving the other
base-labile protecting groups with MeOH/NH3. In this
manner, we were able to obtain excellent yields of the
dinucleoside boranophosphate 27 (B1 ) A, C, G, or T) via
25 and 26 as shown in Table 5.
entry
B1
X
yield of 6 (%)
1
2
3
N6-benzoyladenin-9-yl
N4-benzoylcytosin-1-yl
O6-diphenylcarbamoyl-
N2-phenylacetylguanin-9-yl
N3-benzoylthymin-1-yl
HNEt3
HNEt3
HN(n-Bu)3
85
quant
83
4
HN(n-Bu)3
quant
Although a TSE group is known to be relatively stable
toward fluoride ion,18 it was found that cleavage of 19 to
20 was complete within 1 min upon treatment with
tetrabutylammonium fluoride (TBAF). After purification
by silica gel column chromatography, the corresponding
boranophosphate diesters 20 (B1 ) N3-benzoylthymin-1-
yl) and 20 (B1 ) O6-diphenylcarbamoyl-N2-phenylacetyl-
guanin-9-yl) were obtained in good yields; unexpectedly,
the diesters were not tetrabutylammonium salts, but
tributylammonium salts, of which the identity was
confirmed by H NMR and elemental analysis.19
1
Syn th esis of Dim er s by Usin g F u lly Ba se-P r o-
tected Der iva tives. We next tried to synthesize di-
nucleoside boranophosphates by the use of four kinds of
fully base-protected monomers (4 (B1 ) N6-benzoylad-
enin-9-yl), 4 (B1 ) N4-benzoylcytosin-1-yl), 20 (B1 ) N3-
benzoylthymin-1-yl), and 20 (B1 ) O6-diphenylcarbamoyl-
N2-phenylacetylguanin-9-yl)). When the fully base-pro-
tected monomers were allowed to react with the protected
nucleoside 5 (B1 ) N3-benzoylthymin-1-yl), the yields of
the dimers 6 were highly improved (Table 2; entries 1-4).
Despite the improvement of the coupling yields, the
condensing reagent Bop-Cl gave rise to an HCl salt which
was hardly soluble in the reaction solvent. To solve this
problem, we applied 3-nitro-1,2,4-triazol-1-yl-tris(pyrro-
lidin-1-yl)phosphonium hexafluorophosphate (PyNTP),
recently developed in our laboratory,20 because it conve-
niently releases the soluble nucleophilic catalyst NT in
situ and does not form an insoluble HCl salt. The yields
of dimers obtained with PyNTP and with Bop-Cl as the
coupling reagents were similar (Table 3; entries 1-4).
(14) Sekine, M.; Fujii, M.; Nagai, H.; Hata, T. Synthesis 1987, 12,
1191-1121.
(15) Kamimura, T.; Tsuchiya, M.; Urakami, K.; Koura, K.; Sekine,
M.; Shinozaki, K.; Miura, K.; Hata, T. J . Am. Chem. Soc. 1984, 106,
4552-4557.
(16) Daub, G. W.; van Tamelen, E. E. J . Am. Chem. Soc. 1977, 99,
3526-3528.
(17) Huss, S.; Gosselin, G.; Imbach, J . L. J . Org. Chem. 1988, 53,
499-506.
(18) Wada, T.; Sekine, M. Tetrahedron Lett. 1994, 35, 757-760.
(19) Elucidation of the mechanism for this unprecedented dealkyl-
ation of the quaternary ammonium salt is now in progress.
(20) Wada, T.; Tsuneyama, T.; Saigo, K. Nucleic Acids Res. Suppl.
2001, 1, 187-188.
(21) Sergueeva, Z. A.; Sergueev, D. S.; Shaw, B. R. Nucleosides
Nucleotides 2001, 20, 941-945.
5264 J . Org. Chem., Vol. 69, No. 16, 2004

Novel Method for the Synthesis of BH3-ODNs
TABLE 4. Detr ityla tion of Bor a n op h osp h a te Tr iester
entry
acid
solvent (0.04 M)a
CDCl3
Et3SiH - CDCl3 (1:9, v/v)
Et3SiH - CDCl3 (1:3, v/v)
Et3SiH - CDCl3 (1:1, v/v)
CD3OD (d4)
time required for detritylation
ratio of compdsb 21/22/23/24
1
2
3
4
5
6
7
8
3% DCA
3% DCA
3% DCA
3% DCA
3% DCA
1% TFA
1% TFA
80% AcOH
<1 min
<1 min
<1 min
<1 min
3 h
2 h
2 h
78:8:6:8
89:6:3:2
91:7:2:0
100:0:0:0
100:0:0:0
100:0:0:0
100:0:0:0
100:0:0:0
MeOH - CDCl3 (1:1, v/v)
MeOH - CDCl3 (1:3, v/v)
c
D2O
>14 h
a
b
Concentration of 3. Estimated by 31P NMR (after 30 min). c Heterogeneous.
TABLE 5. Dep r otection of Din u cleosid e Bor a n op h osp h a te
entry
B1 of 27
yield of 27 (%)
1
adenin-9-yl
cytosin-1-yl
guanin-9-yl
thymin-1-yl
75
89
92
86
2
3a
4
a
Methanolic ammonia treatment was carried out for the compounds 25 (B1 ) O6-diphenylcarbamoyl-N2-phenylacetylguanin-9-yl) in
order to remove the Dpc group at the O-6 position of the guanin-9-yl moiety, since the Dpc group was displaced by PhS-.
mmol) in dry THF (70 mL) under argon atmosphere was added
dropwise a 1.1 M solution of BH3‚THF in THF (78 mL, 85.8
mmol) at 0 °C. The mixture was stirred for 2 h at rt and then
cooled to 0 °C. To the mixture was slowly added dry MeOH
(28.4 mL, 700 mmol) at 0 °C. After being stirred for 30 min at
rt, the solution was treated with redistilled Et3N (19.6 mL,
140 mmol) at rt, and stirring at rt was continued for 12 h.
The solution was concentrated to dryness under reduced
pressure and then dried by repeated coevaporation with dry
toluene and CHCl3 to obtain 2 (14.5 g, 91%) as a colorless oil:
1H NMR (CDCl3) δ 12.58 (1H, bs), 3.56 (6H, d, J POCH ) 10.5
Hz), 3.13-3.02 (6H, m), 1.33 (9H, t, J ) 7.5 Hz), 1-0 (3H,
bq); 13C NMR (CDCl3) δ 50.1, 50.0, 45.2, 8.2; 31P NMR (CDCl3)
δ 96.52 (q, J PB ) 136.5 Hz); IR (KBr) 3401, 2986, 2946, 2840,
Con clu sion
We have developed a new efficient method for the
synthesis of boranophosphates. The present new bora-
nophosphotriester method enabled us to synthesize di-
nucleoside borabophosphates with all possible nucleo-
bases, A, C, G, and T. The present strategy essentially
eliminates the troublesome side reactions and thus
should find widespread applications in solid-phase syn-
thesis of unnatural oligonucleotide analogues.
Exp er im en ta l Section 22
Tr ieth yla m m on iu m Dim eth yl Bor a n op h osp h a te 2. To
a solution of dimethyl trimethylsilyl phosphite (12.9 g, 70.8
2362, 1643, 1476, 1399, 1142, 1029, 840, 782, 712, 634 cm-1
.
Anal. Calcd for C8H25BNO3P‚1/5CHCl3: C, 39.56; H, 10.20; N,
5.63. Found: C, 39.40; H, 10.40; N, 5.58.
(22) A general statement describing materials and methods is
provided in the Support Information.
J . Org. Chem, Vol. 69, No. 16, 2004 5265

Shimizu et al.
Tr ieth ylam m on iu m Meth yl 2-(Tr im eth ylsilyl)eth yl Bo-
r a n op h osp h a te 17. To a solution of 14 (34.9 g, 159 mmol) in
dry THF (160 mL) under argon atmosphere was slowly added
a mixture of MeOH (14.2 mL, 352 mmol) and distilled i-Pr2-
NEt (59.7 mL, 352 mmol) at 0 °C. The mixture was allowed to
warm to rt and stirred at rt for 1 h. Then, the mixture was
diluted with CHCl3 (300 mL) and washed with satd NaHCO3
aq (3 × 200 mL), and the aqueous layer was back-extracted
with CHCl3 (4 × 100 mL). The organic layer and washings
were combined, dried over Na2SO4, filtered, and concentrated
to dryness under reduced pressure. The residue was purified
by distillation (3 mmHg, 65-67 °C) to obtain 15 (25.8 g, 77%)
as a colorless oil. To a solution of 15 (25.8 g, 123 mmol) in dry
THF (123 mL) under argon atmosphere was added dropwise
a 1.0 M solution of BH3‚THF in THF (147 mL, 147 mmol) at
0 °C, and the solution was stirred for 1 h at rt. The mixture
was concentrated to dryness under reduced pressure. The
residue was chromatographed on silica gel (100 g) using CH2-
Cl2 as an eluent. The fractions containg dimethyl 2-(trimeth-
ylsilyl)ethyl boranophophate (16) were combined and concen-
trated to dryness under reduced pressure to obtain 16 (27.5
g, quantitative) as a colorless oil. To a solution of 16 (26.9 g,
120 mmol) in dry THF (120 mL) was added a mixture of Et3N
(66.5 mL, 480 mmol) and PhSH (48.9 mL, 480 mmol) at rt.
After being stirred at rt for 24 h, the mixture was concentrated
to dryness under reduced pressure. The residue was chro-
matographed on silica gel (100 g) using a gradient of MeOH
(0-4%) in CH2Cl2 with 0.5% Et3N as an eluent. The fractions
containg triethylammonium methyl 2-(trimethylsilyl)ethyl bo-
ranophosphate (17) were combined and concentrated to dry-
ness under reduced pressure. Excess Et3N was removed by
repeated coevaporation with toluene, and the residue was
dissolved in CHCl3 (100 mL). The solution was washed with
satd NaCl aq (3 × 100 mL), and the aqueous layer was back-
extracted with CHCl3 (3 × 50 mL). The organic layer and
washings were combined, dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure to obtain 17
(31 g, 83%) as a colorless oil: 1H NMR (CDCl3) δ 4.00-3.88
(2H, m), 3.55 (3H, d, J POCH ) 9.0 Hz), 3.13-2.98 (6H, m), 1.31
(9H, t, J ) 7.5 Hz), 1.03 (2H, m), 1-0 (3H, bq), 0.01 (9H, s);
13C NMR (CDCl3) δ 61.2, 50.5, 50.4, 45.5, 20.2, 20.1, 8.7, -1.2;
31P NMR (CDCl3) δ 94.70 (q, J PB ) 139.4 Hz); IR (KBr) 2951,
2895, 2838, 2359, 1475, 1398, 1249, 1177, 1139, 1045, 997, 936,
839, 779, 696 cm-1. Anal. Calcd for C12H35BNO3PSi‚0.5H2O‚
0.25CHCl3: C, 42.02; H, 10.44; N, 4.00. Found: C, 42.04; H,
10.62; N, 3.91.
5′-O-Dim eth oxytr ityl-N6-ben zoyl-2′-d eoxya d en osin -3′-
yl Dim eth yl Bor a n op h osp h a te (3, B1 ) N6-Ben zoyla d -
en in -9-yl). 1 (B1 ) N6-benzoyladenin-9-yl) (197.3 mg, 0.300
mmol) and triethylammonium dimethyl boranophosphate 2
(203 mg, 0.900 mmol) were dried by repeated coevaporation
with dry toluene followed by dry pyridine and finally dissolved
in dry THF (3.00 mL). To the solution were successively added
i-Pr2NEt (0.510 mL, 3.00 mmol), NT (171 mg, 1.50 mmol), and
Bop-Cl (382 mg, 1.50 mmol). After being stirred at rt for 30
min, the mixture was diluted with CHCl3 (10 mL). The solution
was washed with satd NaHCO3 aq (3 × 10 mL), and the
aqueous layer was back-extracted with CHCl3 (3 × 10 mL).
The organic layer and washings were combined, dried over
Na2SO4, filtered, and concentrated to dryness under reduced
pressure. The residue was chromatographed on silica gel (30
g) using a gradient of MeOH (0-4%) in CH2Cl2 as an eluent.
The fractions containing 5′-O-dimethoxytrityl-N6-benzoyl-2′-
deoxyadenosin-3′-yl dimethyl boranophosphate (3, B1 ) N6-
benzoyladenin-9-yl) were combined and concentrated to dry-
ness under reduced pressure to obtain 3 (B1 ) N6-benzoylad-
enin-9-yl) (202 mg, 88%) as a colorless foam: 1H NMR (CDCl3)-
δ 9.03 (1H, bs), 8.72 (1H, s), 8.17 (1H, s), 8.07-7.18 (14H, m),
6.81 (4H, d, J ) 8.1 Hz), 6.52 (1H, t, J ) 7.1 Hz), 5.25 (1H,
m), 4.38 (1H, m), 3.78 (6H, s), 3.73, 3.72 (6H, 2d, J POCH ) 4.5,
6.6 Hz), 3.44 (2H, m), 3.12-2.99 (1H, m), 2.81-2.70 (1H, m),
1-0 (3H, bq); 13C NMR (CDCl3) δ 164.5, 158.6, 152.7, 152.6,
151.5, 149.5, 144.3, 141.4, 141.3, 135.4, 133.6, 132.8, 130.0,
129.9, 128.9, 128.0, 127.8, 127.1, 123.4, 113.3, 113.1, 86.8, 85.3,
84.6, 84.5, 77.6, 63.0, 55.3, 55.2, 55.1, 53.4, 39.0; 31P NMR
(CDCl3) δ 120.0-116.8 (m). IR (KBr) 3420, 2953, 2837, 2400,
1702, 1610, 1582, 1509, 1457, 1300, 1252, 1178, 1072, 1033,
952, 830, 799, 756, 708, 645 cm-1. Anal. Calcd for C40H43
-
BN5O8P‚1/3CHCl3: C, 60.30; H, 5.44; N, 8.72. Found: C, 60.06;
H, 5.50; N, 8.73.
5′-O-Dim et h oxyt r it yl-N4-b en zoyl-2′-d eoxycyt id in -3′-
yl Dim eth yl Bor a n op h osp h a te (3, B1 ) N4-Ben zoylcy-
tosin -1-yl).23 This compound was obtained from 1 (B1 ) N4-
benzoylcytosin-1-yl) as a colorless foam (96% yield) in a similar
manner as 3 (B1 ) N6-benzoyladenin-9-yl).
5′-O-Dim eth oxytr ityl-N3-ben zoylth ym id in -3′-yl Meth yl
2-(Tr im eth ylsilyl)eth yl Bor a n op h osp h a te (19, B1 ) N3-
Ben zoylth ym in -1-yl). 1 (B1 ) N3-benzoylthymin-1-yl) (130
mg, 0.200 mmol) and 17 (187 mg, 0.600 mmol) were dried by
repeated coevaporation with dry toluene followed by dry
pyridine and finally dissolved in dry THF (2.00 mL). To the
solution were successively added i-Pr2NEt (0.510 mL, 3.00
mmol), NT (114 mg, 1.00 mmol), and Bop-Cl (255 mg, 1.00
mmol). After being stirred at rt for 30 min, the mixture was
diluted with CHCl3 (20 mL). The reaction mixture was washed
with satd NaHCO3 aq (3 × 20 mL), and the aqueous layer was
back-extracted with CHCl3 (2 × 20 mL). The organic layer and
washings were combined, dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The residue
was chromatographed on silica gel (30 g) using EtOAc/hexane
(1:1 v/v) as an eluent. The fractions containing 5′-O-dimeth-
oxytrityl-N3-benzoylthymidin-3′-yl methyl 2-(trimethylsilyl)-
ethyl boranophosphate (19, B1 ) N3-benzoylthymin-1-yl) were
combined and concentrated to dryness under reduced pressure
to obtain 19 (B1 ) N3-benzoylthymin-1-yl) (153 mg, 91%) as a
light yellow foam: 1H NMR (CDCl3) δ 7.95, 7.93 (2H, m), 7.72
(1H, s), 7.68-7.21 (12H, m), 6.87 (4H, d, J ) 9.0 Hz), 6.45,
6.42 (1H, dd, J 1′,2′/J 1′,2′′ ) 5.7, 8.7 Hz), 5.21 (1H, t, J ) 7.5 Hz),
4.25 (1H, m), 4.10 (2H, m), 3.80 (6H, s), 3.68, 3.63 (3H, 2d,
J POCH ) 9.0, 12.0 Hz), 3.48-3.44 (2H, m), 2.66-2.57 (1H, m),
2.53-2.41 (1H, m), 1.46 (3H, s), 1.09-0.99 (2H, m), 1-0 (3H,
bq), 0.04, 0.02 (9H, 2s); 13C NMR (CDCl3) δ 168.9, 162.7, 158.8,
158.7, 149.3, 144.1, 135.2, 135.0, 134.9, 131.6, 130.5, 130.2,
130.0, 129.3, 129.1, 128.2, 128.1, 128.0, 127.3, 126.1, 113.6,
113.4, 111.6, 87.3, 85.2, 85.1, 85.0, 84.9, 84.7, 84.6, 66.0, 65.9,
63.3, 63.2, 55.2, 55.1 55.0, 53.1 53.0 39.7, 39.6, 19.7, 19.6, 19.5
19.4, 11.7, -1.6; 31P NMR (CDCl3) δ 118.79-115.48 (m). IR
(KBr) 3448, 2954, 2835, 2397, 1751, 1704, 1664, 1608, 1509,
1445, 1395, 1280, 1253, 1178, 1110, 1034, 987, 834, 762, 701
cm-1. Anal. Calcd for C44H54BN2O10PSi: C, 62.86; H, 6.47; N,
3.33. Found: C, 63.07; H, 6.44; N, 3.27.
5′-O-Dim eth oxytr ityl-O6-d ip h en ylca r ba m oyl-N2-p h en -
yla cetyl-2′-d eoxygu a n osin -3′-yl Meth yl 2-(Tr im eth ylsi-
lyl)eth yl Bor a n op h osp h a te (19, B1 ) O6-Dip h en ylca r -
ba m oyl-N2-p h en yla cetylgu a n in -9-yl).23 This compound was
obtained from
1
(B1 ) O6-diphenylcarbamoyl-N2-phenyl-
acetylguanin-9-yl) as a light yellow foam (80% yield) in a
similar manner as 19 (B1 ) O6-diphenylcarbamoyl-N2-phenyl-
acetylguanin-9-yl).
Tr ieth yla m m on iu m 5′-O-Dim eth oxytr ityl-N6-ben zoyl-
2′-d eoxya d en osin -3′-yl Meth yl Bor a n op h osp h a te (4, B1
) N6-Ben zoyla d en in -9-yl). To a solution of 3 (B1 ) N6-
benzoyladenin-9-yl) (764 mg, 1.00 mmol) in THF (10 mL) were
successively added Et3N (5.54 mL, 40.0 mmol) and PhSH (4.08
mL, 40.0 mmol). After being stirred at rt for 2 h, the mixture
was concentrated to dryness under reduced pressure. The
residue was chromatographed on silica gel (100 g) by using a
gradient of MeOH (0-4%) in CH2Cl2 with 0.5% Et3N as an
eluent. The fractions containing triethylammomium 5′-O-
dimethoxytrityl-N6-benzoyl-2′-deoxyadenosin-3′-yl methyl bo-
ranophosphate (4, B1 ) N6-benzoyladenin-9-yl) were combined
(23) Characterization data are provided in the Supporting Informa-
tion.
5266 J . Org. Chem., Vol. 69, No. 16, 2004

Novel Method for the Synthesis of BH3-ODNs
and concentrated to dryness. Excess Et3N was removed by
repeated coevaporation with toluene, and the residue was
dissolved in CHCl3 (10 mL). The solution was washed with
satd NaCl aq (4 × 20 mL), and the aqueous layer was back-
extracted with CHCl3 (3 × 10 mL). The organic layer and
washings were combined, dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure to obtain 4
(B1 ) N6-benzoyladenin-9-yl) (791 mg, 93%) as a colorless
foam: 1H NMR (CDCl3) δ 9.18 (1H, bs), 8.71 (1H, s), 8.20, 8.19
(1H, 2s), 8.02, 8.05 (2H, 2m), 7.65-7.15 (12H, m), 6.79 (4H, d,
J ) 9.0 Hz), 6.58 (1H, m), 5.14 (1H, m), 4.43 (1H, m), 3.77
(6H, s), 3.55, 3.49 (3H, 2d, J POCH ) 10.8, 11.1 Hz), 3.41 (2H,
m), 3.04 (6H, q, J ) 7.2 Hz), 2.84 (2H, m), 1.31 (9H, t, J ) 7.4
Hz), 1-0 (3H, bq); 13C NMR (CDCl3) δ 164.6, 158.4, 152.6,
152.5 151.5 149.3, 144.5, 141.4, 141.3 135.7 135.6, 133.8, 132.7,
130.1, 130.0, 128.8, 128.2, 127.8, 126.9, 123.1, 113.2, 113.0,
108.9, 86.5, 86.4, 85.8, 84.8, 84.7, 84.5, 74.0, 73.6, 73.4, 63.7,
55.3, 55.2, 55.1, 55.0, 50.2, 45.4, 40.2, 40.0, 8.6, 8.4; 31P NMR
(CDCl3) δ 98.55-93.80 (m). IR (KBr) 3426, 2944, 2838, 2365,
1701, 1611, 1581, 1509, 1459, 1399, 1300, 1252, 1177, 1072,
5′-O-Dim eth oxytr ityl-N6-ben zoyl-2′-d eoxya d en osin -3′-
yl 3′-O,N3-Diben zoylth ym id in -5′-yl Meth yl Bor a n op h os-
p h a te (6, B1 ) N6-Ben zoyla d en in -9-yl, B2 ) N3-Ben zoyl-
th ym in -1-yl). 5 (B2 ) N3-benzoylthymin-1-yl) (45.0 mg, 0.100
mmol) and 4 (B1 ) N6-benzoyladenin-9-yl) (102 mg, 0.120 mol)
were dried by repeated coevaporation with dry toluene followed
by dry pyridine and finally dissolved in dry THF (1.00 mL).
To the solution were successively added i-Pr2NEt (170 µL, 1.00
mmol), NT (34.2 mg, 0.300 mmol), and Bop-Cl (76.4 mg, 0.300
mmol). After being stirred at rt for 20 min, the mixture was
diluted with CHCl3 (10 mL). The solution was washed with
satd NaHCO3 aq (3 × 10 mL), and the aqueous layer was back-
extracted with CHCl3 (3 × 10 mL). The organic layer and
washings were combined, dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The residue
was chromatographed on silica gel (30 g) using a gradient of
MeOH (0-4%) in CH2Cl2 as an eluent. The fractions containg
5′-O-dimethoxytrityl-N6-benzoyl-2′-deoxyadenosin-3′-yl 3′-O,N3-
dibenzoylthymidin-5′-yl methyl boranophosphate (6, B1 ) N6-
benzoyladenin-9-yl, B2 ) N3-benzoylthymin-1-yl) were com-
bined and concentrated to dryness under reduced pressure to
obtain 6 (B1 ) N6-benzoyladenin-9-yl, B2 ) N3-benzoylthymin-
1-yl) (100 mg, 85%) as a colorless foam: 1H NMR (CDCl3) δ
8.99 (1H, bs), 8.65 (1H, m), 8.21, 8.18 (1H, 2s), 8.06-7.16 (25H,
m), 6.86-6.74 (4H, m), 6.61-6.46 (2H, m), 5.57-5.46 (1H, m),
5.37-5.29 (1H, m), 4.57-4.26 (4H, m), 3.91-3.70 (9H, m),
3.55-3.39 (2H, m), 3.27-3.12 (1H, m), 2.84-2.71 (1H, m),
2.65-2.53 (1H, m), 2.40-2.25 (1H, m), 2.05, 2.02 (3H, 2s), 1-0
(3H, bq); 13C NMR (CDCl3) δ 168.7, 166.1, 164.5, 162.6, 158.5,
152.5, 151.5, 149.4, 144.2, 141.7, 141.5, 141.3, 135.3, 135.2,
135.1, 134.5, 133.8, 133.5, 132.8, 131.4, 130.4, 130.0, 129.7,
129.1, 128.9, 128.6, 128.0, 127.9, 127.8, 127.0, 123.5, 113.2,
112.3, 112.1, 86.9, 85.5, 85.3, 84.7, 84.6, 83.1, 79.0, 78.7, 74.9,
74.8, 66.0, 65.6, 63.1, 55.2, 54.4, 54.3, 45.5, 41.4, 38.5, 37.3,
37.0, 12.6; 31P NMR (CDCl3) δ 121.22-117.23 (m); IR (KBr)
3426, 2954, 2839, 2399, 1750, 1705, 1663, 1609, 1582, 1509,
1451, 1297, 1253, 1178, 1098, 1072, 1032, 952, 830, 762, 714,
646 cm-1. Anal. Calcd for C63H61BN7O14P‚2/3CHCl3: C, 60.61;
H, 4.93; N, 7.77. Found: C, 60.56; H, 5.28; N, 7.97.
1033, 946, 831, 794, 756, 710, 646 cm-1. Anal. Calcd for C45H56
-
BN6O8P‚1.5H2O: C, 61.57; H, 6.77; N, 9.57. Found: C, 61.35;
H, 6.51; N, 9.32.
Tr ieth yla m m on iu m 5′-O-Dim eth oxytr ityl-N4-ben zoyl-
2′-d eoxycytid in -3′-yl Meth yl Bor a n op h osp h a te (4, B1
)
N4-Ben zoylcytosin -1-yl).23 This compound was obtained from
3 (B1 ) N4-benzoylcytosin-1-yl) as a colorless foam (92% yield)
in a similar manner as 4 (B1 ) N6-benzoyladenin-9-yl).
Tr ibu tyla m m on iu m 5′-O-Dim eth oxytr ityl-N3-ben zoyl-
th ym id in -3′-yl Meth yl Bor a n op h osp h a te (20, B1 ) N3-
Ben zoylth ym in -1-yl). 19 (B1 ) N3-benzoylthymin-1-yl) (168
mg, 0.200 mmol) was dried by repeated coevaporation with
dry THF followed by dry pyridine and dry toluene and then
dissolved in a 0.5 M solution of TBAF in THF (2.00 mL), which
was predried over MS 4A for 12 h, and the mixture was stirred
at rt for 5 min. The solution was diluted with CHCl3 (10 mL)
and washed with 1 M pH 7.0 phosphate buffer (3 × 10), and
the aqueous layer was back-extracted with CHCl3 (3 × 10 mL).
The organic layer and washings were combined, dried over
Na2SO4, filtered, and concentrated to dryness under reduced
pressure. The residue was chromatographed on silica gel (30
g) using a gradient of MeOH (0-4%) in CH2Cl2 as an eluent.
The fractions containg tributylammonium 5′-O-dimethoxytri-
tyl-N3-benzoylthymidin-3′-yl methyl boranophosphate (20, B1
) N3-benzoylthymin-1-yl) were combined and concentrated to
dryness under reduced pressure to obtain 20 (B1 ) N3-
benzoylthymin-1-yl) (150 mg, 81%) as a colorless foam: 1H
NMR (CDCl3) δ 12.42 (1H, bs), 7.97-7.20 (16H, m), 6.46, 6.43
(1H, dd, J 1′,2′/J 1′,2′′ ) 5.7, 8.1 Hz), 5.21-5.10 (1H, m), 4.29 (1H,
d, J ) 12.0 Hz), 3.79 (6H, s), 3.52, 3.41 (3H, 2d, J POCH ) 12.0,
12.0 Hz), 3.45-3.42 (2H, m), 2.95-2.85 (6H, m), 2.69-2.56
(1H, m), 2.50-2.37 (1H, m), 1.73-1.60 (6H, m), 1.41-1.28 (6H,
m), 1.35 (3H, s), 0.94 (9H, t, J ) 6.0 Hz), 1-0 (3H, bq); 13C
NMR (CDCl3) δ 169.2, 169.1, 162.9, 162.8, 158.6, 158.5, 149.2,
144.3, 144.2, 135.7, 135.6, 135.5, 135.4, 135.2, 134.8, 131.6,
130.5, 130.1, 129.0, 128.2, 128.0, 127.0, 113.3, 111.1, 111.0,
87.0, 86.9, 86.1, 86.0, 85.5, 85.4, 85.0, 84.8, 74.1, 73.9, 73.8,
63.7, 63.6, 55.2, 51.7, 50.0, 49.9, 49.8, 49.7, 45.8, 40.3, 40.2,
25.2, 20.1, 13.6, 11.5, 11.4, 8.5; 31P NMR (CDCl3) δ 98.31-
93.59 (m); IR (KBr) 3448, 2961, 2874, 2838, 2362, 1749, 1703,
1661, 1608, 1509, 1460, 1445, 1392, 1279, 1253, 1178, 1114,
1066, 1034, 992, 829, 792, 763, 688, 648 cm-1. Anal. Calcd.
5′-O-Dim et h oxyt r it yl-N4-b en zoyl-2′-d eoxycyt id in -3′-
yl 3′-O,N3-Diben zoylth ym id in -5′-yl m eth yl Bor a n op h os-
p h a te (6, B1 ) N4-Ben zoylcytosin -1-yl, B2 ) N3-Ben zoyl-
th ym in -1-yl).23 This compound was obtained from 4 (B1 ) N4-
benzoylcytosin-1-yl) and 5 (B2 ) N3-benzoylthymin-1-yl) as a
colorless foam (quantitative) in a similar manner as 6 (B1
N6-benzoyladenin-9-yl, B2 ) N3-benzoylthymin-1-yl).
)
5′-O-Dim eth oxytr ityl-O6-d ip h en ylca r ba m oyl-N2-p h en -
yla cetyl-2′-d eoxygu a n osin -3′-yl 3′-O,N3-Diben zoylth ym i-
d in -5′-yl Meth yl Bor a n op h osp h a te (6, B1 ) O6-Dip h en yl-
ca r b a m oyl-N2-p h en yla cet ylgu a n in -9-yl, B2 ) N3-Ben -
zoylth ym in -1-yl).23 This compound was obtained from 4 (B1
) O6-diphenylcarbamoyl-N2-phenylacetylguanin-9-yl) and 5
(B2 ) N3-benzoylthymin-1-yl) as a colorless foam (83%) in a
similar manner as 6 (B1 ) N6-benzoyladenin-9-yl, B2 ) N3-
benzoylthymin-1-yl).
5′-O-Dim eth oxytr ityl-N3-ben zoylth ym id in -3′-yl 3′-O,N3-
Diben zoylth ym id in -5′-yl Meth yl Bor a n op h osp h a te (6, B1
) B2 ) N3-Ben zoylth ym in -1-yl).23 This compound was
obtained from 4 (B1 ) N3-benzoylthymin-1-yl) and 5 (B2 ) N3-
benzoylthymin-1-yl) as a colorless foam (quantitative) in a
similar manner as 6 (B1 ) N6-benzoyladenin-9-yl, B2 ) N3-
benzoylthymin-1-yl).
5′-O-Dim eth oxytr ityl-N3-ben zoylth ym id in -3′-yl 3′-O,N6-
Diben zoyl-2′-d eoxya d en osin -5′-yl Meth yl Bor a n op h os-
p h a te (6, B1 ) N3-Ben zoylth ym in -1-yl, B2 ) N6-Ben zoyl-
a d en in -9-yl). By repeated coevaporation with dry toluene
followed by dry pyridine, 5 (B2 ) N6-benzoyladenin-9-yl) (45.9
mg, 0.100 mmol) and 20 (B1 ) N3-benzoylthymin-1-yl) (111
mg, 0.120 mol) were dried and finally dissolved in dry CH3-
CN (1.00 mL). To the solution were successively added i-Pr2-
NEt (170 µL, 1.00 mmol) and PyNTP (150 mg, 0.300 mmol).
After being stirred at rt for 20 min, the mixture was diluted
for
C51H69BN3O10P‚0.5H2O: C, 65.52; H, 7.55; N, 4.49.
Found: C, 65.60; H, 7.42; N, 4.65.
Tr ibu tyla m m on iu m 5′-O-Dim eth oxytr ityl-O6-d ip h en -
ylcarbamoyl-N2-phenylacetyl-2′-deoxyguanosin-3′-yl Meth-
yl Bor a n op h osp h a t e (20, B1 ) O6-Dip h en ylca r ba m oyl-
N2-p h en yla cetylgu a n in -9-yl).23 This compound was obtained
from 19 (B1 ) O6-diphenylcarbamoyl-N2-phenylacetylguanin-
9-yl) as a light yellow foam (80% yield) in a similar manner
as 20 (B1 ) N3-benzoylthymin-1-yl).
J . Org. Chem, Vol. 69, No. 16, 2004 5267

Shimizu et al.
with CHCl3 (10 mL). The solution was washed with 1 M pH
7.0 phosphate buffer (3 × 10 mL), and the aqueous layer was
back-extracted with CHCl3 (3 × 10 mL). The organic layer and
washings were combined, washed with satd NaHCO3 aq (3 ×
50 mL), and the aqueous layer was back-extracted with CHCl3
(3 × 100 mL). The organic layer and washings were combined,
dried over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was chromatographed on silica
gel (30 g) using a gradient of MeOH (0-4%) in CH2Cl2 as an
eluent. The fractions containing 5′-O-dimethoxytrityl-N3-ben-
zoylthymidin-3′-yl 3′-O,N6-dibenzoyl-2′-deoxyadenosin-5′-yl
methyl boranophosphate (6, B1 ) N3-benzoylthymin-1-yl, B2
) N6-benzoyladenin-9-yl) were combined and concentrated to
dryness under reduced pressure to obtain 6 (B1 ) N3-ben-
zoylthymin-1-yl, B2 ) N6-benzoyladenin-9-yl) (109 mg, 92%)
as a colorless foam: 1H NMR (CDCl3) δ 8.83-8.79 (1H, bs),
8.32, 8.27 (1H, 2s), 8.10-7.15 (26H, m), 6.90-6.81 (4H, m),
6.67-6.49 (1H, m), 6.48-6.38 (1H, m), 5.77-5.59 (1H, m),
5.32-5.20 (1H, m), 4.49-4.26 (4H, m), 3.80-3.74 (6H, m), 3.68,
3.64 (3H, 2d, J POCH ) 12.0, 11.4 Hz), 3.51-3.41 (2H, m), 3.15-
2.97 (1H, m), 2.86-2.41 (3H, m), 1.47, 1.44 (3H, 2s), 1-0 (3H,
bq); 13C NMR (CDCl3) δ 168.8, 165.8, 164.5, 162.6, 159.1, 158.7,
152.9, 152.6, 151.5, 151.3, 149.6, 149.2, 143.9, 141.2, 141.0,
135.2, 134.9, 133.6, 133.2, 132.6, 131.4, 130.9, 130.3, 129.9,
129.6, 129.0, 128.6, 128.5, 127.9, 127.7, 127.5, 127.1, 125.3,
123.4, 113.3, 111.5, 87.2, 84.9, 84.6, 84.4, 84.2, 83.3, 78.2, 74.8,
65.9, 65.7, 63.1, 55.1, 53.7, 39.5, 39.3, 37.3, 37.0, 29.5, 14.0,
11.7; 31P NMR (CDCl3) δ 121.33-117.45 (m). Anal. Calcd for
75.6, 75.3, 75.1, 73.5, 65.4, 64.3, 63.7, 63.6, 52.8, 49.4, 41.6,
41.5, 40.9, 40.7, 14.5, 14.4, 14.3, 10.9; 31P NMR (D2O) δ 95.78-
90.60 (m); FAB+ m/z calcd for C20H31BN4O11P [M + H]+
545.1820, found 545.1814; UV (H2O) λmax 267 nm; ꢀ267 17800.
Tr ieth yla m m on iu m 2′-Deoxya d en osin -3′-yl Th ym id in -
5′-yl Bor a n op h osp h a te (27, B1 ) Ad en in -9-yl, B2 ) Th ym -
in -1-yl).23 This compound was obtained from 6 (B1 )N6-
benzoyladenin-9-yl, B2 ) N3-benzoylthymin-1-yl) as a colorless
foam (75% yield) in a similar manner as 27 (B1 ) B2 ) thymin-
1-yl).
Tr ieth yla m m on iu m 2′-Deoxycytid in -3′-yl th ym id in -5′-
yl Bor a n op h osp h a te (27, B1 ) Cytosin -1-yl, B2 ) Th ym in -
1-yl).23 This compound was obtained from 6c’t′ (B1 ) N4-
benzoylcytosin-1-yl, B2 ) N3-benzoylthymin-1-yl) as a colorless
foam (89% yield) in a similar manner as 27 (B1 ) B2 ) thymin-
1-yl).
Tr ieth yla m m on iu m 2′-Deoxygu a n osin -3′-yl Th ym id in -
5′-yl Bor a n op h osp h a te (27, B1 ) Gu a n in -9-yl, B2 ) Th ym -
in -1-yl). 6 (B1 ) O6-diphenylcarbamoyl-N2-phenylacetylguanin-
9-yl, B2 ) N3-benzoylthymin-1-yl) (28.1 mg, 20.0 µmol) were
added to a solution of 3% DCA in Et3SiH-CH2Cl2 (1:1 v/v, 2
mL), and the solution was stirred at rt for 1 min. The mixture
was diluted with CH2Cl2 and washed with satd NaHCO3 aq
(3 × 5 mL), and the aqueous layer was back-extracted with
CHCl3 (1 × 5 mL). The organic layer and washings were
combined, dried over Na2SO4, filtered, and concentrated to
dryness under reduced pressure. The residue was dissolved
in concentrated NH3/MeOH (2 mL) and allowed to cool to 0
°C. After being kept at 0 °C for 2 h, the mixture was
concentrated to dryness under reduced pressure. To a solution
of the residue in THF (1 mL) was added a mixture of Et3N
and PhSH (1:1 v/v; 1 mL). After being stirred at rt for 2 h, the
mixture was concentrated to dryness under reduced pressure.
Then, the residue was dissolved in concentrated NH3/MeOH
(20 mL) and heated at 55 °C for 12 h. The reaction mixture
was then allowed to cool to rt and concentrated to dryness
under reduced pressure. The residue was purified by reversed-
phase column chromatography [a linear gradient of acetonitrile
0-15% in 0.1 M triethylammonium acetate buffer (pH 7.0)]
to afford 27 (B1 ) guanin-9-yl, B2 ) thymin-1-yl) (12.4 mg,
92%) as a colorless foam: 1H NMR (D2O) δ 7.88 (1H, s), 7.61,
7.54 (1H, 2s), 6.33-6.12 (2H, m), 5.02-4.93 (1H, m), 4.57-
4.49 (1H, m), 4.30-3.47 (6H, m), 3.26 (6H, q, J ) 8.0 Hz), 2.82-
2.53 (2H, m), 2.36-2.24 (2H, m), 1.89, 1.76 (3H, 2s), 1.24 (9H,
t, J ) 7.5 Hz), 1-0 (3H, bq); 13C NMR (D2O) δ 184.2, 169.1,
169.0, 162.2, 157.0, 154.4, 153.5, 140.4, 140.2, 140.0, 139.8,
139.7, 119.5, 114.3, 114.1, 114.0, 89.2, 88.4, 87.9, 87.8, 87.6,
87.3, 87.0, 76.4, 76.3, 74.0, 73.2, 65.5, 64.3, 64.2, 63.9, 52.8,
49.4, 41.7, 41.5, 41.2, 40.9, 26.0, 14.5, 14.4, 11.0; 31P NMR
(D2O) 96.07-90.74 (m); FAB+ m/z calcd for C20H30BN7O10P [M
+ H]+ 570.1885, found 570.1891; UV (H2O) λmax 256.4 nm; ꢀ256.4
16500.
C
63H61BN7O14P‚CHCl3: C, 59.07; H, 4.80; N, 7.53. Found: C,
59.05; H, 4.92; N, 7.48.
5′-O-Dim et h oxyt r it yl-N3-b en zoylt h ym id in -3′-yl 3′-O-
ter t-Bu tyld im eth ylsilyl-N4-ben zoylcytid in -5′-yl Meth yl
Bor a n op h osp h a te (6, B1 ) N3-Ben zoylth ym in -1-yl, B2
)
N4-Ben zoylcytosin -1-yl).23 This compound was obtained from
20 (B1 ) N3-benzoylthymin-1-yl) and 5 (B2 ) N4-benzoylcy-
tosin-1-yl) as a colorless foam (99%) in a similar manner as 6
(B1 ) N3-benzoylthymin-1-yl, B2 ) N6-benzoyladenin-9-yl).
5′-O-Dim et h oxyt r it yl-N3-b en zoylt h ym id in -3′-yl 3′-O-
b en zoyl-N6-d ip h en ylca r b a m oyl-N2-p h en yla cet yl-2′-d e-
oxygu a n osin -5′-yl Meth yl Bor a n op h osp h a te (6, B1 ) N3-
Ben zoylt h ym in -1-yl, B2 ) N6-Dip h en ylca r b a m oyl-N2-
p h en yla cetylgu a n in -9-yl).23 This compound was obtained
from 20 (B1 ) N3-benzoylthymin-1-yl) and 5 (B2 ) N6-
diphenylcarbamoyl-N2-phenylacetylguanin-9-yl) as a colorless
foam (94%) in a similar manner as 6 (B1 ) N3-benzoylthymin-
1-yl, B2 ) N6-benzoyladenin-9-yl).
Tr ieth yla m m on iu m Th ym id in -5′-yl Th ym id in -3′-yl Bo-
r a n op h osp h a te (27, B1 ) B2 ) Th ym in -1-yl). 6 (B1 ) B2 )
N3-benzoylthymin-1-yl) (58.6 mg, 50 µmol) were added to a
solution of 3% DCA in Et3SiH-CH2Cl2 (1:1 v/v, 5 mL), and
the solution was stirred at rt for 1 min. The mixture was
diluted with CH2Cl2 and washed with satd NaHCO3 aq (3 ×
10 mL), and the aqueous layer was back-extracted with CHCl3
(1 × 10 mL). The organic layer and washings were combined,
dried over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. To a solution of the residue in THF (2 mL)
was added a mixture of Et3N and PhSH (1:1 v/v; 2 mL). After
being stirred at rt for 2 h, the mixture was concentrated to
dryness under reduced pressure. Then, the residue was
dissolved in concentrated NH3/MeOH (50 mL) and heated at
55 °C for 12 h. The reaction mixture was then allowed to cool
to rt and concentrated to dryness under reduced pressure. The
residue was purified by reversed-phase column chromatogra-
phy [a linear gradient of acetonitrile 0-15% in 0.1 M triethyl-
Ack n ow led gm en t. We thank Dr. Takashi Yamash-
ita (Tokyo University of Science) for obtaining mass
spectra. This work was partially supported by a grant
from a Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and
Technology, J apan and by J SPS Research Fellowships
for Young Scientists (M.S.).
ammonium acetate buffer (pH 7.0)] to afford 27 (B1 ) B2
)
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 31P
NMR spectra of 2-4, 6, 17, 19, 20, and 27. FAB-MS spectra
of 27. Experimental details and characterization data for O6-
diphenylcarbamoyl-N2-phenylacetyl-2′-deoxyguanosine and 1.
Characterization data for 3, 19, 4, 20, 6, and 27. This material
is available free of charge via the Internet at http://pubs.acs.org.
thymin-1-yl) (27.7 mg, 86%) as a colorless foam: 1H NMR
(D2O) δ 7.75-7.64 (2H, m), 6.36-6.20 (2H, m), 4.93-4.79 (1H,
m), 4.61-4.53 (1H, m), 4.20-4.12 (2H, m), 4.14-4.05 (1H, m),
3.89-3.74 (2H, m), 3.57 (1H, m), 3.20 (6H, q, J ) 8.0 Hz), 2.55-
2.29 (4H, m), 1.94-1.86 (6H, m), 1.28 (9H, t, J ) 6.0 Hz), 1-0
(3H, bq); 13C NMR (D2O) δ 169.2, 169.1, 169.0, 168.9, 154.3,
154.2, 140.2, 140.0, 114.2, 114.1, 88.5, 88.1, 88.0, 87.9, 87.5,
J O0493875
5268 J . Org. Chem., Vol. 69, No. 16, 2004