Synthesis of ribonucleic guanidine: Replacement of the negative phosphodiester linkages of RNA with positive guanidinium linkages

Article abstract of DOI:10.1016/S0040-4020(01)01185-1

Replacement of the negatively charged phosphodiester linkages of RNA with positively charged guanidinium linkages provides the polycationic ribonucleic guanidine (RNG). RNG is designed to bind strongly to target DNA/RNA through the specific interactions o

Full text of DOI:10.1016/S0040-4020(01)01185-1

TETRAHEDRON
Pergamon
Tetrahedron 58 .2002) 867±879
Synthesis of ribonucleic guanidine: replacement of the negative
phosphodiester linkages of RNA with
positive guanidinium linkages
²
Naoshi Kojima, Istvan E. Szabo and Thomas C. Bruicep
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA
Received 23 May 2001; accepted 26 November 2001
AbstractÐReplacement of the negatively charged phosphodiester linkages of RNA with positively charged guanidinium linkages provides
the polycationic ribonucleic guanidine .RNG). RNG is designed to bind strongly to target DNA/RNA through the speci®c interactions of
nucleobases and the attractive electrostatic interactions of backbones, thus RNG is a putative antisense/antigene agent. Preparation of uridine
and adenine building blocks for the RNG synthesis, as well as the stepwise synthesis of oligomeric RNG are reported. Pentameric homo
uridyl, adenyl, and a pentameric mixed base sequence have been synthesized. The methods were also applied to a convenient solid-phase
synthesis of RNG. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
backbones11 have been synthesized and used for biological
and biochemical studies.
Putative drugs consisting of oligonucleotide analogs
capable of inhibiting cellular processes at the transcriptional
or translational level via recognition and binding to comple-
mentary DNA or RNA are known as antigene and antisense
agents, respectively.1 Important goals in designing anti-
sense/antigene compounds include increasing the binding
af®nity while maintaining the sequence speci®city, and
having resistance to degradation by nucleases. Numerous
structural analogs of DNA/RNA designed to be effective
antisense/antigene agents have been reported.2 Replacement
of the negatively charged phosphodiester linkages with
neutral linkages eliminates the electrostatic repulsion that
exists in natural duplex DNA, thus increased binding af®nity
can be expected. Oligonucleotides linked by methyl-
phosphonate,3 methylenemethylimino .MMI),4 and amides5
are representative of this strategy. An alternative approach
involves replacing the sugar±phosphate backbone entirely,
such as in the case of PNA,6 PHONA,7 or PNAA.8 These
oligonucleotide analogs with modi®ed backbones are
resistant to nuclease degradation. On the other hand,
incorporation of positive charges into oligonucleotides has
been correctly anticipated to increase binding of analogs to
negative DNA/RNA strands. Aminoalkyl linkers attached
through the bases,9 the sugar moieties,10 and the phosphate
We have recently reported the synthesis and binding proper-
ties of deoxyribonucleic guanidine .DNG), wherein the
negatively charged phosphodiester linkages of DNA have
been entirely replaced by positively charged guanidinium
linkages .Fig. 1).12 DNG is anticipated to, and does, bind
strongly to target DNA/RNA because the repulsive electro-
static effects in duplex DNA are replaced by close attractive
electrostatic interactions. A pentameric thymidyl oligomer
of DNG .II), with four positively charged guanidinium
linkages, has been synthesized and shown to bind to
poly.dA) and poly.dA) with unprecedented high af®nity in
Keywords: antisense; oligonucleotides; RNA; catioinic.
Corresponding author. Tel.: 11-805-893-2044; fax: 11-805-893-2229;
p
e-mail: tcbruice@bioorganic.ucsb.edu
Present address: Hokkaido National Industrial Research Institute, 2-17-2-
1 Tsukisamuhigashi, Toyohira-ku, Sapporo 062-8517, Japan.
²
Figure 1.
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020.01)01185-1

868
N. Kojima et al. / Tetrahedron 58 )2002) 867±879
a 2:1 .T/A) complex.12c Since interactions with poly.dG),
poly.dC), and poly.dT) were not observed, II exhibits a
®delity of base-pair recognition. In addition, our recent
studies indicate that the highly positively charged octameric
thymidyl DNG oligomer was able to discriminate between
complementary and noncomplementary base pairs. A single
mismatch at the terminal of complementary DNA lowered
the melting points by approximately 48C, while a mismatch
at the center lowered the Tm by 158C.13 Although the double
helical structure of the duplex formed between DNG and
natural DNA/RNA is unknown, computational molecular
modeling suggests that the DNG/DNA duplex primarily
retains a B-DNA conformation while the DNG/RNA duplex
adopted an A-type structure.12b In addition to these results,
the guanidinium linkage was shown to have nuclease resist-
ance,14 and the positive charges of the backbone may give
rise to cell membrane permeability through electrostatic
attraction of the guanidinium moiety to the negatively
charged phosphate groups of the cell surface. Because of
these potential antisense/antigene aspects of DNG, further
studies on the synthesis and properties of guanidinium
linked oligonucleotides are warranted.
antigene agent because the guanidinium linkages are stable
to both nuclease degradation and intramolecular nucleo-
philic attack by the ribose C20 ±OH. This paper presents
the synthesis of uridine .8,10) and adenine .20,24) building
blocks for the synthesis of RNG. By using these monomers,
the stepwise solution-phase synthesis of uridyl, adenyl, and
mixed sequence RNG oligomers .Ia±Ic) are reported. Also,
these methods have been adapted to more convenient solid-
phase RNG synthesis.16
2. Results and discussion
We have recently established an effective method for the
synthesis of internucleoside guanidinium linkages, which
can be used with both solution phase and solid phase
synthesis.13,17 This method includes the abstraction of the
sulfur atom from an ¯uorenylmethyloxycarbonyl.Fmoc)-
protected thiourea by Hg21 to provide an activated
carbodiimide .Fig. 2) which can react with a free amino
group to give a protected guanido group.18
The Fmoc-protected uridine building block .8), required for
synthesis of the body of the polynucleotide, was obtained
from 30-deoxy-30-.hydroxyamino) uridine derivative 3
.Scheme 1). Compound 3 was prepared from the di-tert-
butyldimethylsilyl .TBS) protected uridine 1 according to
a literature procedure.19 This involves a stereoselective
reduction of the 30-oxime moiety. Initially, the 30-hydroxy
group of compound 1 was oxidized to give 30-keto uridine,
The interesting differences and similarities between the
nature of DNA and RNA prompted us to explore the
synthesis and properties of ribonucleic guanidine .RNG I).
Even though RNA possesses a strong af®nity for DNA, its
susceptibility to the various nucleases and the labile nature
of the phosphodiester backbone limit its applications.15 The
RNG structure should be better suited as an antisense/
Figure 2.
Ê
Scheme 1. Reagents and conditions: .a) .1) CrO3, pyridine, Ac2O, MS 4 A, CH2Cl2, 08C, .2) NH2OH´HCl, pyridine, rt, .3) 90% aqueous TFA, 08C; .b) NaBH4,
AcOH, 58C; .c) .1) H2, 10% Pd/C, 90% aqueous AcOH, rt, .2) CF3CO2Et, Et3N, MeOH, rt; .d) .1) TsCl, pyridine, rt, .2) NaN3, NH4Cl, DMF, 808C; .e) .1) H2S
.gas), 60% aqueous pyridine, rt, .2) MMTrCl, pyridine, rt; .f) NH3/MeOH, rt; .g) Fmoc-NCS, CH2Cl2, rt; .h) .1) MMTrCl, pyridine, rt, .2) NH3/MeOH, rt.

N. Kojima et al. / Tetrahedron 58 )2002) 867±879
869
which was subsequently converted into the 30-oxime
derivative by treatment with hydroxylamine hydrochloride.
The 50-O-TBS group was removed to give compound 2 and
stereoselective reduction of the 30-hydroxylimino group to a
30-a-hydroxylamine, by using NaBH.OAc)3, gave 3.20
Further reduction of compound 3 by catalytic hydrogena-
tion, in aqueous acetic acid, gave the 30-amino derivative,
which was then protected with a tri¯uoroacetyl group to
provide 4. The 50-hydroxy group was converted into an
azide via the 50-O-tosylate. Hydrogen sul®de reduction of
the 50-azido functional group of compound 5 was followed
by protection of the subsequent amine with a 4-mono-
methoxytrityl .MMTr) group, providing 6. MMTr protec-
tion of this amino moiety is ideal because it can be
selectively removed, on RNG synthesis, by brief acid
treatment. Removal of the tri¯uoroacetyl protection on the
30-amine by methanolic ammonia provides a free 30-amino
nucleoside 7, which was allowed to react with ¯uorenyl-
methyloxycarbonyl isothiocyanate .Fmoc-NCS)21 in
dichloromethane to give the desired building block .8). To
place a hydroxy group at the 50-end of the growing RNG
oligomer, a building block with a 50-protected-hydroxy
group was required. This compound 10 was synthesized
from the intermediate 4 by protecting the 50-hydroxy moiety
with a 4-monomethoxytrityl group followed by conversion
of the 30-amino group to a Fmoc-protected 30-thioureido
group.
butyloxycarbonyl .Boc) group providing 14. Next the
50-hydroxy group of 14 was converted into an azido group
through a tosyl intermediate to give 15. The Boc group on
the 30-amine of compound 15 was successfully removed by
either Lewis acid .AlCl3 in CH2Cl2, 08C, 73% yield) or by a
convenient acid treatment .7% TFA in CH2Cl2, room
temperature, 85% yield). At this stage, introduction of a
MMTr group to the di-amino compound, which was
obtained by reduction of compound 16, selectively
derivatizes the 50-amino function. However, the desired
mono-tritylated compound 19 was only obtained in low
yield .35%, data not shown). In this regard, the 30-free-
amino group of compound 16 was protected with an Fmoc
group to give 17. The reduction of the 50-azido group with
hydrogen sul®de, followed by protection with MMTr gave
18 in 81% yield. The Fmoc group was selectively removed
by piperidine±DMF treatment at room temperature to
provide 19, which was then reacted with Fmoc-NCS to
give the adenine internal building block .20). An adenine
building block .24) for the 50-terminus of the RNG oligomer
was also synthesized from 14 as shown in Scheme 2. The
50-end-hydroxy group of 14 was protected with a TBS,
instead of a MMTr group, to provide 21. Next the
N6-amino group of the adenine moiety was benzoylated
giving 22. The removal of the Boc group from the pentose
30-amine was successfully achieved by treating 22 with
AlCl3 in anhydrous dichloromethane at 08C. The resulting
compound 23 was ®nally reacted with Fmoc-NCS to give
the desired 50-terminal-adenine building block .24).
Adenine nucleosides were also modi®ed in a similar fashion
.Schemes 1 and 2). The benzoyl group was selected to block
the N6-amino group of adenine because of its stability to
acidic conditions and ease of removal by ammonia treat-
ment. The 30-hydroxy group of 20,50-O-silylated adenosine
.11)22 was oxidized to its 30-keto derivative and directly
converted into the 30-oxime nucleoside. The TBS group
on the 50-hydroxy position was then selectively removed
by treating with 90% aqueous tri¯uoroacetic acid at 08C
providing 12. Stereoselective reduction of 12 with
NaBH.OAc)3 gave 13 in 76% yield. The resulting
30-hydroxylamino group was reduced to a 30-amino group
by catalytic hydrogenation and then protected with a tert-
2.1. Solution-phase synthesis of RNG
We have developed a solution-phase method for the
synthesis of RNG. Pentameric uridyl-RNG was synthesized
by this method and a general schematic solution-phase
synthesis is outlined in Scheme 3. The synthesis of uridyl
RNG was accomplished via a cyclic process starting with a
condensation reaction between the 30-terminus of the
uridine building block .25a)23 and internal monomer .8),
affording the protected guanido linked dimer 26a. Typi-
cally, a mixed solution of compound 25a and 8 was treated
Scheme 2. Reagents and conditions: .a) .1) H2, 10% Pd/C, 90% aqueous AcOH, rt, .2) .Boc)2O, Na2CO3, THF/H2O, rt; .b) .1) i) TMSCl, pyridine, rt, ii) BzCl,
rt, iii) aqueous NH4OH, rt, .2) TsCl, pyridine, rt, .3) NaN3, NH4Cl, DMF, 808C; .c) method A .for 16 and 23): AlCl3, CH2Cl2, 08C, method B .for 16): TFA,
CH2Cl2, rt; .d) FmocCl, Na2CO3, THF/H2O, rt; .e) .1) H2S .gas), 60% aqueous pyridine, rt, .2) MMTrCl, pyridine, rt; .f) piperidine, DMF, rt; .g) Fmoc-NCS,
CH2Cl2, rt; .h) TBSCl, imidazole, DMF, rt; .i) .1) BzCl, pyridine, rt, .2) aqueous NH4OH, rt.

870
N. Kojima et al. / Tetrahedron 58 )2002) 867±879
Scheme 3. Solution-phase synthesis. Reagents and conditions: .a) coupling: 8 or 20, HgCl2, DIEA, DMF, rt; .b) deblocking: 80% aqueous AcOH, rt;
.c) coupling of 50-terminal monomer: 10 or 24, HgCl2, DIEA, DMF, rt; .d) deprotection .1) 80% aqueous AcOH, rt, .2) 1.0 M TBAF in THF, rt;
.e) deprotection .1) NH3/MeOH, rt, .2) 1.0 M TBAF in THF, rt.
with HgCl2 in DMF in the presence of N,N-
diisopropylethylamine as base. The carbodiimide inter-
mediate, which was generated in situ by the mercury.II)-
mediated abstraction of the sulfur atom from the protected
thiourea .8), was allowed to react with free 50-amino group
of 25a to give a dimer with an Fmoc-protected guanido
group .26a). The resulting guanido moiety was kept
protected until the end of RNG synthesis. Chain extension
from the dimer .26a) followed a cyclic two-step process
involving removal of the MMTr protecting group from the
50-amino terminus with 80% aqueous acetic acid. The
50-free-amino oligomer .27a) was then reacted with another
internal monomer .8) to give a longer RNG oligomer .28a).
Excess amount .2±3 equiv.) of incoming monomer 8 was
used in each coupling reaction. The isolated reaction yields
for these chain extensions were between 77 and 90%. The
monomer with 50-protected-hydroxy group .10) was used
for the 50-terminus of the growing chain to give a fully
protected uridyl RNG oligomer .32a). At the end of each
coupling and deprotection cycle, the product was puri®ed by
silica gel column chromatography, and the structure was
con®rmed by mass spectrometry .listed in Section 4).
monomers. The coupling yields for each step were similar
to those for the coupling of uridine building blocks. Depro-
tection of the adenyl oligomer was accomplished as follows.
The fully protected oligomer .32b) was ®rst treated with
methanolic ammonia at room temperature to remove the
benzoyl protecting group from the adenine N6-amino
group as well as the Fmoc group from the guanido group.
The TBS groups were removed from hydroxyls by treating
with a 1.0 Msolution of tetrabutylammonium ¯uoride in
tetrahydrofuran. The resulting free oligomer was puri®ed
by RP-HPLC to give pentameric adenyl RNG .Ib).
The synthesis of an adenine and uracil mixed sequenced
RNG .Ic) began with the coupling of adenine 30-terminal
building block .25b) with the uridine internal building block
.8). This was followed by coupling with the adenine internal
building block .20), then the uridine internal building block
.8), and ®nally the 50-terminal adenine building block .24).
On completion of the synthesis of the protected pentameric
RNG oligomer .32c), the guanido protecting Fmoc groups
as well as the benzoyl groups on the N6-amino nucleobases
were removed by treating with methanolic ammonia. This
was followed by treatment with tetrabutylammonium
¯uoride to remove TBS groups from both the terminal 30
and 50-terminal hydroxy groups to provide the fully depro-
tected mixed sequence oligomer .Ic). All synthesized RNG
oligomers .Ia±Ic) were analyzed by mass spectrometry.
The observed molecular weights supported their structures
.listed in Section 4).
The ®rst step in deprotection of the fully protected oligomer
.32a) is removal of the MMTr group on the 50-termini
hydroxy by 80% aqueous acetic acid treatment. Then the
Fmoc groups on the guanido group and the silyl groups on
the hydroxy moieties were simultaneously removed by
treating with 1.0 Msolution of tetrabutylammonium
¯uoride in tetrahydrofuran at room temperature. The result-
ing crude oligomer was then analyzed and puri®ed by
RP-HPLC using the solvent system of 3% acetic acid in
CH3CN to give pentameric uridyl RNG oligomer .Ia).
2.2. Solid-phase synthesis of RNG
To synthesize RNG on solid support, it is required that the
30-terminal building block be attached to a support with a
linkage that is cleavable after completion of the synthesis.
The most useful method of cleavage from the solid support
is the one that simultaneously removes both Fmoc and
Pentameric adenyl RNG oligomer .Ib) was also synthesized
by the identical method as mentioned earlier, using a
30-terminal .25b),23 internal .20), and 50-terminal .24)

N. Kojima et al. / Tetrahedron 58 )2002) 867±879
871
presence of 2 mol equiv. of HgCl2 and 4 mol equiv. of
N,N-diisopropylethylamine at room temperature for 4 h.
Preliminary study indicated that this single coupling reac-
tion gave a yield of 77%, as determined from the released
MMTr cation. When the coupling reaction was repeated the
average yield rose to 90%. At this point, a black precipitate
of mercurous sul®de forms. The latter is removed by treat-
ing the CPG resin with a 20% thiophenol solution in DMF.
Next, the unreacted 50-amino group was capped by acyl-
ation with tri¯uoroacetic anhydride. The deblocking/
coupling/capping cycles were repeated two more times
before coupling the 50-terminal monomer 10. The MMTr
groups on the 50-end of the completed RNG oligomer .39)
were removed prior to the cleavage of oligomer from CPG
support, however, the MMTr groups can be left on through-
out the puri®cation steps if desired. Treatment of CPG-
bound RNG oligomer with an NH4OH/EtOH .3:1) mixture
resulted in cleavage of oligomer from the CPG support as
well as removal of Fmoc groups from guanido moieties.
TBS groups were then removed using TBAF to provide
the fully de-protected RNG oligomer, which was puri®ed
by RP-HPLC as mentioned earlier for the solution-phase
method. Final yield of HPLC puri®ed oligomer .Ic)
obtained from the LCAA-CPG bound monomer .36) was
15%. Characterization of uridyl RNG .Ic) synthesized by
the solid-phase method revealed that its spectrum was
identical to the oligomer synthesized by solution-phase
method, and mass spectrometry con®rmed the correct mass.
Scheme 4. Reagents and conditions: .a) TBSCl, AgNO3, pyridine, THF, rt;
.b) succinic anhydride, DMAP, pyridine, rt; .c) LCAA-CPG, 1-.3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, DMF, rt.
benzoyl protecting groups. The solid-phase condensation
cycle was identical to the solution-phase method described
earlier, except for the addition of a thiophenol treatment to
remove mercurous sul®de precipitate that resulted from the
reaction between HgCl2 and the sulfur atom of the thiourea.
Long chain alkylamine controlled pore glass .LCAA-CPG)
was chosen for the solid support. The attachment of
30-terminal nucleoside onto LCAA-CPG followed the
standard procedure for DNA/RNA synthesis .Scheme 4).9
Selective silylation of 50-monomethoxytritylamino deriva-
tive 33 was accomplished using TBSCl, AgNO3, and
pyridine in tetrahydrofuran to give 20-O-protected deriva-
tive 34 in 70% yield.22 After the compound 34 was
converted into 30-succinate 35, it was condensed with
LCAA-CPG to provide solid-support attached 30-terminal
monomer 36 .support loading was 23.5 mmol/g).
3. Conclusion
Pentameric uridyl RNG .Ia) was synthesized on a 2.0 mmol
scale .Scheme 5). 30-terminal-LCAA attached nucleoside
.36) was placed in a solid-phase synthesis vessel and treated
with 3% trichloroacetic acid in dichloromethane to remove
the 50-MMTr substituent. The coupling of the internal
monomer .8) to the uridine loaded solid support was
conducted by treating with 15 mol equiv. of 8 in the
We have developed a method for the synthesis of RNG, a
polycationic analog of RNA in which the phosphodiester
linkages have been replaced with positively charged
guanidinium moieties. The synthesis of modi®ed uridine
and adenine building blocks along with the liquid-phase
and solid-phase synthesis of oligoribonucleotides is
reported. Uridine and adenine building blocks for RNG
synthesis were prepared from 20 and 50-O-protected uridine
and adenosine, respectively. Homo-oligomeric RNG
sequences, consisting of pentameric adenyl and uridyl
tracks, as well as a mixed-base pentamer .50-AUAUA-30)
were synthesized using a solution-phase method. This
method was also applied to standard solid-phase techniques,
resulting in pentameric uridyl RNG. Future plans include
synthesis of the remaining guanosine and cytidine RNG
analogs and greater length mixed sequences to determine
their antisense potential.
Despite the exposure of Fmoc-protecting groups to
50-amines during the course of monomer synthesis and
oligomer coupling, they remained remarkably stable. This
stability was con®rmed by the thorough characterization of
monomer 18 and a mixed RNG uridyl±adenyl Fmoc-
protected dimer 40 .Fig. 3; synthesis not included) resulting
from a single solution-phase coupling. High-resolution mass
1
spectroscopy, H and 13C NMR data for 18 and 40 are
Scheme 5. Solid-phase synthesis. Reagents and conditions: .a) deblocking
3% TCA in CH2Cl2, rt; .b) coupling .1) 8, HgCl2, DIEA, DMF, rt, .2) 20%
thiophenol in DMF, rt; .c) capping CF3CO2Et, DIEA, DMF, rt; .d) coupling
of 50-terminal monomer .1) 10, HgCl2, DIEA, DMF, rt, .2) 20% thiophenol
in DMF, rt; .e) cleavage and deprotection .1) 3% TCA in CH2Cl2, rt, .2)
NH4OH/EtOH, rt, .3) 1.0 MTBAF in THF, rt.
included in Section 4. High-resolution mass spectroscopy
data is also included for the six protected and de-protected
RNG dimers, 26a±c and 27a±c, indicating that Fmoc
groups remain even after the 50-MMTr protecting group is
removed, exposing the free amines. Thus we believe Fmoc

872
N. Kojima et al. / Tetrahedron 58 )2002) 867±879
chloride .3.20 g, 46.0 mmol) and allowed to stir at room
temperature for 20 h. The solvent was removed in vacuo
and residue was dissolved in AcOEt .350 mL), washed
with H2O .150 mL£2) and brine .150 mL), then dried
.Na2SO4) and concentrated in vacuo. A solution of the
residue in aqueous TFA .90%, 30 mL) was stirred at 08C
for 20 min. The solvent was removed in vacuo, co-evapo-
rated with toluene .15 mL£3), and the residue was puri®ed
by silica gel column chromatography .4.5£12 cm, 70%
AcOEt in hexane) to give 2 .1.96 g, 58%) as a white
foam: LRMS .FAB) m/z 372 [.M1H)1, 60%]; HRMS
.FAB) calcd for C15H26N3O6Si .M1H)1 372.1585, found
372.1577; 1H NMR .200 MHz, CDCl3) 7.54 .d, 1H,
Jˆ8.1 Hz), 5.82 .d, 1H, Jˆ8. 1 Hz), 5.60 .d, 1H,
Jˆ6.5 Hz), 5.05 .d, 1H, Jˆ6.5 Hz), 5.03 .m, 1H), 4.16
.dd, 1H, Jˆ1.7, 12.5 Hz), 3.88 .dd, 1H, Jˆ2.0, 12.5 Hz),
0.85 .s, 9H), 0.08 .s, 3H), 0.03 .s, 3H).
Figure 3.
to be effective in protecting the guanido group and we
consider this to be a more elegant and ef®cient synthesis
compared to past methods. Previous RNG oligomers had
to be isolated after each coupling reaction, the result of
which was a thiourea-linkage. The guanidinium-linked
oligomer was then derived from the thiourea-linked
oligomer via oxidation with peracetic acid, followed by
amination with ammonia. In combination with the outlined
solid-phase procedure, the new Fmoc-protected synthesis
requires no isolation after each coupling and exposure of
the completed oligomer to ammonia simultaneously cleaves
the oligomer from the solid support, de-protects the guanido
groups, and will remove N6-benzoyl protecting groups from
any adenyl moieties present. Final exposure to TBAF yields
the completely de-protected, guanidinium-linked oligomer.
4.1.2. 20-O-1tert-Butyldimethylsilyl)-30-1hydroxyamino)-
30-deoxyuridine 13). 2 .1.90 g, 5.11 mmol) was added to
a 58C solution of AcOH .60 mL) containing NaBH4
.390 mg, 21.3 mmol). After the mixture was stirred at the
same temperature for 2 h, the solvent was removed in vacuo.
The residue was dissolved in AcOEt .180 mL), washed with
H2O .60 mL£2) and brine .60 mL), dried .Na2SO4), and
concentrated in vacuo. The residue was puri®ed by silica
gel column chromatography .3.5£10 cm, 15% EtOH in
CHCl3) to give 3 .1.83 g, 96%) as a white foam: LRMS
.FAB) m/z 374 [.M1H)1, 100%]; HRMS .FAB) calcd for
C15H28N3O6Si .M1H)1 374.1747, found 374.1759; 1H
NMR .200 MHz DMSO-d6) 11.35 .br s, 1H), 8.01 .d, 1H,
Jˆ8.2 Hz), 7.59 .s, 1H), 5.81 .d, 1H, Jˆ4.6 Hz), 5.66 .d,
1H, Jˆ8.2 Hz), 5.42 .br s, 1H), 5.25 .br s, 1H), 4.31 .dd, 1H,
Jˆ4.6, 5.7 Hz), 3.98 .m, 1H), 3.70 .dd, 1H, Jˆ2.2,
12.2 Hz), 3.54 .dd, 1H, Jˆ2.5, 12.2 Hz), 3.37 .dd, 1H,
Jˆ4.6, 5.7 Hz), 0.83 .s, 9H), 0.05 .s, 3H), 0.02 .s, 3H).
4. Experimental
4.1. General methods
Physical data were measured as follows: H and 13C NM R
1
spectra were recorded on 200, 400, and 500 MHz instru-
ments using CDCl3 or DMSO-d6 as the solvent with tetra-
methylsilane as an internal standard. Chemical shifts are
reported in parts per million .d), and signals are expressed
as s .singlet), d .doublet), t .triplet), q .quartet), m .multi-
plet), or br .broad). All exchangeable protons were detected
by their disappearance on addition of D2O. TLC was done
on Silica Gel 60 F254 pre-coated plates .EMScience,
Germany). Silica gel used for column chromatography
was Scienti®c Silica Gel .particle size 63±200) .Selecto
Scienti®c, Georgia, USA). RP-HPLC was performed on a
Hewlett Packard 1050 system equipped with a quaternary
solvent delivery system, a UV detector set at 260 nm, and an
Alltech macrosphere RP C8 column.
4.1.3. 20-O-1tert-Butyldimethylsilyl)-30-1tri¯uoroacetamido)-
30-deoxyuridine 14). A mixture of 3 .1.95 g, 5.22 mmol)
and Pd/C .10%, 390 mg) in aqueous AcOH .90%, 50 mL)
was stirred under atmospheric pressure of H2 at room
temperature for 3 h. The catalyst was ®ltered through Celite
and the ®ltrate was concentrated in vacuo. A mixture of the
residue, ethyl tri¯uoroacetate .3.11 mL, 26.1 mmol), and
triethylamine .3.64 mL, 26.1 mmol) in MeOH .50 mL)
was stirred at room temperature for 12 h and the solvent
was removed in vacuo. The residue was puri®ed by silica
gel column chromatography .4.5£10 cm, 6% EtOH in
CHCl3) to give 4 .2.22 g, 94%) as a white foam: LRMS
.FAB) m/z 454 [.M1H)1, 10%]; HRMS .FAB) calcd for
1
C17H27N3F3O6Si .M1H)1 454.1621, found 454.1601; H
4.1.1. 1-[2-O-1tert-Butyldimethylsilyl)-3-deoxy-3-1hydroxy-
imino)-b-d-erythro-pentofuranosyl]uracil 12) from 20,50-
bis-O-1tert-butyldimethylsilyl)uridine
.4.50 mL, 55.2 mmol) was added dropwise to a mixture of
Ê
CrO3 .2.76 g, 27.6 mmol) and 4 A molecular sieves
.powder, 7.0 g) in CH2Cl2 .60 mL) at 08C. After stirring
for 30 min, Ac2O .4.50 mL, 55.2 mmol) was added and
the mixture was allowed to stir at 08C for another 15 min.
A solution of 1 .4.34 g, 9.18 mmol) in CH2Cl2 .20 mL) was
added at room temperature and 2 h later the reaction mixture
was poured into AcOEt .500 mL). After ®ltering through
Celite the ®ltrate was concentrated in vacuo then dissolved
in pyridine .90 mL) containing hydroxylamine hydro-
NMR .400 MHz, CDCl3) 9.46 .br s, 1H), 8.01 .d, 1H,
Jˆ8.1 Hz), 6.93 .br d, 1H, Jˆ7.3 Hz), 5.77 .d, 1H,
Jˆ8.1 Hz), 5.75 .d, 1H, Jˆ1.5 Hz), 4.48 .ddd, 1H, Jˆ5.2,
7.3, 8.0 Hz), 4.44 .dd, 1H, Jˆ1.5, 5.2 Hz), 4.13 .ddd, 1H,
Jˆ8.0, 1.6, 2.2 Hz), 4.08 .dd, 1H, Jˆ1.6, 13.2 Hz), 3.81 .dd,
1H, Jˆ2.2, 13.2 Hz), 2.99 .br s, 1H), 0.94 .s, 9H), 0.26 .s,
3H), 0.17 .s, 3H).
11).
Pyridine
4.1.4. 50-Azido-20-O-1tert-butyldimethylsilyl)-30-1tri¯uoro-
acetamido)-30,50-dideoxyuridine 15). A mixture of 4
.1.55 g, 3.42 mmol) and p-TsCl .1.32 g, 6.90 mmol) in
pyridine .35 mL) was stirred at room temperature for

N. Kojima et al. / Tetrahedron 58 )2002) 867±879
873
24 h. H2O .5 mL) was added to the mixture and the solvent
was removed in vacuo. The residue was dissolved in AcOEt
.200 mL), which was washed with H2O .70 mL), aqueous
NaHCO3 .saturated, 70 mL) and brine .70 mL), then dried
.Na2SO4) and concentrated in vacuo. A solution of the
residue, NaN3 .670 mg, 10.3 mmol) and NH4Cl .730 mg,
13.7 mmol) in DMF .20 mL) was heated at 808C for 1 h.
The mixture was diluted with AcOEt .200 mL) and washed
with H2O .70 mL£3) and brine .70 mL), dried .Na2SO4),
and concentrated in vacuo. The residue was puri®ed by
silica gel column chromatography .3.5£10 cm, 40%
AcOEt in hexane) to give 5 .1.22 g, 75%) as a white
solid: LRMS .FAB) m/z 479 [.M1H)1, 40%]; HRMS
.FAB) calcd for C17H26N6F3O5Si .M1H)1 479.1686,
8.78 .br s, 1H), 7.49±7.19 .m, 12H), 7.36 .d, 1H,
Jˆ8.2 Hz), 6.84±6.81 .m, 2H), 5.66 .s, 1H), 5.56 .d, 1H,
Jˆ8.2 Hz), 4.03 .d, 1H, Jˆ4.5 Hz), 3.97 .ddd, 1H, Jˆ9.5,
3.0, 6.9 Hz), 3.78 .s, 3H), 2.98 .dd, 1H, Jˆ4.5, 9.5 Hz), 2.79
.dd, 1H, Jˆ3.0, 12.7 Hz), 2.33 .dd, 1H, Jˆ7.0, 12.7 Hz),
1.57 .br s, 2H), 0.94 .s, 9H), 0.23 .s, 3H), 0.13 .s, 3H).
4.1.7. 20-O-1tert-Butyldimethylsilyl)-30-[1N-19-¯uorenyl-
methyloxycarbonyl)thiocarbamoyl)amino]-50-14-mono-
methoxytritylamino)-30,50-dideoxyuridine 18). A solution
of 7 .905 mg, 1.44 mmol) in CH2Cl2 .20 mL) was added
dropwise to a solution of ¯uorenylmethyloxycarbonyl
isothiocyanate .445 mg, 1.58 mmol) in CH2Cl2 .30 mL) at
room temperature. After the mixture was stirred for 15 min,
the solvent was removed in vacuo. The residue was puri®ed
by silica gel column chromatography .4.5£14 cm, 40%
AcOEt in hexane) to give 8.1.22 g, 93%) as a white foam:
LRMS .FAB) m/z 910 [.M1H)1, 5%]; HRMS .FAB) calcd
for C51H56N5O7SSi .M1H)1 910.3670, found 910.3698
1
found 479.1690; H NMR .400 MHz, CDCl3) 9.41 .br s,
1H), 7.65 .d, 1H, Jˆ8.2 Hz), 7.00 .br d, 1H, Jˆ7.1 Hz),
5.84 .d, 1H, Jˆ8.2 Hz), 5.82 .d, 1H, Jˆ2.9 Hz), 4.45
.ddd, 1H, Jˆ7.1, 6.2, 7.3 Hz), 4.38 .dd, 1H, Jˆ2.9,
6.2 Hz), 4.15 .ddd, 1H, Jˆ7.3, 2.6, 3.5 Hz), 3.88 .dd, 1H,
Jˆ2.6, 13.5 Hz), 3.70 .dd, 1H, Jˆ3.5, 13.5 Hz), 0.92 .s,
9H), 0.18 .s, 3H), 0.12 .s, 3H).
1
.3.1 ppm); H NMR .400 MHz, DMSO-d6) 11.67 .s, 1H),
11.45 .d, 1H, Jˆ1.9 Hz), 10.12 .d, 1H, Jˆ8.0 Hz), 8.02 .d,
1H, Jˆ8.0 Hz), 7.89 .m, 2H), 7.80 .m, 2H), 7.45±7.14 .m,
16H), 6.83 .m, 2H), 5.69 .d, 1H, Jˆ2.6 Hz), 5.60 .dd, 1H,
Jˆ8.0, 1.9 Hz), 5.06 .ddd, 1H, Jˆ6.1, 8.6, 8.0 Hz), 4.67 .dd,
1H, Jˆ2.6, 6.1 Hz), 4.42±4.34 .m, 2H), 4.27 .t, 1H,
Jˆ7.3 Hz), 4.08 .ddd, 1H, Jˆ8.6, 4.2, 4.1 Hz), 3.70 .s,
3H), 3.05 .t, 1H, Jˆ8.1 Hz), 2.53 .ddd, 1H, Jˆ4.2, 12.8,
8.1 Hz), 2.36 .ddd, 1H, Jˆ4.1, 12.8, 8.1 Hz), 0.82 .s, 9H),
0.07 .s, 3H), 0.02 .s, 3H); 13C NMR .500 MHz, DMSO-d6)
179.9, 163.0, 157.4, 153.5, 150.2, 146.1, 143.2, 143.1,
141.4, 140.7, 140.7, 137.6, 129.6, 128.3, 127.9, 127.7,
127.1, 126.0, 125.4, 125.4, 120.2, 113.0, 101.8, 91.2, 81.0,
73.7, 69.8, 67.4, 56.7, 54.9, 46.0, 44.7, 25.5, 17.6, 25.1,
25.2.
4.1.5.
20-O-1tert-Butyldimethylsilyl)-30-1tri¯uoroacet-
amido)-50-14-monomethoxytritylamino)-30,50-dideoxy-
uridine 16). Hydrogen sul®de gas was bubbled into a
solution of 5 .1.73 g, 3.62 mmol) in aqueous pyridine
.60%, 80 mL) for 15 min. After the mixture was sealed
and stirred at room temperature for 5 h, argon gas was
bubbled through to remove the hydrogen sul®de gas. The
solvent was removed in vacuo, and the residue was
dissolved in AcOEt .200 mL) then washed with H2O
.60 mL£2) and brine .60 mL). The organic layer was
dried .Na2SO4) and concentrated in vacuo. A solution of
the residue and monomethoxytrityl chloride .2.25 g,
7.30 mmol) in pyridine .25 mL) was stirred at room
temperature for 3 h, after which time EtOH .3 mL) was
added to the mixture and the solvent was removed in
vacuo. The residue was dissolved in AcOEt .200 mL),
washed with H2O .60 mL), followed by aqueous NaHCO3
.60 mL) and brine .60 mL), dried .Na2SO4) and concen-
trated in vacuo. The residue was puri®ed by silica gel
column chromatography .4.5£17 cm, 40% AcOEt in
hexane) to give 6 .2.21 g, 84%) as a pale yellow foam:
LRMS .FAB) m/z 725 [.M1H)1, 5%]; 1H NM
.400 MHz, CDCl3) 9.10 .br d, 1H, Jˆ1.6 Hz), 8.45 .d,
1H, Jˆ8.2 Hz), 7.61±7.18 .m, 12H), 6.84±6.80 .m, 2H),
6.61 .br d, 1H, Jˆ8.6 Hz), 5.82 .s, 1H), 5.71 .dd, 1H,
Jˆ8.2, 1.6 Hz), 4.42 .ddd, 1H, Jˆ4.8, 9.9, 8.6 Hz), 4.31
.d, 1H, Jˆ4.8 Hz), 4.08 .ddd, 1H, Jˆ9.9, 2.7, 3.9 Hz),
3.78 .s, 3H), 2.87 .dd, 1H, Jˆ2.7, 14.1 Hz), 2.64 .br s,
1H), 2.22 .dd, 1H, Jˆ3.9, 14.1 Hz), 0.92 .s, 9H), 0.29 .s,
3H), 0.15 .s, 3H).
4.1.8. 30-Amino-20-O-1tert-butyldimethylsilyl)-50-O-1mono-
methoxytrityl)-30-deoxyuridine 19). A mixture of 4
.907 mg, 2.00 mmol) and monomethoxytrityl chloride
.1.54 g, 5.00 mmol) in pyridine .20 mL) was stirred at
room temperature for 20 h. After EtOH .3 mL) was added
to the mixture, the solvent was removed in vacuo. The
residue was dissolved in AcOEt .200 mL), which was
washed with H2O .70 mL), aqueous NaHCO3 .saturated,
70 mL), brine .70 mL), then dried .Na2SO4) and concen-
trated in vacuo. The residue was dissolved in methanolic
ammonia .saturated at 08C, 40 mL) and kept at room
temperature for 5 days. The solvent was removed in vacuo
and the residue was puri®ed by silica gel column chromato-
graphy .4.5£12 cm, 60% AcOEt in hexane) to give 9
.1.09 g, 87%) as a white foam: LRMS .FAB) m/z 630
R
1
[.M1H)1, 5%]; H NMR .400 MHz, CDCl3) 8.89 .br s,
1H), 8.20 .d, 1H, Jˆ8.2 Hz), 7.44±7.25 .m, 12H), 6.87±
6.85 .m, 2H), 5.74 .s, 1H), 5.26 .d, 1H, Jˆ8.2 Hz), 4.10
.d, 1H, Jˆ4.6 Hz), 3.89 .ddd, 1H, Jˆ9.5, 2.0, 2.6 Hz), 3.80
.s, 3H), 3.64 .dd, 1H, Jˆ2.0, 11.4 Hz), 3.53 .dd, 1H, Jˆ4.6,
9.5 Hz), 3.51 .dd, 1H, Jˆ2.6, 11.4 Hz), 1.16 .br s, 2H), 0.92
.s, 9H), 0.25 .s, 3H), 0.16 .s, 3H).
4.1.6. 30-Amino-20-O-1tert-butyldimethylsilyl)-50-14-mono-
methoxytritylamino)-30,50-dideoxyuridine 17). Compound
6 .2.05 g, 1.15 mmol) was dissolved in methanolic
ammonia .saturated at 08C, 100 mL) and kept at room
temperature for 6 days. The solvent was removed in vacuo
and the residue was puri®ed by silica gel column chromato-
graphy .4.5£20 cm, 3% EtOH in CHCl3) to give 7 .1.48 g,
83%) as a white foam: LRMS .FAB) m/z 629 [.M1H)1,
3%]; HRMS .FAB) calcd for C35H45N4O5Si .M1H)1
4.1.9. 20-O-1tert-Butyldimethylsilyl)-30-[1N-19-¯uorenyl-
methyloxycarbonyl)thiocarbamoyl)amino]-50-O-14-mono-
methoxytrityl)-30-deoxyuridine 110). A solution of 9
.1.05 g, 1.67 mmol) in CH2Cl2 .20 mL) was added dropwise
to a solution of ¯uorenylmethyloxycarbonyl isothiocyanate
1
629.3159, found 629.3189; H NMR .400 MHz, CDCl3)

874
N. Kojima et al. / Tetrahedron 58 )2002) 867±879
.520 mg, 1.84 mmol) in CH2Cl2 .30 mL) at room tempera-
ture. After the mixture was stirred at the same temperature
for 15 min, the solvent was removed in vacuo. The residue
was puri®ed by silica gel column chromatography
.4.5£13 cm, 40% AcOEt in hexane) to give 10 .1.47 g,
96%) as a pale yellow foam: LRMS .FAB) m/z 911
[.M1H)1, 15%]; HRMS .FAB) calcd for C51H551N4O8SSi
.M1H)1 911.3510, found 911.3535 .2.7 ppm); H NM R
.400 MHz, DMSO-d6) 11.73 .s, 1H), 11.48 .d, 1H,
Jˆ2.0 Hz), 10.09 .d, 1H, Jˆ7.3 Hz), 7.90 .m, 2H), 7.85 .d,
1H, Jˆ8.3 Hz), 7.81 .m, 2H), 7.46±7.20 .m, 16H), 6.89 .m,
2H), 5.76 .d, 1H, Jˆ2.9 Hz), 5.35 .dd, 1H, Jˆ8.3, 2.0 Hz),
5.14 .dt, 1H, Jˆ6.0, 7.3 Hz), 4.58 .dd, 1H, Jˆ2.9, 6.0 Hz),
4.39 .dd, 1H, Jˆ8.0, 10.2 Hz), 4.35 .dd, 1H, Jˆ7.4, 10.2 Hz),
4.27 .dd, 1H, Jˆ7.4, 8.0 Hz), 4.18 .ddd, 1H, Jˆ7.3, 3.6,
2.2 Hz), 3.72 .s, 3H), 3.44 .dd, 1H, Jˆ3.6, 11.0 Hz), 3.37
.dd, 1H, Jˆ2.2, 11.0 Hz), 0.80 .s, 9H), 0.06 .s, 3H), 0.00 .s,
3H); 13C NMR .500 MHz, DMSO-d6) 180.2, 162.9, 158.3,
153.6, 150.3, 144.1, 143.7, 143.2, 140.8, 140.7, 140.2,
134.4, 130.2, 128.0, 128.0, 127.9, 127.2, 127.1, 127.0,
125.5, 120.2, 113.3, 101.8, 90.0, 86.5, 81.0, 73.8, 67.4, 62.6,
55.6, 55.0, 46.00, 44.9, 25.4, 17.6, 25.2, 25.3.
.dd, 0.5H, Jˆ1.7, 11.0 Hz), 4.08 .dd, 0.5H, Jˆ2.4, 11.7 Hz),
3.94 .m, 0.5H), 3.91 .m, 0.5H), 0.94 .s, 4.5H), 0.86 .s,
4.5H), 0.85 .s, 4.5H), 0.72 .s, 4.5H), 0.13 .s, 1.5H), 0.13
.s, 1.5H), 0.10 .s, 1.5H), 0.03 .s, 1.5H), 0.02 .s, 1.5H), 0.02
.s, 1.5H), 20.10 .s, 1.5H), 20.30 .s, 1.5H). Physical data for
9-[2-O-)tert-butyldimethylsilyl)-3-deoxy-3-)hydroxyimino)-
b-d-erythro-pentofuranosyl]adenine )12): LRMS .FAB)
m/z 395 .M1H)1; HRMS .FAB) calcd for C16H27N6O4Si
1
.M1H)1 395.1863, found 395.1864; H NMR .200 MHz,
DMSO-d6) 8.53 .s, 1H), 8.25 .s, 1H), 7.93 .br s, 2H), 5.90
.d, 1H, Jˆ7.4 Hz), 5.27 .dd, 1H, Jˆ7.4, 1.5 Hz), 4.98 .ddd,
1H, Jˆ1.5, 2.8, 2.0 Hz), 3.82 .dd, 1H, Jˆ2.8, 12.2 Hz), 3.73
.dd, 1H, Jˆ2.0, 12.2 Hz), 0.67 .s, 9H), 20.14 .s, 3H),
20.33 .s, 1H).
4.1.11. 20-O-1tert-Butyldimethylsilyl)-30-1hydroxyamino)-
30-deoxyadenosine 113). To a solution of AcOH .110 mL)
containing NaBH4 .960 mg, 25.3 mmol) was added 12
.3.33 g, 8.44 mmol) at 58C. After the mixture was stirred
at the same temperature for 2 h the solvent was removed in
vacuo. The residue was dissolved in CHCl3 .300 mL),
which was washed with H2O .100 mL£2) and brine
.100 mL), dried .Na2SO4) and concentrated in vacuo. The
residue was puri®ed by silica gel column chromatography
.4.5£14 cm, 20% EtOH in CHCl3) to give 13 .2.76 g, 82%)
as a pale yellow foam: LRMS .FAB) m/z 397 [.M1H)1,
25%]; HRMS .FAB) calcd for C16H29N6O4Si .M1H)1
4.1.10. 9-[2,5-Bis-O-1tert-butyldimethylsilyl)-3-deoxy-3-
1hydroxyimino)-b-d-erythro-pentofuranosyl]adenine
and 9-[2-O-1tert-butyldimethylsilyl)-3-deoxy-3-1hydro-
xyimino)-b-d-erythro-pentofuranosyl]adenine 112) from
20,50-bis-O-1tert-butyldimethylsilyl)adenosine 111). Pyri-
dine .5.82 mL, 72.0 mmol) was added dropwise to a mixture
1
397.2020, found 397.2011; H NMR .200 MHz, DMSO-
d6) 8.39 .s, 1H), 8.12 .s, 1H), 7.65 .s, 1H), 7.40 .br s,
2H), 5.94 .d, 1H, Jˆ5.8 Hz), 5.67 .dd, 1H, Jˆ3.7,
7.6 Hz), 4.87 .dd, 1H, Jˆ5.8, 5.2 Hz), 4.16 .m, 1H), 3.75
.ddd, 1H, Jˆ3.7, 2.5, 12.2 Hz), 3.58±3.51 .m, 2H), 0.74 .s,
9H), 20.14 .s, 3H), 20.27 .s, 3H).
Ê
of CrO3 .3.60 g, 36.0 mmol) and molecular sieves 4 A
.powder, 8.0 g) in CH2Cl2 .80 mL) at 08C. After 30 min
Ac2O .5.82 mL, 72.0 mmol) was added and the mixture
was stirred at 08C for another 15 min. To this mixture was
added a solution of 11 .5.95 g, 12.0 mmol) in CH2Cl2
.40 mL) at room temperature. After the mixture was stirred
at room temperature for a further 2 h, the mixture was
poured into AcOEt .500 mL). The mixture was ®ltered
through Celite pad and the ®ltrate was concentrated in
vacuo. The residue was dissolved in pyridine .150 mL)
4.1.12. 30-1tert-Butyloxycarbonylamino)-20-O-1tert-butyl-
dimethylsilyl)-30-deoxyadenosine 114). A mixture of 13
.2.75 g, 6.94 mmol) and Pd/C .10%, 550 mg) in aqueous
AcOH .90%, 100 mL) was stirred under atmospheric
pressure of H2 at room temperature for 20 h. The catalyst
was ®ltered off with Celite and the ®ltrate was concentrated
in vacuo. To a solution of the residue and Na2CO3 .1.47 g,
13.9 mmol) in THF/H2O .4:1, 100 mL) was added .Boc)2O
.3.03 g, 13.9 mmol) at 08C. After the mixture was stirred at
room temperature for 2 h the solvent was removed in vacuo.
The residue was dissolved in AcOEt .300 mL), which was
washed with H2O .100 mL£2) and brine .100 mL), dried
.Na2SO4) and concentrated in vacuo. The residue was puri-
®ed by silica gel column chromatography .4.5£14 cm, 6%
EtOH in CHCl3) to give 14 .2.22 g, 67%) as a white foam:
LRMS .FAB) m/z 481 [.M1H)1, 35%]; HRMS .FAB)
calcd for C21H37N6O5Si .M1H)1 481.2595, found
containing
hydroxylamine
hydrochloride
.4.17 g,
60.0 mmol), the mixture was stirred at room temperature
for 14 h and the solvent was removed in vacuo. The residue
was dissolved in AcOEt .350 mL), which was washed with
H2O .150 mL£2) and brine .150 mL), then dried .Na2SO4)
and concentrated in vacuo. The residue was puri®ed by
silica gel column chromatography .4.5£14 cm, 60%
AcOEt in hexane) to give 9-[2,5-bis-O-.tert-butyldimethyl-
silyl)-3-deoxy-3-.hydroxyimino)-b-d-erythro-pentofurano-
syl]adenine .5.16 g, 84%) as a white foam. A solution of this
compound .5.15 g, 10.1 mmol) in aqueous TFA .90%,
40 mL) was stirred at 08C for 20 min. The solvent was
removed in vacuo, co-evaporated with toluene .15 mL£3),
and the residue was puri®ed by silica gel column chromato-
graphy .4.5£13 cm, 10% EtOH in CHCl3) to give 12
.3.34 g, 70% from 11) as a white foam. Physical data for
9-[2,5-bis-O-)tert-butyldimethylsilyl)-3-deoxy-3-)hydroxy-
imino)-b-d-erythro-pentofuranosyl]adenine: LRMS .FAB)
m/z 509 .M1H)1; HRMS .FAB) calcd for C22H41N6O4Si2
1
481.2597; H NMR .200 MHz, DMSO-d6) 8.41 .s, 1H),
8.13 .s, 1H), 7.34 .br s, 2H), 7.14 .br d, 1H, Jˆ8.2 Hz),
5.91 .d, 1H, Jˆ4.2 Hz), 5.37 .t, 1H, Jˆ5.5 Hz), 4.56 .dd,
1H, Jˆ4.2, 5.8 Hz), 4.15 .ddd, 1H, Jˆ8.2, 5.8, 1.8 Hz), 4.05
.ddd, 1H, Jˆ1.8, 2.2, 3.0 Hz), 3.70 .ddd, 1H, Jˆ5.5, 2.2,
12.4 Hz), 3.52 .ddd, 1H, Jˆ5.5, 3.0, 12.4 Hz), 1.37 .s, 9H),
0.74 .s, 9H), 20.07 .s, 3H), 20.14 .s, 3H).
1
.M1H)1 509.2728, found 509.2748; H NMR .200 MHz,
CDCl3) 10.64 .br s, 1H), 8.37 .s, 0.5H), 8.36 .s, 0.5H), 8.31
.s, 0.5H), 8.12 .s, 0.5H), 6.13 .d, 0.5H, Jˆ2.7 Hz), 6.11 .d,
0.5H, Jˆ7.0 Hz), 6.03 .br s, 2H), 5.49 .m, 0.5H), 5.18 .m,
0.5H), 5.13 .dd, 0.5H, Jˆ7.0, 1.7 Hz), 4.92 .m, 0.5H), 4.26
4.1.13. 50-Azido-N6-benzoyl-30-1tert-butyloxycarbonyl-
amino)-20-O-1tert-butyldimethylsilyl)-30,50-dideoxyadeno-
sine 115). To a solution of 14 .1.80 g, 3.75 mmol) in
pyridine .50 mL) was added TMSCl .1.43 mL,

N. Kojima et al. / Tetrahedron 58 )2002) 867±879
875
11.3 mmol) at room temperature. After the mixture was
stirred for 30 min, benzoyl chloride .2.18 mL, 18.8 mmol)
was added and stirring was continued at room temperature
for 2 h. The mixture was then cooled in an ice bath and H2O
.10 mL) was added. After 5 min, 28% aqueous ammonia
.20 mL) was added and the mixture was stirred at room
temperature for 30 min. Solvent was removed in vacuo
and the residue dissolved in AcOEt .300 mL), washed
with H2O .100 mL), aqueous NaHCO3 .100 mL) and brine
.100 mL), dried .Na2SO4) and concentrated in vacuo to give
crude N6-benzoyl-30-.tert-butyloxycarbonylamino)-20-O-
[.M1H)1, 15%]; HRMS .FAB) calcd for C23H32N9O3Si
1
.M1H)1 510.2397, found 510.2447; H NMR .400 MHz,
DMSO-d6) 11.23 .br s, 1H), 8.78 .s, 1H), 8.69 .s, 1H), 8.04
.dd, 2H, Jˆ1.3, 7.2 Hz), 7.65 .t, 1H, Jˆ7.2 Hz), 7.55 .t, 2H,
Jˆ7.2 Hz), 6.10 .d, 1H, Jˆ2.6 Hz), 4.68 .dd, 1H, Jˆ2.6,
5.2 Hz), 3.91 .dt, 1H, Jˆ7.8, 4.6 Hz), 3.68 .m, 2H), 3.64
.dd, 1H, Jˆ5.2, 7.8 Hz), 1.72 .br s, 2H), 0.86 .s, 9H), 0.05
.s, 3H), 0.00 .s, 3H).
4.1.15.
50-Azido-N6-benzoyl-20-O-1tert-butyldimethyl-
silyl)-30-19-¯uorenylmethyloxycarbonylamino)-30,50-
dideoxyadenosine 117). To a solution of 16 .1.24 g,
2.43 mmol) and Na2CO3 .515 mg, 4.86 mmol) in THF±
H2O .4:1, 50 mL) was added ¯uorenylmethyloxycarbonyl
chloride .670 mg, 2.60 mmol) at 08C. After the mixture was
stirred at room temperature for 10 min, it was diluted with
AcOEt .150 mL), which was washed with H2O .70 mL£2)
and brine .70 mL). The organic layer was dried .Na2SO4)
and concentrated in vacuo. The residue was puri®ed by
silica gel column chromatography .3.0£14 cm, 50%
AcOEt in hexane) to give 17 .1.65 g, 93%) as a white
.tert-butyldimethylsilyl)-30-deoxyadenosine as
a
pale
yellow foam [LRMS .FAB) m/z 585 [.M1H)1, 20%];
HRMS .FAB) calcd for C28H41N6O6Si .M1H)1 585.2857,
found 585.2854]. To a solution of this compound in pyridine
.40 mL) was added p-TsCl .2.15 g, 11.3 mmol) and the
mixture was stirred at room temperature for 20 h. After
H2O .3 mL) was added the solvent was removed in vacuo.
The residue was dissolved in AcOEt .300 mL), which was
washed with H2O .100 mL), aqueous NaHCO3 .saturated,
100 mL) and brine .100 mL), dried .Na2SO4) and concen-
trated in vacuo. A solution of the residue, NaN3 .1.22 g,
18.8 mmol), and NH4Cl .1.20 g, 22.5 mmol) in DMF
.35 mL) was heated at 808C for 1 h. The mixture was diluted
with AcOEt .300 mL), which was washed with H2O
.100 mL£3) and brine .100 mL), dried .Na2SO4) and
concentrated in vacuo. The residue was puri®ed by silica
gel column chromatography .4.5£12 cm, 50% AcOEt in
hexane) to give 15 .1.62 g, 71%) as a white foam: LRMS
.FAB) m/z 610 [.M1H)1, 15%]; HRMS .FAB) calcd for
C28H40N9O6Si .M1H)1 610.2922, found 610.2921; 1H
NMR .400 MHz, DMSO-d6) 11.24 .br s, 1H), 8.79 .s,
1H), 8.72 .s, 1H), 8.05 .dd, 2H, Jˆ1.3, 7.3 Hz), 7.65 .tt,
1H, Jˆ1.3, 7.3 Hz), 7.55 .dt, 2H, Jˆ1.3, 7.3 Hz), 7.15 .d,
1H, Jˆ8.4 Hz), 6.11 .d, 1H, Jˆ3.8 Hz), 4.81 .dd, 1H,
Jˆ3.8, 6.1 Hz), 4.32 .ddd, 1H, Jˆ6.1, 7.6, 8.4 Hz), 4.22
.ddd, 1H, Jˆ7.6, 2.8, 6.3 Hz), 3.67 .dd, 1H, Jˆ2.8,
13.7 Hz), 3.61 .dd, 1H, Jˆ6.3, 13.7 Hz), 1.40 .s, 9H),
0.79 .s, 9H), 20.01 .s, 3H), 20.12 .s, 3H).
1
foam: LRMS .FAB) m/z 732 [.M1H)1, 30%]; H NM R
.400 MHz, DMSO-d6) 11.24 .br s, 1H), 8.78 .s, 1H), 8.75
.s, 1H), 8.04 .d, 2H, Jˆ7.3 Hz), 7.90 .d, 2H, Jˆ7.4 Hz),
7.72 .d, 1H, Jˆ7.4 Hz), 7.69 .d, 1H, Jˆ7.4 Hz), 7.65 .t,
1H, Jˆ7.3 Hz), 7.55 .t, 2H, Jˆ7.3 Hz), 7.43 .t, 2H,
Jˆ7.4 Hz), 7.34 .t, 2H, Jˆ7.4 Hz), 6.12 .d, 1H,
Jˆ4.3 Hz), 4.86 .dd, 1H, Jˆ4.3, 6.1 Hz), 4.44 .dd, 1H,
Jˆ6.4, 10.5 Hz), 4.36 .dt, 1H, Jˆ6.1, 7.4 Hz), 4.31 .dd,
1H, Jˆ6.7, 10.5 Hz), 4.23 .m, 2H), 3.68 .dd, 1H, Jˆ2.9,
13.4 Hz), 3.61 .dd, 1H, Jˆ6.0, 13.4 Hz), 0.72 .s, 9H), 20.10
.s, 3H), 20.20 .s, 3H).
4.1.16. N6-Benzoyl-20-O-1tert-butyldimethylsilyl)-30-19-
¯uorenylmethyloxycarbonylamino)-50-14-monomethoxy-
tritylamino)-30,50-dideoxyadenosine 118). Hydrogen
sul®de gas was bubbled through a solution of 17 .1.60 g,
2.19 mmol) in aqueous pyridine .60%, 60 mL) for 15 min.
After the mixture was sealed and stirred at room tempera-
ture for 20 h, argon gas was bubbled through to remove the
hydrogen sul®de gas. The solvent was removed in vacuo
and the residue was dissolved in AcOEt .200 mL), which
was washed with H2O .70 mL£2) and brine .70 mL). The
organic layer was dried .Na2SO4) and concentrated in
vacuo. A solution of the residue and monomethoxytrityl
chloride .1.40 g, 4.50 mmol) in pyridine .30 mL) was stir-
red at room temperature for 16 h. After EtOH .5 mL) was
added to the mixture, the solvent was removed in vacuo.
The residue was dissolved in AcOEt .200 mL), which was
washed with H2O .70 mL), aqueous NaHCO3 .70 mL) and
brine .70 mL), dried .Na2SO4) and concentrated in vacuo.
The residue was puri®ed by silica gel column chromato-
graphy .4.0£15 cm, 50% AcOEt in hexane) to give 18
.1.73 g, 81%) as a pale yellow foam: LRMS .FAB) m/z
978 [.M1H)1, 5%]; HRMS .ESI) calcd for C58H59N7O6Si
4.1.14. 30-Amino-50-azido-N6-benzoyl-20-O-1tert-butyl-
dimethylsilyl)-30,50-dideoxyadenosine 116). Method A
.with AlCl3): to a solution of 15 .1.55 g, 2.54 mmol) in
CH2Cl2 .35 mL) was added AlCl3 .1.03 g, 7.70 mmol) at
08C. After the mixture was stirred at the same temperature
for 20 min the mixture was poured into aqueous NaHCO3
.saturated, 100 mL) in an ice bath. The mixture was further
partitioned by the addition of AcOEt .250 mL) then the
organic layer was washed with aqueous NaHCO3 .saturated,
70 mL) and brine .70 mL), dried .Na2SO4) and concentrated
in vacuo. The residue was puri®ed by silica gel column
chromatography .4.5£12 cm, 5% EtOH in CHCl3) to give
16 .944 mg, 73%) as a white foam. Method B .with TFA): to
a solution of 15 .1.76 g, 2.89 mmol) in CH2Cl2 .44 mL) was
added TFA .3.3 mL, 43 mmol) at 08C. After the mixture
was stirred at room temperature for 40 h, aqueous
NaHCO3 .saturated, 70 mL) was added. The mixture was
diluted with CHCl3 .250 mL) and the organic layer was
washed with aqueous NaHCO3 .saturated, 70 mL) and
brine .70 mL), dried .Na2SO4) and concentrated in vacuo.
The residue was puri®ed by silica gel column chromato-
graphy .4.5£12 cm, 5% EtOH in CHCl3) to give 16
.1.25 g, 85%) as a white foam: LRMS .FAB) m/z 510
1
.M1H)1 978.4374, found 978.4333 .4.2 ppm); H NM R
.400 MHz, DMSO-d6) 11.21 .br s, 1H), 8.68 .s, 1H), 8.03
.d, 2H, Jˆ7.3 Hz), 8.00 .s, 1H), 7.90 .d, 2H, Jˆ7.4 Hz),
7.74 .d, 1H, Jˆ7.4 Hz), 7.71 .d, 1H, Jˆ7.4 Hz), 7.65 .t,
1H, Jˆ7.3 Hz), 7.55 .t, 2H, Jˆ7.3 Hz), 7.46±7.16 .m,
2H), 6.82 .m, 2H), 5.97 .d, 1H, Jˆ5.7 Hz), 5.16 .dd, 1H,
Jˆ5.7, 6.4 Hz), 4.76 .m, 1H), 4.45 .dd, 1H, Jˆ6.4,
10.5 Hz), 4.38 .dd, 1H, Jˆ6.7, 10.5 Hz), 4.25 .dd, 1H,

876
N. Kojima et al. / Tetrahedron 58 )2002) 867±879
Jˆ6.4, 6.7 Hz), 4.18 .m, 1H), 3.70 .s, 3H), 2.49 .m, 1H),
2.44 .m, 1H), 2.21 .m, 1H), 0.68 .s, 9H), 20.10 .s, 3H),
20.23 .s, 3H); 13C NMR .500 MHz, DMSO-d6) 165.5,
157.4, 156.1, 151.7, 150.7, 146.3, 146.8, 143.9, 143.8,
140.8, 137.6, 133.3, 132.5, 129.6, 128.8, 128.6, 128.5,
128.3, 127.8, 127.6, 127.1, 126.2, 126.1, 125.1, 125.1,
120.2, 113.1, 89.1, 82.3, 73.0, 69.6, 65.6, 63.5, 59.8, 55.1,
55.0, 53.4, 46.7, 45.2, 40.0, 39.8, 39.7, 39.5, 39.4, 39.2,
39.0, 38.9, 38.7, 30.2, 25.5, 25.4, 25.3, 20.7, 18.6, 17.6,
14.1, 13.5, 25.4, 25.7.
14 .1.63 g, 3.40 mmol), tert-butyldimethylsilyl chloride
.770 mg, 5.10 mmol), and imidazole .700 mg, 10.2 mmol)
in DMF .20 mL) was stirred at room temperature for 1.5 h.
After EtOH .3 mL) was added to the mixture, the mixture
was diluted with AcOEt .200 mL), which was washed with
H2O .70 mL£4) and brine .70 mL). The organic layer was
dried .Na2SO4), and concentrated in vacuo. The residue was
puri®ed by silica gel column chromatography .4.0£10 cm,
60% AcOEt in hexane) to give 21 .1.73 g, 86%) as a white
solid: LRMS .FAB) m/z 595 [.M1H)1, 10%]; HRMS
.FAB) calcd for C27H51N6O5Si2 .M1H)1 595.3449, found
4.1.17. 30-Amino-N6-benzoyl-20-O-1tert-butyldimethylsilyl)-
50-14-monomethoxytritylamino)-30,50-dideoxyadenosine
119). To a solution of 18 .1.13 g, 1.15 mmol) in DMF
.17.5 mL) was added piperidine .0.35 mL, 3.50 mmol) at
room temperature. After the mixture was stirred at room
temperature for 45 min it was taken up in AcOEt
.200 mL), and the solution washed with H2O .70 mL£4)
and brine .70 mL). The organic layer was dried .Na2SO4)
and concentrated in vacuo. The residue was precipitated
from AcOEt/hexane and the precipitate was collected to
give 19 .805 mg, 92%) as a white powder: LRMS .FAB)
595.3432; H NMR .400 MHz, DMSO-d6) 8.32 .s, 1H),
1
8.14 .s, 1H), 7.34 .br s, 2H), 7.14 .d, 1H, Jˆ8.0 Hz), 5.96
.d, 1H, Jˆ3.0 Hz), 4.43 .dd, 1H, Jˆ3.0, 5.3 Hz), 4.19 .ddd,
1H, Jˆ8.0, 5.3, 7.7 Hz), 4.11 .ddd, 1H, Jˆ7.7, 2.2, 2.9 Hz),
3.96 .dd, 1H, Jˆ2.2, 11.8 Hz), 3.76 .dd, 1H, Jˆ2.9,
11.8 Hz), 1.37 .s, 9H), 0.89 .s, 9H), 0.80 .s, 9H), 0.08 .s,
3H), 0.07 .s, 3H), 20.01 .s, 3H), 20.03 .s, 3H).
4.1.20. N6-Benzoyl-30-1tert-butyloxycarbonylamino)-20,50-
bis-O-1tert-butyldimethylsilyl)-30-deoxyadenosine 122). A
mixture of 21 .1.63 g, 2.74 mmol) and benzoyl chloride
.0.96 mL, 8.30 mmol) in pyridine .30 mL) was stirred at
room temperature for 2 h. The mixture was then cooled in
an ice bath and H2O .2 mL) was added. After 5 min, 28%
aqueous ammonia .10 mL) was added and the mixture was
stirred at room temperature for another 30 min. The solvent
was removed in vacuo. The residue was dissolved in AcOEt
.200 mL), which was washed with H2O .70 mL), aqueous
NaHCO3 .70 mL) and brine .70 mL), dried .Na2SO4) and
concentrated in vacuo. The residue was puri®ed by silica gel
column chromatography .4.0£10 cm, 30% AcOEt in
hexane) to give 22 .1.82 g, 95%) as a white solid: LRMS
.FAB) m/z 699 [.M1H)1, 10%]; HRMS .FAB) calcd for
C34H55N6O6Si2 .M1H)1 699.3722, found 699.3711; 1H
NMR .400 MHz, DMSO-d6) 11.23 .s, 1H), 8.76 .s, 1H),
8.62 .s, 1H), 8.04 .d, 2H, Jˆ7.3 Hz), 7.65 .t, 1H, Jˆ
7.3 Hz), 7.55 .t, 2H, Jˆ7.3 Hz), 7.17 .d, 1H, Jˆ8.3 Hz),
6.12 .d, 1H, Jˆ3.0 Hz), 4.56 .dd, 1H, Jˆ3.0, 5.2 Hz),
4.24 .ddd, 1H, Jˆ8.3, 5.2, 7.8 Hz), 4.15 .ddd, 1H, Jˆ7.8,
2.1, 2.9 Hz), 3.97 .dd, 1H, Jˆ2.1, 11.8 Hz), 3.79 .dd, 1H,
Jˆ2.9, 11.8 Hz), 1.38 .s, 9H), 0.88 .s, 9H), 0.81 .s, 9H),
0.07 .s, 6H), 20.01 .s, 3H), 20.03 .s, 3H).
1
m/z 756 [.M1H)1, 5%]; H NMR .400 MHz, DMSO-d6)
11.20 .br s, 1H), 8.62 .s, 1H), 8.03 .d, 2H, Jˆ7.3 Hz), 7.95
.s, 1H), 7.65 .t, 1H, Jˆ7.3 Hz), 7.55 .t, 1H, Jˆ7.3 Hz),
7.46±7.17 .m, 12H), 6.84 .m, 2H), 6.02 .d, 1H,
Jˆ5.1 Hz), 5.05 .t, 1H, Jˆ5.1 Hz), 3.94 .ddd, 1H, Jˆ5.1,
4.4, 3.1 Hz), 3.84 .t, 1H, Jˆ5.1 Hz), 3.71 .s, 3H), 3.46 .dd,
1H, Jˆ10.3, 4.9 Hz), 2.46 .ddd, 1H, Jˆ4.4, 12.6, 10.3 Hz),
2.28 .ddd, 1H, Jˆ3.1, 12.6, 4.9 Hz), 1.80 .br s, 2H), 0.82 .s,
9H), 20.02 .s, 3H), 20.12 .s, 3H).
4.1.18. N6-Benzoyl-20-O-1tert-butyldimethylsilyl)-30-[1N-
19-¯uorenylmethyloxycarbonyl)thiocarbamoyl)amino]-
50-14-monomethoxytritylamino)-30,50-dideoxyadenosine
120). A solution of 19 .1.05 g, 1.39 mmol) in CH2Cl2
.20 mL) was added dropwise to a solution of ¯uorenyl-
methyloxycarbonyl isothiocyanate .430 mg, 1.53 mmol) in
CH2Cl2 .50 mL) at room temperature. After the mixture was
stirred for 10 min the solvent was removed in vacuo. The
residue was puri®ed by silica gel column chromatography
.3.0£15 cm, 60% AcOEt in hexane) to give 20 .1.44 g,
quantitative) as a white foam: LRMS .FAB) m/z 1037
[.M1H)1, 15%]; HRMS .FAB) calcd for C59H61N8O6SSi
.M1H)1 1037.4204, found 1037.4226 .2.1 ppm); 1H NM R
.400 MHz, DMSO-d6) 11.79 .s, 1H), 11.23 .s, 1H), 10.54 .d,
1H, Jˆ5.4 Hz), 8.75 .s, 1H), 8.03 .d, 2H, Jˆ7.4 Hz), 7.92
.d, 2H, Jˆ7.7 Hz), 7.84 .d, 1H, Jˆ7.7 Hz), 7.81 .d, 1H,
Jˆ7.7 Hz), 7.65 .t, 1H, Jˆ7.4 Hz), 7.60 .s, 1H), 7.55 .t,
2H, Jˆ7.4 Hz), 7.50±7.20 .m, 16H), 6.84 .m, 2H), 5.99
.d, 1H, Jˆ6.1 Hz), 5.59 .dd, 1H, Jˆ6.1, 7.0 Hz), 5.48
.ddd, 1H, Jˆ7.0, 3.4, 5.4 Hz), 4.46±4.40 .m, 3H), 4.31 .t,
1H, Jˆ7.1 Hz), 3.85 .br d, 1H, Jˆ8.3 Hz), 3.71 .s, 3H), 2.87
.dd, 1H, Jˆ9.9, 8.3 Hz), 2.21 .d, 1H, Jˆ9.9 Hz), 0.73 .s,
9H), 20.01 .s, 3H), 20.18 .s, 3H); 13C NMR .500 MHz,
DMSO-d6) 180.3, 165.6, 153.7, 151.8, 150.6, 143.2, 143.1,
141.7, 140.8, 140.7, 133.3, 132.5, 128.6, 128.4, 127.9,
127.1, 125.6, 125.5, 125.4, 120.2, 88.8, 83.5, 74.4, 67.4,
62.0, 55.9, 46.0, 40.0, 39.9, 39.7, 39.5, 39.4, 39.2, 39.0,
25.8, 25.3, 18.1, 17.5, 25.4, 25.5, 25.5.
4.1.21.
30-Amino-N6-benzoyl-20,50-bis-O-1tert-butyl-
dimethylsilyl)-30-deoxyadenosine 123). To a solution of
22 .1.36 g, 1.95 mmol) in CH2Cl2 .30 mL) was added
AlCl3 .790 mg, 5.90 mmol) at 08C. After the mixture was
stirred for 45 min, the mixture was poured into aqueous
NaHCO3 .saturated, 100 mL) in an ice bath. The mixture
was diluted with CHCl3 .200 mL) and the organic layer was
washed with aqueous NaHCO3 .saturated, 70 mL) and brine
.70 mL), dried .Na2SO4) and concentrated in vacuo. The
residue was puri®ed by silica gel column chromatography
.3£12 cm, 75% AcOEt in hexane) to give 23 .960 mg, 82%)
as a white foam: LRMS .FAB) m/z 599 [.M1H)1, 80%];
HRMS .FAB) calcd for C29H47N6O4Si2 .M1H)1 599.3197,
1
found 599.3210; H NMR .400 MHz, DMSO-d6) 11.22 .br
s, 1H), 8.75 .s, 1H), 8.62 .s, 1H), 8.04 .dd, 2H, Jˆ1.3,
7.3 Hz), 7.65 .t, 1H, Jˆ7.3 Hz), 7.55 .t, 2H, Jˆ7.3 Hz),
6.09 .d, 1H, Jˆ2.2 Hz), 4.49 .dd, 1H, Jˆ2.2, 4.8 Hz),
3.98 .dd, 1H, Jˆ2.5, 11.5 Hz), 3.83 .dd, 1H, Jˆ3.5,
11.5 Hz), 3.81 .ddd, 1H, Jˆ7.4, 2.5, 3.5 Hz), 3.56 .dd,
4.1.19. 30-1tert-Butyloxycarbonylamino)-20,50-bis-O-1tert-
butyldimethylsilyl)-30-deoxyadenosine 121). A mixture of

N. Kojima et al. / Tetrahedron 58 )2002) 867±879
877
1H, Jˆ4.8, 7.4 Hz), 1.63 .br s, 2H), 0.88 .s, 9H), 0.87 .s,
9H), 0.06 .s, 6H), 0.05 .s, 3H), 0.04 .s, 3H).
2±3 equiv. of the incoming monomer .8,10,20, or 24) was
used for each coupling reactions.
4.1.22. N6-Benzoyl-20,50-bis-O-1tert-butyldimethylsilyl)-
30-[1N-19-¯uorenylmethyloxycarbonyl)thiocarbamoyl)-
amino]-30-deoxyadenosine 124). A solution of 23 .1.05 g,
1.75 mmol) in CH2Cl2 .20 mL) was added dropwise to a
solution of ¯uorenylmethyloxycarbonyl isothiocyanate
.540 mg, 1.93 mmol) in CH2Cl2 .50 mL) at room tempera-
ture. After the mixture was stirred for 10 min, the solvent
was removed in vacuo. The residue was puri®ed by silica
gel column chromatography .3.0£14 cm, 40% AcOEt in
hexane) to give 24 .1.47 g, 95%) as a pale orange foam:
LRMS .FAB) m/z 880 [.M1H)1, 10%]; HRMS .FAB)
calcd for C45H58N7O6SSi2 .M1H)1 880.3708, found
880.3744 .4.1 ppm); 1H NMR .400 MHz, DMSO-d6)
11.73 .s, 1H), 11.27 .s, 1H), 10.30 .d, 1H, Jˆ7.0 Hz),
8.77 .s, 1H), 8.64 .s, 1H), 8.05 .dd, 2H, Jˆ1.3, 7.4 Hz),
7.91 .d, 2H, Jˆ7.5 Hz), 7.83 .d, 1H, Jˆ7.5 Hz), 7.80 .d,
1H, Jˆ7.5 Hz), 7.65 .t, 1H, Jˆ7.4 Hz), 7.56 .t, 2H, Jˆ
7.4 Hz), 7.44 .t, 2H, Jˆ7.5 Hz), 7.35 .t, 2H, Jˆ7.5 Hz),
6.16 .d, 1H, Jˆ3.9 Hz), 5.13 .dt, 1H, Jˆ6.0, 7.0 Hz), 4.95
.dd, 1H, Jˆ3.9, 6.0 Hz), 4.41 .m, 2H), 4.33 .ddd, 1H,
Jˆ7.0, 2.2, 3.5 Hz), 4.29 .t, 1H, Jˆ7.3 Hz), 4.04 .dd, 1H,
Jˆ2.2, 11.5 Hz), 3.94 .dd, 1H, Jˆ3.5, 11.5 Hz), 0.88 .s,
9H), 0.75 .s, 9H), 0.08 .s, 3H), 0.07 .s, 3H), 20.07 .s,
3H), 20.09 .s, 3H); 13C NMR .500 MHz, DMSO-d6)
180.4, 165.5, 157.5, 153.7, 151. 4, 151.0, 150.9, 146.4,
143.2, 140.8, 140.7, 137.6, 133.3, 132.5, 129.8, 128.5,
127.9, 127.8, 127.2, 126.3, 126.1, 125.5, 120.2, 113.1,
89.3, 84.0, 71.8, 69.8, 67.4, 63.5, 59.8, 57.1, 55.0, 46.1,
45.3, 40.0, 39.8, 39.7, 39.5, 39.4, 39.2, 39.0, 30.2, 25.3,
20.7, 17.6, 14.0, 13.5, 25.6.
4.2.4. Deprotection. Fully protected uridyl oligomer 32a
.3 mg, approximately 1 mmol) was dissolved in aqueous
acetic acid .80%, 5.0 mL) and kept at room temperature
for 24 h. The solvent was removed in vacuo and the residue
was taken up in EtOH .3 mL£3) and then evaporated. The
residue was dissolved in 1.0 MTBAF in THF .0.5 mL,
0.5 mmol) at room temperature and the mixture was stirred
at the same temperature for 16 h. Acetic acid .0.3 mL) was
added and the mixture was diluted with H2O .10 mL), which
was subsequently washed with Et2O .5 mL£2). The aqueous
phase was concentrated in vacuo. The residue was puri®ed
by RP-HPLC .Alltech Macrosphere RP C8 column, 0±10%
acetonitrile in 3% aqueous acetic acid) to give pentauridyl
RNG .Ia). For the adenyl .Ib) and mixed-based .Ic)
oligomer, each fully protected oligomer 32b,32c .approxi-
mately 1 mmol) was dissolved in methanolic ammonia
.saturated at 08C, 5 mL) and kept at room temperature for
60 h. After the solvent was removed in vacuo, the residue
was dissolved in 1.0 MTBAF in THF .0.5 mL, 0.5 mmol) at
room temperature and the mixture was stirred at the same
temperature for 16 h. Acetic acid .0.3 mL) was added and
the mixture was diluted with H2O .10 mL), which was
washed with Et2O .5 mL£2). The aqueous phase was
concentrated in vacuo. Each residue was puri®ed by
RP-HPLC .Alltech Macrosphere RP C8 column, 0±40%
acetonitrile in 3% aqueous acetic acid) to give penta-adenyl
RNG .Ib) and mixed-based RNG .Ic).
4.2.5. Characterization. At the end of each coupling and
deblocking step, the compounds were con®rmed by electro-
spray ionization mass spectrometry .ESI-MS), and accurate
mass .HRMS) is provided for 26a±c and 27a±c. The puri-
®ed RNG oligomers .Ia±Ic) were also analyzed by ESI-MS.
4.2. Solution-phase synthesisÐgeneral procedure
4.2.1. Coupling reaction. To a mixture of the uridine
30-terminal building block 25a .104 mg, 0.22 mmol) and
the uridine internal building block 8 .182 mg, 0.20 mmol)
in DMF .6.0 mL) was added N,N-diisopropylethylamine
.87 mL, 0.50 mmol) and HgCl2 .68 mg, 0.25 mmol), and
the mixture was stirred at room temperature for 3 h. The
mixture was ®ltered through Celite pad. The ®ltrate was
diluted with AcOEt .100 mL), which was washed with
H2O .30 mL£4), dried .Na2SO4) and concentrated in
vacuo. The residue was puri®ed by silica gel column
chromatography .2.7£12 cm, 40% AcOEt in hexane) to
give 50-N-MMTr-dimer 26a .220 mg, 82%) as a yellow
foam.
4.2.6. Synthesis of Ia. 26a C72H94N8O12Si3: calcd for
.M1H)1 1347.6371, found 1347.6360 .0.8 ppm); 27a
C52H78N8O11Si3: calcd for .M1H)1 1075.5171, found
1075.5128 .3.9 ppm); 28a C103H131N13O18Si4: calcd for
.M1H)1 1950.89, found 1950.85; 29a C83H115N13O17Si4:
calcd for .M1H)1 1678.77, found 1678.74, calcd for
.M12H)21 839.89, found 839.88; 30a C134H168N18O24Si5:
calcd for .M1H)1 2554.14, found 2554.12, calcd for
.M12H)21
1277.58,
found
1277.56;
31a
C114H152N18O23Si5: calcd for .M1H)1 2282.02, found
2281.96, calcd for .M12H)21 1141.51, found 1141.50,
calcd for .M13H)31 761.35, found 761.34; 32a
C165H204N22O31Si6: calcd for .M1H)1 3158.37, found
3158.30, calcd for .M12H)21 1579.69, found 1579.68; Ia
C49H64N22O22: calcd for .M1H)1 1313.46, found 1313.53,
calcd for .M12H)21 657.24, found 657.27, calcd for
.M13H)31 438.49, found 438.51.
4.2.2. Deblocking reaction. The 50-N-MMTr-dimer 26a
.200 mg, 0.15 mmol) was dissolved in 80% aqueous acetic
acid .3.0 mL) and the mixture was kept at room temperature
for 10 min. The solvent was removed in vacuo, and the
residue was co-evaporated with EtOH and toluene. The
residue was puri®ed by silica gel column chromatography
.2.7£10 cm, 25% EtOH in CHCl3) to give 50-NH2-dimer
27a .153 mg, 96%) as a white solid.
4.2.7. Synthesis of Ib. 26b C88H104N14O10Si3: calcd for
.M1H)1 1601.7439, found 1601.7445 .0.4 ppm); 27b
C68H88N14O9Si3: calcd for .M1H)1 1329.6239, found
1329.6217 .1.7 ppm); 28b C127H146N22O15Si4: calcd for
.M1H)1 2332.05, found 2331.97, calcd for .M12H)21
1166.53, found 1166.65; 29b C107H130N22O14Si4: calcd for
.M1H)1 2059.92, found 2059.61, calcd for .M12H)21
1030.46, found 1030.49; 30b C166H188N30O20Si5: calcd for
4.2.3. Synthesis of longer sequence. Each pentameric RNG
oligomer .Ia±Ic) was synthesized in the similar two-step
cycle as described for the dimer synthesis except that

878
N. Kojima et al. / Tetrahedron 58 )2002) 867±879
.M1H)1 3062.35, found 3062.27, calcd for .M12H)21
1531.68, found 1531.68, calcd for .M13H)31 1021.45,
found 1021.44; 31b C146H172N30O19Si5: calcd for .M1H)1
2790.23, found 2790.16, calcd for .M12H)21 1395.62,
found 1395.61, calcd for .M13H)31 930.75, found
931.08; 32b C191H227N37O25Si7: calcd for .M1H)1
3635.60, found 3635.58, calcd for .M12H)21 1818.30,
found 1818.31, calcd for .M13H)31 1212.54, found
1212.22, calcd for .M14H)41 909.66, found 909.43; Ib
C54H69N37O12: calcd for .M12H)21 714.80, found 714.84,
calcd for .M13H)31 476.87, found 476.55.
1H, Jˆ6.8, 3.1 Hz), 3.90 .ddd, 1H, Jˆ8.1, 5.2, 6.8 Hz), 3.79
.s, 3H), 2.71 .dd, 1H, Jˆ3.1, 12.8 Hz), 2.45 .d, 1H,
Jˆ8.1 Hz), 2.35 .dd, 1H, Jˆ6.8, 12.8 Hz), 2.07 .br s, 1H),
0.93 .s, 9H), 0.18 .s, 3H), 0.13 .s, 3H).
4.2.11. 20-O-1tert-Butyldimethylsilyl)-50-14-monomethoxy-
tritylamino)-30-O-succinyl-50-deoxyuridine
135).
A
mixture of 34 .440 mg, 0.70 mmol), succinic anhydride
.210 mg, 2.10 mmol), and DMAP .86 mg, 0.70 mmol) in
pyridine .10 mL) was stirred at room temperature for 2
days, and the solvent was removed in vacuo. The residue
was dissolved in AcOEt .60 mL), which was washed with
H2O .20 mL£2) and brine .20 mL), dried .Na2SO4) and
concentrated in vacuo. The residue was puri®ed by silica
gel column chromatography .2.5£9 cm, 5% EtOH in
CHCl3) to give 35 .320 mg, 62%) as a white foam: LRMS
.FAB) m/z 730 [.M1H)1, 10%]; HRMS .FAB) calcd for
C39H47N3O9Si .M1H)1 730.3160, found 730.3149; 1H
NMR .400 MHz, CDCl3) 9.35 .br s, 1H), 7.84 .d, 1H,
Jˆ8.2 Hz), 7.45±7.18 .m, 12H), 6.84±6.80 .m, 2H), 5.72
.d, 1H, Jˆ2.6 Hz), 5.64 .d, 1H, Jˆ8.2 Hz), 4.99 .dd, 1H,
Jˆ4.6, 7.3 Hz), 4.37 .dd, 1H, Jˆ2.6, 4.6 Hz), 4.32 .ddd, 1H,
Jˆ7.3, 3.3, 5.2 Hz), 3.78 .s, 3H), 2.74 .dd, 1H, Jˆ3.3,
13.4 Hz), 2.70±2.61 .m, 5H), 2.22 .dd, 1H, Jˆ5.2,
13.4 Hz), 0.89 .s, 9H), 0.12 .s, 3H), 0.06 .s, 3H).
4.2.8. Synthesis of Ic. 26c C80H99N11O11Si3: calcd for
.M1H)1 1474.6905, found 1474.6923 .1.2 ppm); 27c
C60H83N11O10Si3: calcd for .M1H)1 1202.5705, found
1202.5687 .1.5 ppm); 28c C119H141N19O16Si4: calcd for
.M1H)1 2204.99, found 2205.18; 29c C99H125N19O15Si4:
calcd for .M1H)1 1932.88, found 1932.85, calcd for
.M12H)21 966.95, found 967.43; 30c C150H178N24O22Si5:
calcd for .M1H)1 2808.24, found 2808.24, calcd for
.M12H)21
1404.62,
found
1404.62;
31c
C130H162N24O21Si5: calcd for .M1H)1 2536.12, found
2536.09, calcd for .M12H)21 1268.56, found 1269.54,
calcd for .M13H)31 846.38, found 846.72; 32c
C175H217N31O27Si7: calcd for .M1H)1 calcd for
.M13H)31 962.76, found 962.76, found 3381.44, calcd
for .M12H)21 1691.25, found 1691.25, calcd for
.M13H)31 1127.84, found 1127.51, calcd for .M14H)41
846.13, found 846.39; Ic C52H67N31O16: calcd for .M1H)1
1382.54, found 1382.57, calcd for .M12H)21 691.78, found
691.79, calcd for .M13H)31 461.52, found 461.53.
4.2.12. 20-O-1tert-Butyldimethylsilyl)-50-14-monomethoxy-
tritylamino)-50-deoxyuridine LCAA-CPG unit 136). To a
solution of 35 .120 mg, 0.16 mmol) and 1-.3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride .WSC,
31 mg, 0.16 mmol) in DMF .5.0 mL) was added LCAA-
CPG .Sigma, 400 mg) and the mixture was agitated at
room temperature for 4 days. The resin was washed with
DMF .5 mL£2) and pyridine .5 mL£3). To the resin was
added capping solution .0.1 MDMAP in pyridine/acetic
anhydride 9:1, 6.0 mL) and the mixture was agitated at
room temperature for 24 h. The resin was washed with
EtOH .5 mL£3) and acetone .5 mL£3) to give 36
.23.5 mmol/g).
4.2.9. Characterization of r1AgU) dimer 140). HRMS
calcd for C66H96N10O11Si4 .M1H)1 1317.6415, found
1317.6380 .2.7 ppm); .500 MHz, DMSO-d6); 13C NM R
159.6, 152.2, 144.2, 142.6, 140.7, 139.4, 137.4, 132.4,
129.0, 128.9, 128.8, 128.5, 128.4, 128.3, 127.4, 127.3,
127.3, 127.2, 127.2, 126.9, 125.9, 125.1, 125.0, 121.4,
121.4, 121.3, 120.1, 120.0, 119.9, 109.6, 74.6, 73.4, 73.1,
65.9, 46.7, 40.0, 39.8, 39.7, 39.5, 39.3, 39.2, 39.0, 25.7,
25.6, 25.5, 25.4, 25.0, 24.8, 17.9, 17.9, 17.6, 17.6, 17.4,
24.8, 25.1, 25.6, 25.7, 25.9.
4.2.13. Solid-phase synthesis of Ia. LCAA-CPG attached
30-terminal monomer 36 .85 mg, 2.0 mmol) was placed in a
solid-phase synthesis vessel and 3% trichloroacetic acid in
dichloromethane .2.0 mL, 10 s, 3 times) added, the resin
drained and washed with dichloromethane .2.0 mL£3) and
dried under vacuum at room temperature for 30 min. To the
vessel was added 8 .27.3 mg, 0.030 mmol) in DMF
.0.5 mL), followed by a solution of N,N-diisopropylethyl-
amine .120 mM, in DMF, 0.5 mL) and mercury.II) chloride
.60 mMin DMF, 0.5 mL). The vessel was agitated at room
temperature for 4 h. After the CPG resin was washed with
DMF .2.0 mL£3), the same coupling reaction was repeated
to give 37. The CPG resin was washed with DMF
.2.0 mL£3), thiophenol .20% in DMF, 10 s, 3 times), and
DMF .2.0 mL£3). To the vessel was added a mixed solution
of ethyl tri¯uoroacetate/N,N-diisopropylethylamine .1:1,
120 mM, in DMF, 2.0 mL), and the mixture was agitated
at room temperature for 30 min. The CPG resin was washed
with DMF .2.0 mL) and dichloromethane .2.0 mL£3). The
deblocking/coupling/capping cycles were repeated until the
®nal coupling of the 50-terminal monomer 10 yielded 39.
The CPG resin 39 was placed in a glass tube .5 mL), to
which was added 28% NH4OH .1.5 mL) and EtOH
4.2.10. 20-O-1tert-Butyldimethylsilyl)-50-14-monomethoxy-
tritylamino)-50-deoxyuridine 134). To a THF solution of
50-.4-monomethoxytritylamino)-50-deoxyuridine
.33,
1.47 g, 2.85 mmol) and AgNO3 .730 mg, 4.30 mmol) was
added pyridine .1.04 mL, 12.9 mmol) at room temperature.
After the mixture was stirred for 10 min, tert-butyldimethyl-
silyl chloride .650 mg, 4.3 mmol) was added, the mixture
stirred for 20 h. When EtOH .2 mL) was added and the
mixture ®ltered through a Celite pad, the ®ltrate was
concentrated in vacuo. The residue was dissolved in
AcOEt .180 mL), which was washed with H2O .60 mL£2)
and brine .60 mL), dried .Na2SO4) and concentrated in
vacuo. The residue was puri®ed by silica gel column chro-
matography .4.5£15 cm, 30% AcOEt in hexane) to give 34
.1.26 g, 70%) as a white foam: LRMS .FAB) m/z 630
[.M1H)1, 30%]; HRMS .FAB) calcd for C35H44N3O6Si1
1
.M1H)1 629.2921, found 629.2932; H NMR .400 MHz,
CDCl3) 8.61 .br s, 1H), 7.48±7.18 .m, 12H), 7.22 .d, 1H,
Jˆ8.2 Hz), 6.83±6.81 .m, 2H), 5.74 .d, 1H, Jˆ2.8 Hz), 5.63
.d, 1H, Jˆ8.2 Hz), 4.19 .dd, 1H, Jˆ2.8, 5.2 Hz), 4.09 .dd,

N. Kojima et al. / Tetrahedron 58 )2002) 867±879
879
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12 h then ®ltered through glass ®lter. The ®ltrate was
concentrated in vacuo, then dissolved in 1.0 MTBAF in
THF .1.0 mL, 1.0 mmol) and stirred at room temperature
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RP-HPLC of the pentameric uridyl oligomer, before puri®-
cation, has been included .Fig. 4) and is typical of that for
crude all RNG pentamers as puri®ed by this method. Only
two signi®cant absorbencies were noted. The de-protected
uridyl pentamer has the retention time corresponding to
8.024 min, based on the solvent system and ¯ow rate chosen
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Figure 4. RP-HPLC analysis of crude RNG-U5 .0±10% acetonitrile in 3%
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This work was supported by a grant from the National
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