Synthesis and properties of (2′S)- and (2′R)-2′-deoxy-2′-C-methyl oligonucleotides

Article abstract of DOI:10.1016/S0040-4020(01)00745-1

The synthesis of (2′S)- and (2′R)-2′-deoxy-2′-C-methyl-N³-(4-t-butylbenzoyl) uridine, (2′S)-2′-deoxy-2′-C-methyluridine and (2′S)-2′-deoxy-2′-C-methyl-N⁴-isobutyryl cytidine building blocks are here described. The preparation of oligonucleotides carrying these monomers in all positions but 3′-end is presented and the binding affinity between these new fragments and the complementary DNA and RNA sequences is also assessed. (2′R) substituted oligonucleotides did not hybridize with either the complementary DNA or RNA sequences. However, the first derivative of melting curves of hybrids containing (2′S) modified oligonucleotides indicated melting transitions.

Full text of DOI:10.1016/S0040-4020(01)00745-1

TETRAHEDRON
Pergamon
Tetrahedron 57 '2001) 7613±7621
Synthesis and properties of ꢀ20S)- and
ꢀ20R)-20-deoxy-20-C-methyl oligonucleotides
Daniel O. Cicero,a Mariana Gallo,b Philippe J. Neunera and Adolfo M. Iribarrenb,c,p
aIstituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km. 30,600, 00040-Pomezia, Rome, Italy
b
Â
Â
Â
Instituto de Investigaciones en Ingenierõa Genetica yBiologõ a Molecular, Vuelta de Obligado 2490 2P, 1428 Buenos Aires, Argentina
c
Â
Universidad Nacional de Quilmes, Roque Saenz PenÄa 180, Bernal, 1876 Pcia. de Buenos Aires, Argentina
Received 24 May 2001; accepted 20 July 2001
AbstractÐThe synthesis of '20S)- and '20R)-20-deoxy-20-C-methyl-N3-'4-t-butylbenzoyl) uridine, '20S)-20-deoxy-20-C-methyluridine and
'20S)-20-deoxy-20-C-methyl-N4-isobutyryl cytidine building blocks are here described. The preparation of oligonucleotides carrying these
monomers in all positions but 30-end is presented and the binding af®nity between these new fragments and the complementary DNA and
RNA sequences is also assessed. '20R) substituted oligonucleotides did not hybridize with either the complementary DNA or RNA
sequences. However, the ®rst derivative of melting curves of hybrids containing '20S) modi®ed oligonucleotides indicated melting transi-
tions. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
pucker consistent with the A-form geometry of RNA
duplexes. Therefore, the sugar ring puckering seems to
constitute an important structural variable to take into
account in the design of antisense oligonucleotides.5,10,11
We have previously determined the in¯uence of 20-C-
alkyl groups on the sugar conformation of nucleosides in
solution based on NMR analysis.12 The results showed that
the preferred sugar conformation in the case of '20S)-20-
deoxy-20-C-alkylnucleosides is C-30 endo while the '20R)
isomers exist predominantly in the C-20 endo conformation.
These ®ndings could be explained since 20-C alkyl sub-
stituents tend to attain a pseudoequatorial location for steric
reasons while an oxygenated subtituent on C-20 tends to
occupy a pseudoaxial site because of the stabilizing O-40/
O-20 gauche effect.13
The synthesis of modi®ed oligonucleotides has received a
great deal of attention during the last years, due to the very
important applications that this class of compounds has
displayed as antisense fragments. Several modi®cations
have been proposed in order to obtain molecules resistant
to the action of nucleases and that can recognize comple-
mentary sequences.1 Oligonucleotides based on a nuclease
resistant phosphorothioate backbone have proved to be very
potent and selective inhibitors of protein expression in vitro
as well as in vivo.2,3 Despite this therapeutic potential, the
detailed analysis of their whole chemical, biochemical and
biophysical characteristics still indicates that this class of
compounds may not represent the ®nal solution to the
problem of designing functional antisense inhibitors.4
20-Modi®cations have also proved to have interesting
hybridization properties.5 20-O-alkyloligoribonucleotides6,7
which have shown to be resistant to nucleases and to form
stable hybrids are widely used both alone and in combina-
tion with modi®ed phosphate backbones.8,9 The improved
hybridization of 20-F- and 20-O-alkyloligonucleotides to
complementary RNA has been attributed to the tendency
of these electronegative substituents to shift the conforma-
tional equilibrium in the sugar moiety towards C30-endo
As far as we know, there is only one report regarding to the
synthesis and properties of chimeric sequences containing
20-deoxy and 20-C-alkyldeoxynucleotides which exhibited a
reduced binding af®nity for complementary DNA and
RNA.14 Despite these results, '20R)-20-C-alkylnucleotides
have proved to be useful in the design of modi®ed hammer-
head ribozymes.15,16 In order to further investigate the
properties of oligonucleotides carrying these modi®cations
we carried out preliminary attempts to explore the in¯uence
of the 20-methyl group on the orientation of the base as well
on vicinal nucleotides. In view of the results afforded by
these preliminary studies, and since differences between the
hybridization af®nity of oligodeoxynucleotides carrying
20-modi®ed moieties in certain positions and completely
modi®ed sequences has been formerly observed10, we
consider interesting to further investigate the synthesis
and hybridization properties of oligonucleotides carrying
Keywords: antisense; modi®ed oligonucleotides; 20-deoxy-20-C-alkyl-
nucleotides; hybridization properties.
p
Â
Corresponding author. Address: Instituto de Investigaciones en Ingenierõa
Â
Â
Genetica y Biologõa Molecular, Vuelta de Obligado 24902P, 1428
Buenos Aires, Argentina. Tel.: 15411-4783-2871; fax: 15411-4786-
8578; e-mail: airi@dna.uba.ar
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020'01)00 745-1

7614
D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
0
Figure 1. Potential energy as a function of the glycosidic angle c for: 'a) U'2 S)Me, 'b) dU and 'c) rU. 'd) Represents the range of c usually found in natural DNA
duplexes.
20-C-methyl substituents with both arabino and ribo con-
®gurations in all but 30-positions.
and 5b starting from the mixture of '20S)- and '20R)-20-
deoxy-30,50-O-'tetraisopropyldisiloxane-1,3-diyl)-20-C-
methyluridine '1a and 1b).18
Reaction of the mixture of 1a and 1b with 4-t-butylbenzoyl
chloride yielded compounds 2a and 2b. The introduction of
the lipophilic acyl group made chromatographic separation
of both diasteromers possible, and also conferred additional
protection for the lactam function during the longer
coupling times required for 20-substituted nucleotides.19
The disiloxane bridge of the isolated isomers was removed
with tetrabutylammonium ¯uoride to afford compounds 3a
'97 % yield) and 3b '69 % yield). The con®guration at C-20
was assigned by comparing the NMR data of 3a and 3b with
those of '20S)- and '20R)-20-deoxy-20-C-20-methyluridine18
'data not shown).
2. Results and discussion
Preliminary studies were done in order to explore the effect
of the methyl group on the orientation of the base which
could produce structural distortions in the case of the '20S)-
isomer. The energy of different rotamers around the glyco-
sidic bond was analyzed by means of molecular mechanics
calculations. Curves of Fig. 1 show the calculated energy as
a function of the glycosidic angle c, using a grid step of 108;
the curves for natural and 20-C-alkyl-modi®ed uridine
nucleosides present similar shapes in the range 180±2808.
These results suggest that the introduction of the '20S)-
methyl group does not impair the orientation of the base
within the range of glycosidic angles found in natural
duplexes.11
50-O-Dimethoxytrityl derivatives '4a and 4b), as well as 30-
O-phosphoramidites, 5a and 5b, were prepared following
standard procedures.
A further attempt to predict the hybridization properties of
this kind of modi®ed oligonucleotides was made by explor-
ing the interaction of the 20-methyl group with vicinal
nucleotides. The pyrimidine±DNA strand from a DNA±
RNA hybrid constructed using the Builder module of the
program Insight,17 was modi®ed by direct replacement of
the 20-b-hydrogen by a methyl group. An analysis of bump-
ing interactions showed that the 20-methyl group most likely
sterically interferes with the 30-nucleotide in the oligomer,
though these minor interactions would not justify an
absence of hybridization. On the other hand, the substitution
of the 20-a-hydrogen produced strong destabilizing inter-
actions with vicinal nucleotides. To verify experimentally
this preliminary conclusions we decided to synthesize oligo-
nucleotides carrying '20S)- and '20R)-C-methylnucleotides
in all positions but 30-end.
In order to assess the hybridization properties of completely
modi®ed oligonucleotides carrying 20-modi®ed building
blocks in all but 30-position the following fragments were
0
0
synthesized: 'U'2 R)Me)10T 'I), and 'U'2 S)Me)10T 'II), 'where
U'2 R)Meˆ'20R)-20-deoxy-20-C-methyluridine; U'2 S)Meˆ'20S)-
20-deoxy-20-C-methyluridine and Tˆthymidine). Sugar-
modi®ed phosphoramidites 5a and 5b were used as building
blocks in an automated DNA synthesizer. Due to the steric
interference caused by the bulky 20-C-alkyl substituent, the
normal coupling cycle was modi®ed to ensure high coupling
yield of the protected monomers '7 min). Deprotection and
puri®cation were performed as described in Section 3.
0
0
On the other hand, with the goal of analyzing the hybridiza-
tion behavior of a mixed cytidine±uridine sequence carry-
ing '20S)-20-deoxy-20-C-methylnucleotides, the synthesis of
the cytidine building block was performed 'Fig. 3). The
Fig. 2 shows the synthesis of the uridine building blocks 5a

D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
7615
Figure 2. Reaction scheme for the preparation of '20S)- and '20R)-20-deoxy-20-C-methyl-N3-'4-t-butylbenzoyl)uridine building blocks. Reagents: 'i) tetra-
butylammonium bromide, Na2CO3 and 4-t-butylbenzoyl chloride in dichloromethane/H2O '1:1, v/v); 'ii) tetrabutylammonium ¯uoride in tetrahydrofuran; 'iii)
4,40-dimethoxytrityl chloride and triethylamine in pyridine; 'iv) 2-cyanoethoxy N,N-diisopropylaminochlorophosphine and N,N-diisopropylethylamine in 1,2-
dichloroethane.
mixture of compounds 1a and 1b was treated with 2-nitro-
phenol and diazabicyclo[2,2,2]octane in 1,2-dichloro-
ethane/triethylamine to afford the two diasteromers of the
corresponding O4-nitrophenylnucleosides. This mixture
could be resolved by silica gel column chromatography to
afford pure compound 6 which was the common inter-
mediate for the synthesis of the uridine '9) and cytidine
phosphoramidites '12). Using the same conditions described
above for the preparation of the 20-modi®ed homo-
oligomers, the synthesis of 'UCCUCCCUCUUCCU-
absorbance is much smaller than those obtained for the
hybrids T11/dA11 and T11/rA11, but transitions were
evidenced by the ®rst derivatives of the corresponding melt-
ing curves. These results are not due to particular single
strand structures, since the melting curves of the single
oligonucleotides did not show any transition.
According to the ®rst derivatives, the melting points
measured were the following: T11/dA11: 24.08C; II/dA11:
25.98C; T11/rA11: 16.58C; II/rA11: 26.28C. These results
show that only the modi®ed oligonucleotides carrying
'20S)-alkyl groups presented good hybridization properties,
with melting points higher than those measured for the
natural DNA±DNA and DNA±RNA hybrids. It also
suggests that although the stacking interactions seem to be
disturbed by the presence of the methyl group, the hybrids
containing modi®ed uridines are thermally more stable than
the corresponding hybrids carrying thymidines.
CUCC)'2 S)MedC 'III) 'where C'2 S)Meˆ'20S)-20-deoxy-20-
0
0
C-methylcytidine; U'2 S)Meˆ'20S)-20-deoxy-20-C-methyluri-
0
dine and dCˆdeoxycytidine) was performed. No difference
in the yield of the oligomers between the preparation using
lactam protected uridine building block 5b, and the lactam
unprotected 9 were observed.
The thermal stability of the hybrids formed between these
oligonucleotides and the complementary sequences was
tested. Oligonucleotide I did not hybridize with dA11 or
-rA11 sequences. This result is in agreement with that
reported by Schmidt et al. for the case of chimeric oligo-
nucloetides carrying '20R)-20-C-alkyloligonucleotide.14 On
the other hand, fragment II showed a thermal transition with
both dA11 and rA11. In both cases, the observed difference in
In order to assess the hybridization behavior of a mixed
cytidine±uridine sequence, the hybridization behavior of
the mixed sequence III with the complementary RNA
olilgonucleotide was performed. In the case of the hybrid
formed between oligonucleotide III and the corresponding
complementary RNA sequence the melting temperature

7616
D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
Figure 3. Reaction scheme for the synthesis of '20S)-20-deoxy-20-C-methyluridine and '20S)-20-deoxy-20-C-methyl-N4-isobutyrylcytidine building blocks.
Reagents: 'i) mesitylenesulfonyl chloride, dimethylaminopyridine, triethylamine, diazabicyclo[2.2.2] octane and 2-nitrophenol in 1,2-dichloroethane;
'ii) 2-nitro-benzaldoxime and tetramethylguanidine in CH3CN; 'iii) ammonia in tetrahydrofuran; 'iv) isobutyryl chloride in pyridine; 'v) tetrabutylammonium
¯uoride in tetrahydrofuran; 'vi) 4,40-dimethoxytrityl chloride and triethylamine in pyridine; 'vii) 2-cyanoethoxy-N,N-diisopropylaminochlorophosphine and
N,N-diisopropylethylamine in 1,2-dichloroethane.
obtained is lower than that measured for the natural DNA±
RNA duplex of the same sequence, the respective melting
points being 38.8 and 43.08C. As observed earlier, the
difference in absorbance between the hybrid and the melted
strands is smaller in the case of the sugar-modi®ed oligonu-
cleotide. The different melting behavior of II and III is not
surprising since previous reports showed similar variations
depending on the sequence and the length of the modi®ed
oligonucleotides.5
modi®cation is located, whether it is located in a back-
ground of deoxynucleotides or modi®ed nucleotides.9
The results regarding to '20S)-20-deoxy-20-C-alkyloligo-
nucleotides, challenge us to investigate more exhaustively
the properties and stability characteristics of this class of
sugar modi®ed oligonucleotides. We are undertaking the
synthesis of mixed sequences which include purine nucleo-
tides, and further structural studies.
The reason why the '20S)-methyloligodeoxynucleotides
hybridize better than the '20R) analogues could be attributed
to the fact that the '20S)-methyloligodeoxynucleotides have
the right preorganization for ®tting with the complementary
RNA strand,20 since the sugar moieties are locked in the
30-endo conformation.
3. Experimental
3.1. General
20-Deoxy-20-C-alkylnucleosides were synthesized as
previously described.19 Column chromatography was
performed on silica gel 60, particle size 20±45 mm
'Amicon). All the reagents used were of the highest avail-
able purity.
The ¯atter transitions suggest that the presence of the
b-methyl group in¯uences the hybrid structures as observed
in our preliminary analysis. The differences between our
®ndings and those reported by Schmidt et al.14 could be
explained, as previously reported for related modi®cations,
by the fact that the binding af®nity depends on the geometry
of the heteroduplex and therefore on the context where the
1H, 13C and 31P NMR spectra were recorded on a Bruker
AC200 or Bruker AM500 using tetramethyl-silane and
phosphoric acid as the respective references.

D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
7617
1
Oligonucleotides were synthesized on a 380B Applied Bio-
systems synthesizer using b-cyanoethyl phosphoramidite
chemistry. Commercially available 'Millipore) derivatized
CPG-linked to either 20-deoxy or ribonucleosides were
used. Coupling ef®ciency was in all the cases higher than
98%. Deprotection of oligonucleotides was achieved by
treatment with concentrated aqueous ammonia at 558C,
overnight. Oligonucleotides 'trityl-on), which showed a
major peak, were puri®ed by reverse phase HPLC on a
Nova-Pak C-18 column '8£10Radial-Pak Cartridge,
4 mm) using a gradient of 16±60% of acetonitrile in
0.1 M triethylammonium acetate buffer pH 7. Cleavage of
DMTr group was accomplished by treatment with 80%
acetic acid for 30min. The solution was diluted with an
equal volume of water, washed three times with two
volumes of diethyl ether, and the aqueous layer was
lyophilized.
'isopropyls), 10.5 '20-methyl). H NMR spectrum of 2a
'CDCl3): 'd, ppm) 0.81±1.23 'm, 28H, ±CHs, ±CH3s,
0
disiloxane); 0.87 'd, 3H, ±CH3, JCH3,2 ˆ7 Hz); 1.32 's,
9H, ±CH3s, t-butyl); 2.23 'm, 1H, H-20); 3.87 'dd, 1H, H-
50, J5 ,5 ˆ13 Hz; J4 ,5 ˆ1 Hz); 4.00 'dd, 01H, H-500,
0
00
0
0
0
00
0
00
J5 ,5 ˆ13 Hz; J4 ,5 ˆ1 Hz); 4.15 'm, 1H, H-4 ); 4.42 'm,
1H, H-30); 5.72 'd, 1H, H-5, J6,5ˆ8 Hz); 5.95 'd, 1H, H-
10, J1,2ˆ9 Hz); 7.40'd, 2H, t-butylbenzoyl, Jˆ9 Hz); 7.81
'd, 1H, H-6, J6,5ˆ8 Hz); 8.02 'd, 2H, t-butylbenzoyl,
Jˆ9 Hz). 13C NMR spectrum of 2b 'CDCl3): 'd, ppm)
168. 9 't-butylbenzoyl); 161.6 'C-4); 154.8 't-butyl-
benzoyl); 149.0'C-2); 138.5 'C-6); 131.7, 128.8 and
124.2 't-butylbenzoyl); 102.4 'C-5); 85.8, 84.3 'C-10 and
C-40); 72.1 'C-30); 60.3 'C-50); 44.1 'C-20); 34.9, 31.5
't-butylbenzoyl); 17.4±12.3 'isopropyls), 11.1 '20-methyl).
1H NMR spectrum of 2b 'CDCl3): 'd, ppm) 0.80±1.22 'm,
28H, ±CHs, ±CH3s, disiloxane); 0.92 'd, 3H, ±CH3,
0
JCH3,2 ˆ7 Hz); 1.34 's, 9H, ±CH3s, t-butyl); 2.73 'm, 1H,
H-20); 3.75 'dd, 1H, H-50, J5 ,5 ˆ12 Hz; J4 ,5 ˆ1 Hz); 3.96
0
00
0
0
Melting curves were measured on a Cary 3E 'Varian)
spectrophotometer with temperature controller. Hybrids
were previously annealed by heating the solution of the
oligonucleotides 3 mM of each strand in100 mM NaCl,
10mM Tris buffer 'pH ˆ7.1) at 808C for 2 min and subse-
quent cooling for 40min to a ®nal temperature of 5 8C.
The melting curves were obtained by measuring the
absorbance at 260nm, using a heating rate of 0.5 8C/min.
Melting temperatures 'Tm) were determined as the maxi-
mum of the ®rst derivative of the plot of absorbance vs.
temperature.
'dd, 1H, H-500, J5 ,5 ˆ13 Hz; J4 ,5 ˆ1 Hz); 4.01 'm, 1H,
H-40); 4.22 'm, 1H, H-30); 5.81 'd, 1H, H-5, J6,5ˆ8 Hz);
6.25 'd, 1H, H-10, J1,2ˆ8 Hz); 7.38 'd, 2H, t-butylbenzoyl,
Jˆ9 Hz); 7.76 'd, 1H, H-6, J6,5ˆ8 Hz); 8.11 'd, 2H, t-butyl-
benzoyl, Jˆ9 Hz). Anal. calcd for C33H52O7N2Si2 '2a and
2b): C, 61.46; H, 8.13; N, 4.34. Found for 2a: C, 61.50; H,
8.15; N, 4.40; 2b: C, 61.50; H, 8.10; N, 4.45.
0
00
0
00
3.1.2. ꢀ20R)-20-Deoxy-20-C-methyl-N3-ꢀ4-t-butylbenzoyl)
uridine ꢀ3a). 30,50-O-'Tetraisopropyldisiloxane-1,3-diyl)-
'20R)-20-deoxy-20-C-methyl-N3-'4-t-butylbenzoyl) uridine
'2a) '300 mg, 0.46 mmol) was dissolved in dry tetrahydro-
furan '1.4 mL) and tetrabutylammonium ¯uoride '1 mmol)
was added with stirring and exclusion of moisture. After
5 min the reaction mixture was quenched with MeOH/
pyridine/H2O '2.8 mL, 3:3:1 v/v/v), and stirred for 5 min.
Pyridinium form Dowex 50£2±200 resin '15 g) was added
to the solution and the mixture was stirred for 15 min. The
resin was ®ltered off and washed with MeOH '2£2.8 mL).
Combined ®ltrate and washings were evaporated in vacuo
and the crude product was puri®ed by silica gel column,
eluting with a gradient of MeOH from 2 to 5% in dichloro-
methane. Pure title compound was obtained '180mg) in
97% yield. 13C NMR spectrum of 3a 'CDCl3): 'd, ppm)
168.8 't-butylbenzoyl); 162.0'C-4); 154.7 ' t-butylbenzoyl);
148.9 'C-2); 139.0'C-6); 131.7, 128.8, 124.4 ' t-butyl-
benzoyl); 102.4 'C-5); 89.2, 87.8 'C-10 and C-40); 72.9
Microanalysis of previously recrystallized samples was
done using an automatic analyzer 'Fisons Instruments).
Molecular mechanics calculations were performed using
the program Discover 'Biosym). The energy minimizations
were performed in vacuo, using the AMBER force ®eld.
Curves of Fig. 1 were obtained using a grid step of 108.
At each value of the glycosidic angle, the structure was
minimized to an rms energy gradient of 0.001 kcal/mol A,
using a 10kcal/mol harmonic restraint to maintain the
appropriate value of c.
3.1.1. 30,50-O-ꢀTetraisopropyldisiloxane-1,3-diyl)-ꢀ20R)-
and -ꢀ20S)-20-deoxy-20-C-methyl-N3-ꢀ4-t-butylbenzoyl)
uridine ꢀ2a and 2b). To a vigorously stirred mixture of
30,50-O-'tetraisopropyldisiloxane-1,3-diyl)-'20R)- and '20S)-
20-deoxy-20-C-methyluridine '1a, 1b) '30 mg, 0.06 mmol),
tetrabutylammonium bromide '2 mg), Na2CO3 '50mg,
0.47 mmol) and dichloromethane/H2O '2 mL, 1:1, v/v),
was added dropwise 4-t-butylbenzoyl chloride '20mL,
0.10 mmol). The reaction mixture was stirred overnight at
room temperature. The organic phase was separated,
washed with H2O '5 mL) and left with stirring overnight
'in order to obtain the thermodynamically more stable
N3-acyl derivative21). The solvent was evaporated in
vacuo and the residue chromatographied on silica gel
column using as eluant a gradient of petroleum ether/ethyl
acetate 9:1 to 7:1 'v/v). Pure compounds 2a '12 mg,
0.018 mmol) and 2b '19 mg, 0.029 mmol) were obtained.
13C NMR spectrum of 2a 'CDCl3): 'd, ppm) 168.8 't-butyl-
benzoyl); 162.0'C-4); 154.6 ' t-butylbenzoyl); 149.2 'C-2);
138.8 'C-6); 131.7, 128.8, 124.4 't-butylbenzoyl); 102.1
'C-5); 90.4 'C-10); 82.7 'C-40); 68.3 'C-30); 59.5 'C-50);
43.4 'C-20); 34.8 and 31.4 't-butylbenzoyl); 17.3±12.3
0
'C-30); 61.0'C-5 ); 43.9 'C-20); 35.0, 31.5 't-butylbenzoyl),
1
10.3 '20-methyl). H NMR spectrum of 3a 'CDCl3): 'd,
0
ppm) 0.91 'd, 3H, CH3, JCH3,2 ˆ7 Hz); 1.32 's, 9H,
0
±CH3s, t-butyl); 2.21 'm, 1H, H-20); 3.70'm, 2H, H-5 and
H-500); 3.83 'ddd, 1H, H-40, J5 -4 ˆJ5 -4 ˆ4 Hz, J3 -4 ˆ2 Hz);
00
0
0
0
0
0
4.04 'dd, 1H, H-30, J2 ,3 ˆ6 Hz, J3 -4 ˆ2 Hz); 5.02 'brs, 1H,
±OH); 5.21 'brs, 1H, ±OH); 5.65 'd, 1H, H-5, J6,5ˆ8 Hz);
5.75 'd, 1H, H-10, J1,2ˆ9 Hz); 7.39 'd, 2H, t-butylbenzoyl,
Jˆ9 Hz); 7.83 'd, 1H, H-6, J6,5ˆ8 Hz); 8.05 'd, 2H, t-butyl-
benzoyl, Jˆ9 Hz).
0
0
0
0
3.1.3. ꢀ20S)-20-Deoxy-20-C-methyl-N3-ꢀ4-t-butylbenzoyl)
uridine ꢀ3b). 30,50-O-'Tetraisopropyldisiloxane-1,3-diyl)-
'20S)-20-deoxy-20-C-methyl-N3-'4-t-butylbenzoyl)uridine
'2b) '510mg, 0.79 mmol) was desilylated and puri®ed
according to the procedure used to prepare compound 3a
above to yield pure title compound '220mg, 69%). 13C

7618
D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
1
methyl). H NMR spectrum of 4b 'CDCl3): 'd, ppm) 0.94
NMR spectrum of 3b 'CDCl3): 'd, ppm) 168.9 't-butyl-
benzoyl); 161.5 'C-4); 154.9 't-butylbenzoyl); 148.9
'C-2); 139.3 'C-6); 131.7, 128.5, 124.3 't-butylbenzoyl);
102.6 'C-5); 86.0 'C-10); 83.7 'C-40); 73.5 'C-30); 61.9
'C-50); 45.1 'C-20); 34.8, 31.3 't-butylbenzoyl), 11.1
0
'd, 3H, ±CH3, JCH3,2 ˆ8 Hz); 1.32 's, 9H, ±CH3s, t-butyl-
benzoyl); 2.41 'brs, 1H, OH-30); 2.58 'm, 1H, H-20); 3.20
'dd, 1H, H-50, J5 ,5 ˆ12 Hz; J4 ,5 ˆ1 Hz); 3.42 'dd, 1H, H-
0
00
0
00
500, J5 ,5 ˆ12 Hz; J4 ,5 ˆ1 Hz); 3.80, 's, 3H, ±OCH3); 3.82,
0
00
0
00
1
'20-methyl). H NMR spectrum of 3b 'CDCl3): 'd, ppm)
's, 3H, ±OCH3); 4.05 'm, 1H, H-40); 4.11 'm, 1H, H-30);
5.32 'd, 1H, H-5, J5,6ˆ8 Hz); 6.28 'd, 1H, H-10, J1 ,2 ˆ8 Hz);
6.82 'd, 4 H, Ar±OCH3, DMT, Jˆ9 Hz); 7.20±7.35 'm, 11
H, Ar1Ar±OCH3, DMT and Ar, t-butylbenzoyl); 8.05 'd,
1H, H-6, J5,6ˆ8 Hz), 8.09 'd, 2H, t-butylbenzoyl, Jˆ9 Hz).
Anal. calcd for C42H44O8N2: C, 71.57; H, 6.29; N, 3.98.
Found: C, 71.50; H, 6.25; N, 3.95.
0
0
0
0.85 'd, 3H, CH3, JCH3,2 ˆ7 Hz); 1.34 's, 9H, ±CH3s,
t-butyl); 2.48 'm, 1H, H-20); 3.65 'm, 2H, H-50 and H-500);
3.75 'm, 1H, H-40); 3.80'm, 1H, H-3 ); 5.14 'brs, 1H,
0
±OH); 5.35 'brs, 1H, ±OH); 5.63 'd, 1H, H-5, J6,5ˆ8 Hz);
6.18 'd, 1H, H-10, J1,2ˆ8 Hz); 7.41 'd, 2H, t-butylbenzoyl,
Jˆ9 Hz); 8.00 'd, 1H, H-6, J6,5ˆ8 Hz); 8.15 'd, 2H, t-butyl-
benzoyl, Jˆ9 Hz). Anal. calcd for C21H26O6N2 '3a and 3b):
C, 62.67; H, 6.51; N, 6.96. Found for 3a: C, 62.71; H, 6.38;
N, 7.01; 3b: C, 62.75; H, 6.44; N, 6.94.
3.1.6. 50-O-Dimethoxytrityl-ꢀ20R)-20-deoxy-20-C-methyl-
N3-ꢀ4-t-butylbenzoyl)uridine-30-O-ꢀ2-cyanoethyl
N,N-
diisopropylphosphoramidite) ꢀ5a). 50-O-Dimethoxytrityl-
'20R)-20-deoxy-20-C-methyl-N3-'4-t-butylbenzoyl)uridine
'4a) '3.23 g, 5 mmol) was dried by evaporation of CH3CN
and dissolved in dry 1,2-dichloroethane '20mL) containing
N,N-diisopropylethylamine '10mmol) under argon. 2-
Cyanoethoxy-N,N-diisopropylaminochlorophosphine
'6 mmol) was added drop wise with stirring. When TLC
showed complete reaction, the mixture was quenched by
addition of MeOH. After 3 min, dichloromethane '50mL)
was added and the solution was washed with 1 M aqueous
Na2CO3 '70mL) followed by saturated brine '50mL). The
organic phase was dried 'Na2SO4), ®ltered and evaporated
to dryness. The residue was puri®ed by silica gel column,
eluting with 33% ethyl acetate in petrol containing 1%
triethylamine to yield pure title compound '78%). 13C
NMR spectrum of 5a 'CDCl3): 'd, ppm) 168.9/168.7
't-butylbenzoyl); 161.9 'C-4); 158.7/158.6 'CAr±OCH3,
DMTr); 154.2 't-butylbenzoyl); 149.3 'C-2); 144.3/144.2
'Ar, DMTr); 140.2 'C-6); 135.3/135.2, 130.5/130.4/130.3,
128.6/128.4/128.2, 127.9/127.4/127.3 'Ar, DMTr1butyl-
benzoyl); 131.5, 124.4 't-butylbenzoyl); 117.4 'CN,
phops);113.2/113.1 'Ar, DMTr); 102.1 'C-5); 87.0
'C±Ar3, DMTr); 89.2/89.4 'C-10); 82.4 'C-40); 72.0/71.8
'C-30); 60.5 'C-50); 58.0/57.9 '±OCH2, phosph); 55.2/55.1
'±OCH3, DMTr); 44.5/44.4 'C-20); 43.7/43.4 'CHs, phosp);
34.7, 31.3 't-butylbenzoyl); 24.6 'CH3, phosp); 18.9/18.8
3.1.4. 50-O-Dimethoxytrityl-ꢀ20R)-20-deoxy-20-C-methyl-
N3-ꢀ4-t-butylbenzoyl)uridine ꢀ4a). '20R)-20-Deoxy-20-C-
methyl-N3-'4-t-butylbenzoyl) uridine 3a '2 g, 5 mmol)
was dried by evaporation of pyridine in vacuo. Anhydrous
0
pyridine '20mL), triethylamine '10mmol) and 4,4 -
dimethoxytrityl chloride '6 mmol) were added with stirring
and exclusion of moisture. The reaction was checked by
TLC and after completion quenched by addition of MeOH
and evaporated to dryness. The residue was dissolved in
'50mL) and washed with 1 M aqueous NaHCO 3
'2£50mL). The organic layer was dried 'Na 2SO4), ®ltered
and solvent was removed in vacuo. The residue was puri®ed
by column cromatography on silica gel using 50% ethyl
acetate in petroleum ether containing 1% triethylamine as
eluant '63%). 13C NMR spectrum of 4a 'CDCl3): 'd, ppm)
168.9 't-butylbenzoyl); 161.8 'C-4); 158.5 'CAr±OCH3,
DMTr); 154.7 't-butylbenzoyl); 149.3 'C-2); 144.2 'Ar,
DMTr); 140.2 'C-6); 135.3, 130.0, 128.6, 128.0, 127.3
'Ar, DMTr1butylbenzoyl); 131.7, 124.4 't-butylbenzoyl);
113.1 'Ar, DMTr); 102.3 'C-5); 86.6 'C±Ar3, DMTr); 89.3
'C-10); 82.4 'C-40); 70.0 'C-30); 60.2 'C-50); 55.1 '±OCH3,
DMTr); 44.5 'C-20); 34.7, 31.3 't-butylbenzoyl), 10.4 '20-
1
methyl). H NMR spectrum of 4a 'CDCl3): 'd, ppm) 0.95
0
'd, 3H, ±CH3, JCH3,2 ˆ7 Hz); 1.30's, 9H, ±CH s, t-butyl-
3
benzoyl); 2.45 'brs, 1H, OH-30); 2.20'm, 1H, H-2 ); 3.25
0
1
'm, 1H, H-50); 3.45 'm, 1H, H-500); 3.81 's, 6H, ±OCH3);
4.00 'm, 1H, H-40); 4.31 'm, 1H, H-30); 5.45 'd, 1H, H-5,
'CH2±CN, phosp); 10.4/10.5 '20-methyl). H NMR spec-
trum of 5a 'CDCl3): d 'ppm) 0.95/0.99 'd, 3H, ±CH3,
J5,6ˆ8 Hz); 6.15 'd, 1H, H-10, J1 ,2 ˆ8 Hz); 6.80'd, 4H, Ar±
OCH3, DMT, Jˆ9 Hz); 7.21±7.35 'm, 11H, Ar1Ar±OCH3,
DMT and Ar, t-butylbenzoyl); 8.03 'd, 1H, H-6, J5,6ˆ 8 Hz),
8.11 'd, 2H, t-butylbenzoyl, Jˆ9 Hz). Anal. calcd for
C42H44O8N2: C, 71.57; H, 6.29; N, 3.98. Found: C, 71.52;
H, 6.33; N, 4.01.
JCH3,2 ˆ7 Hz); 1.08, 1.15, 1.16, 1.17 'd, 12H, ±CH3,
0
0
0
phosp, JCH3,CHˆ7 Hz); 1.30/1.33 's, 9H, ±CH3s, t-butyl-
benzoyl); 2.28/2.30, 'm, 3H, CH2±CN and H-20); 3.40±
3.45 'm, 2H, ±CHs, phosp); 3.44±3.66 'm, 2H, H-50and
H-500; 3.85 'm, 1H, H-30); 3.79/3.80's, 3H, ±OCH 3);
3.81/3.82 's, 3H, ±OCH3); 3.83/3.86 'm, 1H, ±CH2O,
phosp); 3.91/3.95 'm, 1H, ±CH2O, phosp); 4.40/4.42 'm,
1H, H-40); 5.20/5.25 'd, 1H, H-5, J5,6ˆ8 Hz); 6.20/6.23 's,
1H, H-10); 6.81±6.88, 7.23±7.30, 7.35±7.45 'm, 17H,
Ar1Ar±OCH3, DMT and Ar, t-butylbenzoyl); 7.98/8.02
'd, 1H, H-6, J5,6ˆ8 Hz). 31P NMR spectrum of 5a
'CDCl3): 'd, ppm) 147.20; 149.34. Anal. calcd for
C51H61O9N4P: C, 67.68; H, 6.79; N, 6.19. Found: C,
67.56; H, 7.23; N, 6.25.
3.1.5. 50-O-Dimethoxytrityl-ꢀ20S)-20-deoxy-20-C-methyl-
N3-ꢀ4-t-butylbenzoyl)uridine ꢀ4b). Compound 3b '2 g,
5 mmol) was dimethoxytritylated and worked up as
described for the synthesis of compound 4a above. Pure
compound 4b was obtained as a foam '73%). 13C NMR
spectrum of 4b 'CDCl3): 'd, ppm) 169.3 't-butylbenzoyl);
161.3 'C-4); 158.5 'CAr±OCH3, DMTr); 155.0' t-butyl-
benzoyl); 149.1 'C-2); 143.9 'Ar, DMTr); 141.2 'C-6);
135.1, 129.8, 128.5, 128.0, 127.3 'Ar, DMTr1butyl-
benzoyl); 131.6, 124.4 't-butylbenzoyl); 113.2 'Ar,
DMTr); 101.8 'C-5); 86.3 'C±Ar3, DMTr); 86.1 'C-10);
83.7 'C-40); 72.8 'C-30); 60.9 'C-50); 54.9 '±OCH3,
DMTr); 45.2 'C-20); 34.6, 31.5 't-butylbenzoyl), 10.9 '20-
3.1.7. 50-O-Dimethoxytrityl-ꢀ20S)-20-deoxy-20-C-methyl-
N3-ꢀ4-t-butylbenzoyl) uridine-30-O-ꢀ2-cyanoethyl N,N-
diisopropylphosphoramidite) ꢀ5b). Compound 4b
'3.23 g, 5 mmol) was phosphitylated, worked up and puri-
®ed according to the procedure used to prepare 5a above to

D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
7619
yield the title compound 5b pure '80%). 13C NMR spectrum
of 5b 'CDCl3): 'd, ppm) 169.0/168.9 't-butylbenzoyl);
161.5 'C-4); 158.7/158.6 'CAr±OCH3, DMTr); 154.9
't-butylbenzoyl); 149.2 'C-2); 144.4/144.2 'Ar, DMTr);
141.1 'C-6); 135.3/135.1, 130.5/130.3/130.2, 128.7/128.6/
128.4, 127.7/127.4/127.3 'Ar, DMTr1butylbenzoyl);
131.4, 124.5 't-butylbenzoyl); 117.3 'CN, phops); 113.3/
113.2 'Ar, DMTr); 102.0 'C-5); 86.5 'C±Ar3, DMTr);
86.3/86.2 'C-10); 83.9 'C-40); 72.8/72.5 'C-30); 60.8
'C-50); 57.9/57.6 '±OCH2, phosph); 55.3/55.2 '±OCH3,
DMTr); 45.4/45.2 'C-20); 43.8/43.5 'CHs, phosp); 34.6,
31.5 't-butylbenzoyl); 24.5 'CH3, phosp); 18.9/18.6 'CH2±
CN, phosp); 10.9/10.8 '20-methyl). 1H NMR spectrum of 5b
3.1.9. 30,50-O-ꢀTetraisopropyldisiloxane-1,3-diyl)-ꢀ20S)-
20-deoxy-20-C-methyluridine ꢀ7). A mixture of 2-nitro-
benzaldoxime '880mg, 5.31 mmol) and tetramethylguani-
dine '420ml, 3.32 mmol) in dry CH 3CN '10mL) was
poured over the solid compound 6 '2.02 g, 3.32 mmol)
under anhydrous conditions and stirred at room temperature
for 4 h. The solvent was removed in vacuo to produce a
syrup that was redissolved in dichloromethane '200 mL)
and washed with H2O '3£200 mL). The organic layer was
dried 'Na2SO4), ®ltered and evaporated in vacuo to give an
oil which was puri®ed by column chromatography on silica
gel using dichloromethane/ethyl acetate '10:1 v/v) as eluent.
The title compound was obtained in 93 % yield '1.50g).
The spectroscopic data of this compound were in agreement
with those previously reported.18. Anal. calcd for
C22H40O6N2Si2: C, 54.51; H, 8.32; N, 5.78. Found: C,
54.61; H, 8.35; N, 5.84.
0
'CDCl3): d 'ppm) 0.99/1.02 'd, 3H, ±CH3, JCH3,2 ˆ7 Hz);
1.09, 1.16, 1.17, 1.18 'd, 12H, ±CH3, phosp, JCH3,CHˆ7 Hz);
1.31/1.32 's, 9H, ±CH3s, t-butylbenzoyl); 2.29/2.31, 'm, 2H,
CH2±CN) 2.55/2.60'm, 1H, H-2 ); 3.39±3.44 'm, 2H,
0
±CHs, phosp); 3.45±3.65 'm, 2H, H-50and H-5±); 3.75
'm, 1H, H-30); 3.80's, 3H, ±OCH ); 3.81/3.83 's, 3H,
3.1.10. 50-O-Dimethoxytrityl-ꢀ20S)-20-Deoxy-20-C-methyl-
uridine ꢀ8). 30,50-O-'Tetraisopropyldisiloxane-1,3-diyl)-
'20S)-20-deoxy-20-C-methyluridine '7) '0.95 g, 1.8 mmol)
was desylilated as described above for the synthesis of
compound 3a. The crude product was dimethoxytritylated,
worked up and puri®ed as described for the synthesis of
compound 4a above. Pure compound 8 was obtained as a
foam '96%).13C NMR spectrum of 8 'CDCl3): 'd, ppm)
163.0'C-4); 158.4 'C Ar±OCH3, DMTr); 150.2 'C-2);
144.2 'Ar, DMTr); 141.2 'C-6); 135.2, 129.6, 128.5,
128.0, 127.4 'Ar, DMTr); 113.0 'Ar, DMTr); 101.4 'C-5);
86.4 'C±Ar3, DMTr); 86.1 'C-10); 82.9 'C-40); 72.7 'C-30);
60.4 'C-50); 54.9 '±OCH3, DMTr); 45.1 'C-20); 10.9 '20-
3
±OCH3); 3.83/3.85 'm, 1H, CH2O, phosp); 30.90/3.92 'm,
1H, ±CH2O, phosp); 4.35/4.40'm, 1H, H-4 ); 5.12/5.15
'd, 1H, H-5, J5,6ˆ8 Hz); 6.25/6.29 's, 1H, H-10); 6.80±
6.85, 7.25±7.30, 7.37±7.45 'm, 17H, Ar1Ar±OCH3,
DMT and Ar, t-butylbenzoyl); 8.05/8.09 'd, 1H, H-6,
J5,6ˆ8 Hz). 31P NMR spectrum of 5b 'CDCl3): 'd, ppm)
145.94; 148.55. Anal. calcd for C51H61O9N4P: C, 67.68;
H, 6.79; N, 6.19. Found: C, 67.60; H, 7.83; N, 6.23.
3.1.8. 30,50-O-ꢀTetraisopropyldisiloxane-1,3-diyl)-ꢀ20S)-
20-deoxy-20-C-methyl-4-O-ꢀ2-nitrophenyl) uridine ꢀ6). A
mixture of 30,50-O-'tetraisopropyldisiloxane-1,3-diyl)-
'20R)- and -'20S)-20-deoxy-20-C-methyluridine '1a and 1b)
',1:3,as determined by NMR, 10.1 g, 20.8 mmol) in 1,2-
dichloroethane '50mL) was treated with mesitylenesulfonyl
chloride '6.2 g, 28.5 mmol), dimethylaminopyridine
'930mg, 7.6 mmol) and triethylamine '13.4 mL, 96 mmol).
After stirring for 1 h, diazabicyclo [2.2.2] octane 'DABCO)
'424 mg, 2.8 mmol), 2-nitrophenol '7.92 g, 47 mmol) and
triethylamine '13.4 mL, 96 mmol) were added and stirring
was continued for three more hours. The resulting solution
was diluted with dichloromethane '100 mL) and poured in
1 M aqueous NaHCO3 '500 mL) with vigorous stirring. The
organic layer was washed with H2O '2£150mL), dried
'Na2SO4), ®ltered and evaporated in vacuo leaving a yellow
foam that was puri®ed by column chromatography on silica
gel eluting with petroleum ether/ethyl acetate/dichloro-
methane '10:1:10, v/v/v) to afford the 20S-isomer 6 '5.17 g,
8.5 mmol) and the 20R-isomer '1.3 g, 2.1 mmol). 13C NMR
spectrum of 6 'CDCl3): 'd, ppm) 170.2 'C-4); 154.8 'C-2);
145.0'C-6); 144.7, 141.3, 134.6, 126.3, 125.6, 125.2 '2-nitro-
1
methyl). H NMR spectrum of 8 'CDCl3): 'd, ppm) 1.00
0
0
'd, 3H, ±CH3, JCH3,2 ˆ7 Hz); 2.40'brs, 1H, OH-3 ); 2.55
'm, 1H, H-20); 3.18 'dd, 1H, H-50, J5 ,5 ˆ12 Hz, J5 ,4
ˆ
0
00
00
0
00
0
00
0
0
2 Hz); 3.50'dd, 1H, H-5 , J5 ,5 ˆ12 Hz, J5 ,4 ˆ2 Hz); 3.83
's, 6H, ±OCH3); 4.00 'm, 1H, H-40); 4.12 'dd, 1H, H-30,
0
0
0
0
J3 ,2 ˆJ3 ,4 ˆ6 Hz); 5.30'd, 1H, H-5, J5,6ˆ8 Hz); 6.29 'd,
1H, H-10, J1 ,2 ˆ8 Hz); 6.82 'd, 4H, Ar±OCH3, DMT,
Jˆ9 Hz); 7.20±7.35 'm, 9H, Ar1Ar±OCH3, DMT); 8.05
'd, 1H, H-6, J5,6ˆ8 Hz); 9.35 'brs, 1H, NH). Anal. calcd
for C31H32O7N2: C, 68.37; H, 5.92; N, 5.14. Found: C,
68.45; H, 6.00; N, 5.23.
0
0
3.1.11. 50-O-Dimethoxytrityl-ꢀ20S)-20-deoxy-20-C-methyl-
uridine-30-O-ꢀ2-cyanoethyl-N,N-diisopropylphosphora-
midite) ꢀ9). Compound
8
'2.26, 5 mmol) was
phosphitylated, worked up and puri®ed according to the
procedure used to prepare 5a above to yield the title
compound 9 pure '73%). 13C NMR spectrum of 9
'CDCl3): 'd, ppm) 162.9 'C-4); 158.8/158.7 'CAr±OCH3,
DMTr); 150.3 'C-2); 144.3/144.2 'Ar, DMTr); 141.1
'C-6); 135.3/135.1, 130.5/130.4/130.3, 128.9/128.6/128.4/
128.2, 127.9/127.4/127.3 'Ar, DMTr); 117.4/117.3 'CN,
phops);113.2 'Ar, DMTr); 101.8 'C-5); 87.0 'C±Ar3,
phenyl); 86.7 'C-10); 83.8 'C-40); 72.1 'C-30); 59.6 'C-50);
1
0
43.9 'C-20); 17.1±12.2 'isopropyls), 11.0'2 -methyl). H
NMR spectrum of 6 'CDCl3): 'd, ppm) 0 .80 ±1.10 'm, 28H,
±CHs and ±CH3s, disiloxane); 0.95 'd, 3H, ±CH3,
0
0
0
JCH3,2 ˆ7 Hz); 2.70'm, 1H, H-2 ); 3.80±4.25 'm, 4H, H-3 ,
DMTr); 86.4/86.2 'C-10); 83.3 'C-40); 76.2/76.0'C-3 );
0
H-40, H-50 and H-500); 6.19 'd, 1H, H-5, J5,6ˆ8 Hz); 6.32 'd,
60.7 'C-50); 58.0/57.8 '±OCH2, phosph); 55.3/55.2
'±OCH3, DMTr); 44.8/44.7 'C-20); 43.9/43.3/43.2 'CHs,
phosp); 24.6/24.5 'CH3, phosp); 17.4/17.3 'CH2±CN,
phosp); 11.6/11.4 '20-methyl). 1H NMR spectrum of 9
1H, H-10, J1 ,2 ˆ6 Hz); 7.27 'dd, 1H, H-6, nitrophenyl,
J6,5ˆ9 Hz, J6,4ˆ1 Hz); 7.4 'ddd, 1H, H-4, nitrophenyl,
J4,5ˆ9 Hz, J4,3ˆ9 Hz, J6,4ˆ1 Hz); 7.65 'ddd, 1H, H-5, nitro-
phenyl, J4,5ˆ9 Hz, J5,6ˆ9 Hz, J5.3ˆ1 Hz); 8.13 'dd, 1H, H-3,
nitrophenyl, J4,3ˆ9 Hz, J5,3ˆ1 Hz); 8.25 'd, 1H, H-6,
J5,6ˆ8 Hz). Anal. calcd for C28H43O8N3Si2: C, 55.51; H,
7.15; N, 6.94. Found: C, 55.63; H, 7.25; N, 6.84.
0
0
0
'CDCl3): d 'ppm) 1.00/1.05 'd, 3H, ±CH3, JCH3,2 ˆ7 Hz);
1.06, 1.13, 1.17, 1.18 'd, 12H, ±CH3, phosp, JCH3,CHˆ7 Hz);
2.28/2.29 'm, 2H, CH2±CN); 2.60/2.63 'm, 1H, H-20);
3.39±3.46 'm, 2H, ±CHs, phosp); 3.48±3.61 'm, 2H, H-50

7620
D. O. Cicero et al. / Tetrahedron 57 92001) 7613±7621
0
and H-500); 3.70'm, 1H, H-4 ); 3.81 's, 3H, ±OCH3); 3.80/
3.79 's, 3H, ±OCH3); 3.80/3.85 'm, 1H, CH2O, phosp);
3.91/3.96 'm, 1H, CH2CO, phosp); 4.33/4.41 'm, 1H, H-
30); 5.10/5.16 'd, 1H, H-5, J5,6ˆ8 Hz); 6.29/6.30's, 1H,
H-10); 6.82±6.87, 7.22±7.30, 7.35±7.42 'm, 13H, DMT);
8.03/8.07 'd, 1H, H-6, J5,6ˆ8 Hz); 8.50'brs, 1H, NH). 31P
NMR spectrum of 9 'CDCl3): 'd, ppm) 145.87; 148.51.
Anal. calcd for C40H49O8N4P: C, 64.50; H, 6.63; N, 7.52.
Found: C, 64.55; H, 6.57; N, 7.58.
'brs, 1H, OH-30); 3.54 'm, 2H, H-50 and H-500); 3.75 's, 6H,
±OCH3); 3.81 'm, 1H, H-40); 4.27 'm, 1H, H-30); 6.21 's,
1H, H-10); 6.84 'd, 4H, Ar±OCH3, DMT, Jˆ9 Hz); 6.98 'd,
1H, H-5, J5,6ˆ8 Hz); 7.18±7.40'm, 9H, Ar 1Ar±OCH3,
DMT); 8.02 'd, 1H, H-6, J5,6ˆ8 Hz); 8.30'brs, 1H, NH).
Anal. calcd for C35H39O7N3: C, 64.50; H, 6.41; N, 6.85.
Found: C, 64.57; H, 6.46; N, 6.89.
3.1.14. 50-O-Dimethoxytrityl-ꢀ20S)-20-deoxy-20-C-methyl-
N4-isobutyrylcytidine-30-O-ꢀ2-cyanoethyl-N,N-diiso-
propylphosphoramidite) ꢀ12). Compound 11 '3.01 g,
5 mmol) was phosphitylated, worked up according to the
procedure used to prepare 5a above. The crude product
was puri®ed by column chromatography on silica gel
eluting with 30% ethyl acetate in dichloromethane contain-
ing 1% of triethylamine to yield the title compound 12 pure
'72%). 13C NMR spectrum of 12 'CDCl3): 'd, ppm) 177.0
'CO, isobutyryl); 162.1 'C-4); 158.8/158.7 'CAr±OCH3,
DMTr); 155.3 'C-2); 145.6 'Ar, DMTr); 144.0'C-6);
135.5/135.4, 130.4/130.4/130.3/130.2, 128.6/128.5, 127.9/
127.3/127.1 'Ar, DMTr); 117.3 'CN, phops);113.2 'Ar,
DMTr); 95.9 'C-5); 87.6/87.3 'C±Ar3, DMTr); 86.9
'C-10); 83.3 'C-40); 76.9 'C-30); 61.3/61.1 'C-50); 58.1/
3.1.12. 30,50-O-ꢀTetraisopropyldisiloxane-1,3-diyl)-ꢀ20S)-
20-deoxy-20-C-methyl-N4-isobutyryl cytidine ꢀ10). 30,50-
O-'Tetraisopropyldisiloxane-1,3-diyl)-'20S)-20-deoxy-20-C-
methyl-4-O-'2-nitrophenyl) uridine '6) '2,04 g, 3.36 mmol)
in dry tetrahydrofuran '120mL) was treated at room
temperature with ammonia '10bar) in a stainless steel
bomb for four days. The solvent was evaporated in vacuo,
the residue was dissolved in dry pyridine '30mL) and the
solution cooled in an ice bath. Isobutyrylchloride '1.05 mL,
10.12 mmol) was added with stirring and exclusion of
moisture and after two hours the reaction was completed.
The reaction mixture was evaporated to dryness, redissolved
in dichloromethane '100 mL) and washed with 10%
aqueous NaHCO3 '2£100 mL) and H2O '2£100 mL). The
organic phase was dried 'Na2SO4), ®ltered and evaporated
to dryness. The residue was puri®ed by column chromato-
graphy using dichloromethane/MeOH '99:1, v/v) as eluant
to afford pure compound 10 in 88% yield '1.62 g). 13C NMR
spectrum of 10 'CDCl3): 'd, ppm)177.5 'CO isobutyryl);
162.7 'C-4); 155.4 'C-2); 144.9 'C-6); 96.4 'C-5); 86.7
'C-10); 84.1 'C-40); 72.3 'C-30); 59.7 'C-50); 44.1 'C-20);
58.0'±OCH , phosph); 55.2 '±OCH3, DMTr); 44.6/44.5
2
'C-20); 43.3/43.2/43.1 'CHs, phosp); 36.7 'CH, isobutyryl);
24.6/24.5/24.4 'CH3, phosp); 20.3/20.0 'CH3, isobutyryl);
1
19.0'CH 2±CN, phosp); 11.8/11.6 '20-methyl). H NMR
spectrum of 12 'CDCl3): 'd, ppm) 0.93/0.94 'd, 3H,
0
±CH3, JCH3,2 ˆ7 Hz); 1.00, 1.13 1.16 'd, 12H, ±CH3,
phosp, JCH3,CHˆ7 Hz); 1.20'm, 6H, ±CH 3, isobutyryl);
2.29/2.30'm, 2H, CH ±CN); 2.58 'm, 1H, H-20); 2.79 'm,
2
0
1H, ±CH, isobutyryl); 3.41±3.47 'm, 2H, ±CHs, phosp);
3.48±3.58 'm, 2H, H-50 and H-500); 3.61/3.63 'm, 1H,
36.1, 19.0'isobutyryl); 17.3±12.5 'isopropyls), 11.3 '2 -
methyl). 1H NMR spectrum of 10 'CDCl3): 'd, ppm)
0.80±1.20 'm, 28H, ±CHs and ±CH3s, disiloxane); 0.95
H-40); 3.80's, 3H, ±OCH ); 3.82 's, 3H, ±OCH3); 3.75/
3.87 'm, 1H, CH2O, phops); 3.97/4.04 'm, 1H, CH2O,
3
0
'd, 3H, ±CH3, JCH3,2 ˆ7 Hz); 1.28 'd, 6H, ±CH3, isobutyryl,
0
phosp); 4.21/4.30'm, 1H, H-3 ); 6.39/6.41 'd, 1H, H-10,
0
JCH,CH3ˆ7 Hz); 2.50'm, 1H, H-2 ); 2.65 'q, 1H, ±CH,
isobutyryl, JCH,CH3ˆ7 Hz); 3.65±4.20'm, 3H, H-4 , H-50
0
0
0
J1 -2 ˆ7 Hz); 6.97/7.04 'd, 1H, H-5, J5,6ˆ7 Hz); 6.82±
6.88, 7.25±7.35, 7.38±7.42 'm, 13H, DMT); 8.29 'brs,
1H, NH); 8.34/8.40'd, 1H, H-6, J5,6ˆ7 Hz).
and H-500); 4.25 'm, 1H, H-30); 6.18 's, 1H, H-10); 6.95 'd,
1H, H-5, J5,6ˆ8 Hz); 8.00 'd, 1H, H-6, J5,6ˆ8 Hz); 9.58 'brs,
1H, NH). Anal. calcd for C26H47O6N3Si2: C, 56.39; H, 8.55;
N, 7.59. Found: C, 56.44; H, 8.51; N, 7.49.
31P NMR spectrum of 12 'CDCl3): 'd, ppm) 145.88; 148.39.
Anal. calcd for C44H56O8N5P: C, 64.93; H, 6.94; N, 8.60.
Found: C, 64.98; H, 7.02; N, 8.66.
3.1.13. 50-O-Dimethoxytrityl- ꢀ20S)-20-Deoxy-20-C-methyl-
N4-isobutyrylcytidine ꢀ11). 30,50-O-'Tetraisopropyldisi-
loxane-1,3-diyl)-'20S)-20-deoxy-20-C-methyl-N4-isobutyryl
cytidine '10) was desilylated and worked up according to
procedures used to prepare 3a and the crude product were
dimethoxytritylated according to procedures given for the
synthesis of 4a. The residue was puri®ed by column chro-
matography which was ®rst washed with 50% ethyl acetate
in petrol and then pure compound 11 was eluted by using
2% MeOH in dichloromethane '79%).
Acknowledgements
Â
We thank Consejo Nacional de Investigaciones Cientõ®cas y
Tecnicas-Argentina, Universidad Nacional de Quilmes-
Â
Argentina and International Centre for Genetic Ingeneering
and Biotechnology-Trieste, Italy for ®nancial support.
13C NMR spectrum of 11 'CDCl3): 'd, ppm) 177.1 'CO
isobutyryl); 162.3 'C-4); 158.5 'CAr±OCH3, DMTr); 155.6
'C-2); 143.2 'Ar, DMTr); 145.7 'C-6); 135.1, 129.7, 128.,
128.0, 127.4 'Ar, DMTr); 113.0 'Ar, DMTr); 96.4 'C-5);
87.1 'C-10); 86.4 'C±Ar3, DMTr); 83.6 'C-40); 73.7 'C-30);
61.3 'C-50); 54.9 '±OCH3, DMTr); 45.9 'C-20); 35.9, 18.8
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