2′-C-Methyluridine phosphoramidite: A new building block for the preparation of RNA analogues carrying the 2′-hydroxyl group

Article abstract of DOI:10.1016/S0040-4020(01)00484-7

A new stereoselective synthesis of 2′-C-methyluridine was carried out. The preparation of the corresponding protected phosphoramidite suitable for the automated synthesis of oligonucleotides, including the regioselective protection of the 2′-hydroxyl grou

Full text of DOI:10.1016/S0040-4020(01)00484-7

TETRAHEDRON
Pergamon
Tetrahedron 57 ?2001) 5707±5713
20-C-Methyluridine phosphoramidite: a new building block for the
preparation of RNA analogues carrying the 20-hydroxyl group
a
b
c
a
Â
Mariana Gallo, Edith Monteagudo, Daniel O. Cicero, Hector N. Torres
and Adolfo M. Iribarrena,b,p
a
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Instituto de Investigaciones en Ingenierõa Genetica y Biologõa Molecular, Vuelta de Obligado 2490 2P, 1428Buenos Aires, Argentina
b
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Universidad Nacional de Quilmes, Roque Saenz PenÄa 180, Bernal 1876, Pcia. de Buenos Aires, Argentina
cIstituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km. 30,600, 00040 Pomezia, Rome, Italy
Received 21 November 2000; accepted 2 May 2001
AbstractÐA new stereoselective synthesis of 20-C-methyluridine was carried out. The preparation of the corresponding protected phos-
phoramidite suitable for the automated synthesis of oligonucleotides, including the regioselective protection of the 20-hydroxyl group, is
described here. This new modi®ed nucleotide is expected to generate RNA analogues potentially useful in applications where proper RNA
folding is required since the 20-hydroxyl is conserved. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
moieties by either deoxynucleotides7 or modi®ed nucleo-
tides without this group.8,9 Such ®ndings have been
supported by X-ray diffraction analysis which evidenced
that 20-hydroxyls are involved in hydrogen bondings
responsible for the stabilization of tertiary structures present
in the catalytic core.10,11
The improvements achieved in solid phase synthesis of
oligonucleotides have enabled the development of new
research areas in molecular and structural biology as well
as the search of new therapeutic principles. Progress
concerning RNA synthesis has been delayed due
to problems related to the selective protection of the
20-hydroxyl group, slower rate of coupling and the inherent
fragility of RNA to hydrolytic and enzymatic degradation.
However, recent advances1,2 have allowed the preparation
of proper quantities of RNA suitable for structural studies,
the search of new aptamers and the generation and evalua-
tion of synthetic ribozymes.
Taking into account these facts, we propose a sugar modi-
®ed analogue, namely 20-C-methylnucleotide, which is
expected to provide when substituting a particular ribo-
nucleotide, a 20-hydroxyl group able to make similar inter-
actions than those displayed by the natural moiety. Previous
preparations12,13 of 20-C-methyluridine involved the syn-
thesis of the modi®ed sugar and its subsequent coupling to
the nucleobase which is often a lengthy and/or low yielding
procedure. Another strategy dealt14 with the addition of
organometallic compounds to 20-ketonucleosides but,
since the reaction proceeded from the a-face of the sugar
moiety, the major product obtained through this route was
the 20-C-methylarabinonucleoside. To overcome these
limitations, we report in this paper the stereoselective
preparation of 20-C-methyluridine, the subsequent protec-
tion of the sterically hindered 20-hydroxyl group and the
®nal generation of the corresponding phosphoramidite
suitable for solid phase synthesis.
Since oligoribonucleotides are particularly prone to the
attack of nucleases present both in serum and inside cells,
the development of chemical modi®cations is essential for
their biological applications. 20-O-Alkyloligonucleotides3,4
and other RNA mimics tried so far, such as 20-amine5 and
20-¯uoroligonucleotides,6 lack the 20-hydroxyl group, which
has shown to be essential for RNA folding and, conse-
quently, fundamental for the development of modi®ed
ribozymes, ribozyme inhibitors and RNA aptamers. In
the particular case of ribozymes, the presence of the
20-hydroxyl group in de®ned positions has proved to be
indispensable for maintaining the activity. This requirement
has been analysed through speci®c replacement of RNA
The con®guration at C-20 of this nucleoside was established
using NMR measurements. It is known that the con®gu-
ration determined by NMR, is always highly correlated to
the conformation,15 and both issues must be resolved simul-
taneously. In the present case, the lack of a hydrogen
attached to C-20 reduced the available 1H±1H coupling
constants to just one ?J?H30H40)), and therefore we decided
Keywords: RNA analogues; sugar conformation; 20-C-alkylnucleotides.
Corresponding author. Address: Instituto de Investigaciones en Ingenierõa
Genetica y Biologõa Molecular, Vuelta de Obligado 2490 2P, 1428
Buenos Aires, Argentina. Tel.: 15411-4783-2871; fax: 15411-4786-
8578; e-mail: airi@dna.uba.ar
p
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3
to measure also the J?1H±13C) coupling constants. To this
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020?01)00484-7

5708
M. Gallo et al. / Tetrahedron 57 72001) 5707±5713
Scheme 1. ?i) TIPDSCl2, Py; ?ii) MeOH; ?iii) PCC, Ac2O, CH2Cl2; ?iv) MePh3PBr, THF, BuLi; ?v) Et2O, NH4Cl; ?vi) OsO4, N-methylmorpholine-N-oxide,
t-BuOH, THF; ?vii) NaHSO3 1 M, EtOAc; ?viii) TsCl, Py; ?ix) NaBH4, DMF; ?x) NH4Cl; ?xi) TBAF, THF.
purpose we used a newly developed pulse sequence,16 which
shows high sensitivity and is especially well suited for these
measurements in natural abundance.
tetroxide21 produced the corresponding diol as a diastereo-
meric mixture ?97:3 as determined by NMR). At this stage,
the con®guration at C-20 of the major diasteromer was tenta-
tively assigned as in compound 5, based on the fact that the
attack by osmium tetroxide is more likely to occur from the
less hindered a-face of the sugar ring. This hypothesis was
con®rmed on a later step of the synthesis ?see below).
Subsequent treatment with an excess of p-toluenesulfonyl
chloride yielded the primary tosylated derivative which
could be resolved by column chromatography obtaining
pure 6 ?60%). Finally, reaction of 6 with sodium boro-
hydride22 gave the disiloxane bridged 20-C-methyluridine
?7).
2. Results and discussion
Scheme 1 shows the synthetic route followed for the
preparation of 20-C-methyluridine ?8). Starting from uridine
the regioselective protection of the 30 and 50 hydroxyls was
carried out by using 1,3-dichloro-1,1,3,3-tetraisopropyldi-
siloxane.17 Intermediate 3 could be obtained from 2 by
using either chromium trioxide/pyridine/acetic anhydride18
or dimethyl sulfoxide/oxalyl chloride19 affording similar
yields ?80%). Wittig reaction of 3 following standard pro-
cedures20 gave the corresponding 20-methylene derivative
4 ?66%), which after catalytic oxidation with osmium
Compound 7 was desilylated with tetrabutyl ammonium
¯uoride, to yield 8, which was used to perform the NMR
measurements in order to assign unambiguously the con-
®guration at C-20. For this purpose we measured all the
three-bond coupling constants that are sensitive to both
con®guration at C-20 and the conformation of the molecule.
This includes the coupling constant between H-30 and H-40,
Table 1. Selected three-bond coupling constants ?in Hz) of 20-C methyl-
uridine ?8) in D2O
C10
C20
C30
C40
CH3
H40
1
and a number of heteronuclear H±13C coupling constants.
H10
H30
H40
±
±
3.8
±
±
1.0
±
±
,0.5
0.5
±
±
9.5
±
Table 1 shows the coupling constants measured for
compound 8 in D2O. The relatively high values measured
for J?H30H40) ?9.5 Hz) and for J?H10C30) ?3.8 Hz), as well
±
,0.5
±
,0.5

M. Gallo et al. / Tetrahedron 57 72001) 5707±5713
5709
Scheme 2. ?i) Ac2O, Py; ?ii) DHP, p-TsOH, dioxane; ?iii) NH3, MeOH; ?iv) DMTrCl, DMAP, TEA, Py; ?v) iPr2NEt, CH2Cl2,
[?CH3)2CH]2NP?Cl)OCH2CH2CN.
as the small value for J?H40C20) ?,0.5 Hz), are all clear
indications that 8 exists in solution mainly in the C-30
endo conformation. The small value obtained for
J?H30CH3) ?0.5 Hz) is only compatible with a cis orientation
of the CH3 group respect to H-30, since a relatively high
value ?,4 Hz) would be expected in the case of these
groups being trans diaxial, and thus establishing the
con®guration at C-20.
50-hydroxyls must be previously capped and therefore the
20-protecting group of choice should be stable under the
required conditions for deblocking the 30 and 50 positions.
tert-Butyldimethylsilyl ether is the protecting group used in
routine RNA synthesis but attempts to obtain the corre-
sponding derivative of 7 using either standard ?tert-butyldi-
methylsilyl chloride/triethylamine) or special conditions for
steric hindered alcohols ?tert-butyldimethylsilyl tri¯ate/
crown ether/potassium hydride)25 were unsuccessful. In
order to assess the in¯uence of the bulky 30,50-disiloxane
bridge, the same reaction was carried out on the crude 30,50-
di-O-acetyl derivative 9 ?Scheme 2). This compound was
prepared by acetylation with acetic anhydride/pyridine
?70% from 7) of the free nucleoside 8. Surprisingly,
2 equiv. of the acylating reagent had to be used in order to
avoid peracetylation. 20-tert-Butyldimethylsilylation of 9
did not proceed even after long reaction times ?5 days)
and using 3.5 equiv. of silylating agent.
2D NOESY experiments on nucleoside 8 also corroborate
these results. In fact, a strong NOE was observed between
H-6 and H-30, indicating both a C-30 endo conformation for
the sugar and an anti orientation for the base. The NOE of
the CH3 group is stronger with the H-30 than with the H-10,
a clear indication of a cis relationship between the
20-C-methyl group and H-30.
The C-30 endo conformation, or RNA-like conformation,
adopted by compound 8 con®rms the fact that positioning
the 20-methyl group in a pseudo-equatorial orientation is the
main driving force in de®ning the preferred conformation of
this type of nucleosides,23 as it was already demonstrated for
20-C-alkyl-20-deoxy analogues.24 Additionally, compound 8
exhibits the most favourable spatial arrangement of the
different substituents of the sugar ring: the bulky CH3
group is positioned pseudo-equatorially, the 20-OH group
shows a positive gauche effect with the O-40, the C-50
side chain is pseudo-equatorial, and the base is pseudo-
axial, satisfying the weak anomeric effect.
Another suitable 20-protecting group, due to its chemical
properties and size, is tetrahydropyranyl ether. Reaction of
7
with 3,4-dihydro-2H-pyran/p-toluenesulphonic acid
monoydrate26 did not afford the expected ether even when
a large excess of reagent and higher temperatures were
employed. Finally, the desired product 10 was obtained
when the diacetylated compound 9 was used as the starting
material ?80%). This reaction did not go to completion and
degradation of the starting material was visible by TLC.
Therefore, molar ratio of reagents and reaction time were
optimised and the remaining compound 9 could be recycled
after isolation by column chromatography. Expected
compound 11 was obtained by treating 10 with methanolic
ammonia ?100%). In order to assess the removal of the THP
group during the deprotection conditions used in RNA
synthesis, compound 11 was treated with 0.01N HCl ?pH
2.0) at rt. As expected, the behaviour of compound 11 is
similar to that of the 20-THP-protected uridine and the
observed t1/2 is around 30 min, monitored by TLC.
The next step in the route towards a suitable derivative for
oligonucleotide solid phase synthesis involved the protec-
tion of the 20-hydroxyl of 7. This protecting group must
ful®l general requirements such as stability to trichloro-
acetic acid treatment and towards oxidation conditions
?iodine/water/pyridine) and quantitative removal at the
end of the oligonucleotide synthesis. In order to derivatize
regioselectively the tertiary 20-hydroxyl group, 30- and

5710
M. Gallo et al. / Tetrahedron 57 72001) 5707±5713
Finally, the appropriate building block 13 for solid phase
synthesis was prepared following standard procedures.
Column chromatography was performed by using Silica gel
20±45, 6±35 mm ?Amicon) and Silica Gel 60F 230±400
mesh ?Merck). Analytical thin layer chromatography
?TLC) was conducted on precoated Merck Silica gel
60F254. Components were located by observation of the
plates under UV light, and dimethoxytrityl products were
detected as orange spots by treating the plates with H2SO4/
EtOH 8:2 and heating.
We expect this building block to provide a 20-hydroxyl
group able of mimicking the interactions observed in natural
RNA structures. This assumption is based on our NMR
analysis which revealed that the preferred conformation
adopted by this nucleoside ?C-30 endo) is similar to that
found in RNA, suggesting that both structures position the
20-hydroxyl group in an equivalent orientation.
1
13C-, H- and 31P NMR spectra were recorded in Cl3CD or
DMSO-d6 ?Aldrich), on a 200 and 500 MHz AM Bruker
1
The generation of resistant oligonucleotides carrying
20-hydroxyl groups can be a challenging task in the ®eld
of ribozymes,27,28 since the presence of 20-hydroxyl groups
in speci®c positions has been shown to be essential for the
activity of these molecules.7±9 Alternatively, these modi®ed
nucleic acids fragments can be regarded as potential
therapeutic agents based on short, resistant and high af®nity
inhibitors of ribozymes.29,30 In this case, tertiary interactions
between ribozymes and 20-hydroxyl groups of the inhibitor
are involved in the mechanism of inhibition. Combinatorial
technologies is a new ®eld in which oligonucleotides have
proved their utility. RNA molecules have demonstrated to
have strong af®nity and speci®city towards proteins and
other targets.31,32 Again, chemical modi®cations which
confer enzymatic stability and provide the appropriate
folding are essential for the development of RNA-based
aptamers in therapeutics and diagnostics.
equipment using tetramethylsilane ?0.00 ppm, H NMR)
and Cl3CD ?76.95 ppm, 13C NMR) as internal standards
and H3PO4 ?0.00 ppm, 31P NMR) as external standard.
Chemical shifts ?d, ppm) quoted in the case of multiplets
are measured from the approximated centre.
1H±13C coupling constants were measured applying the
phase-sensitive HMBC experiment described in Ref. 16,
on a 20 mM sample of 8 in D2O, recorded at 400 MHz
and 258C on a Bruker Avance spectrometer equipped with
z-shielded gradient inverse detection probe. The acquisition
times t2 and t1 were 1.3 s ?spectral width 6410 Hz, 8K
complex data points) and 3 ms ?spectral width 20,100 Hz,
64 real data points), respectively. The relaxation delay was
2 s; 80 scans were accumulated per t1 increment.
Compounds
30,50-O-?tetraisopropyldisiloxane-1,3-diyl)-
uridine ?2)17, 30,50-O-?tetraisopropyldisiloxane-1,3-diyl)-
20-oxouridine ?3)18 and 30,50-O-?tetraisopropyldisiloxane-
1,3-diyl)-20-methyleneuridine ?4)20 were synthesized as
previously described, affording satisfactory 1H and 13C
NMR spectra.
At present, the synthesis of chimeric oligonucleotides
containing DNA, RNA or modi®ed nucleotides and the
modi®cation here reported is being undertaken. Deprotec-
tion and puri®cation conditions are also being evaluated.
Further biochemical ?activity of chemically modi®ed
ribozymes) and structural analysis ?NMR) will be carried
out in order to verify the hypothesis that 20-C-methylnucleo-
tides mimic the interactions occurring in natural RNA
structures.
3.1.1.
30,50-O-.Tetraisopropyldisiloxane-1,3-diyl)-20-
hydroxymethyleneuridine .5). Compound 4 ?48.3 g,
100.3 mmol, MW: 482) was dissolved in a mixture of THF
?250 mL), t-butanol ?250 mL) and water ?75 mL);
N-methylmorpholine-N-oxide ?15 g; 128 mmol, 1.2 equiv.)
and OsO4 ?10 mL of a 2.5% w/v, solution in t-butanol;
1 mmol, 0.01 equiv.) were added under ice cooling. The
mixture was stirred for 5 days at 48C and then quenched
with 1 M aqueous NaHSO3 and extracted with ethyl acetate
?2£500 mL). The organic layer was washed with water,
dried ?Na2SO4) and evaporated to obtain crude 5 ?51.7 g),
which was used for the next reaction without further puri®-
cation ?100%, Rf 0.34, petroleum ether/ethyl acetate, 1:2).
13C NMR ?CDCl3) d ?ppm): 12.1, 12.7, 12.8, 13.3 ?isopropyl
CHs); 16.7±17.3 ?isopropyl CH3s); 59.8 ?C-50); 61.4
?±CH2OH); 67.8 ?C-30); 80.1 ?C-20); 81.0 ?C-40); 90.3
?C-10); 101.5 ?C-5); 141.2 ?C-6); 151.3 ?C-2); 164.4 ?C-4).
1H NMR ?CDCl3) d ?ppm):00.80±1.20 ?m, CH?CH3)2); 3.45
3. Experimental
3.1. General methods
Ethyl acetate ?EtOAc), tetrahydrofuran ?THF), methanol,
petroleum ether, triethylamine ?TEA), pyridine ?Py),
dimethylformamide ?DMF), t-butanol ?t-BuOH), diethyl
ether ?Et2O) and HPLC grade methylene chloride
?CH2Cl2), were supplied by J. T. Baker. THF was dried by
re¯uxing over sodium metal using benzophenone as indi-
cator of dryness and then distilled at atmospheric pressure.
DMF was dried by heating at 1008C with calcium hydride
and then distilled under reduced pressure. Pyridine, dioxane
and triethylamine were dried by heating, under re¯ux, with
calcium hydride and distilled at atmospheric pressure.
0
0
0
00
?d, 1H, J6 ,6 ˆ10.1 Hz, H-6 ); 3.65 ?m, 2H, OH-2 , OH-6 );
00
0
0
00
3.90 ?d, 1H, J6 ,6 ˆ10.1 Hz, H-6 ); 3.95±4.40 ?m, 4H, H-3 ,
H-40, H-50 and H-500); 5.60 ?d, 1H, J5,6ˆ7.8 Hz, H-5); 6.00
?s, 1H, H-10); 7.81 ?d, 1H, J5,6ˆ7.8 Hz, H-6); 10.35 ?br s,
1H, NH). Anal. Calcd for C22H40N2O8Si2: C, 51.14; H, 7.80;
N, 5.42. Found: C, 51.21; H, 7.83; N, 5.39.
All other reagents were commercially available ?Fluka,
Aldrich) and uridine was of the best analytical grade
?Pharma Waldhof). All moisture-sensitive reagents were
transferred via a syringe under positive nitrogen pressure
or argon atmosphere, moisture-sensitive reactions were
carried out under positive pressure of nitrogen gas.
3.1.2. 30,50-O-.Tetraisopropyldisiloxane-1,3-diyl)-20-p-
toluenesulfonylmethyleneuridine .6). To a solution of
compound 5 ?51.7 g, 100.3 mmol, MW: 516) in dry pyridine
?130 L), p-toluenesulfonyl chloride ?TsCl) ?38 g, 200 mmol,

M. Gallo et al. / Tetrahedron 57 72001) 5707±5713
5711
2.0 equiv.) was added and the mixture stirred over night.
Then, a second portion of p-toluenesulfonyl chloride
?56 g, 294 mmol, 3 equiv.) was added and the stirring was
continued for 3 h. The solvent was removed in vacuo, the
residue dissolved in ethyl acetate and washed with water,
dried ?Na2SO4) and stripped to obtain the crude. After
puri®cation by silica gel column chromatography eluting
with petroleum ether/ethyl acetate ?1:2) pure 6 was obtained
as a foam, ?60%, Rf 0.80, petroleum ether/ethyl acetate, 1:2).
13C NMR ?CDCl3) d ?ppm): 12.6, 12.7, 12.9, 13.6 ?isopropyl
CHs); 16.7±17.3 ?isopropyl CH3s); 21.6 ?tosyl CH3); 60.9
?C-50); 66.5 ?±CH2OTos); 72.5 ?C-30); 79.3 ?C-20); 81.8
?C-40); 89.5 ?C-10); 102.4 ?C-5); 127.9 ?tosyl C-2); 129.7
?tosyl C-3); 131.6 ?tosyl C-4); 144.8 ?C-6); 145.2 ?tosyl
C-1); 151.8 ?C-2); 163.1 ?C-4). 1H NMR. ?CDCl3) d
?ppm): 0.80±1.20 ?m, CH?CH3)2); 2.35 ?s, 3H, tos0yl CH3);
NMR ?DMSO-d6) d ?ppm): 1.00 ?s,0 3H, ±CH3); 3.79 ?dd,
0 00 0 0 0 0
1H, J5 ,5 ˆ11.8 Hz, J5 ,4 ˆ2Hz, H-5 ); 3.84 ?d, 1H, J3 ,4
ˆ
9.5 Hz, H-30); 3.98 ?m, 2H, H-40 and H-500); 5.05 ?br s, 2H,
OH-30 and OH-20); 5.19 ?br s, 1H, OH-50); 5.84 ?d, 1H,
J5,6ˆ8.2 Hz, H-5); 5.95 ?s, 1H, H-10); 7.86 ?d, 1H, J5,6ˆ
8.2 Hz, H-6). Anal. Calcd for C10H14N2O6: C, 46.51; H,
5.47; N, 10.85. Found: C, 46.54; H, 5.44; N, 10.86.
3.1.5. 30,50-Di-O-acetyl-20-C-methyluridine .9). The syrup
obtained in the previous reaction was suspended in 280 mL
of pyridine and acetic anhydride ?Ac2O) ?9.5 mL,
100 mmol, 1.8 equiv.) was added. The mixture was stirred
overnight and the then 1 mL ?0.2 equiv.) of acylating
reagent were added. After an hour the mixture was cooled
?08C), quenched with 28 mL of methanol and concentrated
in vacuo. The colourless oil obtained was dissolved in
CH2Cl2 ?280 mL) and poured into NaHCO3 ?5£56 mL).
The organic phase dried ?Na2SO4) was evaporated and the
product was puri®ed by silica gel column chromatography,
eluting with CH2Cl2:methanol ?98:2). Pure 9 was obtained
as a foam ?70% from 7, Rf 0.40, CH2Cl2:methanol, 95:5).
13C NMR ?CDCl3) d ?ppm): 20.6a ?±CH3); 20.7a ?acetyl
±CH3s); 62.1 ?C-50); 73.9 ?C-30); 78.1 ?C-20); 78.3 ?C-40);
91.6 ?C-10); 102.7 ?C-5); 139.4 ?C-6); 151.0 ?C-2); 163.3
?C-4); 170.1 ?CO); 170.2 ?CO). 1H NMR ?CDCl3) d ?ppm):
1.20 ?s, 3H, ±CH3); 2.12 ?s, 3H, CH3CO); 2.17 ?s, 3H,
0
00
3.42 ?s, 1H, OH); 3.82 ?d, 1H, J6 ,6 ˆ10.8 Hz, H-6 ); 3.90±
4.50 ?m, 5H, H-30, H-40, H-50, H-500 and H-600); 5.62 ?d, 1H,
J5,6ˆ8.0 Hz, H-5), 5.98 ?s, 1H, H-10); 7,20 ?d, 2H,
JOˆ7.4 Hz, phenyl); 7.60 ?d, 1H, J5,6ˆ8.0 Hz, H-6); 7.70
?d, 2H, JOˆ7.4 Hz, phenyl); 10.35 ?br s, 1H, NH). Anal.
Calcd for C29H46N2O10SSi2: C, 51.92; H, 6.91; N, 4.18.
Found: C, 51.97; H, 6.92; N, 4.21.
3.1.3. 30,50-O-.Tetraisopropyldisiloxane-1,3-diyl)-20-C-
methyluridine .7). Compound 6 ?40.4 g, 60 mmol, MW:
670) was dissolved in 615 mL of anhydrous DMF, then
NaBH4 ?11.75 g, 310 mmol, 5.0 equiv.) was added and the
mixture kept at rt for 2 h. The reaction was quenched by
careful dropwise addition of NH4Cl ?saturated solution) on
the chilled mixture and extracted with ethyl acetate
?2£800 mL). The combined organic layers were washed
with brine ?5£1.4 L), dried ?Na2SO4) and evaporated to
obtain the crude. The product was puri®ed by ¯ash chroma-
tography, eluting with petroleum ether/ethyl acetate ?1:1)
yielding pure 7 as a foam. ?93%, Rf 0.70, petroleum ether/
ethyl acetate, 4:6). 13C NMR ?CDCl3) d ?ppm): 12.5, 12.8,
13.4, 13.6 ?isopropyl CHs); 16.8±17.3 ?isopropyl CH3s);
20.4 ?±CH3); 59.8 ?C-50); 72.7 ?C-30); 78.9 ?C-20), 81.6
?C-40); 90.4 ?C-10); 102.3 ?C-5); 139.6 ?C-6); 150.5 ?C-2);
CH3CO); 3.25 ?s, 1H, OH-20); 4.35±4.45 ?m, 3H, H-40, 50
0
and 500); 4.92 ?d, 1H, J3 ,4 ˆ6.7 Hz, H-3 ); 5.78 ?d, 1H,
J5,6ˆ8.2 Hz, H-5); 6.01 ?s, 1H, H-10); 7.63 ?d, 1H, J5,6ˆ
8.2 Hz, H-6); 9.45 ?br s, 1H, NH). Anal. Calcd for
C14H18N2O8: C, 49.12; H, 5.30; N, 8.18. Found: C, 49.15;
H, 5.34; N, 8.25.
0
0
3.1.6.
30,50-Di-O-acetyl-20-O-tetrahydropyranyl-20-C-
methyluridine .10). 13.40 g of compound 9 ?39.2 mmol,
MW: 342) were added to a solution of 187 mg ?0.1 mmol)
of p-toluenesulfonic acid monohydrate ?p-TsOH) in 133 mL
of 1,4-dioxane and then, 3,4-dihydro-2H-pyran ?DHP)
?52 mL, 572 mmol, 14.6 equiv.) was dropped into the
mixture. After six hours the reaction was completed and
quenched with 133 mL of triethylamine. The mixture was
concentrated and poured into Na2CO3. The product was
puri®ed by silica gel column chromatography, eluting
with CH2Cl2/methanol ?98:2) yielding 10 as an epimeric
mixture ?80%, Rf 0.65, CH2Cl2/methanol, 97:3). 13C NMR
?CDCl3) d ?ppm): 16.6/17.1 ?±CH3); 19.2/19.3, 19.7/20.3,
20.4 ?acetyl ±CH3s and C-3, THP); 24.9/25.2 ?C-4, THP);
31.3/31.5 ?C-2, THP); 61.3/61.5 ?C-50); 62.0/63.1 ?C-5,
THP); 73.2/73.5 ?C-30); 76.8/77.0 ?C-40); 83.3/83.5 ?C-20);
89.7/89.9 ?C-10); 94.5/95.4 ?C-1, THP); 102.1/102.3 ?C-5);
138.5/139.2 ?C-6); 150.1/150.2 ?C-2); 162.9/163.0 ?C-4);
1
163.4 ?C-4). H NMR ?CDCl3) d ?ppm): 0.80±1.20 ?m,
CH?CH3)2); 1.22 ?s, 3H, ±CH3); 2.53 ?s, 1H, OH-20);
3.90±4.15 ?m, 3H, H-4, 50 and 500); 4.21 ?d, 1H, J3 ,4
ˆ
0
0
8.1 Hz, H-30); 5.70 ?d, 1H, J5,6ˆ8.1 Hz, H-5); 6.00 ?s, 1H,
H-10); 7.73 ?d, 1H, J5,6ˆ8.1 Hz, H-6); 8.40 ?br s, 1H, NH).
Anal. Calcd for C22H40N2O7Si2: C, 52.77; H, 8.05; N, 5.59.
Found: C, 52.74; H, 8.02; N, 5.51.
3.1.4. 20-C-Methyluridine .8). A solution of compound 7
?28 g, 56 mmol, MW: 500) in 900 mL of anhydrous THF was
treated with 1 M tetrabutylammonium ¯uoride solution in
THF ?TBAF) ?123 mL, 123 mmol, 2.2 equiv.) at rt. Five
minutes later the reaction mixture was diluted with
0.4 mL of pyridine/methanol/water ?3:3:1) and the solution
was poured into stirred pyridinium form Dowex 50Wx4-200
resin ?5 g) suspended in pyridine/methanol/water ?3:3:1,
33.6 mL). The mixture was stirred for 10 min, the resin
®ltered off and washed with methanol ?3£56 mL).
Combined ®ltrate and washings were evaporated to dryness
in vacuo to produce a crude syrup ?Rf 0.40, CH2Cl2:metha-
nol, 9:1). 13C NMR ?DMSO-d6) d ?ppm): 19.3 ?±CH3); 59.9
?C-50); 72.7 ?C-30); 79.4 ?C-20); 82.1 ?C-40); 92.0 ?C-10);
1
169.8 ?CO); 169.9 ?CO). H NMR ?CDCl3) d ?ppm): 0.85
?m, 1H, H-3 THP); 1.11 ?m, 1H, H-30, THP); 1.19/1.20 ?s,
3H, ±CH3); 1.20±1.90 ?m, 4H, H-4, H-40, H-2 and H-20,
THP); 2.06/2.09 ?s, 3H, CH3CO); 2.10/2.11 ?s, 3H,
CH3CO); 3.20±4.40 ?m, 5H, H-40, H-50, H-500 and, H-5
and H-50, THP); 4.32 ?m, 1H, H-30); 4.95 ?m, 1H, H-1,
THP); 5.72 ?m, 1H, H-5); 5.98/6.44 ?s, 1H, H-10); 7.62
?m, 1H, H-6); 9.97/10.03 ?br s, 1H, NH). Anal. Calcd for
C19H26N2O9: C, 53.52; H, 6.15; N, 6.57. Found: C, 53.48; H,
6.45; N, 6.75.
1
102.6 ?C-5); 141.8 ?C-6); 151.0 ?C-2); 166.4 ?C-4). H
3.1.7. 20-O-Tetrahydropyranyl-20-C-methyluridine .11).

5712
M. Gallo et al. / Tetrahedron 57 72001) 5707±5713
13.63 g of 30,50-di-O-acetyl-20-O-tetrahydropyranyl-20-C-
methyluridine ?10) ?39 mmol, MW: 426) were dissolved
in 140 mL of methanolic ammonia and the mixture was
stirred overnight at rt. The diasteromeric mixture was
isolated after the evaporation of volatiles ?100%, Rf 0.5 in
dichloromethane/methanol, 95:5). 13C NMR ?CDCl3) d
?ppm): 15.7/16.5 ?±CH3); 20.4/20.7 ?C-3, THP); 24.8/25.3
?C-4, THP); 31.8/31.9 ?C-2, THP); 61.9/62.1 ?C-50); 64.1/
64.6 ?C-5, THP); 72.5/73.1 ?C-30); 82.5/82.7 ?C-40); 85.2/
84.4 ?C-20); 90.2 ?C-10); 95.1/95.5 ?C-1, THP); 102.0/102.1
ice bath. b-Cyanoethoxy-N,N-diisopropylaminochloro-
phosphine ?6.1 mL, 27.3 mmol, 1.1 equiv.) was added drop-
wise. The mixture was stirred at rt for 15 min. And then,
since the reaction was not completed, more b-cyanoethoxy
N,N-diisopropylaminochlorophosphine ?1.1 mL, 5 mmol,
0.2 equiv.) was added and the stirring continued for
30 min. Methanol ?0.36 mL), ethyl acetate ?550 mL) and
triethylamine ?28 mL) were subsequently added. And the
resulting solution washed twice with aqueous Na2CO3
?10%) and twice with brine. The organic layer was dried
?Na2SO4) and concentrated in vacuo. The crude product was
puri®ed by column chromatography eluting with petrol
ether/ethyl acetate/triethylamine ?33:66:1) to yield pure 13
as a foam ?72%, Rf 0.6, petrol ether/ethyl acetate, 3:4). 13C
NMR. ?CDCl3) d ?ppm): 17.1/17.8/18.0 ?±CH3); 18.8/18.9/
19.7 ?CH2±CN, phosp); 20.1/20.2/20.3/21.0 ?C-3, THP);
24.6 ?CH3, phosp); 25.3/25.5 ?C-4, THP); 29.7/31.7/31.9/
32.0 ?C-2, THP); 43.4/43.7 ?CHs, phosp); 55.2/55.3
?±OCH3, DMTr); 57.9/58.0 ?±OCH2, phosph); 60.2/60.4/
60.5 ?C-50); 61.5/62.7 ?C-5, THP); 75.8/75.9/76.0/76.2
?C-30); 80.6/80.7 ?C-40); 83.8/84.0 ?C-20); 87.2 ?C±Ar3,
DMTr); 89.2/89.3/89.5/89.6 ?C-10); 93.6/93.8/94.9 ?C-1,
THP); 102.1/102.3 ?C-5); 113.2/113.3 ?Ar, DMTr); 117.4
?CN, phops);127.2/127.3, 128.0, 128.6/128.7, 130.2/130.5,
135.2/135.3 ?Ar, DMTr); 140.2/140.5 ?C-6); 144.3 ?Ar,
DMTr); 150.6 ?C-2); 158.8 ?CAr±OCH3, DMTr), 163.1/
1
?C-5); 140.2 ?C-6); 150.8 ?C-2); 164.7 ?C-4). H NMR
?CDCl3) d ?ppm): 1.28 ?m, 1H, H-30, THP); 1.27/1.30 ?s,
3H, ±CH3); 1.57 ?m, 3H, H-30, H-4 and H-40, THP); 1.85
?m, 2H, H-2 and H-20, THP); 3.37±4.05 ?m, 5H, H-40, H-50,
H-500 and, H-5 and H-50, THP); 4.07/4.15 ?m, 1H, H-30);
4.94/5.12 ?m, 1H, H-1, THP); 5.73/5.74 ?d, 1H, J5,6ˆ9.0 Hz,
H-5); 6.03/6.51 ?s, 1H, H-10); 7.93/8.02 ?d, 1H, J5,6ˆ9.0 Hz,
H-6). Anal. Calcd for C15H22N2O7: C, 52.63; H, 4.48; N,
8.18. Found: C, 52.66; H, 4.49; N, 8.20.
3.1.8. 50-O-Dimethoxytrityl-20-O-tetrahydropyranyl-20-
C-methyluridine .12). Dried compound 11 ?10.95 g,
32 mmol, MW: 342) was dissolved in anhydrous pyridine
?250 mL). Triethylamine ?6.75 mL, 48.4 mmol, 1.5
equiv.), 4-dimethylaminopyridine ?DMAP) ?213 mg, 1.7
mmol, 0.05 equiv.) and 4,40-dimethoxytrityl chloride
?DMTrCl) ?14.58 g, 43 mmol, 1.3 equiv.) were added with
stirring and exclusion of moisture, and the reaction mixture
was stirred at rt. After 2 h the reaction was not completed,
therefore more DMTrCl ?24.30 g, 71 mmol, 2.2 equiv.) and
triethylamine ?11.2 mL, 80 mmol, 2.5 equiv.) were added
and the stirring was continued for 5 h. The reaction was
quenched with an equivalent volume of water and extracted
with ethyl ether ?2£1 L), dried ?Na2SO4) and evaporated.
The crude product ?Rf 0.45, methanol/CH2Cl2, 97:3) was
puri®ed by column chromatography eluting with petroleum
ether/ethyl acetate/triethylamine ?66:33:1) to yield pure 12
as a foam ?78%). 13C NMR ?CDCl3) d ?ppm): 15.9/16.9
?±CH3); 20.7/21.0 ?C-3, THP); 24.9/25.0 ?C-4, THP);
29.7/32.0 ?C-2, THP); 55.3 ?±OCH3, DMTr); 60.6 ?C-50);
64.4/64.6 ?C-5, THP); 73.4/74.1 ?C-30); 81.4/82.1 ?C-40);
84.2/84.3 ?C-20); 87.2/87.8 ?C±Ar3, DMTr); 90.4 ?C-10);
95.3/95.7 ?C-1, THP); 102.0/102.1 ?C-5); 113.3/113.2 ?Ar,
DMTr); 127.1, 128.0/128.3, 130.2/130.3, 135.2, 135.4/
135.5 ?Ar, DMTr); 140.2/140.4 ?C-6); 144.4/144.5 ?Ar,
DMTr); 150.7 ?C-2); 158.7 ?CAr±OCH3, DMTr), 163.4/
1
163.2 ?C-4). H NMR ?CDCl3) d ?ppm): 1.10 ?m, 1H,
H-30, THP); 1.18 ?m, 12H, CH3, phosp), 1.26 ?s, 3H,
±CH3); 1.55 ?m, 3H, H-30, H-4and H-40, THP); 2.28/2.34
?m, 2H, H-2 and H-20, THP); 2.63 ?m, 2H, CH2±CN,
phosp), 3.41±4.42 ?m, 16H, H-30, H-40, H-50, H-500,
±OCH3, DMTr, OCH2 and CHs phosp and, H-5 and H-50,
THP) 3.37±4.05 ?m, 5H,); 4.07/4.15 ?m, 1H, H-30); 4.94/
5.12?m, 1H, H-1, THP); 5.73/5.74 ?d, 1H, J5,6ˆ9.0 Hz,
H-5); 6.03/6.51 ?s, 1H, H-10); 7.93/8.02 ?d, 1H, J5,6ˆ
9.0 Hz, H-6). 31P NMR ?CDCl3) d ?ppm): 151.09/151.13/
151.18/151.22. Anal. Calcd for C45H57N4O10P: C, 63.97; H,
6.80; N, 6.63. Found: C, 64.25; H, 6.95; N, 6.75.
Acknowledgements
Â
We thank Consejo Nacional de Investigaciones Cientõ®cas y
Tecnicas-Argentina, Universidad Nacional de Quilmes-
Â
Argentina and International Centre for Genetic Engineering
and BiotechnologyÐTrieste, Italy for ®nancial support.
1
163.5 ?C-4). H NMR ?CDCl3) d ?ppm): 0.88 ?m, 1H,
H-3, THP); 1.32 ?s, 3H, ±CH3); 1.52 ?m, 3H, H-30, H-4
and H-40, THP); 1.70/1.85 ?m, 2H, H-2 and H-20, THP);
3.54±4.17 ?m, 9H, H-30, H-40, H-50, H-500, ±OCH3, DMTr
and, H-5 and H-50, THP); 5.02/5.06 ?d, 1H, J5,6ˆ9.0 Hz,
H-5); 5.21/5.33 ?m, 1H, H-1, THP); 6.04/6.50 ?s, 1H,
H-10); 6.85 ?m, 4H, DMTr), 7.20±7.45 ?m, 9H, DMTr),
8.18/8.21 ?d, 1H, J5,6ˆ9.0 Hz, H-6). Anal. Calcd for
C36H40N2O9: C, 67.07; H, 6.25; N, 4.35. Found: C, 67.47;
H, 5.53; N, 4.39.
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