Synthesis of 2′-C-α-(hydroxyalkyl) and 2′-C-α- alkylcytidine phosphoramidites: Analogues for probing solvent interactions with RNA

Article abstract of DOI:10.1021/jo062002t

(Chemical Equation Presented) Nucleoside analogues bearing 2′-C-α-(hydroxyalkyl) and 2′-C-α-alkyl substitutes have numerous applications in RNA chemistry and biology. In particular, they provide a strategy to probe the interaction between the 2′-hydroxyl

Full text of DOI:10.1021/jo062002t

Synthesis of 2′-C-r-(Hydroxyalkyl) and 2′-C-r-Alkylcytidine
Phosphoramidites: Analogues for Probing Solvent Interactions with
RNA
Nan-Sheng Li* and Joseph A. Piccirilli*
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology and
Department of Chemistry, UniVersity of Chicago, 929 East 57th Street, Chicago, Illinois 60637
nli@uchicago.edu; jpicciri@uchicago.edu
ReceiVed September 28, 2006
Nucleoside analogues bearing 2′-C-R-(hydroxyalkyl) and 2′-C-R-alkyl substitutes have numerous
applications in RNA chemistry and biology. In particular, they provide a strategy to probe the interaction
between the 2′-hydroxyl group of RNA and water. To incorporate these nucleoside analogues into
oligonucleotides for studies of the group II intron (Gordon, P. M.; Fong, R.; Deb, S.; Li, N.-S.; Schwans,
J. P.; Ye, J.-D.; Piccirilli, J. A. Chem. Biol. 2004, 11, 237), we synthesized six new phosphoramidite
derivatives of 2′-deoxy-2′-C-R-(hydroxyalkyl)cytidine (36: R ) -(CH2)2OH; 38: R ) -(CH2)3OH;
40: R ) -(CH2)4OH) and 2′-deoxy-2′-C-R-alkylcytidine (37: R ) -CH2CH3; 39: R ) -(CH2)2CH3;
41: R ) -(CH2)3CH3) from cytidine or uridine via 2′-C-R-allylation, followed by alkene and alcohol
transformations. Phosphoramidites 36 and 37 were prepared from cytidine in overall yields of 14% (10
steps) and 7% (11 steps), respectively. Phosphoramidites 38 and 39 were prepared from uridine in overall
yields of 30% (10 steps) and 13% (11 steps), respectively. Phosphoramidites 40 and 41 were synthesized
from uridine in overall yields of 21% (13 steps) and 25% (14 steps), respectively.
Introduction
functional groups may allow stereocontrolled nucleobase ex-
change,5 conjugation of biophysical probes or biological mol-
ecules,6 or introduction of structural constraints.7
Nucleotides bearing hydroxyalkyl and alkyl substituents
provide a way to reveal the role of water molecules important
for nucleic acid function. The substrate specificity of SMUG1,
a uracil DNA glycosylase, provides a biological illustration of
this approach.8 (Figure 1). The enzyme deglycosylates uridine
residues selectively over thymidine residues because the methyl
group displaces an important water molecule in the active site.
Nucleoside analogues bearing 2′-C-R-hydroxyalkyl substit-
uents have numerous applications in biomedical science and
biophysical and mechanistic chemistry. As components of gene
regulatory agents (antisense and siRNAs), they retain the ability
to form Watson-Crick base pairs but confer enhanced resistance
to nuclease degradation.1 Within the context of 2′-5′-linked RNA
hairpin loops, the 2′-hydroxymethyl substituent lengthens the
sugar-phosphate backbone, thereby enhancing thermal stabil-
ity.2 Hydroxyalkyl nucleosides also provide valuable precursors
to access the corresponding alkyl derivatives3 or to append
amino, thiol, and carboxylate groups to nucleic acids.4 These
(4) Sukeda, M.; Shuto, S.; Sugimoto, I.; Ichikawa, S.; Matsuda, A. J.
Org. Chem. 2000, 65, 8988.
(5) Fehring, V.; Knights, S.; Chan, M.-Y.; O’Neil, I. A.; Cosstick, R.
Org. Biomol. Chem. 2003, 1, 123.
(6) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San
Diego, CA, 1995.
(7) Goodwin, J. T.; Osborne, S. E.; Scholle, E. J.; Glick, G. D. J. Am.
Chem. Soc. 1996, 118, 5207.
(1) Pavey, J. B. J.; Lawrence, A. J.; Potter, A. J.; Cosstick, R.; O’Neil,
I. A. Tetrahedron Lett. 1998, 39, 6967.
(2) Peng, C. G.; Damha, M. J. Nucleic Acids Res. 2005, 33, 7019.
(3) Mesmaeker, A. D.; Lebreton, J.; Hoffmann, P.; Freier, S. M. Synlett
1993, 677.
10.1021/jo062002t CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/18/2007
1198
J. Org. Chem. 2007, 72, 1198-1210

Analogues for Probing SolVent Interactions with RNA
FIGURE 1. Pyrimidine specificity of SMUG1 deglycosylase. Adapted from ref 8 with permission from Elsevier. (A) Uracil makes hydrogen
bonds without displacing the active site water molecule. (B) Hydroxymethyluracil makes the same specific hydrogen bonds as uracil. The
hydroxymethyl group displaces the water molecule but supplants the lost hydrogen bonds. (C) Thymidine binds less favorably because the methyl
group displaces the water molecule but cannot supplant the lost hydrogen bonds.
FIGURE 3. RNAs with site-specific 2′-C-R-alkyl or 2′-C-R-hydroxy-
FIGURE 2. (A) SER reaction catalyzed by group II intron RNA. (B)
Hypothetical model showing how ethanol could stimulate the reactivity
of E12′HE2. Removal of the splice junction 2′-OH might disrupt solvent
organization and allowing binding of ethanol.
lalkylcytidine.
cytosine residue at the splice junction. We therefore constructed
a series of substrates containing hydroxyalkyl groups [(CH2)n-
OH; n ) 1-4] at the 2′-position (Figure 3). As the SER reaction
exhibited sensitivity to the substituent volume, we also synthe-
sized the corresponding deoxygenated substrates containing
straight chain alkyl groups [(CH2)nH; n ) 1-4] (Figure 3).
Together, the analogues allow us to quantitate the effect of
extending the hydroxyl group away from the sugar ring in one-
carbon steps successively, thereby revealing whether the 2′-
hydroxyl group imparts its functional contribution via inductive
effects or via space interactions with solvent. The synthesis of
2′-C-R-methyl and hydroxymethyl cytidine phosphoramidites
was described previously.12,13 Here, we describe the preparation
of the six remaining phosphoramidites (n ) 2-4).
A 5-hydoxymethyl substituent supplants this water molecule,
allowing SMUG1 to deglycosylate 5-hydroxymethyl uridine
residues. Thus, enhanced activity upon hydroxyalkyl substitution
(5-hydroxymethyluridine in the case of SMUG1) relative to the
corresponding alkyl substitution (thymidine) implicates the
localization of an important water molecule in the absence of
any substitution (uridine).
The synthetic work described herein was specifically inspired
by investigation of the spliced exons reopening (SER) reaction
catalyzed by the group II intron RNA. In the SER reaction, an
RNA substrate whose sequence corresponds to spliced exons
undergoes hydrolysis at the splice junction phosphodiester
(Figure 2A).9 A 2′-deoxycytosine residue at the splice junction
reduces the rate of the SER reaction by 10-fold, implying that
the 2′-hydroxyl group imparts a functional contribution to
catalysis.10 Using a series of nucleotide analogues that span a
range of chemical diversity, we showed that the reaction rate
inversely correlates with the substituent hydrophobicity,11 sug-
gesting that the 2′-hydroxyl group may interact with a water
molecule. Consistent with this observation, we observed that
ethanol, but not methanol, 2-propanol, or tert-butyl alcohol,
modestly rescues the deoxynucleotide substrate.11 Possibly the
space vacated by the hydroxyl group allows the ethanol to bind
to the ribozyme-substrate complex via hydrophobic interactions
with the deoxyribose ring so as to localize or replace an active
site water molecule (Figure 2B). This led us to investigate
whether we might observe stimulation of the SER reaction by
attaching an alcohol moiety covalently to the 2′-carbon of the
Results and Discussion
Grotli and Undheim prepared the 2′-C-R-allyl-2′-deoxy-
nucleoside analogues of A, C, G, and U via ultraviolet light-
induced radical allylation and derivatized them to the corre-
sponding phosphoramidites.14 Beigelman et al. used an AIBN-
initiated radical allylation reaction to obtain the phosphoramidite
derivatives of 2′-allyl-2′-deoxycytidine and 2′-allyl-2′-deox-
yuridine.15 As the procedure of Grotli and Undheim required a
quartz tube, a 125 W Hg UV lamp, and long photolysis time
(ca. 4 days), we opted to use Beigelman et al.’s AIBN-initiated
radical allylation procedure for the synthesis of our target
phosphoramidites.15
1. 2′-C-r-(Hydroxyethyl)cytidine and 2′-C-r-Ethylcytidine.
Cytidine derivatives bearing 2′-ethyl and 2′-hydroxyethyl groups
have not been prepared previously, but the corresponding uridine
analogues and their phosphoramidites have been prepared.
Mesmaeker et al. prepared 2′-C-R-ethyldeoxyuridine and the
(8) Wibley, J. E. A.; Waters, T. R.; Haushalter, K.; Verdine, G. L.; Pearl,
L. Mol. Cells 2003, 11, 1647.
(9) Jarrell, K. A.; Dietrich, R. C.; Perlman, P. S. Mol. Cell. Biol. 1988,
8, 2361.
(10) Griffin, E. A.; Qin, P. Z.; Michels, W. J.; Pyle, A. M. Chem. Biol.
1995, 2, 761.
(11) Gordon, P. M.; Fong, R.; Deb, S.; Li, N.-S.; Schwans, J. P.; Ye,
J.-D.; Piccirilli, J. A. Chem. Biol. 2004, 11, 237.
(12) Li, N.-S.; Piccirilli, J. A. J. Org. Chem. 2003, 68, 6799.
(13) Li, N.-S.; Piccirilli, J. A. J. Org. Chem. 2004, 69, 4751.
(14) Grotli, M.; Undheim, K. Acta Chem. Scand. 1995, 49, 217.
(15) Beigelman, L.; Karpeisky, A.; Matulic-Adamic, J.; Haeberli, P.;
Sweedler, D.; Usman, N. Nucleic Acids Res. 1995, 23, 4434.
J. Org. Chem, Vol. 72, No. 4, 2007 1199

Li and Piccirilli
SCHEME 1 a
a (i) (1) t-Bu2SiCl2, AgNO3, DMF, 0 °C, 30 min; (2) Et3N, rt, 5 min; (ii) (1) PhOCSCI, DMAP, CH2Cl2, 0 °C to rt, 16 h; (2) AcCl, 0 °C, 1 h; (iii)
allyltributylstannane, AlBN, benzene, reflux, 4 h, 65% or toluene, reflux, 6 h, 59%; (iv) NalO4, OsO4 (cat), acetone/water (6:1), rt, overnight; (v) (1) NaBH4,
MeOH, 0 °C, 30 min; (2) AcCl, DMAP, CH2Cl2, 0 °C, 30 min; (vi), Et3N-3HF, Et3N, THF, rt, 30 min.
corresponding phosphoramidite from 3′,5′-O-(1,1,3,3-tetraiso-
propyldisiloxyane-1,3-diyl)uridine via a reaction sequence that
included UV-initiated radical allylation, oxidative cleavage,
reduction, and deoxygenation. This reaction sequence gave the
phosphoramidite in 10 steps and 7.6% overall yield.3 Lawrence
et al. used the olefin oxidative cleavage product from Mes-
maeker’s sequence, a 2′-C-R-(2-oxyethyl)uridine derivative, to
prepare 2′-C-R-(hydroxyethyl)deoxyuridine and its phosphora-
midite.16,17 Additionally, Matsuda and co-workers reported
access to a 2′-C-R-(hydroxyethyl)uridine derivative in low yield
from the corresponding 2′-phenylseleno-3′-O-vinylsilyluridine
derivative by an intramolecular radical reaction.4
We accessed 2′-C-R-(hydroxylethyl)cytidine and 2′-C-R-
ethylcytidine from cytidine using reactions analogous to those
of Mesmaker et al. and Lawrence et al. for the preparation of
the corresponding uridine derivatives. To allow cytidine trans-
formation via this strategy, we chose protecting groups judi-
ciously. We protected the exocyclic amino group as the
acetamide for two reasons: (1) we had observed that the 2′-
thionoester of both 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxyane-
1,3-diyl)-N4-benzoylcytidine and 3′,5′-O-(1,1,3,3-tetraisopro-
pyldisiloxyane-1,3-diyl)cytidine (lacking N4-protection) resists
AIBN-initiated radical 2′-allylation,15,18 and (2) acetamide
protection has been used previously for radical 2′-deoxygen-
ation19 and radical 2′-allylation (up to 50% yield has been
reported using AIBN as initiator).15 We protected the 3′- and
5′-hydroxyl groups as the di-tert-butylsilanediyl ether rather than
as the 1,1,3,3-tetraisopropyldisiloxyane-1,3-diyl ether based on
the lower cost of the reagent (di-tert-butyldichlorosilane) and
because its removal occurs under milder conditions.20,21 To allow
incorporation of 2′-hydroxyethylcytidine into oligonucleotides
during solid-phase synthesis, we blocked the hydroxyethyl group
as the acetate ester, a strategy that has allowed incorporation
of 2′-hydroxylmethyluridine into RNA previously.1 Although
nucleobase deprotection conditions that follow solid-phase
synthesis remove the acetyl group, residues bearing 2′-hydroxy-
lalkyl groups within RNA exhibit greater stability against base-
catalyzed phosphodiester cleavage than do ribonucleotides.1
Scheme 1 shows the synthesis of 2′-C-R-(acetoxyethyl)-N4-
acetyl-2′-deoxycytidine (6) via the 2′-allyl derivative. Using a
modified literature procedure, we obtained 3′,5′-O-(di-tert-
butylsilanediyl)cytidine (1) in quantitative yield from cytidine
and di-tert-butyldichlorosilane in the presence of silver nitrate
(the reaction was carried out at 0 °C for 30 min rather than at
23 °C for 4 min).22 This alternative procedure improved the
yield by ∼10%. Reaction of 1 with phenyl chlorothionoformate
(16) Lawrence, A. J.; Pavey, J. B. J.; O’Neil, I. A.; Cosstick, R.
Tetrahedron Lett. 1995, 36, 6341.
(17) Lawrence, A. J.; Pavey, J. B. J.; Cosstick, R.; O’Neil, I. A. J. Org.
Chem. 1996, 61, 9213.
(19) Robins, M. J.; Wilson, J. S.; Hansske, F. J. Am. Chem. Soc. 1983,
105, 4059.
(20) Serebryany, V.; Beigelman, L. N. Tetrahedron Lett. 2002, 43, 1983.
(21) Tang, X.-Q.; Liao, X.; Piccirilli, J. A. J. Org. Chem. 1999, 64, 747.
(22) Furusawa, K.; Ueno, K.; Katsura, T. Chem. Lett. 1990, 97.
(18) Rhei, S. O.; Pfleiderer, W. Nucleosides Nucleotides 1992, 11, 835.
1200 J. Org. Chem., Vol. 72, No. 4, 2007

Analogues for Probing SolVent Interactions with RNA
SCHEME 2 a
SCHEME 3 a
a (i) TBAF, THF, rt, 10 min; (ii) 10% Pd/C, H2, MeOH, rt, 24 h; (iii)
(1) t-Bu2SiCl2, AgNO3, DMF, 0 °C, 30 min, (2) Et3N, rt, 10 min; (iv) (1)
2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, 60
h; (2) NH4OH, rt, 3 h; (v) CH3COCl, DMAP, CH2Cl2, 0 °C, 30 min; (vi)
Et3N-3HF, Et3N, THF, rt, 1 h.
a (i) NaBH4, MeOH, 0 °C to rt, 30 min; (ii) I2, PPH3, imidazole, benzene/
acetonitrile (4:1), 0 °C to rt, 2 h; (iii) n-Bu3SnH/AlBN, benzene, THF,
sonication, 0 °C, 1 h; (iv) Et3N-3HF, Et3N, THF, rt, 1 h.
gave the 2′-phenoxythiocarbonyl ester, which was then acety-
lated to give the N4-acetylcytidine derivative 2 in 75% yield.
Radical AIBN-initiated allylation of 2 with allyltributylstannane
in refluxing benzene gave the 2′-R-allylcytidine derivative 3 in
65% yield. From refluxing toluene, a slightly lower yield (59%)
was obtained. This approach gives superior yields of protected
2′-R-allylcytidine (49% overall yield from cytidine) at less
expense than does the literature procedure for preparing the
analogous compound with the 3′,5′-O-(1,1,3,3-tetraisopropyl-
disiloxyane-1,3-diyl) protecting group (overall 35% yield from
cytidine). Continuing with the synthesis of 6, osmium tetroxide-
catalyzed sodium periodate oxidation converted 3 to the 2′-(2-
oxoethyl)cytidine derivative 4 in 74% yield. Sodium borohydride
reduction followed by acetylation gave the bisacetyl cytidine
derivative 5 in 58% yield. Desilylation with triethylamine-
trihydrofluoride complex gave the 2′-C-R-(hydroxyethyl)cytidine
derivative 6 in 88% yield.
C-R-propyluridine, which was obtained from 2′-C-R-allyluridine
via palladium-catalyzed hydrogenation.26 We prepared the
starting material, 2′-C-R-allyl-3′,5′-O-(1,1,3,3-tetraisopropyld-
isiloxyane-1,3-diyl)uridine, in three steps from uridine15 and
attempted to transform it to N4-acetyl-2′-C-R-propylcytidine as
described by Cicero at al. (hydrogenation, desilylation, and
amination).26 Our attempts to hydrogenate 2′-C-R-allyl-3′,5′-
O-(1,1,3,3-tetraisopropyldisiloxyane-1,3-diyl)uridine (treatment
of 11 with 10% Pd/C under hydrogen at room temperature for
19 h) gave incomplete hydrogenation and some desilylation,
however. We found that the reverse reaction sequence, desily-
lation14 followed by palladium-catalyzed hydrogenation, pro-
ceeded more smoothly to give 2′-C-R-propyluridine (13) in 79%
yield from 11 (Scheme 3). Simultaneous protection of the 3′
and 5′-hydroxyl groups using di-tert-butyldichlorosilane gave
the bis-silyl ether 14 in 51% yield.22 Transformation to the N4-
acetylcytidine derivative 15 was achieved in 79% yield using a
two-step reaction sequence: amination using 2,4,6-triisopropy-
lbenzenesulfonyl chloride and ammonium hydroxide27 followed
by acetylation. Desilylation with the triethylamine-trihydrof-
luoride complex gave N4-acetyl-2′-C-R-propylcytidine (16) in
85% yield.
The synthesis of 2′-C-R-(hydroxypropyl)cytidine has not been
reported, but syntheses for the corresponding uridine and
thymidine analogues have been reported. Xi et al. prepared 2′-
hydroxypropylthymidine via an intramolecular radical reaction
from 2′-phenylseleno-3′-O-allyldimethylsilylthymidine.28 Fe-
hring et al. prepared the 2′-C-R-(hydroxypropyl)uridine deriva-
tive 17 in 63% yield from 11 by hydroboration using a borane-
dimethyl sulfide complex, followed by oxidation with triethyl-
amine-N-oxide.5 We attempted direct hydroboration of the 2′-
allylcytidine derivative 3 using 9-BBN and sodium perborate
(a generally efficient hydroboration/oxidation sequence29,30) but
We used intermediate 4 to prepare N4-acetyl-2′-deoxy-2′-C-
R-ethylcytidine (10) (Scheme 2). Reduction of 4 with sodium
borohydride gave the 2′-hydroxyethylcytidine derivative 7 in
31% yield and the corresponding deacetylated product 7a in
24% yield. Incubation with a mixture of iodine, triphenylphos-
phine, and imidazole converted 7 to the corresponding 2′-C-R-
(iodoethyl)cytidine derivative 8 in 74% yield.23 Ultrasound-
initiated radical dehalogenation with tributyltin hydride converted
8 to the 2′-C-R-ethylcytidine derivative 9 in 91% yield.24
Without ultrasound irradiation, the reaction in refluxing toluene
gave a ∼1:1 mixture of 7 and 9, based on TLC analysis. The
attempt to convert 7 to 9 with Mesmaeker et al.’s method
(tosylation, followed by AIBN-initiated reduction) failed.3
Subsequent desilylation of 9 with triethylamine-trihydrofluoride
complex gave the 2′-C-R-ethylcytidine derivative 10 in 99%
yield.
2. 2′-C-r-Propylcytidine and 2′-C-r-(Hydroxypropyl)-
cytidine. The susceptibility of the cytosine ring to reduction
under hydrogenation conditions precludes preparation of 2′-C-
R-propylcytidine directly from 2′-C-R-allylcytidine.13,25 Cicero
et al. prepared 2′-C-R-propylcytidine by transformation of 2′-
(26) Cicero, D. O.; Neuner, P. J. S.; Franzese, O.; D’Onofrio, C.;
Iribarren, A. M. Bioorg. Med. Chem. Lett. 1994, 4, 861.
(27) Iino, T.; Yoshimura, Y.; Matsuda, A. Tetrahedron 1994, 50, 10397.
(28) Xi, Z.; Agback, P.; Plavec, J.; Sandstrom, A.; Chattopadhyaya, J.
Tetrahedron 1992, 48, 349.
(23) Corey, E. J.; Nagata, R. Tetrahedron Lett. 1987, 28, 5391.
(24) Nakamura, E.; Machii, D.; Inubushi, T. J. Am. Chem. Soc. 1989,
111, 6849.
(29) Pelter, A.; Smith, K.; Brown, H. C. Borane Reagents; Academic
Press: San Diego, CA, 1988.
(30) Kabalka, G. W.; Li, N.-S.; Pace, R. D. Synth. Commun. 1995, 25,
2135.
(25) Christensen, L. F.; Broom, A. D. J. Org. Chem. 1972, 37, 3398.
J. Org. Chem, Vol. 72, No. 4, 2007 1201

Li and Piccirilli
SCHEME 4 a
a (i) (1) 9-BBN, THF, rt, 4 h; (2) NaBO3-4H2O, rt, 2 h; (ii) Ac2O, DMAP, Et3N, CH2Cl2, 0 °C to rt, 1 h; (iii) (1) 2,4,6-triisopropylbenzenesulfonyl
chloride, DMAP, Et3N, CH3CN, rt, 48 h; (2) NH4OH, rt, 3 h; (iv) acetyl chloride, DMAP, CH2Cl2, 0 °C to rt, 1 h; (v) Et3N-3HF, Et3N, THF, rt, 2 h.
SCHEME 5 a
a (i) Dess-Martin periodinane, CH2Cl2, 0 °C, 3 h; (ii) Ph3P+MeBr-, s-BuLi, THF, -78 °C to rt, 20 h; (iii) (1) 9-BBN, THF, rt, 2 h, (2) NaBO3-4H2O,
rt, 2 h; (iv) Ac2O, DMAP, Et3N, CH2Cl2, 0 °C to rt, 1 h; (v) (1) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, 66 h; (2) NH4OH, rt,
3 h; (vi) CH3COCl, DMAP, CH2Cl2, 0 °C, 30 min; (vii) Et3N-3HF, Et3N, THF, rt, 2 h.
obtained no corresponding 2′-C-R-hydroxypropylcytidine de-
rivative. It is possible that the borane reagent reduced the N4-
acetamide.31 We therefore prepared 2′-C-R-acetoxypropyl-N4-
acetylcytidine (20) via its uridine analogue (Scheme 4). We
improved upon the procedure of Fehring et al. for the preparation
of 17 from 11 (76% yield versus 63% yield) by conducting the
hydroboration and the oxidation with 9-BBN and sodium
perborate, respectively. Protection of the hydroxyl group with
acetic anhydride gave 2′-C-R-(acetoxypropyl)uridine derivative
18 in 94% yield. Transformation to the N4-acetylcytidine
derivative 19 was achieved in 82% yield by the two-step reaction
sequence: amination with 2,4,6-triisopropylbenzenesulfonyl
chloride and ammonium hydroxide27 followed by N4-acetylation.
Subsequent desilylation with the triethylamine-trihydrofluoride
complex gave 2′-C-R-acetoxypropyl-N4-acetylcytidine (20) in
quantitative yield (>99% yield).
3. 2′-C-r-(Hydroxybutyl)cytidine and 2′-C-r-Butylcyti-
dine. Scheme 5 shows the synthesis of 2′-C-R-(acetoxybutyl)-
N4-acetylcytidine (25). The 2′-C-R-hydroxypropyl chain of 17
was oxidized to the corresponding aldehyde using Dess-Martin
periodinane and subsequently extended by one carbon via Wittig
olefination to give the 2′-C-R-(3-butenyl)uridine derivative 21
in 68% yield. Hydroboration with 9-BBN followed by oxidation
with sodium perborate gave the corresponding 2′-C-R-(4-
hydroxybutyl)uridine 22 in 88% yield. Acetylation of the
hydroxyl group with acetic anhydride gave the 2′-C-R-(4-
acetoxybutyl)uridine derivative 23 (93% yield), which was
transformed to the corresponding N4-acetylcytidine derivative
(31) Lane, C. F. Chem. ReV. 1976, 76, 773.
1202 J. Org. Chem., Vol. 72, No. 4, 2007

Analogues for Probing SolVent Interactions with RNA
SCHEME 6 a
a (i) I2, PPh3, imidazole, C6H6/CH3CN (4:1), 0 °C, 45 min; (ii) n-Bu3SnH, AIBN, toluene, sonication, 0 °C, 3 h; (iii) (1) 2,4,6-triisopropylbenzenesulfonyl
chloride, DMAP, Et3N, CH3CN, rt, 53 h, (2) NH4OH, rt, 3 h; (iv) CH3COCl, DMAP, CH2Cl2, 0 °C, 30 min; (v) Et3N-3HF, Et3N, THF, rt, 2 h.
a
TABLE 1. Synthesis of Phosphoramidites (36-41)
SCHEME 7
starting
overall
material
phosphoramidites
steps
yield (%)
cytidine
cytidine
uridine
uridine
uridine
uridine
36
37
38
39
40
41
10
11
10
11
13
14
14
7
30
13
21
25
(20), and 2′-propylcytidine (16) with DMTrCl in the presence
of DMAP gave the corresponding 5′-DMTr-protected cytidine
derivatives (30-33) in 76-89% yield. We prepared 5′-O-
DMTr-2′-acetoxybutylcytidine (34) and 5′-O-DMTr-2′-butyl-
cytidine (35) from 2′-acetoxybutylcytidine (25) and 2′-
propylcytidine (29), in 98 and 89% yield, respectively, by the
addition of silver nitrate to accelerate the reaction. Phosphity-
lation with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
converted 30-35 quantitatively to the corresponding phos-
phoramidite derivatives 36-41. On the basis of the NMR
spectra, we estimate the purity of these phosphoramidites to be
g95%. The small amount of impurity has little or no effect on
solid-phase oligonucleotide synthesis. The overall yields of 36-
41 from cytidine or uridine are summarized in Table 1.
a (i) For 30-33, DMTrCl, DMAP, pyridine, rt, 20-27 h, 76-89% yield;
for 34 and 35, DMTrCl, DMAP, AgNO3, pyridine, rt, 48 h, 89-98% yield;
(ii) (i-Pr)2NP(Cl)OCH2CH2CN, i-Pr2NEt, 1-methylimidazole, CH2Cl2, 0 °C
to rt, 1-2 h, TLC indicated quantitative conversion; purity estimated at
95%.
24 in 84% yield. Desilylation with the triethylamine-trihydrof-
luoride complex gave 2′-C-R-(acetoxybutyl)-N4-acetylcytidine
(25) in quantitative yield (>99% yield).
Scheme 6 shows the synthesis of N4-acetyl-2′-C-R-butylcy-
tidine (29). We first converted 2′-C-R-(4-hydroxybutyl)uridine
22 to the 2′-C-R-(iodobutyl)uridine derivative 26 in 94% yield
using iodine/triphenylphosphine/imidazole at 0 °C for 45 min.
Longer reaction times and a higher temperature (room temper-
ature) significantly decreased the yield of 26, possibly reflecting
formation of the triphenylphosphonium iodide salt. Ultrasound
irradiation of 26 in the presence of tributyltin hydride induced
radical dehalogenation to give 2′-C-R-butyluridine derivative
27 in 97% yield. Subsequent transformation to the corresponding
N4-acetylcytidine derivative 28 as described earlier was achieved
in 90% yield. Desilylation with the triethylamine-trihydrof-
luoride complex gave N4-acetyl-2′-C-R-butylcytidine (29) in
95% yield.
Summary
Hydroxyalkyl nucleotides serve as valuable analogues for
investigating how 2′-hydroxyl groups impart function within
RNA molecules. They extend the hydroxyl group away from
the ribose ring by successive methylene units. Together with
the corresponding alkyl substituents as “deoxynucleotide con-
trols”, these analogues provide a means to ascertain whether a
2′-hydroxyl group contributes to function via inductive effects
or via through-space interactions with solvent. We have
synthesized two series of cytidine phosphoramidites bearing 2′-
C-R-(CH2)nOH and -(CH2)nH substituents, respectively, and
have used these phosphoramidites to prepare substrates for the
SER reaction catalyzed by the group II intron. The phosphora-
midites couple with the same efficiency as commercial phos-
phoramidites.11
4. Phosphoramidite Derivatives. Scheme 7 shows the
synthesis of the phosphoramidite derivatives from modified
cytidines (6, 10, 16, 20, 25 and 29). Treatment of 2′-acetoxy-
ethylcytidine (6), 2′-ethylcytidine (10), 2′-acetoxypropylcytidine
J. Org. Chem, Vol. 72, No. 4, 2007 1203

Li and Piccirilli
mL) was heated to 100 °C under argon. A solution of AIBN (116
mg, 0.71 mmol) in toluene (11 mL) was added slowly over 30
min, followed by refluxing for 6 h. The solvent was removed, and
the residue was purified by silica gel chromatography, eluting with
30% hexane in ethyl acetate to give the product as a white foam:
Construction of the nucleosides entailed classic alkene and
alcohol transformations. We accessed 2′-hydroxyethylcytidine
from protected 2′-C-R-allylcytidine via oxidative cleavage
followed by borohydride reduction. We accessed hydroxypropyl
and hydroxybutylcytidine from the corresponding uridine
derivatives by amination of the pyrimidine ring. Hydroboration/
oxidation of protected 2′-C-R-allyluridine provided hydrox-
ypropyluridine. Subsequent oxidation, Wittig olefination, and
hydroboration/oxidation gave hydroxybutyluridine. For the alkyl
series, we accessed ethylcytidine and butylcytidine from hy-
droxylethylcytidine and hydroxylbutyluridine, respectively. 2′-
Propylcytidine was obtained by direct hydrogenation of 2′-C-
R-allyluridine, followed by amination.
Several improvements to existing transformations emerge
from this work. (1) We developed an alternative, more
economical strategy to access protected 2′-allylcytidine, a key
intermediate in nucleoside chemistry. (2) We developed an
efficient two-step sequence involving mild reaction conditions
(iodination followed by ultrasound dehalogenation) to deoxy-
genate the hydroxyalkyl groups. (3) We improved access to
hydroxylpropyluridine using 9-BBN/sodium perborate rather
than borane-dimethyl sulfide complex/triethylamine-N-oxide
for hydroboration/oxidiation of protected 2′-C-R-allyluridine.
(4) We improved access to propyluridine by removing the TIPS
group from 2′-allyluridine before hydrogenation. These results
may also allow more facile and efficient synthesis of purine
nucleosides bearing 2′-C-R-alkyl and hydroxyalkyl substitutents.
1
746 mg (59% yield). H NMR (CDCl3/TMS) δ 10.15 (brs, 1H),
7.70 (d, 1H, J ) 7.6 Hz), 7.46 (d, 1H, J ) 7.6 Hz), 6.00 (m, 1H),
5.87 (d, 1H, J ) 1.6 Hz), 5.16 (dd, 1H, J ) 1.0, 9.0 Hz), 5.08 (m,
1H), 4.50 (dd, 1H, J ) 4.4, 8.8 Hz), 4.21 (m, 1H), 4.05-3.95 (m,
2H), 2.70 (m, 1H), 2.49 (m, 1H), 2.30 (s, 3H), 2.35-2.25 (m, 1H),
1.06 (s, 9H), 1.02 (s, 9H); 13C NMR (CDCl3) δ 171.2, 163.0, 154.7,
143.7, 135.7, 117.0, 96.9, 91.4, 76.03, 76.00, 67.7, 47.4, 31.2, 27.4,
27.1, 24.9, 22.7, 20.4; HRMS calcd for C22H36N3O5Si [MH+]
450.2424, found 450.2432.
N4-Acetyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-deoxy-2′-C-r-(2-
oxoethyl)cytidine (4). To a solution of 3 (411 mg, 0.92 mmol) in
a mixed solvent of acetone/water ) 6:1 (v/v) (28 mL), osmium
tetraoxide (4 wt % in water, 56 µL, 9.2 µmol) was added. After
the reaction mixture was stirred at room temperature for 5 min,
sodium periodate (413 mg, 1.93 mmol) was added, and the mixture
was stirred overnight at room temperature in the dark. TLC showed
that the starting material had disappeared. The mixture was diluted
with ether, washed with brine, and dried over MgSO4. The solvent
was removed, and the residue was purified by silica gel chroma-
tography, eluting with 10% hexane in ethyl acetate to give the
1
product as a white foam: 0.308 g (74% yield). H NMR (CDCl3/
TMS) δ 10.50 (brs, 1H), 9.80 (brs, 1H), 7.82 (d, 1H, J ) 7.4 Hz),
7.50 (d, 1H, J ) 7.4 Hz), 5.76 (s, 1H), 4.52 (m, 1H), 4.24 (m, 1H),
4.06 (m, 1H), 3.91 (m, 1H), 3.12 (m, 1H), 2.92 (m, 1H), 2.80 (m,
1H), 2.30 (s, 3H), 1.03 (s, 9H), 1.00 (s, 9H); 13C NMR (CDCl3) δ
199.8, 171.5, 163.1, 154.9, 143.2, 96.9, 91.1, 75.9, 75.1, 67.3, 43.0,
40.8, 27.2, 26.9, 24.8, 22.5, 20.2; HRMS calcd for C21H33N3O6-
NaSi [MNa+] 474.2036, found 474.2034.
Experimental Section
N4-Acetyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-O-(phenoxythio-
carbonyl)cytidine (2). To the solution of 3′,5′-O-(di-tert-butylsi-
lanediyl)cytidine (1)22 (7.54 g, 19.7 mmol) and 4-(dimethylamino)-
pyridine (8.43 g, 69.0 mmol) in dry dichloromethane (150 mL)
under argon at 0 °C, phenyl chlorothionoformate (3.18 mL, 23.0
mmol) was added. The reaction mixture was warmed to room
temperature and stirred at room temperature for 16 h. TLC showed
that no 1 remained in the reaction mixture. The reaction mixture
was cooled with an ice bath to 0 °C. Acetyl chloride (1.78 mL,
25.0 mmol) was added, and the mixture was stirred at 0 °C for 1
h. TLC showed that the reaction was complete. The reaction was
quenched with methanol (1.0 mL). After being stirred at room
temperature for 10 min, the mixture was washed sequentially with
water, saturated aqueous NaHCO3, and brine. The solvent was
removed, and the residue was purified by silica gel chromatography,
eluting with 40% hexane in ethyl acetate to give the product as a
light yellow foam: 8.25 g (75% yield). 1H NMR (CDCl3/TMS) δ
7.71 (d, 1H, J ) 7.6 Hz), 7.51 (d, 1H, J ) 7.6 Hz), 7.42 (m, 2H),
7.31 (t, 1H, J ) 6.4 Hz), 7.13 (dd, 2H, J ) 7.13, 1.0 Hz), 6.07 (d,
1H, J ) 5.2 Hz), 5.89 (s, 1H), 4.53 (dd, 1H, J ) 4.8, 8.8 Hz), 4.45
(dd, 1H, J ) 4.8, 9.2 Hz), 4.16 (m, 1H), 4.08 (m, 1H), 2.30 (s,
3H), 1.09 (s, 18H); 13C NMR (CDCl3) δ 193.7, 171.5, 163.6, 154.4,
153.4, 144.9, 129.5, 126.6, 121.8, 97.4, 92.5, 82.1, 75.2, 75.0, 67.1,
27.2, 27.0, 24.9, 22.7, 20.3; HRMS calcd for C26H35N3O7NaSiS
[MNa+] 584.1863, found 584.1894.
2′-C-r-(2-Acetoxyethyl)-N4-acetyl-3′,5′-O-(di-tert-butylsi-lane-
diyl)-2′-deoxycytidine (5). To a solution of 4 (0.177 g, 0.39 mmol)
in methanol (3.0 mL) at 0 °C under argon, sodium borohydride
(13 mg, 0.35 mmol) was added. The reaction mixture was stirred
at 0 °C for 30 min. The reaction mixture was quenched with
saturated aqueous ammonium chloride, extracted with ethyl acetate,
and dried over MgSO4. The solvent was removed, and the residue
was dried under vacuum to give the residue as a white foam. The
white foam was dissolved into dry dichloromethane (10 mL). To
the resulting solution at 0 °C, 4-(dimethylamino)pyridine (191 mg,
1.56 mmol) and acetyl chloride (55 µL, 0.78 mmol) were added.
After the reaction mixture was stirred at 0 °C for 30 min, TLC
showed that the reaction was complete. The reaction was quenched
with methanol (1.0 mL), and the mixture was stirred at room
temperature for 10 min. The solvent was removed, and the residue
was purified by silica gel chromatography, eluting with 10% hexane
in ethyl acetate to give the product as a white foam: 112 mg (58%
yield). 1H NMR (CDCl3/TMS) δ 10.49 (brs, 1H), 7.77 (d, 1H, J )
7.6 Hz), 7.49 (d, 1H, J ) 7.6 Hz), 5.88 (d, 1H, J ) 0.75 Hz), 4.52
(dd, 1H, J ) 4.9, 9.2 Hz), 4.35 (t, 2H, J ) 6.9 Hz), 4.16 (m, 1H),
4.06 (m, 1H), 3.95 (m, 1H), 2.49 (m, 1H), 2.31 (s, 3H), 2.22 (m,
1H), 2.05 (s, 3H), 1.90 (m, 1H), 1.05 (s, 9H), 1.02 (s, 9H); 13C
NMR (CDCl3) δ 171.4, 170.8, 163.0, 154.6, 143.2, 96.8, 91.4, 75.8,
75.7, 67.5, 62.4, 44.7, 27.3, 27.0, 25.9, 24.8, 22.6, 20.9, 20.2; HRMS
calcd for C23H38N3O7Si [MH+] 496.2479, found 496.2500.
2′-C-r-(2-Acetoxyethyl)-N4-acetyl-2′-deoxycytidine (6). To a
solution of 5 (0.189 g, 0.382 mmol) in THF (10 mL), triethylamine
(0.70 mL, 5.0 mmol) and triethylamine-trihydrofluoride (0.31 mL,
1.9 mmol) were added. The mixture was stirred at room temperature
for 30 min. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 7.5% methanol in
chloroform to give product as a white foam: 0.120 g (88% yield).
1H NMR (CD3OD/TMS) δ 8.43 (d, 1H, J ) 7.6 Hz), 7.41 (dd, 1H,
J ) 4.4, 7.6 Hz), 6.16 (d, 1H, J ) 8.0 Hz), 4.32 (dd, 1H, J ) 2.4,
5.6 Hz), 4.13-4.04 (m, 3H), 3.80 (m, 2H), 2.41 (m, 1H), 2.18 (s,
3H), 2.10 (m, 1H), 1.96 (s, 3H), 1.78 (m, 1H); 13C NMR (CD3OD)
N4-Acetyl-2′-C-r-allyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-deoxy
cytidine (3). Reaction in Dry Benzene. A mixture of thionoester
2 (3.18 g, 5.66 mmol) with allyltributylstannane (7.04 mL, 22.7
mmol) in benzene (40 mL) was degassed by bubbling argon through
the solution for 1 min. AIBN (483 mg, 2.83 mmol) was added,
and the mixture was heated to reflux for 4 h under argon. TLC
showed that the reaction was complete. The solvent was removed,
and the residue was purified by silica gel chromatography, eluting
with 30% hexane in ethyl acetate to give the product as a white
foam: 1.66 g (65% yield).
Reaction in Dry Toluene. A mixture of 2 (1.586 g, 2.83 mmol)
with allyltributylstannane (6.40 mL, 20.6 mmol) in dry toluene (45
1204 J. Org. Chem., Vol. 72, No. 4, 2007

Analogues for Probing SolVent Interactions with RNA
δ 173.0, 172.7, 164.1, 158.2, 146.7, 98.5, 91.1, 89.0, 73.3, 63.8,
63.0, 48.4, 24.7, 24.6, 20.8; HRMS calcd for C15H22N3O7 [MH+]
356.1458, found 356.1466.
49.7, 27.3, 27.1, 24.8, 22.7, 20.3, 19.9, 12.2; HRMS calcd for
C21H36N3O5Si [MH+] 438.2424, found 438.2413.
N4-Acetyl-2′-deoxy-2′-C-r-ethylcytidine (10). To a solution of
9 (89 mg, 0.20 mmol) in THF (10 mL), triethylamine-trihydro-
fluoride (0.17 mL, 1.0 mmol) and triethylamine (0.42 mL, 3.0
mmol) were added. The mixture was stirred at room temperature
for 1 h. TLC showed that the reaction was complete. The solvent
was removed, and the residue was purified by silica gel chroma-
tography, eluting with 7% methanol in chloroform to give the
N4-Acetyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-deoxy-2′-C-r-(2-
hydroxyethyl)cytidine (7) and 2′-Deoxy-3′,5′-O-(di-tert-butylsi-
lanediyl)-2′-C-r-(2-hydroxyethyl)cytidine (7a). To a solution of
4 (639 mg, 1.42 mmol) in methanol (20 mL) at 0 °C under argon,
sodium borohydride (54 mg, 1.42 mmol) was added. The reaction
mixture was stirred at 0 °C for 30 min. TLC showed that the
reaction was complete. The reaction mixture was quenched with
saturated aqueous ammonium chloride, extracted with ethyl acetate,
and dried over MgSO4. The solvent was removed, and the residue
was purified by silica gel chromatography, eluting with ethyl acetate
to give compound 7 as a white foam: 198 mg (31% yield). 7: 1H
NMR (CDCl3/TMS) δ 10.51 (brs, 1H), 7.88 (d, 1H, J ) 7.6 Hz),
7.56 (d, 1H, J ) 7.6 Hz), 6.01 (s, 1H), 4.60-4.53 (m, 2H), 4.12-
3.97 (m, 3H), 3.87 (m, 1H), 2.64 (m, 1H), 2.28 (s, 3H), 2.15 (m,
1H), 1.72 (m, 1H), 1.03 (s, 9H), 1.01 (s, 9H); 13C NMR (CDCl3)
δ 171.4, 163.0, 155.4, 143.0, 97.2, 90.9, 76.3, 75.1, 67.7, 61.0,
46.4, 27.7, 27.3, 27.0, 24.7, 22.6, 20.3; HRMS calcd for C21H35N3O6-
NaSi [MH+] 476.2193, found 476.2173.
A byproduct, 2′-deoxy-3′,5′-O-(di-tert-butylsilanediyl)-2′-C-r-
(2-hydroxyethyl)cytidine (7a) (155 mg, 24% yield), was also
purified by eluting the column with 7.5% methanol in chloroform.
The structure of 7a was confirmed by 1H and 13C NMR. 1H NMR
(CD3OD/CDCl3/TMS) δ 7.50 (d, 1H, J ) 7.5 Hz), 5.93 (d, 1H, J
) 7.5 Hz), 5.91 (s, 1H), 4.49 (dd, 1H, J ) 4.9, 9.2 Hz), 4.18 (dd,
1H, J ) 8.0, 9.2 Hz), 4.05 (m, 1H), 3.94 (m, 1H), 3.87 (m, 1H),
3.82 (m, 1H), 2.53 (m, 1H), 2.16 (m, 1H), 1.70 (m, 1H), 1.05 (s,
9H), 1.02 (s, 9H); 13C NMR (CD3OD/CDCl3) δ 165.6, 156.2, 139.4,
94.8, 90.4, 75.4, 75.3, 67.2, 60.0, 45.2, 28.2, 26.6, 26.4, 22.1, 19.8.
Both 7 and 7a could be converted to 5 by acetylation (see the
description earlier in the Experimental Section for the preparation
of 5).
N4-Acetyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-deoxy-2′-C-r-(2-
iodoethyl)cytidine (8). To a solution of 7 (180 mg, 0.40 mmol) in
a solvent mixture of benzene (12 mL) and CH3CN (3 mL),
triphenylphosphine (524 mg, 2.0 mmol) and imidazole (136 mg,
2.0 mmol) were added. The resulting mixture was cooled to 0 °C,
and iodine (508 mg, 2.0 mmol) was then added. After the reaction
mixture was stirred at room temperature for 3 h, TLC showed that
the reaction was complete. The mixture was diluted with dichlo-
romethane and washed with brine. The organic layer was concen-
trated, and the residue was purified by silica gel chromatography,
eluting with 45% ethyl acetate in hexane to give the product as a
foam: 167 mg (74% yield). 1H NMR (CDCl3/TMS) δ 10.62 (brs,
1H), 7.76 (d, 1H, J ) 7.6 Hz), 7.47 (d, 1H, J ) 7.6 Hz), 5.84 (s,
1H), 4.51 (m, 1H), 4.15 (m, 1H), 4.04 (m, 1H), 3.92 (m, 1H), 3.48
(m, 2H), 2.43 (m, 2H), 2.33 (s, 3H), 2.15 (m, 1H), 1.07 (s, 9H),
1.02 (s, 9H); 13C NMR (CDCl3) δ 171.5, 163.1, 154.7, 143.1, 97.2,
90.7, 76.0, 75.7, 67.4, 48.8, 31.4, 27.3, 27.0, 24.9, 22.6, 20.3, 3.44;
HRMS calcd for C21H35N3O5SiI [MH+] 564.1391, found 564.1411.
1
product as a white foam: 60 mg (99% yield). H NMR (CD3OD/
TMS) δ 8.42 (d, 1H, J ) 7.6 Hz), 7.44 (d, 1H, J ) 7.6 Hz), 6.15
(d, 1H, J ) 8.4 Hz), 4.31 (dd, 1H, J ) 2.0, 5.6 Hz), 4.01 (m, 1H),
3.77 (m, 2H), 2.25-2.12 (m, 1H), 2.17 (s, 3H), 1.74 (m, 1H), 1.32
(m, 1H), 0.94 (t, 3H, J ) 7.6 Hz); 13C NMR (CD3OD) δ 173.1,
164.2, 158.4, 146.8, 98.5, 91.3, 89.1, 73.5, 63.3, 48.4, 24.5, 18.5,
12.4; HRMS calcd for C13H20N3O5 [MH+] 298.1403, found
298.1390.
2′-Deoxy-2′-C-r-propyluridine (13). To a solution of 2′-C-R-
allyl-2′-deoxyuridine 1214 (442 mg, 1.65 mmol) in methanol (20
mL) under argon, palladium on carbon (10 wt %, 176 mg, 0.165
mmol) was added. The reaction was then carried out under a
hydrogen atmosphere with a hydrogen balloon at room temperature
for 24 h. The catalyst was removed by filtration, and the solvent
was removed by evaporation. The residue was purified by silica
gel chromatography, eluting with 2% methanol in ethyl acetate to
give 2′-deoxy-2′-C-R-propyluridine (13)26 as a white foam: 360
mg (81% yield). 13C NMR (D2O) δ 165.9, 151.9, 141.7, 102.8,
88.4, 86.8, 72.2, 61.8, 46.9, 25.4, 19.9, 13.3; HRMS calcd for
C12H18N2O5Na [MNa+] 293.1113, found 293.1100.
3′,5′-O-(Di-tert-butylsilanediyl)-2′-deoxy-2′-C-r-propyluri-
dine (14). To a solution of 13 (324 mg, 1.20 mmol) in dry DMF
(20 mL) at 0 °C, silver nitrate (448 mg, 2.64 mmol) was added,
followed by dropwise addition of di-tert-butyldichlorosilane (279
µL, 1.32 mmol) with vigorous stirring. After the mixture was stirred
at 0 °C for 30 min, triethylamine (368 µL, 2.64 mmol) was added,
and the reaction mixture was stirred at room temperature for 10
min. The solvent was evaporated under reduced pressure, and the
residue was purified by silica gel chromatography, eluting with 25%
ethyl acetate in hexane to give product as a white foam: 252 mg
(51% yield). 1H NMR (CDCl3/TMS) δ 9.89 (brs, 1H), 7.24 (d, 1H,
J ) 8.0 Hz), 5.86 (s, 1H), 5.79 (d, 1H, J ) 8.0 Hz), 4.43 (dd, 1H,
J ) 4.8, 9.2 Hz), 4.17 (m, 1H), 3.96 (m, 1H), 3.80 (m, 1H), 2.27
(m, 1H), 1.80 (m, 1H), 1.53 (m, 1H), 1.40 (m, 2H), 1.10-0.87 (m,
21H); 13C NMR (CDCl3) δ 163.4, 150.0, 139.6, 103.0, 90.1, 76.6,
75.4, 67.4, 47.0, 29.0, 27.3, 27.0, 22.6, 20.5, 20.3, 14.2; HRMS
calcd for C20H35N2O5Si [MH+] 411.2315, found 411.2307.
N4-Acetyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-deoxy-2′-C-r-pro-
pylcytidine (15) and 2′-Deoxy-2′-C-r-propylcytidine (15a). Tri-
ethylamine (0.161 mL, 1.16 mmol) was added to a mixture of 14
(239 mg, 0.58 mmol), 4-(dimethylamino)pyridine (142 mg, 1.16
mmol), and 2,4,6-triisopropylbenzenesulfonyl chloride (351 mg,
1.16 mmol) in dry CH3CN (15 mL). After the reaction mixture
was stirred at room temperature for 60 h, concentrated ammonium
hydroxide (28%, 20 mL) was added slowly into the reaction
mixture. The mixture was stirred at room temperature for 3 h. The
solvent was removed by evaporation, and the aqueous phase was
extracted with dichloromethane. The organic layers were combined,
washed with brine, and dried over MgSO4. The solvent was
removed, and the residue (pale yellow foam) was dried under
vacuum overnight.
N4-Acetyl-3′,5′-O-(di-tert-butylsilanediyl)-2′-deoxy-2′-C-r-eth-
ylcytidine (9). A solution of 8 (133 mg, 0.236 mmol) and AIBN
(16 mg, 0.10 mmol) in a mixed solvent of benzene (10 mL) and
THF (10 mL) was cooled in an ice bath (0 °C) under argon. The
mixture was irradiated with ultrasound (40 kHz, 100 w) by a
Bransonic 2510 R-MT sonicator. n-Bu3SnH (125 µL, 0.47 mmol)
was slowly injected into the solution. The reaction was complete
in 1 h as monitored by TLC. The solvent was removed, and the
residue was purified by silica gel chromatography, eluting with 30%
hexane in ethyl acetate to give the product as a white foam: 94
A portion of the yellow foam (∼25%, ∼0.145 mmol) was
dissolved to THF (5.0 mL) and treated with TBAF (0.30 mL, 1.0
M in THF, 0.30 mmol) at room temperature for 1 h. The solvent
was removed, and the residue was dissolved into water (7.0 mL).
The resulting solution was washed with chloroform (3 × 5 mL).
The aqueous layer was evaporated, and the residue was purified
by silica gel chromatography, eluting with 10% methanol in ethyl
acetate to give 2′-deoxy-2′-C-R-propylcytidine (15a):26 25 mg (64%
yield from 14). 13C NMR (CD3OD) δ 167.4, 158.7, 143.1, 96.6,
1
mg (91% yield). H NMR (CDCl3/TMS) δ 10.62 (brs, 1H), 7.74
(d, 1H, J ) 7.6 Hz), 7.50 (d, 1H, J ) 7.6 Hz), 5.91 (d, 1H, J ) 1.6
Hz), 4.50 (dd, 1H, J ) 4.8, 8.8 Hz), 4.14 (m, 1H), 4.02 (m, 1H),
3.94 (m, 1H), 2.30 (s, 3H), 2.23 (m, 1H), 1.93 (m, 1H), 1.57 (m,
1H), 1.13 (t, 3H, J ) 7.2 Hz), 1.06 (s, 9H), 1.02 (s, 9H); 13C NMR
(CDCl3) δ 171.5, 163.0, 154.7, 143.5, 97.0, 91.3, 76.3, 75.8, 67.7,
J. Org. Chem, Vol. 72, No. 4, 2007 1205

Li and Piccirilli
90.6, 88.8, 74.0, 63.6, 50.4, 27.5, 21.8, 14.6; HRMS calcd for
C12H19N3O4Na [MNa+] 292.1273, found 292.1262.
NMR (CDCl3) δ 171.0, 164.0, 150.2, 139.5, 101.5, 89.8, 82.8, 68.1,
64.1, 60.1, 48.1, 26.3, 22.1, 21.0, 17.4, 17.3, 17.2, 17.1, 16.9, 16.83,
16.79, 16.7, 13.2, 13.0, 12.8, 12.4; HRMS calcd for C26H46N2O8-
NaSi2 [MNa+] 593.2690, found 593.2721.
The remaining portion of the yellow foam (∼75%, ∼0.435
mmol) was dissolved into dry dichloromethane (15 mL) under
argon. To the resulting solution at 0 °C, 4-(dimethylamino)pyridine
(213 mg, 1.74 mmol) and acetyl chloride (62 µL, 0.87 mmol) were
added sequentially. After the reaction mixture was stirred at 0 °C
for 30 min, the reaction was quenched with methanol (1.0 mL)
and stirred at room temperature for 10 min. The solvent was
removed, and the residue was purified by silica gel chromatography,
eluting with 50% ethyl acetate in hexane to give product 15 as a
white foam: 155 mg (79% yield from 14). 1H NMR (CDCl3/TMS)
δ 10.27 (brs, 1H), 7.74 (d, 1H, J ) 7.6 Hz), 7.48 (d, 1H, J ) 7.6
Hz), 5.89 (s, 1H), 4.50 (dd, 1H, J ) 4.4, 9.2 Hz), 4.13 (m, 1H),
4.00 (m, 1H), 3.94 (m, 1H), 2.35-2.25 (m, 1H), 2.28 (s, 3H), 1.84
(m, 1H), 1.66 (m, 1H), 1.40 (m, 2H), 1.07 (s, 9H), 1.02 (s, 9H),
0.95 (t, 3H, J ) 7.0 Hz); 13C NMR (CDCl3) δ 171.2, 162.9, 154.8,
143.5, 96.9, 91.7, 76.2, 75.9, 67.7, 47.8, 28.9, 27.4, 27.1, 24.7, 22.7,
20.7, 20.3, 14.2; HRMS calcd for C22H38N3O5Si [MH+] 452.2581,
found 452.2571.
N4-Acetyl-2′-deoxy-2′-C-r-propylcytidine (16). To a solution
of 15 (153 mg, 0.34 mmol) in THF (10 mL), triethylamine-
trihydrofluoride (0.28 mL, 1.7 mmol) and triethylamine (0.70 mL,
5.0 mmol) were added. The reaction was stirred at room temperature
for 1 h. The solvent was removed, and the residue was purified by
silica gel chromatography, eluting with 7% methanol in chloroform
to give the product as a white foam: 89 mg (85% yield). 1H NMR
(CD3OD/TMS) δ 8.42 (d, 1H, J ) 7.6 Hz), 7.43 (d, 1H, J ) 7.6
Hz), 6.15 (d, 1H, J ) 8.4 Hz), 4.28 (dd, 1H, J ) 2.0, 5.2 Hz), 4.01
(m, 1H), 3.76 (m, 2H), 2.25 (m, 1H), 2.18 (s, 3H), 1.72 (m, 1H),
1.45-1.25 (m, 3H), 0.89 (t, 3H, J ) 6.8 Hz); 13C NMR (CD3OD)
δ 173.1, 164.2, 158.5, 146.8, 98.5, 91.4, 89.2, 73.7, 63.3, 51.6,
27.6, 24.5, 21.8, 14.6; HRMS calcd for C14H22N3O5 [MH+]
312.1559, found 312.1545.
2′-C-r-(3-Acetoxypropyl)-N4-acetyl-2′-deoxy-3′,5′-O-(1,1,3,3-
tetraisopropyldisiloxyane-1,3-diyl)cytidine (19). Triethylamine
(0.21 mL, 1.5 mmol) was added to a mixture of 18 (441 mg, 0.77
mmol), 4-(dimethylamino)pyridine (183 mg, 1.50 mmol), and 2,4,6-
triisopropylbenzenesulfonyl chloride (454 mg, 1.50 mmol) in dry
CH3CN (20 mL). After the reaction mixture was stirred at room
temperature for 48 h, concentrated ammonium hydroxide (28%,
30 mL) was slowly added, and the mixture was stirred at room
temperature for 3 h. The solvent was removed by evaporation, and
the aqueous phase was extracted with chloroform. The organic
layers were combined, washed with brine, and dried over MgSO
.
4
The solvent was removed by rotary evaporation, and the residue
(pale yellow foam) was dried overnight under vacuum. The pale
yellow foam was dissolved in dry dichloromethane (25 mL). To
the resulting solution at 0 °C, 4-(dimethylamino)pyridine (376 mg,
3.08 mmol) was added, followed by dropwise addition of acetyl
chloride (109 µL, 1.54 mmol). After the reaction mixture was stirred
at 0 °C for 30 min, the reaction was quenched with methanol (1.0
mL) and stirred at room temperature for 10 min. The solvent was
removed, and the residue was purified by silica gel chromatography,
eluting with 30% hexane in ethyl acetate to give the product as a
white foam: 386 mg (82% yield). 1H NMR (CDCl3/TMS) δ 10.40
(brs, 1H), 8.33 (d, 1H, J ) 7.4 Hz), 7.43 (d, 1H, J ) 7.4 Hz), 5.79
(s, 1H), 4.39 (m, 1H), 4.24 (m, 1H), 4.13 (m, 2H), 4.00 (m, 2H),
2.28 (s, 3H), 2.24 (m, 1H), 2.04 (s, 3H), 2.03-1.75 (m, 3H), 1.52
(m, 1H), 1.15-0.90 (m, 28H); 13C NMR (CDCl3) δ 171.13, 171.09,
163.0, 154.9, 144.3, 96.2, 90.2, 82.8, 67.6, 64.3, 59.9, 48.0, 26.6,
24.8, 22.5, 20.9, 17.5, 17.4, 17.3, 17.2, 17.0, 16.9, 16.83, 16.80,
13.3, 13.1, 12.8, 12.5; HRMS calcd for C28H49N3O8NaSi2 [MNa+]
634.2956, found 634.2925.
2′-C-r-(3-Acetoxypropyl)-N4-acetyl-2′-deoxycytidine (20). To
a solution of 19 (376 mg, 0.615 mmol) in THF (15 mL),
triethylamine-trihydrofluoride (0.51 mL, 3.1 mmol) and triethyl-
amine (1.39 mL, 10.0 mmol) were added. The reaction was stirred
at room temperature for 2 h. The solvent was removed, and the
residue was purified by silica gel chromatography, eluting with 7.5%
methanol in chloroform to give the product as a white foam: 227
mg (>99% yield). 1H NMR (CD3OD/CDCl3/TMS) δ 8.37 (d, 1H,
J ) 7.5 Hz), 7.46 (d, 1H, J ) 7.5 Hz), 6.07 (d, 1H, J ) 7.6 Hz),
4.33 (dd, 1H, J ) 2.7, 5.7 Hz), 4.10-4.00 (m, 3H), 3.82 (m, 2H),
2.30 (m, 1H), 2.22 (s, 3H), 2.03 (s, 3H), 1.85-1.65 (m, 3H), 1.45
(m, 1H); 13C NMR (CD3OD/CDCl3) δ 172.1, 171.7, 162.7, 156.9,
145.9, 97.7, 91.0, 87.6, 71.9, 64.8, 62.2, 50.0, 26.6, 24.5, 21.0, 20.9;
HRMS calcd for C16H23N3O7Na [MNa+] 392.1434, found 392.1426.
2′-Deoxy-2′-C-r-(3-butenyl)-3′,5′-O-(1,1,3,3-tetraisopropyldi-
siloxyane-1,3-diyl)uridine (21). Under argon, Dess-Martin pe-
riodinane (1.27 g, 3.0 mmol) was dissolved into dry dichlo-
romethane (15 mL) and cooled to 0 °C. 2′-Hydroxypropyluridine
17 (1.06 g, 2.0 mmol) in dichloromethane (20 mL) was then added.
After the mixture was stirred at 0 °C for 2 h, TLC showed that the
reaction was not yet complete (∼5-10% starting material re-
mained). Additional Dess-Martin periodinane (127 mg, 0.30 mmol)
was then added, and the reaction mixture was stirred at 0 °C for 1
h. TLC showed that the reaction was complete. The reaction mixture
was diluted with ether (50 mL) and washed with saturated aqueous
NaHCO3 (50 mL) containing Na2S2O3‚5H2O (5.2 g, 21.0 mmol).
The aqueous layer was extracted with ether (3 × 15 mL). The
combined organic layers were sequentially washed with saturated
NaHCO3 and brine and dried over anhydrous MgSO4. The solvent
was removed by rotary evaporation, and the residue was dried under
vacuum to give the crude aldehyde as a white foam, which was
used for the following Wittig reaction without further purification.
In a separate flask containing a suspension of triphenylmethylphos-
phonium bromide (2.86 g, 8.0 mmol) in THF (15 mL) at -78 °C
under argon, s-butyllithium (6.15 mL, 1.3 M in cyclohexane, 8.0
2′-Deoxy-2′-C-r-(3-hydroxypropyl)-3′,5′-O-(1,1,3,3-tetraiso-
propyldisiloxyane-1,3-diyl)uridine (17). To a solution of 2′-
allyluridine 11 (1.91 g, 3.70 mmol) in THF (30 mL) under argon,
9-BBN (1.81 g, 7.40 mmol) was added. After the reaction mixture
was stirred at room temperature for 4 h, sodium perborate
tetrahydrate (3.42 g, 22.3 mmol) and water (7.0 mL) were added,
and the mixture was stirred at room temperature for 2 h. The
reaction mixture was extracted with ethyl acetate, and the organic
layers were combined and dried over MgSO4. Following removal
of solvent by rotary evaporation, the residue was purified by silica
gel chromatography, eluting with 30% hexane in ethyl acetate to
give product (17)5 as a white foam: 1.48 g (76% yield). 13C NMR
(CDCl3) δ 163.7, 150.9, 139.5, 101.9, 89.4, 82.8, 67.7, 62.5, 59.9,
48.4, 30.3, 21.7, 17.5, 17.4, 17.3, 17.2, 17.03, 16.96, 16.9, 16.8,
13.4, 13.0, 12.9, 12.4; HRMS calcd for C24H44N2O7NaSi2 [MNa+]
551.2585, found 551.2606.
2′-C-r-(3-Acetoxypropyl)-2′-deoxy-3′,5′-O-(1,1,3,3-tetraiso-
propyldisiloxyane-1,3-diyl)uridine (18). To a solution of 17 (544
mg, 1.03 mmol) in dichloromethane (10 mL), 4-(dimethylamino)-
pyridine (126 mg, 1.03 mmol) and triethylamine (0.72 mL, 5.1
mmol) were added. Under argon, the mixture was cooled to 0 °C,
and acetic anhydride (0.12 mL, 1.3 mmol) was added. The ice bath
was removed, and the reaction mixture was stirred at room
temperature for 1 h. TLC showed that the reaction was complete.
The reaction was then quenched with methanol (1.0 mL). After 5
min, the solvent was removed, and the residue was dissolved in
chloroform. The chloroform solution was washed sequentially with
water and brine and dried over MgSO4. Following removal of
solvent by evaporation, the residue was purified by silica gel
chromatography, eluting with 50% ethyl acetate in hexane to give
1
product as a white foam: 550 mg (94% yield). H NMR (CDCl3/
TMS) δ 10.23 (brs, 1H), 7.84 (d, 1H, J ) 8.4 Hz), 5.78 (s, 1H),
5.71 (d, 1H, J ) 8.4 Hz), 4.45 (m, 1H), 4.20-4.05 (m, 3H), 3.99
(dd, 1H, J ) 2.7, 13.2 Hz), 3.91 (m, 1H), 2.20 (m, 1H), 2.05 (s,
3H), 2.03-1.75 (m, 3H), 1.48 (m, 1H), 1.15-0.90 (m, 28H); 13C
1206 J. Org. Chem., Vol. 72, No. 4, 2007

Analogues for Probing SolVent Interactions with RNA
mmol) was added slowly. The mixture was allowed to warm slowly
to -30 °C in 30 min and cooled to -78 °C again. A THF (10 mL)
solution of the aldehyde (∼2.0 mmol) was then added slowly under
argon. The reaction mixture was allowed to warm to room
temperature and stirred for 20 h. The mixture was diluted with
dichloromethane (100 mL), and the solution was washed sequen-
tially with water, saturated ammonium chloride, and brine and dried
over MgSO4. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 20% ethyl acetate in
hexane to give the product as a white foam: 0.716 g (68% yield).
1H NMR (CDCl3/TMS) δ 10.04 (brs, 1H), 7.81 (d, 1H, J ) 8.0
Hz), 5.83 (m, 2H), 5.71 (d, 1H, J ) 8.0 Hz), 5.06 (d, 1H, J ) 16.0
Hz), 4.98 (d, 1H, J ) 8.0 Hz), 4.44 (m, 1H), 4.14 (m, 1H), 3.99
(dd, 1H, J ) 4.0, 12.0 Hz), 3.90 (m, 1H), 2.42-2.10 (m, 3H), 1.94
(m, 1H), 1.50 (m, 1H), 1.10-0.95 (m, 28H); 13C NMR (CDCl3) δ
164.1, 150.2, 139.7, 138.0, 115.1, 101.6, 89.0, 83.0, 68.4, 60.3,
47.7, 31.1, 24.8, 17.5, 17.33, 17.27, 17.2, 17.0, 16.92, 16.87, 16.8,
13.3, 13.0, 12.9, 12.4; HRMS calcd for C25H45N2O6Si2 [MH+]
525.2816, found 525.2802.
pressure, and the aqueous layer was extracted with chloroform. The
organic layers were combined, washed with brine, and dried over
MgSO4. The solvent was removed, and the residue (pale yellow
foam) was dried under vacuum overnight. The pale yellow foam
was dissolved into dry dichloromethane (10 mL). To the resulting
solution at 0 °C, 4-(dimethylamino)pyridine (71 mg, 0.58 mmol)
and acetyl chloride (21 µL, 0.29 mmol) were added. After the
reaction mixture was stirred at 0 °C for 30 min, the reaction was
quenched with methanol (0.5 mL) and stirred at room temperature
for 10 min. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 30% hexane in ethyl
acetate to give the product as a white foam: 76 mg (84% yield).
1H NMR (CDCl3/TMS) δ 10.55 (brs, 1H), 8.33 (d, 1H, J ) 7.6
Hz), 7.42 (d, 1H, J ) 7.4 Hz), 5.77 (s, 1H), 4.40 (m, 1H), 4.23 (m,
1H), 4.08 (m, 2H), 4.02-3.90 (m, 2H), 2.28 (s, 3H), 2.24 (m, 1H),
2.04 (s, 3H), 1.82-1.42 (m, 6H), 1.15-0.90 (m, 28H); 13C NMR
(CDCl3) δ 171.2, 163.1, 154.9, 144.3, 96.2, 90.5, 82.8, 67.6, 64.3,
59.9, 48.2, 28.8, 26.0, 24.8, 24.0, 21.0, 17.5, 17.4, 17.3, 17.2, 17.0,
16.94, 16.86, 16.8, 13.3, 13.1, 12.8, 12.4; HRMS calcd for
C29H51N3O8NaSi2 [MNa+] 648.3112, found 648.3144.
2′-Deoxy-2′-C-r-(4-hydroxybutyl)-3′,5′-O-(1,1,3,3-tetraisopro-
pyldisiloxyane-1,3-diyl)uridine (22). To a solution of 21 (602 mg,
1.15 mmol) in anhydrous THF (20 mL) at 0 °C, 9-BBN (842 mg,
3.45 mmol) was added. After the reaction mixture was stirred at
room temperature for 2 h, sodium perborate tetrahydrate (3.19 g,
20.7 mmol) and water (5.0 mL) were added. The mixture was then
stirred at room temperature for 2 h. The mixture was extracted with
ethyl acetate, and the organic layers were combined and dried over
MgSO4. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 35% hexane in ethyl
acetate to give the product as a white foam 547 mg (88% yield).
1H NMR (CDCl3/TMS) δ 10.98 (brs, 1H), 7.96 (d, 1H, J ) 8.0
Hz), 5.79 (s, 1H), 5.72 (d, 1H, J ) 8.0 Hz), 4.42 (m, 1H), 4.21 (m,
1H), 3.98 (m, 1H), 3.90 (m, 1H), 3.69 (brs, 3H), 2.21 (m, 1H),
1.87 (m, 2H), 1.70-1.30 (m, 4H), 1.11-0.95 (m, 28H); 13C NMR
(CDCl3) δ 164.1, 150.9, 139.6, 101.7, 88.9, 82.6, 67.4, 62.2, 59.8,
48.8, 33.0, 25.2, 23.9, 17.4, 17.3, 17.2, 17.1, 16.9, 16.82, 16.78,
16.7, 13.2, 13.0, 12.7, 12.3; HRMS calcd for C25H47N2O7Si2 [MH+]
543.2922, found 543.2909.
2′-C-r-(4-Acetoxybutyl)-2′-deoxy-3′,5′-O-(1,1,3,3-tetraisopro-
pyldisiloxyane-1,3-diyl)uridine (23). To a solution of 2′-hydroxy-
butyluridine 22 (174 mg, 0.32 mmol) in dichloromethane (10 mL),
4-(dimethylamino)pyridine (39 mg, 0.32 mmol) and triethylamine
(0.22 mL, 1.6 mmol) were added. The mixture was cooled to 0
°C, and acetic anhydride (45 µL, 0.48 mmol) was added. After the
reaction mixture was stirred at room temperature for 1 h, the
reaction was quenched with methanol (1.0 mL). The solvent was
removed by rotary evaporation, and the residue was dissolved into
chloroform. The chloroform solution was sequentially washed with
water and brine and dried over MgSO4. The solvent was removed,
and the product was purified by silica gel chromatography, eluting
with 50% ethyl acetate in hexane as a white foam: 174 mg (93%
yield). 1H NMR (CDCl3/TMS) δ 10.21 (brs, 1H), 7.84 (d, 1H, J )
8.0 Hz), 5.77 (d, 1H, J ) 1.2 Hz), 5.71 (d, 1H, J ) 8.0 Hz), 4.44
(m, 1H), 4.20-4.05 (m, 3H), 3.98 (dd, 1H, J ) 2.4, 13.2 Hz), 3.90
(m, 1H), 2.20 (m, 1H), 2.05 (s, 3H), 1.90 (m, 1H), 1.80-1.40 (m,
5H), 1.15-0.90 (m, 28H); 13C NMR (CDCl3) δ 171.1, 164.1, 150.2,
139.6, 101.5, 89.0, 82.9, 68.2, 64.2, 60.2, 48.4, 28.6, 25.4, 23.7,
21.0, 17.4, 17.3, 17.2, 17.1, 16.9, 16.87, 16.83, 16.7, 13.3, 13.0,
12.8, 12.4; HRMS calcd for C27H48N2O8NaSi2 [MNa+] 607.2847,
found 607.2839.
2′-C-r-(4-Acetoxybutyl)-N4-acetyl-2′-deoxycytidine (25). To a
solution of 24 (68 mg, 0.11 mmol) in THF (5.0 mL), triethylamine-
trihydrofluoride (89 µL, 0.55 mmol) and triethylamine (0.22 mL,
1.6 mmol) were added. The reaction mixture was stirred at room
temperature for 2 h. TLC showed that the reaction was complete.
The solvent was removed, and the residue was purified by silica
gel chromatography, eluting with 7.5% methanol in chloroform to
give the product as a white foam: 42 mg (>99% yield). 1H NMR
(CD3OD/TMS) δ 8.43 (d, 1H, J ) 7.4 Hz), 7.44 (d, 1H, J ) 7.4
Hz), 6.15 (d, 1H, J ) 8.4 Hz), 4.30 (dd, 1H, J ) 1.6, 5.2 Hz), 4.02
(m, 3H), 3.77 (m, 2H), 2.26 (m, 1H), 2.18 (s, 3H), 2.00 (s, 3H),
1.75 (m, 1H), 1.60 (m, 2H), 1.45-1.30 (m, 3H); 13C NMR (CD3-
OD) δ 173.1, 164.2, 158.5, 146.8, 98.5, 91.3, 89.1, 73.6, 65.5, 63.3,
51.7, 29.8, 25.1, 25.0, 24.6, 20.8; HRMS calcd for C17H26N3O7
[MH+] 384.1771, found 384.1781.
2′-Deoxy-2′-C-r-(4-iodobutyl)-3′,5′-O-(1,1,3,3-tetraisopropyl-
disiloxyane-1,3-diyl)uridine (26). To a solution of 22 (320 mg,
0.59 mmol), triphenylphosphine (787 mg, 3.0 mmol), and imidazole
(204 mg, 3.0 mmol) in a solvent mixture of benzene (32 mL) and
CH3CN (8.0 mL) at 0 °C, iodine (761 mg, 3.0 mmol) was added.
The reaction mixture was stirred at 0 °C for 45 min. TLC showed
that the reaction was complete. The mixture was oxidized with
hydrogen peroxide (50%, 0.5 mL) at room temperature for 10 min.
The product was extracted with dichloromethane, washed sequen-
tially with saturated aqueous NaHCO3, saturated aqueous Na2S2O3,
and brine, and dried over MgSO4. The solvent was removed, and
the residue was purified by silica gel chromatography, eluting with
30% ethyl acetate in hexane to give the product as a white foam:
0.361 g (94% yield). 1H NMR (CDCl3/TMS) δ 9.56 (brs, 1H), 7.88
(d, 1H, J ) 8.1 Hz), 5.80 (d, 1H, J ) 1.3 Hz), 5.75 (dd, 1H, J )
1.9, 8.1 Hz), 4.47 (m, 1H), 4.18 (m, 1H), 4.02 (dd, 1H, J ) 2.7,
13.2 Hz), 3.93 (m, 1H), 3.26 (t, 2H, J ) 6.8 Hz), 2.21 (m, 1H),
2.00-1.40 (m, 6H), 1.15-0.95 (m, 28H); 13C NMR (CDCl3) δ
163.7, 150.2, 139.7, 101.6, 89.2, 83.0, 68.2, 60.2, 48.4, 33.6, 28.4,
24.8, 17.5, 17.4, 17.3, 17.2, 17.1, 17.01, 16.96, 16.9, 13.4, 13.1,
12.9, 12.5, 6.6; HRMS calcd for C25H45N2O6NaSi2I [MNa+]
675.1759, found 675.1758.
2′-Deoxy-2′-C-r-butyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxy-
ane-1,3-diyl)uridine (27). A solution of 2′-R-iodobutyluridine 26
(487 mg, 0.747 mmol) and AIBN (25 mg, 0.15 mmol) in dry toluene
(30 mL) under argon was cooled to 0 °C and irradiated with
ultrasound (40 kHz, 100 W) by a Bransonic 2510 R-MT sonicator.
n-Bu3SnH (387 µL, 1.46 mmol) was slowly injected into the
solution, and the reaction was irradiated at 0 °C for 1 h. TLC
showed that the reaction was not complete (∼50% conversion).
To the mixture, additional AIBN (25 mg, 0.15 mmol) and n-Bu3-
SnH (387 µL, 1.46 mmol) were added. After the mixture was
irradiated at 0 °C for an additional 2 h, TLC showed that the reaction
was complete. The solvent was removed, and the residue was
2′-C-r-(4-Acetoxybutyl)-N4-acetyl-2′-deoxy-3′,5′-O-(1,1,3,3-
tetraisopropyldisiloxyane-1,3-diyl)cytidine (24). Triethylamine
(56 µL, 0.40 mmol) was added to a mixture of 23 (85.0 mg, 0.145
mmol), 4-(dimethylamino)pyridine (49 mg, 0.40 mmol), and 2,4,6-
triisopropylbenzenesulfonyl chloride (121 mg, 0.40 mmol) in dry
CH3CN (5.0 mL). After the mixture was stirred at room temperature
for 66 h, concentrated ammonium hydroxide (28%, 5 mL) was
slowly added, and the mixture was stirred at room temperature for
3 h. The solvent was removed by rotary evaporation under reduced
J. Org. Chem, Vol. 72, No. 4, 2007 1207

Li and Piccirilli
purified by silica gel chromatography, eluting first with hexane to
remove the tin compounds, following with 30% hexane in ethyl
acetate to give the product as a white foam: 379 mg (97% yield).
1H NMR (CDCl3/TMS) δ 9.48 (brs, 1H), 7.81 (d, 1H, J ) 8.1
Hz), 5.79 (d, 1H, J ) 1.9 Hz), 5.70 (d, 1H, J ) 8.1 Hz), 4.44 (m,
1H), 4.13 (dd, 1H, J ) 2.4, 13.2 Hz), 3.99 (dd, 1H, J ) 2.8, 13.2
Hz), 3.90 (m, 1H), 2.14 (m, 1H), 1.86 (m, 1H), 1.57 (m, 1H), 1.50-
1.30 (m, 4H), 1.15-0.95 (m, 28H), 0.92 (t, 1H, J ) 7.3 Hz); 13C
NMR (CDCl3) δ 163.8, 150.1, 139.8, 101.6, 89.2, 83.1, 68.5, 60.4,
48.5, 29.5, 25.4, 22.8, 17.5, 17.4, 17.3, 17.2, 17.02, 17.96, 16.9,
16.8, 13.9, 13.4, 13.1, 12.9, 12.5; HRMS calcd for C25H47N2O6Si2
[MH+] 527.2973, found 527.2977.
to give the product as a light yellow foam: 0.167 g (76% yield).
1H NMR (CDCl3/TMS) δ 10.06 (brs, 1H), 8.19 (d, 1H, J ) 7.5
Hz), 7.40 (d, 2H, J ) 7.5 Hz), 7.35-7.18 (m, 7H), 7.16 (d, 1H, J
) 7.5 Hz), 6.85 (d, 4H, J ) 8.8 Hz), 6.35 (d, 1H, J ) 7.6 Hz),
4.43 (m, 1H), 4.22 (m, 2H), 4.16-4.05 (m, 1H), 3.78 (s, 6H), 3.47
(m, 2H), 2.56 (brs, 1H), 2.38 (m, 1H), 2.21 (s, 3H), 2.17 (m, 1H),
1.94 (s, 3H), 1.88 (m, 1H); 13C NMR (CDCl3) δ 171.2, 171.0, 162.7,
158.5, 155.9, 144.9, 144.2, 135.2, 135.1, 129.9, 127.9, 127.0, 113.2,
97.3, 89.4, 86.9, 86.0, 72.5, 63.6, 62.9, 55.1, 48.5, 24.7, 23.4, 20.9;
HRMS calcd for C36H40N3O9 [MH+] 658.2765, found 658.2751.
N4-Acetyl-2′-deoxy-5′-O-(dimethoxytrityl)-2′-C-r-ethylcyti-
dine (31). Compound 10 (69 mg, 0.23 mmol) was coevaporated
with dry pyridine (2 × 3 mL) and redissolved into dry pyridine
(10 mL). To the resulting solution, 4-(dimethylamino)pyridine (56
mg, 0.46 mmol) and DMTrCl (156 mg, 0.46 mmol) were added.
After the reaction mixture was stirred at room temperature for 15
h, TLC showed that the reaction was not complete. Additional
4-(dimethylamino)pyridine (56 mg, 0.46 mmol) and DMTrCl (156
mg, 0.46 mmol) were added to the reaction mixture. The mixture
was stirred at room temperature until TLC indicated that the reaction
was complete (∼6 h). The solvent was removed, and the residue
was purified by silica gel chromatography, eluting with 3%
methanol in chloroform to give the product as a light yellow
foam: 0.122 g (89% yield). 1H NMR (CDCl3/TMS) δ 10.13 (brs,
1H), 8.09 (d, 1H, J ) 7.6 Hz), 7.41 (d, 2H, J ) 8.8 Hz), 7.35-
7.25 (m, 6H), 7.23 (m, 1H), 7.18 (d, 1H, J ) 7.6 Hz), 6.85 (d, 4H,
J ) 8.8 Hz), 6.36 (d, 1H, J ) 7.6 Hz), 4.43 (m, 1H), 4.21 (m, 1H),
3.78 (s, 6H), 3.45 (m, 2H), 2.18 (s, 3H), 2.14 (m, 1H), 1.82 (m,
1H), 1.50 (m, 1H), 0.97 (t, 3H, J ) 7.4 Hz); 13C NMR (CDCl3) δ
171.0, 162.6, 158.5, 155.8, 145.0, 144.2, 135.3, 135.1, 130.0, 128.0,
127.9, 127.0, 113.2, 97.2, 89.4, 86.8, 85.8, 72.5, 63.5, 55.1, 53.1,
24.6, 17.2, 12.2; HRMS calcd for C34H38N3O7 [MH+] 600.2710,
found 600.2728.
N4-Acetyl-2′-C-r-butyl-2′-deoxy-3′,5′-O-(1,1,3,3-tetraisopro-
pyldisiloxyane-1,3-diyl)cytidine (28). Triethylamine (185 µL, 1.33
mmol) was added to a mixture of 27 (269 mg, 0.51 mmol),
4-(dimethylamino)pyridine (162 mg, 1.33 mmol), and 2,4,6-
triisopropylbenzenesulfonyl chloride (403 mg, 1.33 mmol) in dry
CH3CN (15 mL). After the mixture was stirred at room temperature
for 53 h, concentrated ammonium hydroxide (28%, 17.5 mL) was
added slowly, and the mixture was stirred at room temperature for
3 h. The solvent was removed by evaporation under vacuum, and
the aqueous phase was extracted with chloroform. The organic
layers were combined, washed with brine, and dried over MgSO4.
The solvent was removed, and the residue (pale yellow powder)
was dried under vacuum overnight. The pale yellow solid powder
was dissolved into dry dichloromethane (20 mL). To the resulting
solution at 0 °C, 4-(dimethylamino)pyridine (125 mg, 1.02 mmol)
and acetyl chloride (72.5 µL, 1.02 mmol) were added. After the
reaction mixture was stirred at 0 °C for 30 min, the reaction was
quenched with methanol (1.0 mL) and stirred at room temperature
for 10 min. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 50% hexane in ethyl
1
acetate to give product as a white foam: 260 mg (90% yield). H
2′-C-r-(3-Acetoxypropyl)-N4-acetyl-2′-deoxy-5′-O-(dimethoxy-
trityl)cytidine (32). Nucleoside 20 (207 mg, 0.56 mmol) was
coevaporated with dry pyridine (2 × 5 mL) and then dissolved
into dry pyridine (10 mL). To the resulting pyridine solution,
4-(dimethylamino)pyridine (137 mg, 1.12 mmol) and DMTrCl (383
mg, 1.12 mmol) were added. The reaction mixture was stirred at
room temperature for 18 h. TLC indicated that the reaction was
not complete (∼90% conversion). Additional DMTrCl (38 mg, 0.11
mmol) was added to the reaction mixture. The reaction mixture
was stirred at room temperature for an additional 6 h. The reaction
was quenched with MeOH (1.0 mL). The solvent was removed,
and the residue was purified by silica gel chromatography, eluting
with 3% methanol in chloroform to give the product as a light
yellow foam: 323 mg (86% yield). 1H NMR (CDCl3/TMS) δ 10.05
(brs, 1H), 8.14 (d, 1H, J ) 7.6 Hz), 7.40 (d, 2H, J ) 7.2 Hz),
7.35-7.20 (m, 7H), 7.18 (d, 1H, J ) 7.6 Hz), 6.85 (d, 4H, J ) 8.8
Hz), 6.30 (d, 1H, J ) 6.8 Hz), 4.42 (m, 1H), 4.19 (m, 1H), 4.04
(m, 2H), 3.79 (s, 6H), 3.47 (m, 2H), 2.25 (m, 1H), 2.19 (s, 3H),
1.99 (s, 3H), 1.85 (m, 1H), 1.74 (m, 2H), 1.50 (m, 1H); 13C NMR
(CDCl3) δ 171.2, 170.9, 162.6, 158.6, 155.8, 144.9, 144.1, 135.3,
135.1, 129.9, 128.0, 127.9, 127.0, 113.2, 97.2, 89.5, 86.9, 85.6,
72.1, 64.2, 63.3, 55.1, 50.7, 26.6, 24.6, 20.9, 20.6; HRMS calcd
for C37H41N3O9Na [MNa+] 694.2741, found 694.2723.
NMR (CDCl3/TMS) δ 10.87 (brs, 1H), 8.32 (d, 1H, J ) 7.5 Hz),
7.43 (d, 1H, J ) 7.4 Hz), 5.81 (s, 1H), 4.38 (m, 1H), 4.22 (m, 1H),
3.99 (m, 1H), 3.96 (m, 1H), 2.30 (s, 3H), 2.20 (m, 1H), 1.88 (m,
1H), 1.63 (m, 1H), 1.49 (m, 2H), 1.35 (m, 2H), 1.15-0.80 (m,
31H); 13C NMR (CDCl3) δ 171.5, 163.2, 154.9, 144.3, 96.3, 90.5,
82.9, 67.8, 60.1, 48.3, 29.6, 25.8, 24.7, 22.8, 17.5, 17.4, 17.3, 17.2,
17.0, 16.9, 16.83, 16.80, 13.9, 13.3, 13.1, 12.8, 12.4; HRMS calcd
for C27H50N3O6Si2 [MH+] 568.3238, found 568.3213.
N4-Acetyl-2′-C-r-butyl-2′-deoxycytidine (29). To a solution of
28 (171 mg, 0.30 mmol) in THF (10 mL), triethylamine-
trihydrofluoride (245 µL, 1.50 mmol) and triethylamine (0.63 mL,
4.5 mmol) were added. The reaction mixture was stirred at room
temperature for 2 h. TLC showed that the reaction was complete.
The solvent was removed, and the residue was purified by silica
gel chromatography, eluting with 6% methanol in chloroform to
1
give the product as a white solid: 93 mg (95% yield). H NMR
(CD3OD/TMS) δ 8.39 (d, 1H, J ) 7.6 Hz), 7.45 (d, 1H, J ) 7.4
Hz), 6.15 (d, 1H, J ) 8.6 Hz), 4.34 (m, 1H), 4.10 (m, 1H), 3.79
(m, 2H), 2.35-2.20 (m, 1H), 2.25 (s, 3H), 1.71 (m, 1H), 1.45-
1.20 (m, 5H), 0.86 (t, 3H, J ) 6.9 Hz); 13C NMR (CD3OD) δ 174.2,
163.8, 158.3, 146.9, 99.0, 91.2, 88.7, 73.6, 63.0, 51.0, 30.3, 24.8,
24.7, 23.5 14.2; HRMS calcd for C15H24N3O5 [MH+] 326.1716,
found 326.1710.
2′-C-r-(2-Acetoxyethyl)-N4-acetyl-2′-deoxy-5′-O-(dimethoxy-
trityl)cytidine (30). Nucleoside 6 (119 mg, 0.335 mmol) was
coevaporated with dry pyridine (2 × 5 mL) and dried under vacuum
for 30 min. To a solution of dried 6 in dry pyridine (10 mL),
4-(dimethylamino)pyridine (82 mg, 0.67 mmol) and DMTrCl (227
mg, 0.67 mmol) were added. After the reaction mixture was stirred
at room temperature for 15 h, TLC indicated that the reaction was
not complete (only ∼10% conversion). To the reaction mixture,
additional 4-(dimethylamino)pyridine (82 mg, 0.67 mmol) and
DMTrCl (227 mg, 0.67 mmol) were added. The reaction mixture
was stirred at room temperature until the reaction was complete
(∼5 h). The solvent was removed, and the residue was purified by
silica gel chromatography, eluting with 3% methanol in chloroform
N4-Acetyl-2′-deoxy-5′-O-(dimethoxytrityl)-2′-C-r-propylcyti-
dine (33). Nucleoside 16 (86 mg, 0.28 mmol) was coevaporated
with dry pyridine (2 × 2 mL) and then dissolved into dry pyridine
(10 mL). To the resulting pyridine solution, 4-(dimethylamino)-
pyridine (81 mg, 0.66 mmol) and DMTrCl (281 mg, 0.83 mmol)
were added. The reaction mixture was stirred at room temperature
for 24 h. TLC indicated that the reaction was not complete (∼90%
conversion). To the reaction mixture, additional 4-(dimethylamino)-
pyridine (38 mg, 0.31 mmol) and DMTrCl (28 mg, 0.088 mmol)
were added. The reaction mixture was stirred at room temperature
for an additional 3 h. The reaction was quenched with MeOH (1.0
mL). The solvent was removed, and the residue was purified by
silica gel chromatography, eluting with 3% methanol in chloroform
1208 J. Org. Chem., Vol. 72, No. 4, 2007

Analogues for Probing SolVent Interactions with RNA
to give the product as a light yellow foam: 139 mg (80% yield).
1H NMR (CDCl3/TMS) δ 10.16 (brs, 1H), 8.11 (d, 1H, J ) 7.6
Hz), 7.40 (m, 2H), 7.35-7.18 (m, 7H), 7.16 (d, 1H, J ) 7.6 Hz),
6.85 (d, 4H, J ) 8.4 Hz), 6.39 (d, 1H, J ) 8.0 Hz), 4.41 (m, 1H),
4.20 (m, 1H), 3.79 (s, 6H), 3.50-3.45 (m, 3H), 2.25 (m, 1H), 2.19
(s, 3H), 1.77 (m, 1H), 1.55-1.30 (m, 3H), 0.89 (t, 3H, J ) 6.8
Hz); 13C NMR (CDCl3) δ 171.0, 162.7, 158.6, 155.8, 145.0, 144.2,
135.4, 135.2, 130.0, 128.03, 127.95, 127.1, 113.2, 97.3, 89.4, 86.9,
86.0, 73.0, 63.6, 55.2, 51.2, 26.1, 24.7, 20.8, 14.1; HRMS calcd
for C35H39N3O7Na [MNa+] 636.2686, found 636.2684.
the phosphoramidite derivative as a white foam: 163 mg (100%
conversion, g95% purity). 31P NMR (CD3CN) δ 153.7, 152.1;
HRMS calcd for C45H56N5O10NaP [MNa+] 880.3663, found
880.3644.
N4-Acetyl-2′-deoxy-5′-O-(dimethoxytrityl)-2′-C-r-ethylcyti-
dine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (37). Ac-
cording to the above procedure, the reaction mixture containing
31 (106 mg, 0.177 mmol), N,N-diisopropylethylamine (82 µL, 0.47
mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (83
mg, 0.35 mmol), and 1-methylimidazole (8.0 µL, 0.10 mmol) was
stirred at room temperature for 1 h. The phosphoramidite 37 was
purified by silica gel chromatography to give a white foam: 141
mg (100% conversion, g95% purity). 31P NMR (CD3CN) δ 153.8,
151.8; HRMS calcd for C43H54N5O8NaP [MNa+] 822.3608, found
822.3597.
2′-C-r-(4-Acetoxybutyl)-N4-acetyl-2′-deoxy-5′-O-(dimethoxy-
trityl)cytidine (34). Nucleoside 25 (42 mg, 0.11 mmol) was
coevaporated with dry pyridine and dried under vacuum for 4 h.
Under argon, to a solution of dried nucleoside 33 in pyridine (5.0
mL), 4-(dimethylamino)pyridine (53 mg, 0.44 mmol) and DMTrCl
(148 mg, 0.44 mmol) were added. After the reaction mixture was
stirred at room temperature overnight, TLC indicated that only a
small amount of product had formed. To the reaction mixture, silver
nitrate (19 mg, 0.11 mmol) was added, and the mixture was stirred
at room temperature for 48 h. TLC showed that no starting material
remained. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 3% methanol in
chloroform to give the product as a light yellow foam: 73 mg (98%
2′-C-r-(3-Acetoxypropyl)-N4-acetyl-2′-deoxy-5′-O-(dimethoxy-
trityl)cytidine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite
(38). According to the above procedure, a reaction mixture
containing 32 (162 mg, 0.241 mmol), N,N-diisopropylethylamine
(113 µL, 0.65 mmol), 2-cyanoethyl N,N-diisopropylchlorophos-
phoramidite (107 µL 0.48 mmol), and 1-methylimidazole (12.0 µL,
0.15 mmol) was stirred at room temperature for 2 h. The
phosphoramidite 38 was purified by silica gel chromatography to
1
yield). H NMR (CDCl3/TMS) δ 9.97 (brs, 1H), 8.10 (d, 1H, J )
7.6 Hz), 7.40 (d, 2H, J ) 7.2 Hz), 7.35-7.20 (m, 7H), 7.18 (d,
1H, J ) 7.6 Hz), 6.85 (d, 4H, J ) 8.8 Hz), 6.32 (d, 1H, J ) 7.6
Hz), 4.40 (m, 1H), 4.19 (m, 1H), 4.08 (m, 1H), 4.00 (m, 1H), 3.79
(s, 6H), 3.47 (m, 2H), 2.23 (m, 1H), 2.20 (s, 3H), 2.02 (s, 3H),
1.75 (m, 1H), 1.60 (m, 2H), 1.46 (m, 3H); 13C NMR (CDCl3) δ
171.4, 170.9, 162.5, 158.6, 155.8, 145.0 144.2, 135.3, 135.1, 130.0,
128.01, 127.95, 127.1, 113.2, 97.2, 89.4, 86.9, 85.6, 72.5, 64.1,
63.5, 55.2, 51.3, 28.7, 24.7, 24.0, 23.5, 20.9; HRMS calcd for
C38H43N3O9Na [MNa+] 708.2897, found 708.2909.
give a white foam: 210 mg (100% conversion, g95% purity). 31
P
NMR (CD3CN) δ 151.0, 149.2; HRMS calcd for C46H58N5O10NaP
[MNa+] 894.3819, found 894.3824.
N4-Acetyl-2′-deoxy-5′-O-(dimethoxytrityl)-2′-C-r-propylcyti-
dine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (39). Ac-
cording to the above procedure, a reaction mixture containing 33
(109 mg, 0.178 mmol), N,N-diisopropylethylamine (78 µL, 0.45
mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (84
mg, 0.36 mmol), and 1-methylimidazole (8.0 µL, 0.10 mmol) was
stirred at room temperature for 2 h. The phosphoramidite 39 was
purified by silica gel chromatography to give a white foam: 145
mg (100% conversion, g95% purity). 31P NMR (CD3CN) δ 151.2,
149.2; HRMS calcd for C44H56N5O8NaP [MNa+] 836.3764, found
836.3749.
N4-Acetyl-2′-C-r-butyl-2′-deoxy-5′-O-(dimethoxytrityl)cyti-
dine (35). To a solution of 2′-C-R-butylcytidine 29 (88 mg, 0.27
mmol) in dry pyridine (5.0 mL), 4-(dimethylamino)pyridine (132
mg, 1.08 mmol) and DMTrCl (386 mg, 1.08 mmol) were added.
After the reaction mixture was stirred at room temperature
overnight, TLC indicated that only a small amount of product had
formed. To the reaction mixture, silver nitrate (51 mg, 0.30 mmol),
DMTrCl (549 mg, 1.62 mmol), and 4-(dimethylamino)pyridine (198
mg, 1.62 mmol) were added. The mixture was stirred at room
temperature for 48 h. The reaction was quenched with methanol
(1.0 mL). The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 3% methanol in
chloroform to give the product as a light yellow foam: 150 mg
2′-C-r-(4-Acetoxybutyl)-N4-acetyl-2′-deoxy-5′-O-(dimethoxy-
trityl)cytidine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite
(40). According to the above typical procedure, a reaction mixture
containing 34 (57 mg, 0.083 mmol), N,N-diisopropylethylamine (32
mg, 44 µL, 0.25 mmol), dry dichloromethane (5.0 mL), 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite (39 mg, 0.17 mmol),
and 1-methylimidazole (4.0 µL, 0.05 mmol) was stirred at room
temperature for 1 h. The reaction was quenched with methanol (0.25
mL). The phosphoramidite 40 was purified by silica gel chroma-
tography to give a white foam: 74 mg (100% conversion, g95%
purity). 31P NMR (CD3CN) δ 151.3, 149.2; HRMS calcd for
C47H60N5O10NaP [MNa+] 908.3976, found 908.3965.
1
(89% yield). H NMR (CDCl3/TMS) δ 10.09 (brs, 1H), 8.08 (d,
1H, J ) 7.6 Hz), 7.40 (m, 2H), 7.35-7.18 (m, 7H), 7.15 (d, 1H,
J ) 7.6 Hz), 6.84 (d, 4H, J ) 8.8 Hz), 6.35 (d, 1H, J ) 8.0 Hz),
4.41 (m, 1H), 4.19 (m, 1H), 3.78 (s, 6H), 3.44 (m, 2H), 2.21 (m,
1H), 2.19 (s, 3H), 1.78 (m, 1H), 1.50-1.20 (m, 5H), 0.85 (t, 3H,
J ) 7.0 Hz); 13C NMR (CDCl3) δ 171.0, 162.7, 158.6, 155.8, 145.0,
144.2, 135.3, 135.2, 130.0, 128.02, 127.95, 127.1, 113.2, 97.2, 89.4,
86.9, 85.9, 72.8, 63.6, 55.2, 51.3, 29.8, 24.7, 23.6, 22.7, 13.9; HRMS
calcd for C36H41N3O7Na [MNa+] 650.2842, found 650.2836.
2′-C-r-(2-Acetoxyethyl)-N4-acetyl-2′-deoxy-5′-O-(dimethoxy-
trityl)cytidine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite
(36). Typical procedure for the preparation of phosphoramidites:
To a cooled (0 °C) solution of 30 (125 mg, 0.19 mmol) and N,N-
diisopropylethylamine (89 µL, 0.51 mmol) in dry dichloromethane
(10 mL) under argon, 2-cyanoethyl N,N-diisopropylchlorophos-
phoramidite (90 mg, 0.38 mmol) and 1-methylimidazole (8.0 µL,
0.10 mmol) were added. The reaction mixture was warmed to room
temperature and stirred until TLC indicated that the reaction was
complete (1 h). The reaction was then quenched with methanol
(0.5 mL). After being stirred at room temperature for 5 min, the
reaction mixture was evaporated under reduced pressure, and the
residue was purified by silica gel chromatography, eluting with 4%
acetone in dichloromethane containing 0.5% triethylamine to give
N4-Acetyl-2′-C-r-butyl-2′-deoxy-5′-O-(dimethoxytrityl)cyti-
dine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (41). Ac-
cording to the above typical procedure, a mixture containing 35
(75 mg, 0.12 mmol), N,N-diisopropylethylamine (41 mg, 56 µL,
0.32 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
(56 mg, 0.24 mmol), and 1-methylimidazole (5.0 µL, 0.06 mmol)
in dry dichloromethane (5.0 mL) was stirred at room temperature
for 1 h. The reaction was quenched with methanol (0.25 mL). The
phosphoramidite 41 was purified to give a white foam: 99 mg
(100% conversion, g95% purity). 31P NMR (CD3CN) δ 153.8,
151.6; HRMS calcd for C45H58N5O8NaP [MNa+] 850.3921, found
850.3916.
J. Org. Chem, Vol. 72, No. 4, 2007 1209

Li and Piccirilli
Supporting Information Available: 1H NMR and 13C NMR
spectra of new compounds 2-10, 14-16, and 18-41; 13C NMR
spectra of known compounds 12, 13, 15a, and 17; and 31P NMR
spectra of phosphoramidites 36-41. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This article is dedicated to Prof. Min-
Zhi Deng on the occasion of his 65th birthday. N.-S.L. is a
research specialist and J.A.P. is an investigator at the Howard
Hughes Medical Institute. We thank J. Ye, J. Lu, Q. Dai, S.
Koo, R. Fong, and C. Lea for helpful discussions and critical
comments on the manuscript.
JO062002T
1210 J. Org. Chem., Vol. 72, No. 4, 2007