Synthesis of pyridine, pyrimidine and pyridinone C-nucleoside phosphoramidites for probing cytosine function in RNA

Article abstract of DOI:10.1021/jo9016919

(Chemical Equation Presented) In the structures of the HDV ribozyme a cytosine nucleobase resides at the active site poised to participate directly in catalysis. Defining the functional role of the nucleobase requires nucleoside analogues that perturb the

Full text of DOI:10.1021/jo9016919

pubs.acs.org/joc
Synthesis of Pyridine, Pyrimidine and Pyridinone C-Nucleoside
Phosphoramidites for Probing Cytosine Function in RNA
Jun Lu, Nan-Sheng Li, Selene C. Koo, 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
jpicciri@uchicago.edu
Received August 3, 2009
In the structures of the HDV ribozyme a cytosine nucleobase resides at the active site poised to
participate directly in catalysis. Defining the functional role of the nucleobase requires nucleoside
analogues that perturb the functional groups in a strategic manner. Herein, we have developed
efficient methods for the synthesis of five C-nucleoside phosphoramidite derivatives that, when used
in combination, provide strategies for probing the potential functional role of cytosine’s keto group
and imino nitrogen. Phosphoramidites 15a and 15b were synthesized in 11 steps starting from 2-
amino-5-bromopyrimidine (1a) and 2-amino-5-bromopyridine (1b), respectively, with overall yields
of 10.8% and 6.6%, respectively. Phosphoramidite 21 was prepared from intermediate 11b in seven
steps with an overall yield of 33.7%. Phosphoramidites 23 and 25 were prepared from 2,4-diamino-
5-(β-D-ribofuranosyl)-1,3-pyrimidine (22) and pseudoisocytidine (24), respectively, with an overall
yield of 15.9% (six steps) and 37.9% (four steps), respectively. These phosphoramidites were
incorporated into oligonucleotides by solid-phase synthesis.
Introduction
described below and elsewhere, C-glycoside linkages also under-
lie novel strategies to investigate RNA structure and function.
In the HDV ribozyme, the nucleobase of a cytosine residue
participates directly in catalysis as a general acid.4 One
mechanistic model that combines data from crystallo-
graphy,5 Raman spectroscopy,6 and chemogenetic suppres-
sion4c proposes that the cytosine engages in multiple inter-
actions during catalysis (Figure 1). The imino nitrogen bears
a proton that transfers to the leaving group during the
reaction.4c The N4 exocyclic amino group participates in a
network of hydrogen bonds in the ribozyme,7 and the
exocyclic O2-keto group interacts with a magnesium-bound
water molecule,5 which may protonate (or donate a hydro-
gen bond to) a nonbridging oxygen at the scissile phosphate
C-Nucleosides are an important class of compounds distin-
guished by a carbon-carbon glycosidic bond.1 Many C-nucleo-
sides possess antiviral and antineoplastic activities and are of
interest in medicinal chemistry.2 In addition, the C-glycoside
linkage expands the range of substituent patterns available to the
aglycon heterocycle, engendering nucleosides with arrangements
of hydrogen-bond donors and acceptors that are distinct from
those of the natural nucleosides. For example, a carbon-carbon
glycosidic linkage enables access to a cytidine analogue bearing
a donor-donor-acceptor hydrogen-bonding pattern.3 This
hydrogen-bonding pattern facilitates the formation of a DNA
triplex by circumventing the need for cytidine protonation. As
(1) Miller, P. S. Antisense/Antigene Oligonucleotides. In Bioorganic
Chemistry-Nucleic Acids; Hecht, S. M., Ed.; Oxford University Press: New
York, Oxford, 1996; pp 347-374.
(2) (a) Franchetti, P.; Cappellacci, L.; Griffantini, M.; Barzi, A.; Nocen-
tini, G.; Yang, H. Y.; O’Connor, A.; Jayaram, H. N.; Carrell, C.; Goldstein,
B. M. J. Med. Chem. 1995, 38, 3829–3837. (b) Walker, J. A.; Liu, W.; Wise, D.
S.; Drach, J. C.; Townsend, L. B. J. Med. Chem. 1998, 41, 1236–1241. (c)
Guntaka, R. V.; Varma, B. R.; Weber, K. T. Int. J. Biochem. Cell Biol. 2003,
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2000, 287, 1493–1497. (b) Shih, I. H.; Been, M. D. Proc. Natl. Acad. Sci. U.S.
A. 2001, 98, 1489–1494. (c) Das, S. R.; Piccirilli, J. A. Nat. Chem. Biol. 2005,
1, 45–52. (d) Cerrone-Szakal, A. L.; Siegfried, N. A.; Bevilacqua, P. C. J. Am.
Chem. Soc. 2008, 130, 14504–14520.
(5) Chen, J. H.; Gong, B.; Bevilacqua, P. C.; Carey, P. R.; Golden, B. L.
Biochemistry 2009, 48, 1498–1507.
ꢀ
ꢀ
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567–574.
(7) Das, S. R.; Koo, S. C.; Piccirilli, J. A. Unpublished results.
(3) (a) Hutter, D.; Benner, S. A. J. Org. Chem. 2003, 68, 9839–9842. (b)
von Krosigk, U.; Benner, S. A. J. Am. Chem. Soc. 1995, 117, 5361–5362.
DOI: 10.1021/jo9016919
r
Published on Web 09/30/2009
J. Org. Chem. 2009, 74, 8021–8030 8021
2009 American Chemical Society

Lu et al.
JOCFeatured Article
proposed for the HDV ribozyme. As shown previously, the
nucleobase of pseudoisocytidine (ΨisoC) (in the N3H tau-
tomeric state;9 Figure 3) mimics CH+ residues without the
need for protonation.10 This property provides a basis for
biochemical detection of CH+ residues within RNA: at
CH+ residues that make important contributions to RNA
function, ΨisoC substitution would be expected to enhance
function over the wild-type RNA at higher pH compared to
lower pH due to a greater fraction of the wild type ribozyme
lacking the N3 proton at higher pH relative to the variant
ribozyme. Strobel and colleagues obtained proof-of-concept
for this biochemical signature in the context of a variant of
the Tetrahymena group I intron ribozyme.11
The APo derivative complements ΨisoC in this assay as it
mimics CH+ and circumvents potential effects from the N1
imino nitrogen,9 including formation of the N1H tautomer
and the capacity for protonation (Figure 3). The biochemical
signature from ΨisoC substitution becomes less predictable
when CH+ serves a role in general acid catalysis, transfer-
ring the imino proton to the leaving group during the
reaction. The N3H tautomer in the neutral form has little
capacity for proton transfer due to its weak acidity; however,
protonation of the nucleobase to form ΨisoCH+ could
provide suitable general acid catalysis. Consequently, the
biochemical signature for ΨisoC substitution at C residues
that act as general acids will depend upon the pH-dependent
contribution of ΨisoCH+ to the reaction. In contrast, APo
substitution would be expected to impair catalysis severely at
all pH values as the nucleobase has little capacity for proton
transfer and has no additional imino nitrogens where pro-
tonation could enhance this capacity.
FIGURE 1. Proposed model for interactions of C75 in the HDV
ribozyme. The N3 proton of C75 protonates the 50-oxygen of the scissile
phosphate. The O2 keto oxygen interacts with an outersphere water
ligand of a divalent metal ion (Mg2+, gray sphere) near the active
site. That water ligand has been proposed to act as a general base that
activates the 20-hydroxyl nucleophile of the reaction. The N4
amino group of C75 interacts with other functional groups (A) in the
ribozyme.
or activate the 20-hydroxyl group for nucleophilic attack.
Synthetic access to the cytidine analogues in Figure 2 would
engender experimental strategies to probe nucleobase inter-
actions. We describe here the synthesis of five phosphora-
midite derivatives for C-nucleoside analogues of cytidine
(Figure 2). When used in combination, these derivatives
enable strategies for delineating the importance of the keto
and imino groups of cytosine residues within RNA.
Strategies To Test Cytidine Nucleobase Interactions. As
the active-site cytidine purportedly uses all of its substituents
to engage in catalytic interactions, testing specific interac-
tions in this model requires design and synthesis of multiple,
carefully designed nucleoside analogues that, ideally, modify
one functional group while introducing minimal changes
elsewhere in the molecule. Synthetic access to the cytidine
analogues in Figure 2 would engender experimental tests for
nucleobase interactions in the manner described below.
(a). Testing the Role of the Keto Group. Ribofuranoside
derivatives of aminopyridine (AP), aminopyrimidine (ΨAPy),
and diaminopyrimidine (ΨDAPy) provide a strategy to probe
the functional importance of the O2 keto group at specific
cytosine residues within RNA. They retain cytosine’s
H2N-CdN group and either remove the keto oxygen (AP
and ΨAPy) or replace it with an amino group (ΨDAPy). To
accommodate these modifications while remaining electrostati-
cally neutral, the heterocycle must be linked to ribose through a
carbon-carbon bond. The new glycosidic linkage in ΨAPy and
ΨDAPy places N1 in the position equivalent to C5 of cytidine.
Comparison of effects from the ΨAPy versus AP modification
provides an independent control for the presence of this nitrogen.
(b). Testing the Role of Cytidine’s Imino Nitrogen. Some
RNA species require protonation of cytidine’s nucleobase
(CH+) for function. These CH+ residues may serve a
structural role, allowing formation of specific RNA confor-
mations through tertiary interactions, or a catalytic role,
transferring its N3 proton via general acid catalysis, as
Here, we present the synthesis of the phosphoramidite
derivatives for C-nucleoside analogues (Figure 2) to enable
functional tests for contributions from the keto and imino
groups at specific cytosine residues within RNA.
Results and Discussion
Synthetic routesforC-nucleosides are usually classified into
two major approaches: (1) introduction of a functional group
at the anomeric position of a sugar derivative followed by the
construction of a heterocyclic base12 and (2) direct attachment
of a preformed aglycone unit to an appropriate carbohydrate
moiety followed by dehydroxylation.13 We chose the second
(9) Cytidine numbering used.
(10) In solution about 4% of psuedoisocytidine exists as the N1H
tautomer, while 96% exits as the N3H tautomer, corresponding to a free
energy difference of 2 kcal/mol (see ref 8c).
(11) Oyelere, A. K.; Kardon, J. R.; Strobel, S. A. Biochemistry 2002, 41,
3667–3675.
(12) (a) Chu, C. K.; Wempen, I.; Watanabe, K. A.; Fox, J. J. J. Org. Chem.
1976, 41, 2793–2797. (b) Chu, C. K.; Reichman, U.; Watanabe, K. A.; Fox, J.
J. J. Org. Chem. 1977, 42, 711–714. (c) Sato, T.; Hayakawa, Y.; Noyori, R.
Bull. Chem. Soc. Jpn. 1984, 57, 2515–2525. (d) Townsend, L. B. Chemistry of
Nucleosides and Nucleotides; Plenum Press: New York, 1994; pp 421-535.
(e) Popsavin, M.; Torovic, L.; Svircev, M.; Kojic, V.; Bogdanovic, G.; Popsavin,
V. Bioorg. Med. Chem. Lett. 2006, 16, 2773–2776. (f) Kamath, V. P.; Zhang, J.;
Morris, P. E.; Babu, S. Bioorg. Med. Chem. Lett. 2006, 16, 2662–2665.
(13) (a) Watanabe, K. A. In The Chemistry of Nucleosides and Nucleotides;
Towsend, L. B., Ed.; Plenum: New York, 1994; Vol. 3, pp 421-535. (b) Zhou, J.;
Yang, M.; Schneller, S. W. Tetrahedron Lett. 2004, 45, 8233–8234. (c) Wu, Q.;
Simons, C. Synthesis 2004, 1533–1553. and references cited therein. (d) Benner,
S. A.; Wellington, K. W. Nucleosides, Nucleotides Nucl. Acids 2006, 25, 1309–
1333. (e) Urban, M.; Pohl, R.; Klepetarova, B.; Hocek, M. J. Org. Chem. 2006,
71, 7322–7328. (f) Sun, Z.; Ahmed, S.; McLaughlin, L. W. J. Org. Chem. 2006,
71, 2922–2925. (g) Joubert, N.; Pohl, R.; Klepetarova, B.; Hocek, M. J. Org.
Chem. 2007, 72, 6797–6805. (h) Peyron, C.; Navarre, J. M.; Dubreuil, D.;
Vierling, P.; Benhid, R. Tetrahedron Lett. 2008, 49, 6171–6174.
(8) (a) Chen, D. L.; McLaughlin, L. W. J. Org. Chem. 2000, 65, 7468–
7474. (b) Miller, J. M.; Blackburn, A. C.; Shi, Y.; Melzak, A. J.; Ando, H. Y.
Electrophoresis 2002, 23, 2833–2841. (c) Kan, L.-S.; Lin, W.-C.; Yadav, R.
D.; Shih, J. H.; Chao, I. Nucleosides Nucleotides 1999, 18, 1091–1093. (d)
Barlin, G. B.; Pfleiderer, W. J. Chem. Soc. B 1971, 1425–1432. (e) Albert, A.;
Goldacre, R.; Phillips, J. J. Chem. Soc. 1948, 2240–2249.
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FIGURE 2. Nucleotide analogues for testing the role of cytidine’s keto and imino groups. Atoms are numbered relative to cytidine.
C-nucleosides 5a and 5b in yields of 43% and 29%, respectively.
1
We observed only β-configured products as confirmed by H
NMR-NOE spectroscopy, indicating that the glycosylation
reaction proceeded stereoselectively. Desilylation of 5a and 5b
with Bu4NF 3H2O (TBAF) in THF gave the corresponding N,
3
N-dibenzyl-protected C-nucleosides 6a and 6b. NOESY analy-
sis further confirmed the β-anomeric configuration (Figure 4).
Relevant 1H NMR spectroscopic data for compounds 6a
and 6b are listed in Table 1 with through-space proton-pro-
ton interactions based on NOESY spectra as shown in
Figure 4. The assignments in Table 1 are based primarily
on two-dimensional 1H-1H COSY spectra. From the
NOESY spectrum of the β-anomer 6a and 6b, we observe
that H-4 and H-50 interact strongly with H-20, while H-20
interacts strongly with H-30. Additionally, H-10 interacts
strongly with H-6 and H-40 but does not interact with H-20
FIGURE 3. Tautomeric and protonated forms of pseudoisocyti-
and H-30. These NOESY spectroscopic data support the
dine. Cytidine numbering used.
β-anomeric configurational assignments.
approach because of its flexibility and potential for higher
yields. Previously, several 20-deoxy-C-nucleoside phosphora-
midites that bear a pyrimidine,8a pyridine,13e,f,14 or a pyridi-
none3a,15 ring as nucleobase have been synthesized. To the
best of our knowledge, the synthesis of the corresponding
C-ribonucleoside phosphoramidites has not been reported.
Synthesis of 2-Amino-5-(β-D-ribofuranosyl)pyridine, 2-
Amino-5-(β-D-ribofuranosyl)-1,3-pyrimidine, and Their Phos-
phoramidites. The synthesis of the C-nucleosides 8a and 8b
(Scheme 1) started from the commercially available 2-amino-
5-bromopyrimidine (1a) or 2-amino-5-bromopyridine (1b)
FIGURE 4. Through-space proton-proton interactions indicated
by the NOESY spectra of 6a (X = N) and 6b (X = CH).
and the readily obtainable 5-[(tert-butyl)diphenylsilyl]-2,3-
isopropylidene-D-ribono-l,4-lactone (3).16 Protection of the
amino group in 1a or 1b with benzyl bromide gave good
yields of compounds 2a and 2b (2a: 92%; 2b: 96%), which
are suitable for coupling with the sugar component. The
coupling essentially followed the syntheses of C-glycosides
reported by Kraus et al.17 Bromide-lithium exchange of 2 with
n-BuLi at -78 °C and in situ reaction with 1,4-lactone 3
furnished hemiacetals 4a and 4b, which were subsequently
dehydroxylated with Et3SiH in the presence of a strong Lewis
TABLE 1. 1H NMR Spectra (CD3OD) of C-Nucleoside 6a and 6b
compd H-10
H-20
H-30
H-40
H-50
H-4
6a
6b
4.61 d 3.95 m 4.10 m 3.97 m 3.75 dd, 3.69 dd 8.44 s
4.51 d 3.79 m 3.96 m 3.85 m 3.62 dd, 3.56 dd 6.41 s
To prepare the corresponding parent nucleosides, 6a and 6b
were first acylated by treatment with acetic anhydride in
acetonitrile to form 7a and 7b. Attempts to debenzylate 7a
and 7b using 10% Pd-C/H2, 20% Pd(OH)2-C/H2, or BCl3
gave unsatisfactory yields. Ammonium cerium(IV) nitrate
(CAN)18 in wet acetonitrile at room temperature for 24 h
removed only one benzyl group. However, when the tempera-
ture was increased to 50 °C, CAN in wet acetonitrile removed
acid (BF3 Et2O) from -78 °C to room temperature. We found
3
that these reaction conditions were effective in removing the
20,30-O-isopropylidine group, affording the corresponding
(14) (a) Hsieh, H. P.; McLaughlin, L. W. J. Org. Chem. 1995, 60, 5356–
5359. (b) Hildbrand, S.; Leumann, C. Angew. Chem., Int. Ed. Engl. 1996, 35,
1968–1970. (c) Hildbrand, S.; Blaser, A.; Parel, S. P.; Leumann, C. J. J. Am.
Chem. Soc. 1997, 119, 5499–5511.
(15) Yang, Z.; Hutter, D.; Sheng, P.; Sismour, A. M.; Benner, S. A.
Nucleic Acids Res. 2006, 34, 6095–6101.
(16) Piccirilli, J. A.; Krauch, T.; MacPherson, L. J.; Benner, S. A. Helv.
Chim. Acta 1991, 74, 397–406.
(17) Kraus, G. A.; Molina, M. T. J. Org. Chem. 1988, 53, 752–753.
(18) Reese, C. B.; Wu, Q. Org. Biomol. Chem. 2003, 1, 3160–3172.
J. Org. Chem. Vol. 74, No. 21, 2009 8023

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SCHEME 1. Synthesis of 2-Amino-5-(β-D-ribofuranosyl)pyridine and 2-Amino-5-(β-D-ribofuranosyl)-1,3-pyrimidine
SCHEME 2. Synthesis of 2-Amino-5-(β-D-ribofuranosyl)pyridine and 2-Amino-5-(β-D-ribofuranosyl)-1,3-pyrimidine Phosphoramidites
both benzyl groups from 7a within 24 h and from 7b within 1 h
to give C-nucleosides 2-amino-5-(β-D-ribofuranosyl)-1,3-pyri-
midine (8a, 71% yield) and 2-amino-5-(β-D-ribofuranosyl)-
pyridine (8b, 86% yield), respectively.
acetonitrile at 50 °C gave 11a (69% yield) and 11b (72% yield),
respectively. Protection of the exocyclic amino group of 11a and
11b with N,N-dimethylformamide dimethyl acetal gave com-
pounds 12a (93% yield) and 12b (85% yield). Pyridinium poly-
(hydrogen fluoride) removed the di-tert-butylsilylene group
efficiently to afford 13a and 13b in yields of 83% and 80%,
respectively. No migration of the 20-O-TBSgrouptothe30-oxygen
was observed under the reaction conditions.19 Using standard
procedures, 13a and 13b were converted efficiently via 14a and 14b
to the corresponding phosphoramidites 15a and 15b.
Next, we prepared suitably protected phosphoramidites of 8a
and 8b starting from synthetic intermediates 6a and 6b
(Scheme 2). To allow regioselective protection of the 20-hydroxyl
group as the 20-O-TBS ether, 6a and 6b were reacted with di-tert-
butylsilyl bis(trifluoromethanesulfonate) (t-Bu2Si(OTf)2) in
DMF to give the corresponding 30,50-O-(di-tert-butylsilanediyl)
derivatives 9a (79% yield) and 9b (82% yield). Subsequent
reaction of 9a and 9b with tert-butyldimethylsilyl chloride
(TBSCl) gave silyl ethers 10a (86% yield) and 10b (93% yield),
respectively. Debenzylation of 10a and 10b with CAN in wet
Synthesis of 1-Deazacytidine Phosphoramidite. In 2002,
Sollogoub et al. reported the synthesis of 1-deazacytidine
(19) Trost, B. M.; Caldwell, C. G. Tetrahedron Lett. 1981, 22, 4999–5002.
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SCHEME 3. Synthesis of 1-Deazacytidine Phosphoramidite
SCHEME 4. Synthesis of 2,4-Diamino-5-(β-D-ribofuranosyl)-1,3-pyrimidine and Pseudoisocytidine Phosphoramidites
through the coupling of a PMB (p-methoxybenzyl)-protected 2-
amino-5-bromopyridine with perbenzylated ribonolactone fol-
lowed by transformation of the pyridine ring to the desired
substituted pyridinone.20 However, synthesis of the correspond-
ing phosphoramidite has not been reported. Starting from 11b,
we developed an efficient synthetic route to synthesize 1-deaza-
cytidine phosphoramidite as shown in Scheme 3. Compound
11b was oxidized to the corresponding N-oxide 16 with m-CPBA
(m-chloroperoxybenzoic acid) in 90% yield.21 Subsequent
refluxing in acetic anhydride for 2 h accomplished the Katada
rearrangement.22 Treatment with methanolic sodium hydroxide
gave 17 in 59% yield (two steps). To prepare the 1-deazacytidine
phosphoramidite 21, the pivaloyloxymethyl (POM) was used as
a N-protecting group for the pyridinone ribonucleoside.23 With-
out protection, the synthesis of the phosphoramidite was
unsuccessful. Treatment of 17 with a mixture of chloromethyl
(22) Katada, M. J. Pharm. Soc. Jpn. 1947, 67, 51–52.
(23) (a) Li, S.; Nair, M. G.; Edwards, D. M.; Kisliuk, R. L.; Gaumont, Y.;
Dev, I. K.; Duch, D. S.; Humphreys, J.; Smith, G. K.; Ferone, R. J. Med.
Chem. 1991, 34, 2746–54. (b) Araki, L.; Harusawa, S.; Yamaguchi, M.;
Yonezawa, S.; Taniguchi, N.; Lilley, D.M. J.; Zhao, Z.; Kurihara, T.
Tetrahedron Lett. 2004, 45, 2657–2661. (c) Araki, L.; Harusawa, S.;
Yamaguchi, M.; Yonezawa, S.; Taniguchi, N.; Lilley, D.M. J.; Zhao, Z.;
Kurihara, T. Tetrahedron 2005, 61, 11976–11985.
(20) Sollogoub, M.; Fox, K. R.; Powersa, V. E. C.; Brown, T. Tetrahedron
Lett. 2002, 43, 3121–3123.
(21) Sato, N.; Miwa, N.; Suzuki, H.; Sakakibara, T. J. Heterocycl. Chem.
1994, 31, 1229–1233.
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TABLE 2. Mass of 30-ACCGAGAGGGAAUC*GGU-50 and 30-AUC*GG-50
30-ACCGAGAGGGAAUC*GGU-50
30-AUC*GG-50
C*
C (wt)
AP
APO
ΨisoC
C (wt)
ΨAPy
ΨDAPy
calcd mass
MALDI mass
5525.8
5525.0a
5508.8
5509.5a
5524.8
5525.6a
5526.8
5526.5a
1568.3
1566.5b
1552.3
1550.7b
1567.3
1566.0b
aPositive (M þ Hþ). bNegative (M - Hþ).
pivaloate and K2CO3 in DMF produced the 1-N-POM deriva-
tive 18 in 97% yield. The di-tert-butylsilylene group of 18 was
then removed by treatment with pyridinium poly(hydrogen
fluoride) to afford 19, which was subsequently converted via
20 to the corresponding POM-protected 1-deazacytidine phos-
phoramidite 21 using standard procedures.
which to investigate the structural and energetic contribution of
functionally important cytidine residues in RNA.
Experimental Section
5-Bromo-2-dibenzylaminopyrimidine (2a). To a solution of 2-
amino-5-bromopyrimidine (1a) (2.12 g, 12.2 mmol) in dry DMF
(40 mL) under argon was added NaH (877 mg, 36.6 mmol), and
the mixture was stirred at 0 °C. After the hydrogen gas genera-
tion had ceased, benzyl bromide (5.21 g, 30.5 mmol) was added,
and the mixture was stirred at 0 °C for 2 h. The reaction was
quenched with MeOH (10 mL); the solvent was removed, and
the residue was partitioned between CH2Cl2 (60 mL) and H2O
(60 mL). The organic layer was washed with brine and dried over
anhydrous Na2SO4. After the solvent was removed, the residue
was purified by silica gel chromatography, eluting with 0-3%
ethyl acetate in hexane, to give the product 2a as a white foam:
3.98 g (92% yield); 1H NMR (CD3Cl) δ 8.33 (s, 2H), 7.22-7.32
(m, 12 H), 4.82 (s, 4H); 13C NMR (CD3Cl): δ 160.7, 158.2, 137.9,
128.7, 127.6, 127.3, 106.2, 49.5; HRMS (FABþ) m/z calcd for
C18H17BrN3 [M þ Hþ] 354.0606, found 354.0614.
Synthesis of 2,4-Diamino-5-(β-D-ribofuranosyl)-1,3-pyri-
midine and Pseudoisocytidine Phosphoramidites. We prepared
2,4-diamino-5-(β-D-ribofuranosyl)-1,3-pyrimidine12b,24 (22) in
eight steps from D-ribose and converted it to phosphoramidite
23 (Scheme 4) in six steps with 15.9% overall yield. We also
prepared phosphoramidite 25 from commercially available
pseudoisocytidine (24) in four steps with 37.9% overall yield.
We subsequently incorporated these five new C-nucleoside
phosphoramidites into the RNA oligonucleotides as described
before.25 The C-nucleoside phosphoramidites (∼100 mg) were
dissolved in dry acetonitrile (1 mL) and coupled twice during the
appropriate synthesis cycle; sequences were 30-ACCGAGAGG-
GAAUC*GGU-50 [C*: 15b (AP), 21 (APo), 25 (ψDAPy)] and
30-AUC*GG-50 [C*: 15a (ΨAPy), 23 (ΨisoC)]. Based on trityl
yield data, coupling efficiencies throughout oligonucleotide
synthesis were excellent (data not shown). Following standard
oligonucleotide deprotection conditions [3:1 NH4OH/EtOH,
55 °C, 2-4 h (for 21 NH3/MeOH in sealed tube, 55 °C, 24 h);
5-Bromo-2-dibenzylaminopyridine (2b). Compound 2b was
prepared from 2-amino-5-bromopyridine (1b) (8.65 g, 50 mmol),
NaH (3.6 g, 150 mmol), and benzyl bromide (21.4 g, 125 mmol)
as described for 2a. Silica gel chromatography (0-3% ethyl
acetate in hexane) of the residue yielded 16.96 g (96% yield) of
2b as a white foam: 1H NMR (CD3Cl) δ 8.20 (dd, 1H,
J = 0.4, 2.4 Hz), 7.41 (dd, 1 H, J = 2.8, 9.2 Hz), 7.19-7.33 (m,
10 H), 6.34 (dd, 1 H, J = 0.4, 9.2 Hz), 4.75 (s, 4H); 13C NMR
(CD3Cl) δ 157.3, 148.7, 139.8, 138.0, 128.8, 127.3, 127.1, 107.6,
106.8, 51.3; HRMS (FABþ) m/z calcd for C19H18BrN2 [M þ Hþ]
353.0653, found 353.0659.
5-[50-O-(tert-Butyldiphenylsilyl)-β-D-ribofuranosyl]-2-diben-
zylamino-1,3-pyrimidine (5a). To a solution of 5-bromo-2-di-
benzylaminopyrimidine 2a (1.417 g, 4.0 mmol) in dry THF (30
mL) was added n-butyllithium (3.0 mL, 1.6 M solution in
hexane, 4.8 mmol) at -78 °C under argon, and the mixture
was stirred for 1 h. A solution of lactone 3 (1.42 g, 3.3 mmol) in
THF (10 mL) was added at -78 °C. The mixture was stirred for
2 h and then allowed to warm to room temperature over 3 h. The
reaction was quenched with H2O (40 mL), and the mixture was
extracted with Et2O (3 ꢀ 80 mL). The organic layer was washed
with brine, dried over anhydrous Na2SO4, and concentrated.
The resulting yellow residue 4a was dried and dissolved in dry
CH2Cl2 (20 mL), Et3SiH (1.60 mL, 10.0 mmol) was added at
TEA 3HF/NMP, 65 °C, 1.5 h] and purification, MALDI mass
3
spectra were obtained for 30-ACCGAGAGGGAAUC*GGU-50
[C*:15b (AP), 21 (APo), 25 (ΨDAPy)] and 30-AUC*GG-50 [C*:
15a (ΨAPy), 23 (ΨisoC)]. Each gave the peaks expected from the
calculated molecular exact mass (Table 2). The structures of
modified oligonucleotides were also confirmed by enzymatic
digestion according to established protocol.26
In summary, we have described efficient methods for the
synthesis of phosphoramidites of five cytidine analogues. The
phosphoramidites of 2-amino-5-(β-D-ribofuranosyl)pyrimidine
and 2-amino-5-(β-D-ribofuranosyl)pyridine were prepared with
10.8% and 6.6% overall yield, respectively, from lactone 3 and
the corresponding 2-amino-5-bromopyrimidine (1a) or 2-amino-
5-bromopyridine (1b). 1-Deazacytidine phosphoramidite 21 was
prepared from 11b via Katada rearrangement of N-oxide 16
with an overall yield of 33.7%. The other two C-nucleoside
phosphoramidites 23 and 25 were prepared from their
corresponding nucleoside derivatives, 2,4-diamino-5-(β-D-ribo-
furanosyl)-1,3-pyrimidine (22) and pseudoisocytidine, with over-
all yields of 37.9% and 15.9%, respectively. Using solid-
phase methods, we achieved efficient incorporation of these five
new C-nucleoside phosphoramidites into RNA oligonucleotides.
This work expands the spectrum of nucleoside analogues with
-78 °C, and the mixture was stirred for 10 min. BF3 Et2O (1.27
3
mL, 10.0 mmol) was added dropwise. The reaction mixture was
allowed to warm to room temperature overnight and quenched
with satd NaHCO3 (10 mL). The mixture was partitioned
between Et2O (60 mL) and H2O (60 mL). The organic layer
was washed with brine and dried over anhydrous Na2SO4. The
sodium sulfate was filtered off, and the solvent was removed by
evaporation under vacuum. The residue was purified by silica
gel chromatography, eluting with 1% methanol in dichloro-
methane, to give the product 5a as a white foam: 922 mg (43%
(24) Ohrui, H.; Jones, G. H.; Moffatt, J. G.; Maddox, M. L.; Christensen,
A. T.; Byram, S. K. J. Am. Chem. Soc. 1975, 97, 4602–4613.
(25) Sun, S.; Yoshida, A.; Piccirilli, J. A. RNA 1997, 3, 1352–1363.
(26) Andrus, A.; Kuimelis, R. G. Base composition analysis of nucleo-
sides using HPLC. In Current Protocols in Nucleic Acid Chemistry; Beaucage,
S. L., Ed.; Wiley: New York, 2001; unit 10.6.
1
yield); H NMR (CD3Cl) δ 8.37 (s, 2H), 7.65-7.68 (m, 4H),
7.34-7.42 (m, 6H), 7.20-7.29 (m, 10H), 4.85 (dd, 4H, J = 15.8,
20.5 Hz), 4.58 (d, 1H, J = 7.1 Hz), 4.21 (t, 1H, J = 4.3 Hz), 4.04
(m, 2H), 3.80 (d, 2H, J = 4.0 Hz), 1.06 (s, 9H); 13C NMR
8026 J. Org. Chem. Vol. 74, No. 21, 2009

Lu et al.
JOCFeatured Article
(CD3Cl) δ 162.4, 156.9, 138.2, 135.7, 133.1, 133.0, 130.0, 129.9,
128.6, 127.9, 127.6, 127.1, 120.7, 84.8, 80.6, 76.8, 72.3, 64.3, 49.3,
27.0, 19.4; HRMS (FABþ) m/z calcd for C39H44N3O4Si [M þ
Hþ] 646.3101, found 646.3117.
(d, 1H, J = 2.4 Hz), 7.41 (dd, 1H, J = 2.4, 8.8 Hz), 7.19-7.40
(m, 10H), 6.48 (d, 1H, J = 8.8 Hz), 5.30 (m, 1H), 5.10 (m, 1H),
4.88 (d, 1H, J = 7.2 Hz), 4.78 (s, 4H), 4.37 (m, 1H), 4.25-4.29
(m, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H); 13C NMR
(CD3OD) δ 170.7, 169.9, 169.8, 159.1, 147.0, 138.2, 135.7, 128.7,
128.6, 127.1, 121.1, 106.1, 80.4, 80.0, 75.8, 71.9, 63.9, 51.1, 20.9,
20.8, 20.7; HRMS (FABþ) m/z calcd for C30H33N2O7 [M þ Hþ]
533.2288, found 533.2291.
5-[50-O-(tert-Butyl)diphenylsilyl-β-D-ribofuranosyl]-2-diben-
zylaminopyridine (5b). Compound 5b was prepared from 2b
(2.83 g, 8 mmol), lactone 3 (3.41 g, 8 mmol), and n-butyllithium
(6.0 mL, 1.6 M solution in hexane, 9.6 mmol) as described for 5a.
Silica gel chromatography (10% ethyl acetate in hexane) of the
2-Amino-5-(β-D-ribofuranosyl)-1,3-pyrimidine (8a). Ammo-
nium cerium(IV) nitrate (586 mg, 1.07 mmol) was added to a
solution of 7a (114 mg, 0.214 mmol) in acetonitrile-water (98:2
v/v, 10 mL), and the mixture was stirred overnight at 50 °C
under Ar. The reaction was quenched with 1 N NaOH until the
pH reached a value of about 8 and stirred at room temperature
for 1 h. The solvent was removed, and the residue was purified
by silica gel chromatography, eluting with 20% methanol in
dichloromethane, to give the product 8a as a white foam: 35 mg
1
residue yielded 1.50 g (29% yield) of 5b as a white foam: H
NMR (CD3Cl) δ 8.18 (s, 1H), 7.65-7.63 (m, 4H), 7.19-7.46 (m,
16H), 6.40 (d, 1H, J = 7.2 Hz), 4.77 (s, 4H), 4.63 (d, 1H, J = 7.2
Hz), 4.26 (m, 1H), 3.99-4.02 (m, 2H), 3.85 (m, 1H), 1.05 (s, 9H);
13C NMR (CD3Cl) δ 158.7, 146.8, 138.3, 136.3, 136.2, 135.8,
135.7, 133.2, 133.1, 130.0, 129.9, 128.7, 128.1, 127.9, 127.8,
127.2, 127.16, 127.12, 123.1, 123.0, 84.5, 82.4, 82.4, 77.1, 72.4,
64.4, 51.1, 27.0, 19.4; HRMS (FABþ) m/z calcd for
C40H45N2O4Si [M þ Hþ] 645.3149, found 645.3151.
1
(71% yield); H NMR (CD3COD) δ 8.33 (s, 2H), 4.56 (d, 1H,
J = 7.6 Hz), 4.08 (m, 1H), 3.95 (m, 1H), 3.89 (m, 1 H), 3.75 (dd, 1
H, J = 3.6, 12.0 Hz), 3.68 (dd, 1 H, J = 4.4, 12.0 Hz); 13C NMR
(CD3COD) δ 164.4, 158.2, 123.8, 87.0, 81.0, 78.5, 73.0, 63.5;
HRMS (FABþ) m/z calcd for C9H14N3O4 [M þ Hþ] 228.0984,
found 228.0988.
2-Amino-5-(β-D-ribofuranosyl)pyridine (8b). Compound 8b
was prepared from 7b (205 mg, 0.39 mmol) and ammonium
cerium(IV) nitrate (1.05 g, 1.95 mmol) as described for 8a. Silica
gel chromatography (20% methanol in dichloromethane) of the
residue yielded 75 mg (86% yield) of 8b as a white foam:1H
NMR (CD3COD) δ 7.89 (d, 1H, J = 2.0 Hz), 7.79 (dd, 1H, J =
2.0, 8.8 Hz), 6.80 (d, 1H, J = 9.2 Hz), 4.60 (d, 1H, J = 7.6 Hz),
4.08 (m, 1H), 3.99 (m, 1H), 3.88 (m, 1H), 3.77 (dd, 1H, J = 3.6,
11.6 Hz), 3.70 (dd, 1H, J = 4.4, 12.0 Hz); 13C NMR (CD3OD) δ
158.3, 140.8, 140.3, 126.4, 112.4, 86.9, 82.3, 78.3, 72.9, 63.5;
HRMS (FABþ) m/z calcd for C10H15N2O4 [M þ Hþ] 227.1032,
found 227.1031.
5-β-D-Ribofuranosyl-2-dibenzylamino-1,3-pyrimidine (6a). To
the solution of 5a (1.81 g, 2.80 mmol) in THF (40 mL) was added
Bu4NF 3H2O (1.10 g, 4.20 mmol). The mixture was stirred
3
at room temperature for 6 h. TLC showed that the reaction
was complete. Solvent was removed, and the resulting residue
was purified by silica gel chromatography, eluting with 5%
methanol in dichloromethane, to give product as a white foam.
6a (1.03 g, 90% yield): 1H NMR (CD3OD) δ 8.44 (s, 2H),
7.16-7.27 (m, 10H), 4.82 (s, 4H), 4.61 (d, 1H, J = 7.6 Hz), 4.10
(m, 1H), 3.97 (m, 1H), 3.95 (m, 1H), 3.75 (dd, 1H, J = 3.8, 12.0
Hz), 3.69 (dd, 1H, J = 4.5, 12.0 Hz); 13C NMR (CD3COD) δ
163.4,158.1, 139.4, 129.4, 128.5, 128.0, 122.8, 86.8, 81.3, 78.3,
73.0, 63.5, 50.4; HRMS (FABþ) m/z calcd for C23H26N3O4 [M þ
Hþ] 408.1923, found 408.1937.
5-β-D-Ribofuranosyl-2-dibenzylaminopyridine (6b). Compound
6b was prepared from 5b (0.583 g, 0.90 mmol) and TBAF (1.50
mL, 1 M in THF, 1.5 mmol) as described for 6a. Silica gel
chromatography (5% methanol in dichloromethane) of the resi-
due yielded 295 mg (80% yield) of 6b as a white foam: 1H NMR
(CD3COD) δ 8.04 (d, 1H, J = 2.0 Hz), 7.40 (dd, 1H, J = 2.0, 9.0
Hz), 7.03-7.14 (m, 10H), 6.41 (d, 1H, J = 9.0 Hz), 4.61 (s, 4H),
4.51 (d, 1H, J = 7.0 Hz), 3.96 (m, 1H), 3.85 (m, 1H), 3.79 (m, 1H),
3.62 (dd, 1H, J = 4.0, 12.0 Hz), 3.56 (dd, 1H, J = 4.5, 12.0 Hz);
13C NMR (CD3OD) δ 159.8, 147.5, 139.6, 137.7, 129.7, 128.2,
128.1, 125.1, 107.7, 86.5, 83.3, 78.4, 73.1, 63.7, 52.4; HRMS
(FABþ) m/z calcd for C24H27N2O4 [M þ Hþ] 407.1971, found
407.1979.
5-[30,50-O-(Di-tert-butylsilylene)-β-D-ribofuranosyl]-2-diben-
zylamino-1,3-pyrimidine (9a). Compound 6a (0.998 g, 2.45
mmol) was dried three times by azeotropic evaporation of
pyridine and then was dissolved in dry DMF (20 mL). t-Bu2Si-
(OTf)2 (1.10 g, 2.5 mmol) was added to the solution at 0 °C under
Ar. The solution was stirred for 30 min and warmed to room
temperature for 15 min. Then Et3N (1.02 mL, 7.35 mmol) was
added and the mixture stirred for another 10 min. The solvent
was removed, and the residue was purified by silica gel chroma-
tography, eluting with 2% methanol in dichloromethane, to give
1
5-(20,30,50-Tri-O-acetyl-β-D-ribofuranosyl)-2-dibenzylamino-
1,3-pyrimidine (7a). To the solution of 6a (89 mg, 0.218 mmol) in
CH3CN (10 mL) were added Et3N (214 μL, 1.53 mmol), DMAP
(27 mg, 0.218 mmol), and Ac2O (74 μL, 0.785 mmol) at 0 °C. The
mixture was stirred at 0 °C for 1 h and then warmed to room
temperature for 1 h. TLC showed that the reaction was com-
plete. The reaction mixture was quenched with MeOH (1 mL),
and the solvent was removed. The residue was purified by silica
gel chromatography, eluting with 1% methanol in dichloro-
methane, to give the product as a white foam. 6a (114 m g, 98%
the product 9a as a white foam: 1.06 g (79% yield); H NMR
(CDCl3) δ 8.31 (s, 2H), 7.21-7.32 (m, 10H), 4.88 (m, 5H), 4.48
(m, 1H), 4.17 (d, 1H, J = 4.2 Hz), 3.99-4.02 (m, 3H), 2.63 (s,
1H, disappeared with D2O), 1.05 (s, 9H), 1.07 (s, 9H); 13C NMR
(CDCl3) δ 162.2, 156.6, 138.0, 128.5, 127.6, 127.4, 120.7, 85.0,
77.8, 75.7, 74.4, 68.0, 49.2, 27.4, 27.3, 22.7, 20.4; HRMS (FABþ)
m/z calcd for C31H42N3O4Si [M þ Hþ] 548.2945, found
548.2958.
5- [30,50-O-(Di-tert-butylsilylene)-β-D-ribofuranosyl]-2-diben-
zylaminopyridine (9b). Compound 9b was prepared from 6b
(2.645 g, 6.50 mmol) and t-Bu2Si(OTf)2 (2.92 g, 6.63 mmol) as
described for 9a. Silica gel chromatography (10% ethyl acetate
in hexane) of the residue yielded 2.92 g (82% yield) of 9b as a
1
yield): H NMR (CD3Cl) δ 8.38 (s, 2H), 7.22-7.32 (m, 10H),
5.32 (m, 1H), 5.12 (m, 1H), 4.87 (s, 4H), 4.86 (m, 1 H), 4.38 (m,
1H), 4.27-4.31 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H);
13C NMR (CD3Cl) δ 170.6, 169.9, 169.8, 162.7, 156.9, 138.0,
128.6, 127.6, 127.2, 118.9, 80.2, 78.8, 75.6, 71.8, 63.7, 49.2, 20.9,
20.7, 20.6; HRMS (FABþ) m/z calcd for C29H32N3O7 [M þ Hþ]
534.2240, found 534.2231.
1
white foam: H NMR (CDCl3) δ 8.17 (d, 1H, J = 2.4 Hz),
7.19-7.30 (m, 10H), 6.43 (d, 1H, J = 8.8 Hz), 4.90 (m, 1H), 4.77
(d, 4H, J = 1.6 Hz), 4.46 (m, 1H), 4.12 (d, 1H, J = 4.4 Hz),
3.98-4.01 (m, 3H), 1.06 (s, 9H), 1.04 (s, 9H); 13C NMR (CDCl3)
δ 158.5, 146.5, 138.3, 136.0, 128.7, 127.1, 123.2, 105.8, 86.9, 78.0,
76.0, 74.4, 68.2, 51.1, 27.5, 27.4, 22.8, 20.5; HRMS (FABþ) m/z
calcd for C32H43N2O4Si [M þ Hþ] 547.2992, found 547.2998.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-dibenzylamino-1,3-pyrimidine (10a). To a
solution of 9a (1.06 g, 1.94 mmol) in dry pyridine (40 mL) were
5-(20,30,50-Tri-O-acetyl-β-D-ribofuranosyl)-2-dibenzylami-
nopyridine (7b). Compound 7b was prepared from 6b (167 mg,
0.41 mmol), DMAP (50 mg, 0.41 mmol), and Ac2O (150 mg,
1.47 mmol) as described for 7a. Silica gel chromatography (1%
methanol in dichloromethane) of the residue yielded 207 mg
(95% yield) of 7b as a white foam: 1H NMR (CD3COD) δ 8.19
J. Org. Chem. Vol. 74, No. 21, 2009 8027

Lu et al.
JOCFeatured Article
added 1H-imidazole (3.95 g, 58.1 mmol) and t-BuMe2SiCl (7.29
g, 48.4 mmol). The mixture was stirred overnight and then
evaporated to a syrup. The residue was partitioned between
CH2Cl2 and H2O; the organic layer was washed with water
followed by brine and dried over anhydrous Na2SO4. The
sodium sulfate was filtered off, and the solvent was removed by
evaporation under vacuum. The residue was purified by silica gel
chromatography, eluting with 5% ethyl acetate in hexane, to give
the product 10a as a white foam: 1.103 g (86% yield); 1H NMR
(CDCl3) δ 8.29 (s, 2H), 7.21-7.31 (m, 10H), 4.87 (d, 4H,
J = 2.4 Hz), 4.78 (s, 1H), 4.47 (m, 1H), 4.15 (d, 1H, J = 4.8
Hz), 4.10 (m, 1H), 3.88-3.97 (m, 2H), 1.05 (s, 9H), 1.04 (s, 9H),
0.93 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H); 13C NMR(CDCl3) δ 162.4,
156.5, 138.1, 128.7, 127.6, 127.2, 121.5, 87.0, 77.9, 77.7, 74.1, 68.5,
49.3, 27.7, 27.2, 26.1, 22.9, 20.5, 18.5, -4.0, -4.7; HRMS
(FABþ) m/z calcd for C37H56N3O4Si2 [M þ Hþ] 662.3809, found
662.3825.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-dibenzylaminopyridine (10b). Compound
10b was prepared from 9b (2.02 g, 3.70 mmol), 1H-imidazole
(7.50 g, 110.32 mmol), and t-BuMe2SiCl (13.90 g, 92.29 mmol) as
described for 10a. Silica gel chromatography (5% ethyl acetate in
hexane) of the residue yielded 2.26 g (93% yield) of 10b as a white
foam: 1H NMR (CDCl3) δ 8.14 (d, 1H, J = 2.4 Hz), 7.20-7.32
(m, 11H), 6.45 (d, 1H, J = 8.8 Hz), 4.81 (s, 1H), 4.79 (s, 4H,), 4.46
(m, 1H), 4.13 (d, 1H, J = 4.8Hz), 4.08(m, 1H), 3.95 (m, 1H), 3.90
(m, 1H), 1.04 (s, 9H), 1.03 (s, 9H), 0.92 (s, 9H), 0.11 (s, 3H), 0.10
(s, 3H); 13C NMR (CDCl3) δ 158.4, 146.2, 138.2, 135.7, 128.6,
127.0, 123.7, 105.8, 88.7, 77.7, 73.9, 68.5, 51.0, 27.5, 27.1, 26.0,
22.8, 20.4, 18.4, -4.1, -4.8; HRMS (FABþ) m/z calcd for
C38H57N2O4Si2 [M þ Hþ] 661.3857, found 661.3867.
dimethyl acetal (1.5 mL, 11.2 mmol), and the reaction mix-
ture was stirred at room temperature overnight. The sol-
vent was removed, and the residue was purified by silica gel
chromatography, eluting with 2% methanol in dichloro-
methane, to give the product 12a as a white foam: 558 mg
(93% yield); 1H NMR (CDCl3) δ 8.65 (s, 1H), 8.41 (s, 2H), 4.84
(s, 1H), 4.50 (m, 1H), 4.08-4.14 (m, 2H), 3.98 (m, 1H), 3.87 (m,
1H), 3.17 (s, 3H), 3.15 (s, 3H), 1.05 (s, 9H), 1.04 (s, 9H), 0.93 (s,
9H), 0.12 (s, 3H), 0.11 (s, 3H); 13C NMR (CDCl3) δ 166.6,
158.2, 156.2, 126.3, 86.6, 77.8, 77.5, 74.2, 68.3, 41.1, 35.1, 27.5,
27.1, 26.0, 22.8, 20.4, 18.4, -4.1, -4.9; HRMS (FABþ) m/z
calcd for C26H49N4O4Si2 [M þ Hþ] 537.3292, found 537.3301.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-N-(dimethylformamidino)pyridine (12b).
Compound 12b was prepared from 11b (390 mg, 0.81 mmol)
and N,N-dimethylformamide dimethyl acetal (1.13 mL, 8.10
mmol) as described for 12a. Silica gel chromatography (2%
methanol in dichloromethane) of the residue yield 370 mg (85%
yield) of 12b as a white foam: 1H NMR (CDCl3) δ 8.42 (s, 1H),
8.18 (d, 1H, J = 2.0 Hz), 7.43 (dd, 1H, J = 2.4, 8.4 Hz), 6.93 (d,
1H, J = 8.4 Hz), 4.87 (s, 1H), 4.49 (m, 1H), 4.08-4.12 (m, 2H),
4.00 (m, 1H), 3.90 (m, 1H), 3.08 (s, 6H), 1.05 (s, 9H), 1.04 (s, 9H),
0.92 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); 13C NMR (CDCl3) δ
161.9, 158.5, 146.1, 135.6, 129.5, 117.9, 88.6, 78.0, 77.5, 74.1,
68.5, 40.8, 34.8, 27.6, 27.2, 26.1, 22.8, 20.5, 18.4, -4.0, -4.8;
HRMS (FABþ) m/z calcd for C27H50N3O4Si2 [M þ Hþ]
536.3340, found 536.3348.
5-[20-O-(tert-Butyldimethylsilyl)-β-D-ribofuranosyl]-2-N-(di-
methylformamidino)-1,3-pyrimidine (13a). HF pyridine (445
3
μL, 3.42 mmol) was carefully diluted with pyridine (0.50 mL)
and then added dropwise to a solution of 12a (344 mg, 0.64
mmol) in THF (10 mL) at 0 °C. The mixture was warmed to
room temperature and stirred for 10 min. TLC showed that the
reaction was complete. The reaction mixture was diluted with
pyridine (2 mL). The mixture was partitioned between CH2Cl2
and H2O; the organic layer was washed with 5% aq NaHCO3
followed by brine and dried over anhydrous Na2SO4. The
sodium sulfate was filtered off, and the solvent was removed
by evaporation under vacuum. The residue was purified by
silica gel chromatography, eluting with 5% methanol in di-
chloromethane, to give the product 13a as a white foam: 212
mg (83% yield); 1H NMR (CDCl3) δ 8.65 (s, 1H), 8.53 (s, 2H),
4.62 (d, 1H, J = 7.1 Hz), 4.15 (m, 1H), 4.12 (m, 1H), 4.06 (m,
1H), 3.93 (dd, 1H, J = 3.2, 11.8 Hz), 3.81 (dd, 1H, J = 3.7, 11.8
Hz), 3.17 (s, 3H), 3.15 (s, 3H), 0.87 (s, 9H), -0.04 (s, 3H), -0.15
(s, 3H); 13C NMR (CDCl3) δ 166.8, 158.3, 157.1, 125.8, 85.5,
80.5, 78.8, 72.3, 62.9, 41.2, 35.1, 27.5, 18.0, -4.7, -4.9; HRMS
(FABþ) m/z calcd for C18H33N4O4Si [M þ Hþ] 397.2271,
found 397.2268.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-amino-1,3-pyrimidine (11a). Ammonium
cerium(IV) nitrate (4.52 g, 8.25 mmol) was added to a solution
of 10a (1.09 g, 1.65 mmol) in acetonitrile-water (98:2 v/v, 50 mL)
and stirred overnight at 50 °C under Ar. The reaction was
quenched with 1 N NaOH until the pH reached about 8. The
mixture was partitioned between Et2O and H2O; the organic
layer was washed with brine and dried over anhydrous Na2SO4.
The sodium sulfate was filtered off, and the solvent was removed
by evaporation under vacuum. The residue was purified by silica
gel chromatography, eluting with 15% ethyl acetate in hexane
and then 2% methanol in dichloromethane, to give the product
11a as a white foam: 546 mg (69% yield); 1H NMR (CDCl3) δ
8.21 (s, 2H), 5.33 (s, 2H, disappearedwith D2O), 4.76 (s, 1H), 4.78
(m, 1H), 4.06-4.13 (m, 2H), 3.95 (m, 1H), 3.84 (m, 1H), 1.05 (s,
9H), 1.04 (s, 9H), 0.91 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H); 13CNMR
(CDCl3) δ 162.9, 156.6, 123.3, 86.7, 77.7, 77.6, 74.2, 68.4, 27.6,
27.2, 26.1, 22.9, 20.5, 18.5, -4.0, -4.8; HRMS (FABþ) m/z calcd
for C23H44N3O4Si2 [M þ Hþ] 482.2870, found 482.2878.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-aminopyridine (11b). Compound 11b was
prepared from 10b (2.04 g, 3.08 mmol) and ammonium cerium-
(IV) nitrate (8.45 g, 15.40 mmol) as described for 11a. Silica gel
chromatography (2% methanol in dichloromethane) of the
5-[20-O-(tert-Butyldimethylsilyl)-β-D-ribofuranosyl]-2-N-(di-
methylformamidino)pyridine (13b). Compound 13b was pre-
pared from 12b (100 mg, 0.19 mmol) and HF pyridine (130
3
μL, 0.95 mmol) as described for 13a. Silica gel chromatography
(5% methanol in dichloromethane) of the residue yielded 59 mg
(80% yield) of 13b as a white foam: 1H NMR (CDCl3) δ 8.41 (s,
1H), 8.21 (d, 1H, J = 1.6 Hz), 7.57 (dd, 1H, J = 2.4, 8.4 Hz), 6.97
(d, 1H, J = 8.4 Hz), 4.62 (d, 1H, J = 6.8 Hz), 4.05-4.10 (m, 2H),
3.99 (m, 1H), 3.89 (dd, 1H, J = 3.2, 12.0 Hz), 3.78 (dd, 1H, J =
4.0, 12.0 Hz), 3.09 (s, 6H), 0.86 (s, 9H), -0.08 (s, 3H), -0.18 (s,
3H); 13C NMR (CDCl3) δ 162.3, 155.7, 147.1, 136.3, 128.5,
117.9, 85.3, 82.4, 78.8, 72.1, 63.0, 40.9, 34.8, 25.7, 18.0, -4.7,
-5.1; HRMS (FABþ) m/z calcd for C19H34N3O4Si [M þ Hþ]
396.2319, found 396.2326.
1
residue yielded 1.06 g (72% yield) of 11b as a white foam: H
NMR (CDCl3) δ 8.01 (d, 1H, J = 2.4 Hz), 7.32 (dd, 1H, J = 2.4,
8.8 Hz), 6.49 (d, 1H, J = 8.8 Hz), 4.80 (s, 1H), 4.50 (b, 2H), 4.47
(m, 1H), 4.06-4.12 (m, 2H), 3.97 (m, 1H), 3.86 (m, 1H), 1.05 (s,
9H), 1.04 (s, 9H), 0.92(s, 9H), 0.11(s, 3H), 0.10(s, 3H); 13C NMR
(CDCl3) δ 158.2, 146.3, 135.9, 125.8, 108.6, 88.6, 77.9, 77.6, 74.1,
68.5, 27.6, 27.2, 26.1, 22.9, 20.5, 18.5,-4.0, -4.8; HRMS (FABþ)
m/z calcd for C24H45N2O4Si2 [M þ Hþ] 481.2918, found
481.2922.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-N-(dimethylformamidino)-1,3-pyrimidine
(12a). To a solution of compound 11a (540 mg, 1.12 mmol) in
methanol (20 mL) was added N,N-dimethylformamide
5-[20-O-(tert-Butyldimethylsilyl)-50-O-(4,4-dimethoxytrityl)-β-
D-ribofuranosyl]-2-N-(dimethylformamidino)-1,3-pyrimidine (14a).
To a solution of 13a (209 mg, 0.527 mmol) in dry pyridine (10 mL)
was added dimethoxytrityl chloride (535 mg, 1.581 mmol). The
mixture was stirred at room temperature overnight. The reaction
8028 J. Org. Chem. Vol. 74, No. 21, 2009

Lu et al.
JOCFeatured Article
was quenched with methanol (1.0 mL) and partitioned between
CH2Cl2 and H2O. The organic layer was washed with 5% aq
NaHCO3 followed by brine and dried over anhydrous Na2SO4.
The sodium sulfate was filtered off, and the solvent was removed
by evaporation under vacuum. The residue was purified by silica
gel chromatography, eluting with 1% MeOH/CH2Cl2 þ 0.5%
Et3N, to give the product 14a as a white foam: 294 mg (80% yield);
1H NMR (CDCl3) δ 8.68 (s, 1H), 8.58 (s, 2H), 7.20-7.47 (m, 9 H),
6.82 (d, 4H, J = 8.4 Hz), 4.66 (d, 1H, J = 8.0 Hz), 4.19-4.23 (m,
2H), 4.11 (m, 1H), 3.78 (s, 6H), 3.48 (dd, 1H, J = 2.8, 10.4 Hz),
3.25 (dd, 1H, J = 3.2, 10.4 Hz), 3.18 (s, 3H), 3.14 (s, 3H), 2.77 (d,
1H, J = 2.0 Hz, disappeared with D2O), 0.87 (s, 9H), -0.04 (s,
3H), -0.13 (s, 3H); 13C NMR (CDCl3) δ 167.0, 158.6, 158.4,
157.1, 144.9, 136.0, 135.9, 130.2, 130.1, 128.2, 128.0, 126.9, 125.8,
113.33, 113.31, 86.4, 84.7, 79.6, 79.0, 73.1, 64.1, 55.4, 41.2, 35.2,
25.8, 18.0, -4.6, -4.9; HRMS (FABþ) m/z calcd for C39H51N4O6-
Si [M þ Hþ] 699.3578, found 699.3583.
dry CH2Cl2 (10 mL) was added m-CBPA (94 mg, 0.54 mmol), and
thereactionmixturewasstirredatroomtemperatureforovernight
under Ar. The solvent was removed by evaporation under
vacuum, and the residue was purified by silica gel chromatography,
eluting with 2-5% MeOH in dichloromethane, to give the
product 16 as a white foam: 160 mg (90% yield); 1H NMR
(CDCl3) δ 8.09 (d, 1H, J = 2.0 Hz), 7.03 (dd, 1H, J = 2.0, 8.8
Hz), 6.77 (d, 1H, J = 8.4 Hz), 5.82 (b, 2H), 4.75 (s, 1H), 4.47 (m,
1H), 4.05-4.11 (m, 2H), 3.97 (m, 1H), 3.80 (m, 1H), 1.03 (s, 9H),
1.02 (s, 9H), 0.93 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H); 13C
NMR (CDCl3) δ 149.5, 135.7, 126.9, 126.5, 109.7, 87.2, 77.8,
77.3, 74.2, 68.3, 27.6, 27.2, 26.1, 22.8, 20.5, 18.4, -3.9, -4.7;
HRMS (FABþ) m/z calcd for C24H45N2O5Si2 [M þ Hþ] 497.2867,
found 497.2872.
6-Acetamido-3-[20-O-(tert-butyldimethylsilyl)-30,50-O-(di-tert-
butylsilylene)-β-D-ribofuranosyl]-1H-pyridin-2-one (17). To a
solution of 5-[20-O-(tert-butyldimethylsilyl)-30,50-O-(di- tert-
butylsilylene)-β-D-ribofuranosyl]-2-aminopyridine 1-oxide (16)
(160 mg, 0.32 mmol) in Ac2O (5 mL) was added Et3N (161 μL,
1.12 mmol), and the reaction mixture was stirred at 140 °C for 2
h. The solvent was removed by evaporation under vacuum, and
the residue was dissolved in MeOH (5 mL). NaOH (38 mg, 0.96
mmol) was added, and the reaction mixture was stirred at room
temperature for 2 h under Ar. The solvent was removed by
evaporation under vacuum, and the residue was purified by
silica gel chromatography, eluting with 3% MeOH in dichloro-
methane, to give the product 17 as a white foam: 102 mg (59%
yield); 1H NMR (CDCl3) δ 10.58 (b, 1H), 7.40 (d, 1H, J = 8.0
Hz), 6.19 (d, 1H, J = 7.6 Hz), 4.88 (s, 1H), 4.46 (m, 1H), 4.25 (m,
1H), 4.08 (m, 1H), 3.90-3.95 (m, 2H), 2.17 (s, 3H), 1.02 (s, 9H),
1.01 (s, 9H), 0.89 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H); 13C NMR
(CDCl3) δ 171.7, 160.0, 143.3, 140.6, 122.9, 93.7, 86.2, 77.3, 75.6,
74.0, 68.5, 27.7, 27.2, 26.1, 24.5, 22.8, 20.5, 18.4, -3.9, -4.8;
HRMS (FABþ) m/z calcd for C26H47N2O6Si2 [M þ Hþ]
539.2973, found 539.2977.
5-[20-O-(tert-Butyl)dimethylsilyl-50-O-(4,4-dimethoxytrityl)-
β-D-ribofuranosyl]-2-N-(dimethylformamidino)pyridine (14b).
Compound 14b was prepared from 13b (132 mg, 0.33 mmol)
and dimethoxytrityl chloride (339 mg, 1.00 mmol) as described
for 14a. Silica gel chromatography (1% methanol in dichlor-
omethane þ 0.5% Et3N) of the residue yielded 224 mg (96%
yield) of 14b as a white foam: 1H NMR (CDCl3) δ 8.43 (s, 1H),
8.25 (d, 1H, J = 2.4 Hz), 7.71 (dd, 1H, J = 2.4, 8.0 Hz),
7.18-7.49 (m, 9H), 6.93 (d, 1H, J = 8.0 Hz), 6.82 (m, 4H), 4.68
(d, 1H, J = 7.6 Hz), 4.16-4.20 (m, 2H), 4.10 (m, 1H), 3.783 (s,
3H), 3.780 (s, 3H), 3.46 (dd, 1H, J = 3.2, 10.4 Hz), 3.26 (dd,
1H, J = 3.6, 10.0 Hz), 3.09 (s, 6H), 2.80 (d, 1H, J = 2.4 Hz),
0.85 (s, 9H), -0.09 (s, 3H), -0.18 (s, 3H); 13C NMR (CDCl3) δ
162.5, 158.6, 155.6, 147.3, 145.0, 136.3, 136.2, 136.0, 130.3,
130.2, 128.8, 128.3, 127.9, 126.9, 118.0, 113.3, 86.4, 84.5, 81.5,
79.2, 73.1, 64.3, 55.3, 40.9, 34.8, 25.8, 18.1, -4.7, -5.0; HRMS
(FABþ) m/z calcd for C40H52N3O6Si [M þ Hþ] 698.3625,
found 698.3630.
5-[20-O-(tert-Butyl)dimethylsilyl-30-O-(2-cyanoethyl-N,N-diiso-
propylphosphino)-50-O-(4,4-dimethoxytrityl)-β-D-ribofuranosyl]-2-
N-(dimethylformamidino)-1,3-pyrimidine (15a). To a solution of
14a (95 mg, 0.136 mmol) in dry dichloromethane (5 mL) under
argon were added N,N-diisopropylethylamine (117 μL, 0.68
mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
(95 mg, 0.41 mmol), and 1-methylimidazole (11 μL, 0.136
mmol). The reaction mixture was stirred at room temperature
for 1 h. TLC indicated that the reaction was complete. The
reaction mixture was quenched with MeOH (1 mL) and stirred
for 5 min. After the solvent was removed, the residue was
purified by silica gel chromatography, eluting with 0-5%
acetone in dichloromethane containing 0.5% triethylamine, to
give the corresponding phosphoramidite 15a as a white foam:
118 mg (96% yield); 31P NMR (CD3CN) δ 153.2, 150.6; HRMS
(FABþ) m/z calcd for C48H68N6O7PSi [M þ Hþ] 899.4656,
found 899.4637.
5-[20-O-(tert-Butyldimethylsilyl)-30-O-(2-cyanoethyl-N,N-diiso-
propylphosphino)-50-O-(4,4-dimethoxytrityl)-β-D-ribofuranosyl]-
2-N-(dimethylformamidino)pyridine (15b). Compound 15b
was prepared from 14b (117 mg, 0.17 mmol), N,N-diisopropy-
lethylamine (155 μL, 0.90 mmol), 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite (128 mg, 0.55 mmol), and 1-methyli-
midazole (16 μL, 0.20 mmol) as described for 15a. Silica
gel chromatography (0-5% acetone in dichloromethane þ
0.5% Et3N) of the residue yielded 132 mg (88% yield) of 15b
as a white foam: 31P NMR (CD3CN) δ 150.6, 148.2; HRMS
(FABþ) m/z calc for C49H69N5O7PSi [M þ Hþ] 898.4704, found
898.4707.
6-Acetamido-3-[20-O-(tert-butyldimethylsilyl)-30,50-O-(di-tert-
butylsilylene)-β-D-ribofuranosyl]-1-[(pivaloyloxy)methyl]pyridin-
2-one (18). To a solution of 17 (214 mg, 0.40 mmol) in DMF (10
mL) were added K2CO3 (83 mg, 0.60 mmol) and chloromethyl
pivaloate (73 mg, 0.48 mmol), and the reaction mixture was
stirred at 90 °C for 4 h. The solvent was removed by evaporation
under vacuum, and the residue was purified by silica gel
chromatography, eluting with 1% MeOH in dichloromethane,
to give the product 18 as a white foam: 223 mg (86% yield); 1H
NMR (CDCl3) δ 7.83 (d, 1H, J = 7.6 Hz), 7.78 (s, 1H), 7.63 (d,
1H, J = 8.0 Hz), 6.13 (d, 1H, J = 5.6 Hz), 6.01 (d, 1H, J = 5.6
Hz), 5.04 (s, 1H), 4.51 (m, 1H), 4.12-4.18 (m, 2H), 4.02 (m, 1H),
3.89 (m, 1H), 2.21 (s, 3H), 1.16 (s, 9H), 1.04 (s, 18H), 0.94 (s, 9H),
0.14 (s, 3H), 0.13 (s, 3H); 13C NMR (CDCl3) δ 177.4, 168.6,
156.1, 148.2, 139.4, 118.5, 107.4, 84.5, 81.9, 77.0, 76.6, 73.9, 68.5,
38.9, 27.7, 27.2, 27.0, 26.0, 24.8, 22.8, 20.4, 18.4, -3.9, -5.0;
HRMS (FABþ): m/z calcd for C32H57N2O8Na Si2 [M þ Naþ]
675.3473, found 675.3475.
6-Acetamido-3-[20-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-
1-[(pivaloyloxy)methyl]pyridin-2-one (19). Compound 19 was
prepared from 18 (274 mg, 0.42 mmol) and HF pyridine (279
3
μL, 2.10 mmol) as described for 13a. Silica gel chromatography
(3% methanol in dichloromethane) of the residue yielded 192 mg
(89% yield) of 19 as a white foam: 1H NMR (CDCl3 þ D2O) δ.17
(s, 1H), 7.84 (d, 1H, J = 8.8 Hz), 7.68 (d, 1H, J = 8.8 Hz), 6.10 (d,
1H, J = 5.6 Hz), 5.98 (d, 1H, J= 5.6 Hz), 4.78 (d, 1H, J= 7.0 Hz),
4.32 (m, 1H), 4.12 (m, 1H), 4.02 (m, 1H), 3.91 (dd, 1H, J = 2.8,
12.0 Hz), 3.78 (dd, 1H, J = 3.6, 12.0 Hz), 2.21 (s, 3H), 1.17 (s, 9H),
0.86 (s, 9H), -0.04 (s, 3H), -0.14 (s, 3H); 13C NMR (CDCl3) δ
177.7, 168.8, 157.3, 148.7, 142.1, 116.4, 107.2, 84.9, 82.0, 81.9,
76.0, 71.8, 62.8, 38.9, 27.0, 25.7, 24.8, 18.0, -4.7, -5.1; HRMS
(FABþ) m/z calcd for C24H41N2O8Si [M þ Hþ] 513.2632, found
513.2641.
5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-aminopyridine 1-Oxide (16). To a solution
of 5-[20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilylene)-
β-D-ribofuranosyl]-2-aminopyridine 11b (173 mg, 0.36 mmol) in
J. Org. Chem. Vol. 74, No. 21, 2009 8029

Lu et al.
JOCFeatured Article
6-Acetamido-3-[50-O-(4,4-dimethoxytrityl)-20-O-(tert-butyl-
dimethylsilyl)-β-D-ribofuranosyl]-1-[(pivaloyloxy)methyl]pyridin-
2-one (20). Compound 20 was prepared from 19 (192 mg, 0.37
mmol) and dimethoxytrityl chloride (382 mg, 1.12 mmol) as
described for 14a. Silica gel chromatography (1% methanol in
dichloromethane þ 0.5% Et3N) of the residue yielded 283 mg
21 was prepared from 20 (96 mg, 0.12 mmol), N,N-diisopropyl-
ethylamine (109 μL, 0.63 mmol), 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (90 mg, 0.39 mmol), and 1-methylimidazole
(11 μL, 0.14 mmol) as described for 15a. Silica gel chromatography
(0-5% acetone in dichloromethane þ 0.5% Et3N) of the residue
yielded 94 mg (79% yield) of 21 as a white foam: 31P NMR
(CD3CN) δ 148.7, 149.6; HRMS (FABþ) m/z calcd for
C54H76N4O11NaPsi 1037.4837, found 1037.4846.
1
(93% yield) of 20 as a white foam: H NMR (CDCl3) δ 7.99
(d, 1H, J = 8.0 Hz), 7.22-7.81 (m, 11H), 6.86 (d, 4H, J = 8.8
Hz), 6.14 (d, 1H, J = 5.2 Hz), 5.95 (d, 1H, J = 5.2 Hz), 5.10 (d,
1H, J = 5.2 Hz), 4.23 (m, 1H), 4.14 (m, 2H), 3.82 (s, 6H), 3.51
(dd, 1H, J = 2.4, 10.4 Hz), 3.39 (dd, 1H, J = 4.0, 10.4 Hz), 2.62
(d, 1H, J = 6.0 Hz), 2.22 (s, 3H), 1.20 (s, 9H), 0.93 (s, 9H), 0.06
(s, 3H), 0.04 (s, 3H); 13C NMR (CDCl3) δ 177.4, 168.5, 158.6,
156.9, 148.3, 145.0, 140.4, 136.1, 136.0, 130.3, 130.2, 128.3,
127.9, 126.9, 118.0, 113.2, 107.5, 86.4, 83.0, 82.2, 79.0, 78.0,
72.0, 63.8, 55.3, 38.9, 27.0, 25.8, 24.8, 18.1, -4.6, -5.1; HRMS
(FABþ) m/z calcd for C45H59N2O10NaSi [M þ Naþ] 837.3758,
found 837.3767.
Acknowledgment. S.C.K. was supported in part by the
Medical Scientist Training Program (5 T32 GM07281). We
thank Q. Dai, R. Sengupta, E. Leung, K. Sundaram, and
N. Tuttle for helpful discussions and critical comments on
the manuscript.
Supporting Information Available: Proton, carbon, and
phosphorus NMR spectra as appropriate for all new com-
pounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
6-Acetamido-3-[50-O-(4,4-dimethoxytrityl)-30-O-(2-cyanoethyl-
N,N-diisopropylphosphino)-20-O-(tert-butyl)dimethylsilyl-β-D-ribo-
furanosyl]-1-[(pivaloyloxy)methyl]pyridin-2-one (21). Compound
8030 J. Org. Chem. Vol. 74, No. 21, 2009