Synthesis of 2′-C-difluoromethylribonucleosides and their enzymatic incorporation into oligonucleotides

Article abstract of DOI:10.1021/jo0507542

Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of these analogues, 2′-C-β- difluoromethylribonucleosides. We constructed

Full text of DOI:10.1021/jo0507542

Synthesis of 2′-C-Difluoromethylribonucleosides and Their
Enzymatic Incorporation into Oligonucleotides
Jing-Dong Ye, Xiangmin Liao,† and Joseph A. Piccirilli*
Howard Hughes Medical Institute, Departments of Biochemistry & Molecular Biology and Chemistry,
The University of Chicago, 5841 South Maryland Avenue, MC 1028, Chicago, Illinois 60637
jpicciri@uchicago.edu
Received April 14, 2005
Nucleosides bearing a branched ribose have significant promise as therapeutic agents and
biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of
these analogues, 2′-C-â-difluoromethylribonucleosides. We constructed the glycosylating agent 4
in three steps from 1,3,5-tri-O-benzoyl-R-D-ribofuranose 1. The key steps included nucleophilic
addition of difluoromethyl phenyl sulfone to 2-ketoribose 2 followed by mild and efficient reductive
desulfonation. Ribofuranose 4 glycosylated bis(trimethylsilyl)uracil directly, giving difluoromethyl-
uridine 7 efficiently after deprotection. Conversion of 4 to the corresponding ribofuranosyl bromide
allowed efficient access to C, A, and G analogues. A related approach starting from methyl
D-ribofuranose offered synthetic entry into the diastereomeric manifold, 2′-C-R-difluoromethyl-
arabino-R-pyrimidine. To incorporate 2′-C-â-difluoromethyluridine into an oligodeoxynucleotide we
converted 7 to the bisphosphate and carried out successive ligation reactions using T4 RNA ligase
and T4 DNA ligase. Analogous to natural RNA linkages, the resulting oligonucleotide undergoes
hydroxide-catalyzed backbone scission at the difluoromethyluridine residue via internal transpho-
sphorylation.
Introduction
adenosine and the corresponding 7-deazaadenosine ana-
logue potently inhibit Hepatitis C Virus (HCV) RNA
replication in vivo.10,11 Within oligonucleotides, 2′-
branched nucleotides impart increased nuclease resis-
tance in biological media and have allowed artificial
regulation of gene expression by antisense approaches,12-16
self-cleaving RNA motifs,17 or the combination thereof.18
Over the past decade 2′-C-branched nucleosides have
garnered much attention for their potential anticancer
and antiviral activities.1-9 For example, 2′-C-â-methyl-
† Present address: GlaxoSmithKline, UP 1110, 1250 S. Collegeville
Road, Collegeville, PA 19426.
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J. R.; Jarvi, E. T.; Matthews, D. P.; Resvick, R. J.; Wagner, E.
Biochemistry 1996, 35, 8381-91.
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Stahlhut, M. W.; Eldrup, A. B.; Bhat, B.; Hall, D.; Simcoe, A. L.;
LaFemina, R.; Rutkowski, C. A.; Wolanski, B.; Yang, Z.; Migliaccio,
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10.1021/jo0507542 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/26/2005
7902
J. Org. Chem. 2005, 70, 7902-7910

Synthesis of 2′-C-Difluoromethylribonucleosides
SCHEME 1a
In this context, 2′-C-difluoromethyl ribonucleosides rep-
resent important synthetic targets as fluorination of
nucleosides may enhance their therapeutic activities.19-22
2′-C-Branched nucleosides also serve as valuable probes
to explore biomolecular structure and function of protein
and RNA enzymes.4,5,23,24 For example, 2′-C-methyluri-
dine has been used to investigate nucleases,23 ribonucleo-
tide reductase,5 and RNA polymerase.25 2′-C-Methylcy-
tidine26 and 2′-C-R-difluoromethyl-arabinouridine27 have
been used to investigate the ai5γ group II intron ri-
bozyme and the hammerhead ribozyme, respectively. Our
interest in 2′-C-branched ribonucleosides arises from
their possible value as mechanistic agents with which
to vary the pKa of the 2′-hydroxyl group. A series of
ribonucleotides bearing substituents of increasing electron-
withdrawing power (CH3, CH2F, CHF2, CF3) might allow
such pKa variation, thereby enabling the application of
physical organic approaches to study RNA-mediated
biological processes.
Previous work has described the synthesis of several
analogues in this series.28-30 Here we aim to develop a
reliable synthesis of 2′-C-â-difluoromethylribonucleo-
sides. Two general strategies prevail for the synthesis of
2′-C-branched ribonucleosides: (a) a linear approach
starting from natural nucleosides and (b) a convergent
approach in which nucleobases are glycosylated with the
appropriately modified sugar. The linear approach often
suffers from poor stereoselectivity during installation of
the branch. The convergent approach offers greater
stereocontrol during installation of the branch by starting
with a sugar bearing suitable substituents configured
with the appropriate stereochemistry. The convergent
approach also offers greater flexibility in that a variety
of nucleobases may be glycosylated, saving synthetic
steps overall. Here we develop a convergent approach for
the first synthesis of 2′-C-â-difluoromethylribonucleosides
and their diastereomeric counterparts, 2′-C-R-difluoro-
a Conditions: (a) Dess-Martin periodinane, CH2Cl2, rt, 48 h,
98%; (b) PhSO2CF2H, LiHMDS, THF, -78 to 0 °C, 2 h, 64%; (c)
(i) 5% Na-Hg, Na2HPO4, MeOH, THF, H2 atmosphere, 0 °C, 45
min; (ii) BzCl, DMAP, Et3N, CH2Cl2, rt, 5 h, 61% in two steps; (d)
bis(trimethylsilyl)uracil, SnCl4, CH3CN, reflux, 2 days, 78%; (e)
NH3, MeOH, 4 °C, 2 days, 95%.
methyl-arabino-R-nucleosides. We exploit an enzymatic
ligation procedure to incorporate the uridine analogue
site-specifically into an oligodeoxynucleotide.
Results and Discussion
Synthesis of 2′-C-â-Difluoromethylribonucleo-
sides. Diethylaminosulfur trifluoride (DAST) under mild
conditions converts aldehydes, including those appended
to nucleosides, to a difluoromethyl group.22,31,32 On this
basis we prepared 2′-C-â-formyluridine as a putative
precursor to 2′-C-difluoromethyluridine via DAST-medi-
ated fluorination. We obtained 1,3,5-tri-O-benzoyl-2-oxo-
R-D-erythro-pentofuranose 2 as previously described33 by
oxidation of 1,3,5-tri-O-benzoyl-R-D-ribofuranose 1 with
Dess-Martin periodinane. Vinylation and glycosylation
generated 2′,3′,5′-tri-O-benzoyl-2′-vinylribouridine as de-
scribed by Harry-O’kuru et al.34 Subsequent oxidation
with OsO4/KIO4 gave 2′-C-formyluridine. Treatment of
this compound with DAST under various conditions gave
none of the desired product, possibly due to the steric
hindrance of the uracil base (Liao, X.; Piccirilli, J. A.
Unpublished results).
Sabol and McCarthy prepared 2′-C-R-difluoromethyl-
arabinocytidine in two steps from 2′-ketocytidine.35 Ad-
dition of difluoromethyl phenyl sulfone in the presence
of lithium hexamethyldisilazane in THF/HMPA gave 2′-
C-R-(phenylsulfonyl)difluoromethyl-arabinocytidine. Sub-
sequent reduction with SmI2 converted the phenylsul-
fonyl difluoromethyl group to a difluoromethyl group.
Inspired by this report, we investigated the reaction of
ketoribose 2 with difluoromethyl phenyl sulfone under
the same conditions. The reaction consumed starting
material but yielded none of the desired product. How-
ever, in the absence of HMPA the reaction gave sulfone
3 in reasonably good yield (64%, Scheme 1). 19F-1H NOE
(14) Milligan, J. F.; Matteucci, M. D.; Martin, J. C. J. Med. Chem.
1993, 36, 1923-37.
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J. Org. Chem, Vol. 70, No. 20, 2005 7903

Ye et al.
â-face, respectively. Glycosylation of bis(trimethylsilyl)-
uracil with 4 in the presence of trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) at room temperature
resulted in recovered starting material only. Using the
stronger Lewis acid SnCl4, the glycosylation reaction gave
uridine 6 in about 5% yield after 2 days at room
temperature. Under the same conditions at reflux in CH3-
CN (83 °C), the yield of 6 improved significantly (78%).
The stereochemistry was verified by 19F-1H NOE experi-
ments. Upon irradiation of the 19F nuclei of the difluo-
romethyl group, strong NOE signals occur for CF2H (δ
6.58 ppm), 3′-H (δ 6.08 ppm), and 6-H (δ 7.57 ppm). In
NOESY spectra, 1′-H (δ 6.97 ppm) exhibits strong cross
peaks with 4′-H (δ 5.01 ppm) but not with CF2H or 3′-H.
Debenzoylation of 6 with saturated ammonia in methanol
at 4 °C for 2 days provided 2′-C-â-difluoromethyluridine
7 in 95% yield. CD spectroscopy further confirmed the â
anomeric configuration: 7 exhibits a positive Cotton
effect between 260 and 280 nm, as expected for a
pyrimidine nucleoside in the â anomeric configuration.43
As observed for bis(trimethylsilyl)uracil, TMSOTf proved
insufficient in the glycosylation of persilylated cytosine,
adenine, and guanine (appropriately protected) with
difluoromethylribose 4, even at high temperature (120
°C). In the presence of the more activated Lewis acid
SnCl4, 4 decomposed, yielding no desired product, similar
to a previous report.44 Possibly SnCl4 forms a “σ complex”
with the stronger nucleobases, leading to undesired side
reactions.42 As a consequence of these observations, we
investigated transformation of 4 to the more active
difluoromethylribofuranosyl bromide 8. At 80 °C in the
presence of 30% HBr/acetic acid 4 gives the bromide 8
in 65% yield (Scheme 2). NOESY experiments supported
the assigned stereochemistry, showing strong cross peaks
between CF2H (δ 6.11 ppm) and 1′-H (δ 6.99 ppm) as well
as 3′-H (δ 5.43 ppm).
In the presence of HgO/HgBr2,29 bromide 8 glycosylates
persilylated N4-benzoylcytosine and N6-benzoyladenine
to give exclusively the corresponding â-nucleoside deriva-
tives 9a and 9b in 65% and 50% yield, respectively.
NOESY experiments revealed the stereochemistry of 9a
and 9b: 1′-H (δ 6.09 ppm for 9a, 6.37 ppm for 9b)
exhibits much stronger cross peaks with 4′-H (δ 4.75 ppm
for 9a, 4.78 ppm for 9b) than with 3′-H (δ 5.63 ppm for
9a, 6.25 ppm for 9b). Additionally, 1′-H in 9b lacks strong
cross peaks with CF2H (in 9a, these two peaks are too
close to determine which cross peaks occur). Debenzoyl-
ation of 9a and 9b in the same manner as described for
6 affords the parent nucleosides 10a and 10b in 96% and
93% yield, respectively.
For the synthesis of the guanosine derivative we
conducted the glycosylation reaction with N2-acetyl-O6-
diphenylcarbamoylguanine to minimize formation of the
undesired N-7 isomer.45,46 Persilylated N2-acetyl-O6-
diphenylcarbamoylguanine was exposed to 8 in the
presence of HgO/HgBr2 to give 9c in 31% yield. NOESY
experiments verified the stereochemistry of 9c. 1′-H (δ
FIGURE 1. Hydrogen pressure shifts the reduction equilib-
rium.
experiments with 3 indicated that the phenylsulfonyl
difluoromethyl group added stereoselectively to the â-face
of the sugar. Irradiation of 19F nuclei resulted in strong
NOE signals for the 1-H (δ 7.08 ppm) and 3-H (δ 6.03
ppm) heterocyclic protons, suggesting that 1-H, 3-H, and
the phenylsulfonyl difluoromethyl group reside on the
same side of the ribofuranose ring.
Reduction of 3 with SmI2 in THF/HMPA36 consumed
the starting material but gave no desired product.
Without HMPA the reaction produced a major product
that lacked both the phenylsulfonyl difluoromethyl group
and the 1-C-benzoyl group. To circumvent this apparent
over-reduction, we tested alternative mild reducing agents.
Among the reagents tested, including Raney Ni, Mg-
MeOH-Na2HPO4,37 [(Cp2NiAlH2)-Li+]2,38 and Na(Hg)-
MeOH-Na2HPO4,27,39 only Na(Hg)-MeOH-Na2HPO4
appeared suitable. Exposure of 3 to this reagent at -10
to 0 °C for 2 h followed by treatment with benzoyl
chloride resulted in 15% difluoromethylribose 4 and 20%
unreduced adduct 5. Longer reaction time resulted in
destruction of the sugar moiety. Using THF as solvent
and stoichiometric equivalents of methanol and Na-Hg
improved the yields of both 4 (25%) and 5 (30%).
Strikingly, we observed that under a hydrogen atmo-
sphere (hydrogen balloon) reduction completes in about
40 min as monitored by TLC, and the yield of 4 improves
to 61% (â/R ) 3/4). The hydrogen pressure may shift the
equilibrium for the redox reaction between sodium and
methanol, minimizing depletion of sodium and thereby
favoring reduction of the phenyl sulfone group (Figure
1). To the best of our knowledge, this is the first
demonstration that hydrogen pressure can assist in
single-electron-transfer reduction by Na-Hg-MeOH-
Na2HPO4.
We tested difluoromethylribose 4 as a glycosylating
agent for bis(trimethylsilyl)uracil. Ribose derivatives
usually glycosylate persilylated nucleobases rapidly and
stereoselectively to yield the â-nucleosides.40-42 However,
4 exhibits significantly weaker glycosylating power than
the corresponding ribose derivative, presumably reflect-
ing both the strong electron-withdrawing character and
the steric bulk of the difluoromethyl group, which would
destabilize the putative carbocation intermediate and
hinder the addition of the nucleobases at the desired
(36) Kunzer, H.; Stahnke, M.; Sauer, G. Tetrahedron Lett. 1991, 32,
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Wang, B. S. L.; Luh, T.-Y. J. Chem. Soc., Chem. Commun. 1988, 53,
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Nucleotides 1995, 14, 799-801.
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Robins, R. K.; Eyring, H. J. Am. Chem. Soc. 1970, 92, 3872-81.
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Jr. Nucleic Acids Res. 1995, 23, 4913-21.
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61, 9207-12.
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60.
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6.
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7904 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis of 2′-C-Difluoromethylribonucleosides
SCHEME 2a
a Conditions: (a) 30% HBr in AcOH, 80 °C, 5 h, 65%; (b) persilylated nucleobases, HgO/HgBr2, toluene, 80-90 °C, 2-5 h, 31-65%; (c)
for 10a and 10b, NH3, MeOH, 4 °C, 2 days, 93-96%; for 10c, NH3/H2O/MeOH, 60 °C, overnight, 55%.
SCHEME 3a
a Conditions: (a) (i) (t-Bu)2Si(Tf)2, 0 °C to rt, 15 min; (ii) Et3N, 5 min, 59%; (b) Dess-Martin periodinane, overnight, 91%; (c) PhSO2CF2H,
LiHMDS, THF/HMPA (10/1), -78 °C, 2 h, 63%; (d) SmI2 (4 equiv), THF/HMPA (20:1), 0 °C, 30 min, 74%; (e) BzCl, DMAP, Et3N, 5 h, 95%;
(f) pyridinium poly(hydrogen fluoride), 0 °C to rt, 15 min, 95%; (g) BzCl, DMAP, Et3N, 5 h, 97%; (h) (i) 30% HBr in AcOH, 75 °C, 4 h; (ii)
persilylated nucleobases, HgO/HgBr2, 36 h, 48-55% in two steps; (i) NH3, MeOH, 4 °C, 2 days, 84-90%.
SCHEME 4a
6.23 ppm) exhibits strong cross peaks with 4′-H (δ 4.78
ppm) but not with CF2H (δ 5.86 ppm). Incubation of 9c
at 60 °C overnight in NH3/H2O/MeOH followed by
preparative HPLC gave the parent guanosine nucleoside
10c in 55% yield.
Synthesis of 2′-C-r-Difluoromethyl-arabino-r-py-
rimidines. During the synthesis of 2′-C-â-difluorometh-
ylribonucleosides an efficient synthesis of 2′-C-R-difluo-
romethyl-arabino-R-pyrimidines emerged, which we briefly
describe here (Scheme 3). The strategy parallels that
used above but starts from ribose and proceeds through
ketone 13. Treatment of D-ribose with sulfuric acid in
methanol gave the methyl D-ribose 11 as an anomeric
mixture.47 Simultaneous protection of the 3- and 5-hy-
droxyl groups with di-tert-butylsilyl bis(trifluoromethane-
sulfonate) (DTBS)48 afforded the bis(silyl)ether 12 after
careful silica gel chromatography. Subsequent oxidation
with Dess-Martin periodinane afforded ketone 13. Nu-
cleophilic addition of difluoromethyl phenyl sulfone in the
presence of lithium hexamethyldisilazane converted 13
exclusively to sulfone 14 in 61% yield. In the presence of
HMPA, reduction of the sulfone 14 with SmI2 generated
the difluoromethylribose derivative 15. The stereochem-
istry of compound 15 was established by 19F-1H NOE
experiments. When the 19F nuclei of the difluoromethyl
group were irradiated, strong NOE signals occur for
a Conditions: (a) diphosphoryl chloride, 0 °C to rt, 4 h, 50%.
CF2H (δ 6.00 ppm) and 1-H (δ 5.05 ppm) but not for 3-H.
These observations indicate that the difluoromethyl
phenyl sulfone adds to the ketone 13 on the R face.
To install the stereodirecting acyl group for subsequent
glycosylation, we converted 15 to 16 with benzoyl chloride
in the presence of DMAP and Et3N. Removal of the DTBS
protecting group with pyridinium poly(hydrogen fluoride)
(PPHF)48 followed by benzoylation afforded the tribenzoyl
difluoromethyl sugar 18. Direct coupling of 18 with
persilylated bases in the presence of SnCl4 in acetonitrile
gave none of the expected products, even after refluxing
for 1 week. We therefore converted 18 to the more
reactive 1-bromide derivative by incubating 18 in 30%
HBr in acetic acid at 75 °C for 4 h. The bromide derivative
readily glycosylated persilylated 4-N-benzoylcytosine and
uracil in the presence of HgO/HgBr2 to give nucleoside
derivatives 19a and 19b in 48% and 55% yield, respec-
tively. Debenzoylation with ammonia in methanol at 0
°C for 2 days afforded the arabinopyrimidines 20a,b in
(47) Barker, R.; Fletcher, H. G. J. Org. Chem. 1961, 26, 4605.
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J. Org. Chem, Vol. 70, No. 20, 2005 7905

Ye et al.
SCHEME 5
80-90% yields. We made no attempt to prepare the
corresponding purine analogues. The stereochemistry of
arabinonucleosides was revealed by NOESY and 19F-
1H NOE experiments. For 19a, 1′-H (δ 7.09 ppm) exhib-
ited strong NOE cross peaks with 3′-H (δ 6.43 ppm) but
not with 4′-H (δ 4.94 ppm) nor with CF2H (δ 6.19 ppm).
Lack of NOE signals for 1′-H and 3′-H in the 19F-1H NOE
experiments of compounds 19a,b and 20a,b is also
consistent with the structure assignment. Furthermore,
CD spectra of 20a,b show a negative Cotton effect in
260-280 nm, confirming the R anomeric configuration.
Enzymatic Incorporation of 2′-C-â-Difluorometh-
yluridine into Oligonucleotides. Use of 2′-â-modified
nucleosides for biochemical investigations of nucleic acid
structure and function requires incorporation of these
analogues into oligonucleotides. Although we could pre-
pare oligonucleotides containing the methyl analogue
using standard phosphoramidite chemistry, the phos-
phoramidite of the trifluoromethyl analogue proved
exceedingly difficult to access (Dai, Q.; Piccirilli, J. A.
Unpublished results). To develop a unified and reliable
approach to incorporate these analogues we exploited an
enzymatic route using 3′,5′-bisphosphorylated derivatives
and T4 RNA ligase.49,50 We chose uridine analogues to
develop a representative incorporation strategy. Difluo-
romethyluridine 3′,5′-bisphosphate 21 (Scheme 4) was
synthesized from uridine 7 using a slightly modified
version of the method described by Barrio et al.50 Reac-
tions were conducted at room temperature instead of -10
to -15 °C to compensate for the lower reactivity of
uridine analogues toward phosphorylation.51 The reaction
mixture was purified by ion-exchange chromatography
on a DEAE-cellulose column, eluting with a linear
gradient of 0.15-0.5 M triethylammonium bicarbonate
(TEAB, pH 8.0). NMR showed that only one product, 3′,5′-
bisphosphate 21 (confirmed by its action as a nucleotide
donor for RNA ligation catalyzed by T4 RNA ligase) was
isolated. We observed no phosphorylation of the 2′-
hydroxyl group, presumably because of the electron-
withdrawing and steric effects of the difluoromethyl
group.
of the reaction mixture followed by electrophoresis through
ananalytical20%denaturingpolyacrylamidegel(DPAGE),
which revealed a conversion yield of 90% (based on the
(dN)12 acceptor). To verify the presence of the modified
nucleoside we synthesized a shorter oligonucleotide
(rCCGAAAU2′CF H) using a similar strategy and confirmed
2
the molecular weight by MALDI mass spectroscopy
(calcd, 2237; found, 2235-2238). To synthesize the full-
length chimeric RNA we incubated the 13mer (dN)12-
rU2′CF H, the donor oligonucleotide pd(ACACGCAA-
2
GATG), and a complementary DNA template in the
presence of T4 DNA ligase overnight at 16 °C (Scheme
5). The resulting 25mer ligation product (dN)12rU2′CF H
-
(dN)12 was purified by 20% DPAGE. We 5′-32P label2ed
the chimeric RNA 25mer and a chemically synthesized
25mer containing natural nucleotides, *p(dN)12rU(dN)12,
with γ-32P-ATP and T4 polynucleotide kinase and purified
them by 20% DPAGE. The two oligonucleotides migrate
with the same electrophoretic mobility and give essen-
tially the same P1 nuclease digestion patterns, except
at the position bearing the modification (see Supporting
Information).
In base-catalyzed cleavage of RNA the 2′-hydroxyl
group acts as a nucleophile, attacking the adjacent
phosphorus center to yield 2′,3′-cyclic phosphate and 5′-
hydroxyl termini. We tested whether the tertiary hy-
droxyl group of 2′-C-â-difluoromethyluridine could facili-
tate RNA cleavage under alkaline conditions. Cleavage
of the 32P-labeled 25mers was initiated with the addition
of 0.1 N KOH. Aliquots were neutralized with aqueous
HCl and separated by 20% DPAGE. At pH 13, both
oligonucleotides react at a similar rate to give a single,
faster-migrating species (Figure 2). These results indicate
that the tertiary hydroxyl group in 2′-C-â-difluoromethyl-
uridine participates in base-catalyzed internal transpho-
sphorylation.
Conclusions
In summary, we established synthetic entry into a new
class of branched ribonucleosides bearing a 2′-C-â-di-
fluoromethyl group. We accessed the analogues of U, C,
Bisphosphate 21 (donor) was first ligated to the 5′-
oligodeoxynucleotide 12mer dCTGTCACCGAAA ((dN)12,
acceptor) using T4 RNA ligase in conjunction with an ATP
regeneration system (Scheme 5).52 Following this initial
ligation the 3′-terminal phosphate group of the ligation
product was removed with calf intestine alkaline phos-
phatase (CIAP). The resulting dephosphorylated 13mer
(dN)12rU2′CF H was analyzed by 32P labeling of an aliquot
2
(49) England, T. E.; Uhlenbeck, O. C. Biochemistry 1978, 17, 2069-
76.
(50) Barrio, J. R.; Barrio, M. C.; Leonard, N. J.; England, T. E.;
Uhlenbeck, O. C. Biochemistry 1978, 17, 2077-81.
(51) Simoncsits, A.; Tomasz, J. Biochim. Biophys. Acta 1975, 395,
74-9.
(52) Brennan, C. A.; Manthey, A. E.; Gumport, R. I. Methods
Enzymol. 1983, 100, 38-52.
FIGURE 2. Cleavage of 5′-32P-labeled (*) oligonucleotides with
0.1 N potassium hydroxide at 23 °C.
7906 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis of 2′-C-Difluoromethylribonucleosides
successively. After stirring for 5 h at ambient temperature,
the reaction mixture was poured into 200 mL of ethyl ether
and washed with saturated aqueous ammonium chloride and
brine. The organic layer was dried over magnesium sulfate,
concentrated under reduced pressure, and purified by silica
gel chromatography, eluting with 12% ethyl acetate in hex-
anes, to give anomeric mixture 4 (2.9 g, 61% overall yield from
A, and G convergently from a common intermediate,
difluoromethylribofuranose 4, prepared by difluoromethyl
phenyl sulfone addition to ketoribose 2 followed by
reduction to generate the difluoromethyl group. To ac-
complish the latter step we described a mild and efficient
reduction procedure involving sodium amalgam under a
hydrogen atmosphere. This finding could broaden the
effectiveness and applicability of sodium amalgam as a
reducing agent and of the sulfone addition/reduction
sequence as a strategy to access synthetic targets con-
taining a difluoromethyl group. Standard Hilbert-
Johnson glycosylation conditions activated 4 sufficiently
to generate the uridine nucleoside derivative, while
synthesis of the C, A, and G analogues required conver-
sion of 4 to the more reactive ribofuranosyl bromide. An
analogous reaction sequence starting from methyl-D-
ribofuranose resulted in the synthesis of 2′-C-R-difluo-
romethyl-arabino-R-pyrimidines, diastereomeric ana-
logues configured with the opposite stereochemistry at
both C2′ and C1′. To incorporate the 2′-C-â-difluoro-
methyluridine into oligonucleotides, we exploited an
enzymatic ligation procedure which shall also allow
incorporation of the corresponding methyl, fluoromethyl,
and trifluoromethyl analogues. A 2′-C-â-difluoromethyl-
nucleoside within an oligonucleotide mediates backbone
scission using its 2′-hydroxyl group in the same manner
as natural nucleotides.
1
3, â/R ) 3/4) as a white foam. H NMR (CDCl3) δ 7.12-8.15
(m, 36H, R, â isomer), 7.06 (s, 0.75H, R isomer), 6.87 (dd, J )
53.7, 56.8 Hz, 0.75H, R isomer), 6.8 (t, J ) 55.7 Hz, 1H, â
isomer), 6.62 (d, J ) 8.0 Hz, 0.75H, R isomer), 6.12 (d, J ) 4.8
Hz, 1H, â isomer), 4.79-4.88 (m, 2.75H, R, â isomer), 4.77 (dd,
J ) 12.1, 3.8 Hz, 0.75H, R isomer), 4.69 (dd, J ) 5.9, 11.7 Hz,
1H, â isomer), 4.56 (dd, J ) 4.4, 12.2 Hz, 0.75H, R isomer);
13C NMR (CDCl3) δ 166.14, 165.99, 164.92, 164.89, 164.69,
164.56, 164.21, 163.97, 133.81, 133.21, 130.12, 130.08, 129.98,
129.92, 129.87, 129.83, 129.76, 129.59, 128.77, 128.67, 128.60,
128.50, 128.36, 128.33, 128.18, 111.79 (t, J ) 251.1 Hz), 110.75
(t, J ) 249.5 Hz), 96.20, 96.16, 94.79, 85.58 (t, J ) 23.1 Hz),
83.04 (t, J ) 23.4 Hz), 81.75, 79.14, 69.68, 69.34, 69.27, 63.19,
63.01; 19F NMR (CDCl3) δ -123.6 (d, J ) 297.4 Hz), -128.8
(d, J ) 301.2 Hz), -129.7 (d, J ) 301.2 Hz), -131.7 (d, J )
297.4 Hz); HRMS calcd for C34H26O9NaF2 [M + Na]+ 639.1443,
found 639.1444.
2′,3′,5′-Tri-O-benzoyl-2′-C-â-difluoromethyluridine (6).
A stirred suspension of uracil (1.1 g, 9.7 mmol) and ammonium
sulfate (40 mg) in 1,1,1,3,3,3-hexamethyldisilazane (40 mL)
was heated at reflux under argon until a clear solution formed
(about 1 h). The clear solution was evaporated under vacuum
to remove the excess 1,1,1,3,3,3-hexamethyldisilazane and
further dried under high vacuum (<0.1 mmHg) for 1 h. The
crude bis(trimethylsilyl)uracil obtained was dissolved in dry
acetonitrile (40 mL) and transferred via cannula to a two-
necked round-bottom flask (equipped with a water condenser)
containing difluoromethylribofuranose 4 (2 g, 3.24 mmol).
Under an argon atmosphere SnCl4 (1.14 mL, 9.72 mmol) was
added in one portion with vigorous stirring and exclusion of
moisture. The resulting homogeneous light yellow solution was
stirred under reflux for 2 days. When TLC indicated that 4
was completely consumed, the reaction was quenched carefully
by the addition of 50 mL of saturated aqueous sodium
bicarbonate and stirred for an additional 15 min. The mixture
was extracted with methylene chloride, and the organic phase
was washed with brine and dried over magnesium sulfate.
After evaporation of the solvent the residue was purified by
silica gel chromatography, eluting with 30% ethyl acetate in
hexanes, to give 7 (1.5 g, 78% yield) as a white foam. 1H NMR
(CDCl3) δ 9.69 (br, 1H), 8.06 (d, J ) 8.5 Hz, 2H), 8.02 (d, J )
8.5 Hz, 2H), 7.80 (d, J ) 8.4 Hz, 2H), 7.58 (d, J ) 8.3 Hz, 1H),
7.18-7.56 (m, 7H), 7.23 (t, J ) 8.0 Hz, 2H), 6.97 (s, 1H), 6.58
(t, J ) 54.8 Hz, 1H), 6.11 (d, J ) 7.6 Hz, 1H), 5.62 (d, J ) 8.3
Hz, 1H), 5.01 (m, 1H), 4.92 (dd, J ) 2.8, 12.6 Hz, 1H), 4.65
(dd, J ) 4.2, 12.6 Hz, 1H); 13C NMR (CDCl3) δ 165.9, 165.0,
164.7, 163.2, 150.2, 139.8, 134.1, 133.8, 133.5, 130.1, 129.8,
129.5, 129.2, 128.6, 128.4, 128.3, 127.8, 113.0 (t, J ) 249.3 Hz),
102.1, 86.8, 84.4 (t, J ) 20.6 Hz), 79.6, 68.2, 62.3; 19F NMR
(CDCl3) δ -128.1 (d, J ) 301.2 Hz), -130.5 (d, J ) 301.2 Hz);
HRMS calcd for C31H25O9N2F2 [M + H]+ 607.1528, found
607.1529.
Experimental Section
1,3,5-Tri-O-benzoyl-2-C-â-(benzenesulfonyl)difluoro-
methyl-r-D-ribofuranose (3). Under an argon atmosphere
lithium hexamethyldisilazane (LiHMDS, 1.0 M in THF, 52 mL,
52 mmol) was added dropwise to a stirred solution of ketori-
bose 2 (10 g, 22 mmol) and difluoromethyl phenyl sulfone (3.8
mL, 5.0 g, 26 mmol) in anhydrous THF at -78 °C. After
stirring at -78 °C for 2 h, the reaction was stirred at 0 °C for
an additional 30 min followed by quenching with 200 mL of
saturated aqueous ammonium chloride. The organic phase was
separated, and the aqueous phase was extracted with ethyl
acetate (2 × 200 mL). The combined organic phases were
washed with brine, dried over magnesium sulfate, concen-
trated, and purified by silica gel chromatography, eluting with
25% ethyl acetate in hexanes, to give 3 (9.0 g, 64% yield) as a
white foam. 1H NMR (CDCl3) δ 8.15 (d, J ) 8.0 Hz, 2H), 8.10
(d, J ) 8.0 Hz, 2H), 7.99 (dd, J ) 8.5, 9.5 Hz, 4H), 7.45-7.78
(m, 10H), 7.35 (t, J ) 7.8 Hz, 2H), 7.10 (s, 1H), 6.05 (d, J )
7.0 Hz, 1H), 4.79 (m, 1H), 4.76 (dd, J ) 13.5, 3.5 Hz, 1H), 4.56
(dd, J ) 5.0, 12.0 Hz, 1H), 3.91 (br, 1H); 13C NMR (CDCl3) δ
166.0, 164.5, 164.0, 135.7, 133.8, 133.0, 132.6, 130.6, 130.4,
130.0, 129.9, 129.6, 129.3, 129.1, 128.8, 128.6, 128.5, 128.4,
128.2, 119.4 (t, J ) 297.4 Hz), 94.5, 81.5 (t, J ) 22.6 Hz), 79.3,
70.3, 62.6; 19F NMR (CDCl3) δ -106.3 (d, J ) 248.5 Hz), -108.0
(d, J ) 248.5 Hz); HRMS calcd for C33H26O10SNaF2 [M + Na]+
675.1112, found 675.1111.
1,2,3,5-Tetra-O-benzoyl-2-C-â-difluoromethyl-D-ribo-
furanose (4). To a stirred mixture of sulfone 3 (5.0 g, 7.7
mmol) and sodium hydrogen phosphate (6.6 g, 46 mmol) in
anhydrous THF at 0 °C under hydrogen atmosphere was added
methanol (1.9 mL, 46 mmol) followed by 5% sodium-mercury
amalgam (21 g, 46 mmol). After stirring at 0 °C for 1 h, the
solution was carefully decanted into chilled saturated aqueous
ammonium chloride (200 mL) and extracted with ethyl acetate
(2 × 200 mL). The combined organic phases were washed with
brine, dried over magnesium sulfate, and concentrated. The
residue was dried under high vacuum and dissolved in dry
dichloromethane (125 mL). To the resulting solution at 0 °C
were added N,N-(dimethylamino)pyridine (0.9 g, 7.4 mmol),
triethylamine (11 mL), and benzoyl chloride (4.0 mL, 35 mmol)
2′-C-â-Difluoromethyluridine (7). Ammonia was bubbled
into a solution of tribenzoyluridine 6 (500 mg, 0.83 mmol) in
methanol (25 mL) at 0 °C for 0.5 h, and the resulting solution
was stirred at 4 °C for 2 days. After TLC indicated the reaction
was complete, the reaction solution was concentrated under
vacuum, and the residue was purified by silica gel chroma-
tography, eluting with 10% methanol in chloroform, to give 7
1
(231 mg, 95% yield) as a white powder. H NMR (CD3OD) δ
8.06 (d, J ) 8.1 Hz, 1H), 6.04 (s, 1H), 5.86 (t, J ) 54.7 Hz,
1H), 5.69 (d, J ) 8.1 Hz, 1H), 4.49 (d, J ) 9.1 Hz, 1H), 3.99
(dd, J ) 1.9, 12.5 Hz, 1H), 3.94 (m, 1H), 3.79 (dd, J ) 2.6,
12.5 Hz, 1H); 13C NMR (CD3OD) δ 166.1, 152.2, 143.3, 116.3
(t, J ) 246.0 Hz), 102.3, 91.2, 83.5, 81.0 (t, J ) 20.5 Hz), 68.3,
J. Org. Chem, Vol. 70, No. 20, 2005 7907

Ye et al.
60.2; 19F NMR (CD3OD) δ -132.1 (d, J ) 298.4 Hz), -133.2
(d, J ) 298.4 Hz); HRMS calcd for C10H13O6N2F2 [M + H]+
295.0742, found 295.0742.
separated, washed with brine, and dried over magnesium
sulfate. After evaporation of the solvent, the residue was
purified by silica gel chromatography, eluting with 40% ethyl
acetate in hexanes, to give 9b (352 mg, 50% yield) as pale
yellow foam. 1H NMR (CDCl3) δ 9.07 (s, 1H), 8.83 (s, 1H), 8.15
(s, 1H), 8.08 (d, J ) 8.3 Hz, 2H), 8.03 (t, J ) 7.1 Hz, 4H), 7.62
(q, J ) 7.4 Hz, 2H), 7.52-7.57 (m, 3H), 7.49 (t, J ) 7.85 Hz,
2H), 7.41 (t, J ) 7.85 Hz, 2H), 6.36 (s, 1H), 6.25 (d, J ) 8.3
Hz, 1H), 5.91 (t, J ) 53.7 Hz, 1H), 4.90 (dd, J ) 2.4, 11.7 Hz,
1H), 4.72-4.79 (m, 2H); 13C NMR (CDCl3) δ 166.2, 165.4, 164.5,
153.0, 151.4, 149.7, 141.9, 133.9, 133.6, 133.4, 132.8, 129.9,
129.7, 129.3, 128.9, 128.6, 128.5, 127.8, 123.0, 113.7 (t, J )
251.5 Hz), 90.8, 82.8 (t, J ) 19.8 Hz), 78.2, 70.9, 62.9; 19F NMR
(CDCl3) δ -127.6 (d, J ) 297.4 Hz), -130.4 (d, J ) 297.4 Hz).
3′,5′-Di-O-benzoyl-2-N-acetyl-6-O-diphenylcarbamoyl-
2′-C-â-difluoromethylguanosine (9c). 2-N-Acetyl-6-O-diphen-
ylcarbamoylguanine (0.64 g, 1.6 mmol) was coevaporated three
times with anhydrous 1,2-dichloroethane (10 mL) under
vacuum. Absolute 1,2-dichloroethane (15 mL) and bis(tri-
methylsilyl)acetamide (0.81 mL, 3.3 mmol) were added at room
temperature. The mixture was stirred at 80 °C until the base
dissolved (∼15 min). Solvent was evaporated, and the residue
was further dried under high vacuum (<0.1 mmHg) for 1 h.
The crude bis(trimethylsilyl) 2-N-acetyl-6-O-diphenylcarbam-
oylguanine obtained was dissolved in anhydrous toluene (15
mL) and transferred via cannula to a double-necked round-
bottom flask (equipped with a water condenser) containing
bromide 8 (0.25 g, 0.53 mmol). To this solution was added HgO
(120 mg) and HgBr2 (120 mg). The resulting reaction mixture
was heated at 80 °C under an argon atmosphere for 2 h. When
TLC indicated that 8 was completely consumed, the reaction
was cooled to room temperature and quenched by addition of
methanol (10 mL) and water (5 mL). The resulting mixture
was stirred for an additional 15 min and extracted with
methylene chloride. The organic phase was separated, washed
with brine, and dried over magnesium sulfate. After evapora-
tion of the solvent the residue was purified by silica gel
chromatography, eluting with 40% ethyl acetate in hexanes,
to give 9c (128 mg, 31% yield) as a pale yellow foam. 1H NMR
(CDCl3) δ 8.23 (br, 1H), 8.10 (s, 1H), 8.07 (d, J ) 8.4 Hz, 2H),
8.01 (d, J ) 8.4 Hz, 2H), 7.61 (t, J ) 7.4 Hz, 1H), 7.54 (t, J )
7.4 Hz, 1H), 7.24-7.45 (m, 14H), 6.27 (s, 1H), 6.12 (s, 1H),
5.86 (t, J ) 54.3 Hz, 1H), 4.94 (dd, J ) 3.7, 12.1 Hz, 1H), 4.77
(m, 1H), 4.69 (dd, J ) 5.4, 12.1 Hz, 1H), 2.29 (s, 3H); 19F NMR
(CDCl3) δ -131.5 (d, J ) 293.6 Hz), -132.5 (d, J ) 293.6 Hz);
HRMS calcd for C40H33O9N6F2 [M + H]+ 779.2277, found
779.2274.
3,5-Di-O-benzoyl-2-C-â-difluoromethyl-r-D-1-ribofura-
nosyl Bromide (8). A stirred mixture of difluoromethylribo-
furanose 4 (2 g, 3.2 mmol) and 30% HBr in acetic acid (15 mL)
was stirred and heated to 80 °C for 5 h in a dry, sealed heavy-
wall pressure tube. After it was cooled the reaction mixture
was diluted with methylene chloride (100 mL) and transferred
to a separation funnel containing ice. The organic layer was
washed subsequently with ice water (45 mL), ice-cooled
saturated aqueous sodium bicarbonate (2 × 45 mL), and ice
water (45 mL). The organic layer was dried over magnesium
sulfate, concentrated, and purified by silica gel chromatogra-
phy, eluting with 10% ethyl acetate in hexanes, to give 8 (1 g,
1
65% yield) as a yellowish foam. H NMR (CDCl3) δ 8.08 (d, J
) 7.2 Hz, 2H), 8.01 (d, J ) 7.2 Hz, 2H), 7.62 (t, J ) 7.4 Hz,
1H), 7.55 (t, J ) 7.4 Hz, 1H), 7.48 (t, J ) 8.0 Hz, 2H), 7.40 (t,
J ) 8.0 Hz, 2H), 6.98 (s, 1H), 6.11 (dd, J ) 54.4, 55.8 Hz, 1H),
5.43 (d, J ) 6.0 Hz, 1H), 4.83 (m, 1H), 4.77 (dd, J ) 3.7, 12.2
Hz, 1H), 4.63 (dd, J ) 5.2, 12.3 Hz, 1H).
3′,5′-Di-O-benzoyl-4-N-benzoyl-2′-C-â-difluoromethyl-
cytidine (9a). A stirred suspension of 4-N-benzoylcytosine (1.0
g, 4.8 mmol) and ammonium sulfate (18 mg) in 1,1,1,3,3,3-
hexamethyldisilazane (25 mL) was heated at reflux under
argon until a clear solution formed in about 1 h. The clear
solution was evaporated under vacuum to remove the excess
1,1,1,3,3,3-hexamethyldisilazane and further dried under high
vacuum (<0.1 mmHg) for 1 h. The crude bis(trimethylsilyl)
4-N-benzoylcytosine obtained was dissolved in dry toluene (25
mL) and transferred via cannula to a two-necked round-bottom
flask (equipped with a water condenser) containing bromide
8 (0.57 g, 1.2 mmol). To this solution was added HgO (0.5 g)
and HgBr2 (0.5 g). The reaction mixture was heated at 90 °C
under an argon atmosphere for 5 h. When TLC indicated that
8 was completely consumed, the reaction was cooled to room
temperature and quenched by addition of methanol (10 mL)
and water (5 mL). The resulting mixture was stirred for an
additional 15 min and extracted with methylene chloride. The
organic phase was separated, washed with brine, and dried
over magnesium sulfate. After evaporation of the solvent, the
residue was purified by silica gel chromatography, eluting with
1% methanol in chloroform, to give 9a (0.47 g, 65% yield) as a
1
pale yellow foam. H NMR (CDCl3) δ 9.01 (br, 1H), 8.07 (m,
5H), 7.90 (d, J ) 7.4 Hz, 2H), 7.68 (d, J ) 7.0 Hz, 1H), 7.41-
7.64 (m, 9H), 6.09 (s, 1H), 6.08 (t, J ) 54.6 Hz, 1H), 5.63 (s,
1H), 4.88 (dd, J ) 2.9, 12.2 Hz, 1H), 4.75 (m, 1H), 4.69 (dd, J
) 5.0, 12.2 Hz, 1H); 13C NMR (CDCl3) δ 166.1, 165.4, 163.0,
156.5, 144.5, 133.7, 133.5, 133.3, 132.7, 129.9, 129.6, 129.2,
129.0, 128.5, 127.6, 115.4 (t, J ) 249.9 Hz), 97.2, 92.1, 78.9,
78.1 (t, J ) 21.6 Hz), 70.9, 62.4; 19F NMR (CDCl3) δ -129.4
(d, J ) 297.4 Hz), -132.3 (d, J ) 297.4 Hz); HRMS calcd for
C31H26O8N3F2 [M + H]+ 606.1688, found 606.1665.
2′-C-â-Difluoromethylcytidine (10a). A solution of 9a
(200 mg, 0.33 mmol) in ammonia-saturated methanol (10 mL)
was stirred at 4 °C for 2 days. When TLC indicated that the
reaction was complete, the solvent was removed under vacuum,
and the residue was purified by silica gel chromatography,
eluting with 10% methanol in ethyl acetate, to give 10a (93
1
mg, 96% yield) as a white powder. H NMR (CD3OD) δ 8.05
3′,5′-Di-O-benzoyl-6-N-benzoyl-2′-C-â-difluoromethyl-
adenosine (9b). A stirred suspension of 6-N-benzoyladenine
(0.8 g, 3.4 mmol) in 1,1,1,3,3,3-hexamethyldisilazane (10 mL)
and dry pyridine (5 mL) was heated at reflux under argon until
a clear solution formed (∼1 h). The clear solution was
evaporated under vacuum to remove the excess 1,1,1,3,3,3-
hexamethyldisilazane and pyridine. The residue was further
dried under high vacuum (<0.1 mmHg) for 1 h. The crude bis-
(trimethylsilyl) 6-N-benzoyladenine obtained was dissolved in
dry toluene (15 mL), which was transferred via cannula to a
double-necked round-bottom flask (equipped with a water
condenser) containing bromide 8 (0.53 g, 1.1 mmol). To this
solution was added HgO (440 mg) and HgBr2 (440 mg). The
resulting reaction mixture was heated at 90 °C under an argon
atmosphere for 5 h. When TLC indicated that 8 was completely
consumed, the reaction was cooled to room temperature and
quenched by addition of methanol (10 mL) and water (5 mL).
The resulting mixture was stirred for an additional 15 min
and extracted with methylene chloride. The organic phase was
(d, J ) 7.5 Hz, 1H), 6.03 (s, 1H), 5.89 (d, J ) 7.5 Hz, 1H), 5.86
(t, J ) 55.1 Hz, 1H), 4.49 (d, J ) 8.9 Hz, 1H), 4.00 (dd, J )
1.9, 12.5 Hz, 1H), 3.94 (m, 1H), 3.82 (dd, J ) 2.8, 12.5 Hz,
1H); 13C NMR (CD3OD) δ 167.5, 158.2, 143.4, 116.0 (t, J )
245.5 Hz), 95.8, 91.9, 83.3, 80.8 (t, J ) 21.0 Hz), 68.0, 60.3;
19F NMR (CD3OD) δ -131.4 (d, J ) 284.2 Hz), -133.9 (d, J )
284.2 Hz); HRMS calcd for C10H14O5N3F2 [M + H]+ 294.0902,
found 294.0903.
2′-C-â-Difluoromethyladenosine (10b). A solution of 9b
(180 mg, 0.28 mmol) in ammonia-saturated methanol (10 mL)
was stirred at 4 °C for 2 days. When TLC indicated that the
reaction was complete, the solvent was removed under vacuum,
and the residue was purified by silica gel chromatography,
eluting with 14% methanol in chloroform, to give 10b (83 mg,
93% yield) as a white powder. 1H NMR (CD3OD) δ 8.44 (s,
1H), 8.19 (s, 1H), 6.21 (s, 1H), 5.66 (t, J ) 54.3 Hz, 1H), 4.82
(d, J ) 8.9 Hz, 1H), 4.06 (m, 2H), 3.88 (dd, J ) 2.7, 12.5 Hz,
1H); 19F NMR (CD3OD) δ -131.2 (d, J ) 293.6 Hz), -136.5
7908 J. Org. Chem., Vol. 70, No. 20, 2005

Synthesis of 2′-C-Difluoromethylribonucleosides
(d, J ) 293.6 Hz); HRMS calcd for C11H14O4N5F2 [M + H]+
of ∼150 mL of the SmI2 solution. After 10 min at room
temperature the reaction mixture was poured into saturated
aqueous ammonium chloride (100 mL). The organic phase was
separated, and the aqueous phase was extracted with ethyl
acetate (2 × 60 mL). The combined organic phases were
washed with brine, dried over magnesium sulfate, concen-
trated, and purified by silica gel chromatography, eluting with
14% ethyl acetate in hexanes, to afford 15 (0.81 g, 74% yield)
318.1014, found 318.1013.
2′-C-â-Difluoromethylguanosine (10c). Guanosine 9c (50
mg, 0.064 mmol) was dissolved in methanol (3 mL) and 33%
aqueous ammonia (3 mL) and heated at 60 °C overnight. When
TLC indicated that the reaction was complete, the reaction
mixture was concentrated, dissolved in water (5 mL), washed
with chloroform (2 × 5 mL), concentrated again, and purified
by reverse-phase HPLC (gradient triethylammonium acetate/
acetonitrile/water) to give 10c (12 mg, 55% yield) as a white
powder. 1H NMR (CD3OD) δ 7.84 (s, 1H), 5.92 (s, 1H), 5.63 (t,
J ) 53.0 Hz, 1H), 4.61 (d, J ) 8.5 Hz, 1H), 3.94 (m, 1H), 3.88
1
as a white foam. H NMR (CDCl3) δ 6.05 (dd, J ) 53.5, 56.5,
1H), 5.08 (s, 1H), 4.40 (dd, J ) 5.0, 9.0 Hz, 1H), 4.11 (dd, J )
4.6, 9.8 Hz, 1H), 3.93 (m, 1H), 3.81 (m, 1H), 3.64 (s, 3H), 3.39
(s, 1H), 1.08 (s, 9H), 1.01 (s, 9H); 13C NMR (CDCl3) δ 114.4 (t,
J ) 246.0 Hz), 100.5, 83.5, 77.3 (t, J ) 21.6 Hz), 71.8, 68.2,
57.7, 27.3, 26.9; 19F NMR (CDCl3) δ -130.4 (d, J ) 296.4 Hz),
-137.4 (d, J ) 296.4 Hz); HRMS calcd for C15H28F2O5Si [M +
H]+ 355.1752, found 355.1751.
(dd, J ) 3.0, 12.5 Hz, 1H), 3.77 (dd, J ) 1.5, 12.5 Hz, 1H); 19
F
NMR (CD3OD) δ -130.7 (d, J ) 297.4 Hz), -135.0 (d, J )
297.4 Hz); UV (H2O) λmax 209.7, 252.0 nm; HRMS calcd for
C11H14O5N5F2 [M + H]+ 334.0963, found 334.0964.
Methyl 2-C-r-Difluoromethyl-2-O-benzoyl-3,5-O-di-tert-
butylsilanediyl-â-D-arabinofuranoside (16). To a stirred
solution of 4-(dimethylamino)pyridine (0.37 g, 3.0 mmol) in dry
methylene chloride (20 mL) was added dry triethylamine (4.7
mL) followed by benzoyl chloride (0.69 mL, 5.9 mmol). After 5
min a solution of compound 15 (0.90 g, 2.5 mmol) in dry
methylene chloride (50 mL) was added. The reaction was kept
at room temperature overnight. When TLC showed no starting
material remaining, the reaction mixture was diluted with
methylene chloride (50 mL), washed with water (3 × 80 mL),
dried over magnesium sulfate, concentrated, and purified by
silica gel chromatography, eluting with 14% ethyl acetate in
Methyl 3,5-O-Di-tert-butylsilanediyl-â-D-ribofurano-
side (12). Methyl â-D-ribofuranose 11 (5.0 g, 30.5 mmol) was
rendered anhydrous by repeated coevaporation with dry
pyridine and finally dissolved in dry DMF at 0 °C. To this cold
solution under argon was added slowly di-tert-butylsilyl ditri-
flate (13.7 g, 16.6 mL, 31.1 mmol) with vigorous stirring. After
being stirred at room temperature for 15 min, the reaction
mixture was treated with dried triethylamine (13 mL, 93
mmol). The reaction mixture was stirred for an additional 5
min and concentrated to dryness under reduced pressure. The
residue was purified by silica gel chromatography, eluting with
14% ethyl acetate in hexanes, to afford 12 as a white solid
(5.5 g, 59% yield). 1H NMR (CDCl3) δ 4.92 (s, 1H), 4.43 (dd, J
) 4.5, 8.5 Hz, 1H), 3.95-4.10 (m, 5H), 3.42 (s, 3H), 1.09 (s,
9H), 1.04 (s, 9H); LRMS (positive-ion FAB) calcd for C14H28O5-
Si [M + H]+ 305, found 305.
1
hexanes, to afford 16 (1.11 g, 95% yield) as a white foam. H
NMR (CDCl3) δ 7.48-8.11 (m, 5H), 6.73 (t, J ) 52.5 Hz, 1H),
5.38 (s, 1H), 4.66 (d, J ) 9.5 Hz, 1H), 4.45 (dd, J ) 4.5, 9.0
Hz, 1H), 3.94-4.06 (m, 2H), 3.51 (s, 3H), 1.12 (s, 9H), 1.06 (s,
9H).
Methyl 3,5-O-Di-tert-butylsilanediyl-2-oxo-â-D-erythro-
ribofuranoside (13). Compound 12 (0.98 g, 3.2 mmol) in dry
methylene chloride (5 mL) was added to a solution of Dess-
Martin periodinane (2.29 g, 4.8 mmol) in dry methylene
chloride (15 mL) at 0 °C. The mixture was allowed to warm to
room temperature and stirred overnight. The solvent was
removed under reduced pressure, and the residue was tritu-
rated with diethyl ether (30 mL). After filtration through a
pad of magnesium sulfate the organic solution was stirred with
an equal volume of Na2S2O3‚5H2O (3.7 g) in saturated aqueous
sodium bicarbonate until the organic layer became clear. The
organic layer was separated, washed with brine, dried, and
evaporated. The residue was purified by silica gel chromatog-
raphy, eluting with 11% ethyl acetate in hexanes, to afford
Methyl 2-C-r-Difluoromethyl-2-O-benzoyl-â-D-arabino-
furanoside (17). HF-pyridine (1.6 mL, 61 mmol) was care-
fully mixed with pyridine (8 mL) at 0 °C. This cold mixture
was added dropwise to a stirred solution of 16 (1.1 g, 2.4 mmol)
in THF (10 mL). After being stirred at room temperature for
10 min, the reaction mixture was diluted with pyridine (30
mL) and then methylene chloride (100 mL). The resulting
organic solution was washed with water (120 mL) and aqueous
sodium bicarbonate (100 mL), dried over sodium sulfate,
concentrated, and purified by silica gel chromatography,
eluting with 50% ethyl acetate in hexanes, to afford 17 (0.74
g, 95%) as a white solid. 1H NMR (CDCl3) δ 7.49-8.08 (m,
5H), 6.48 (dd, J ) 53.5, 55.5 Hz, 1H), 5.26 (s, 1H), 4.85 (dd, J
) 3.0, 7.0 Hz, 1 H), 4.26 (dd, J ) 3.3, 7.0 Hz, 1H), 3.99 (b,
1H), 3.90 (m, 1H), 3.75 (m, 1H), 3.60 (s, 3H), 2.27 (b, 1H).
Methyl 2-C-r-Difluoromethyl-2,3,5-tri-O-benzoyl-â-D-
arabinofuranoside (18). To a stirred solution of 4-(dimethyl-
amino)pyridine (0.74 g, 6.0 mmol) in dry methylene chloride
(50 mL) was added dry triethylamine (9.4 mL), followed by
benzoyl chloride (1.4 mL, 12 mmol). After 5 min a solution of
compound 17 (0.72 g, 2.3 mmol) in dry methylene chloride (20
mL) was added. The reaction was kept at room temperature
overnight, and TLC showed that no starting material re-
mained. The reaction mixture was diluted with methylene
chloride (50 mL), washed with water (3 × 80 mL), dried over
sodium sulfate, concentrated, and purified by silica gel chro-
matography, eluting with 20% ethyl acetate in hexanes, to
afford 18 (1.2 g, 97% yield) as a white foam. 1H NMR (CDCl3)
δ 8.11 (d, J ) 8.5 Hz, 2H), 8.08 (d, J ) 8.5 Hz, 2H), 8.03 (d, J
) 8.1 Hz, 2H), 7.57 (t, J ) 7.5 Hz, 2H), 7.42-7.49 (m, 5H),
7.32 (t, J ) 7.8 Hz, 2H), 6.74 (t, J ) 55.0 Hz, 1H), 6.33 (d, J
) 6.0 Hz, 1H), 5.55 (s, 1H), 4.61-4.71 (m, 3H), 3.39 (s, 3H);
13C NMR (CDCl3) δ 166.0, 165.3, 165.0, 133.7, 133.6, 133.0,
130.1, 129.9, 129.6, 128.5, 128.2, 112.7 (t, J ) 252.5 Hz), 102.5,
84.5 (t, J ) 20.1 Hz), 77.9, 65.5, 55.8; 19F NMR (CDCl3) δ
-127.5 (d, J ) 301.1 Hz), -130.2 (d, J ) 301.1 Hz); HRMS
calcd for C28H25F2O8 [M + H]+ 527.1518, found 527.1517.
1
13 as a clear oil (0.89 g, 91% yield). H NMR (CDCl3) δ 4.74
(d, J ) 10.0 Hz, 1H), 4.52 (m, 1H), 3.74-4.38 (m, 3H), 3.57 (d,
J ) 5.0 Hz, 3H), 1.07 (m, 18H); LRMS (positive-ion FAB) calcd
for C14H26O5Si [M + H]+ 303, found 303.
Methyl 2-C-r-(Benzenesulfonyl-1′,1′-difluoro)methyl-
3,5-O-di-tert-butylsilane-diyl-â-D-arabinofuranoside (14).
To a stirred solution of 13 (1.5 g, 4.8 mmol) and difluoromethyl
phenyl sulfone (1.1 g, 0.73 mL, 6.8 mmol) in dry THF/HMPA
(10/1, 70 mL) at -78 °C under argon was added dropwise
LiHMDS (1.0 M in THF, 8 mL). After stirring at -78 °C for 2
h, the reaction was quenched with saturated aqueous am-
monium chloride (30 mL). The organic phase was separated,
and the aqueous phase was extracted with ethyl acetate (2 ×
30 mL). The combined organic phases were washed with brine
(30 mL), dried over sodium sulfate, concentrated, and purified
by silica gel chromatography, eluting with 14% ethyl acetate
in hexanes, to afford 14 (1.5 g, 63% yield) as a white foam. 1H
NMR (CDCl3) δ 8.02 (d, J ) 7.5 Hz, 2H), 7.79 (t, J ) 7.5 Hz,
1H), 7.65 (t, J ) 8.0 Hz, 2H), 5.57 (s, 1H), 4.41 (dd, J ) 5.0,
9.0 Hz, 1H), 4.21 (d, J ) 10 Hz, 1H), 3.90-3.95 (m, 2H), 3.79-
3.83 (m, 1H), 3.77 (s, 3H), 1.04 (s, 9H), 0.95 (s, 9H).
Methyl 2-C-r-Difluoromethyl-3,5-O-di-tert-butylsilane-
diyl-â-D-arabinofuranose (15). To a mixture of 14 (1.5 g,
3.0 mmol) and dry HMPA (10 mL) at 0 °C under argon was
added slowly commercial SmI2 in THF (0.1 M) with vigorous
stirring. The reaction mixture became dark blue after addition
4-N-Benzoyl-1-(2-C-r-difluoromethyl-3,5-di-O-benzoyl-
r-D-arabinofuranosyl)cytosine (19a). To a pressure tube
J. Org. Chem, Vol. 70, No. 20, 2005 7909

Ye et al.
containing 18 (0.45 g, 0.86 mmol) was added a solution of HBr
in acetic acid (30%, 10 mL). The tube was sealed and heated
to 75 °C for 2 h with stirring. After cooling to room tempera-
ture, the reaction mixture was evaporated under reduced
pressure. The residue was dissolved in ethyl acetate (15 mL),
washed with brine, dried over sodium sulfate, concentrated
under vacuum, and subsequently dissolved in dry benzene (10
mL). With exclusion of moisture, this solution was added to a
solution of 4-N-benzoyl bis(trimethylsilyl)cytosine (5 mL,
prepared in situ from 4-N-benzoyl cytosine (0.59 g, 0.26 mmol),
1,1,1,3,3,3-hexamethyldisilazane (10 mL), and 22 mg am-
monium sulfate) in dry benzene. To this clear solution were
added HgO (0.28 g) and HgBr2 (0.28 g), and the reaction
mixture was stirred at room temperature under argon for 36
h. The reaction was diluted with ethyl acetate (15 mL), washed
with aqueous potassium iodide (3 × 10 mL) and brine (10 mL),
dried over sodium sulfate, concentrated, and purified by silica
gel chromatography, eluting with 50% ethyl acetate in hex-
anes, to afford 19a (0.25 g, 48%) as a white foam. 1H NMR
(CDCl3) δ 9.06 (s, 1H), 8.08 (d, J ) 7.5 Hz, 2H), 8.07 (d, J )
7.5 Hz, 1H), 7.92 (d, J ) 7.5 Hz, 2H), 7.76 (d, J ) 7.6 Hz, 2H),
7.71 (d, J ) 7.3 Hz, 1H), 7.63 (t, J ) 7.6 Hz, 1H), 7.34-7.56
(m, 8H), 7.09 (s, 1H), 6.43 (s, 1H), 6.19 (t, J ) 53.9 Hz, 1H),
4.93 (m, 2H), 4.79 (dd, J ) 4.7, 11.0 Hz, 1H). 13C NMR (CDCl3)
δ 166.2, 165.2, 163.1, 156.9, 144.5, 133.9, 133.3, 133.1, 132.9,
129.8, 129.7, 129.5, 128.8, 128.6, 128.4, 128.3, 127.9, 114.9 (t,
J ) 249.8 Hz), 97.1, 92.5, 83.8, 81.3 (t, J ) 21.8 Hz), 78.2,
64.6, 60.4; 19F NMR (CDCl3) δ -131.06; HRMS calcd for
C31H26F2O8N3 [M + H]+ 606.1688, found 606.1688.
1-(2-C-r-Difluoromethyl-3,5-di-O-benzoyl-r-D-arabino-
furanosyl)uracil (19b). Compound 19b was synthesized by
the similar procedure used for 19a with a yield of 55% as a
white foam. 1H NMR (CDCl3) δ 10.20 (s, 1H), 8.12 (d, J ) 7.5
Hz, 2H), 7.98 (d, J ) 7.5 Hz, 2H), 7.66-7.46 (m, 8H), 6.47 (s,
1H), 6.13 (t, J ) 55.0 Hz, 1H), 5.86 (d, J ) 5.0 Hz, 1H), 5.78
(t, J ) 10.5 Hz, 2H), 4.92 (d, J ) 4.0 Hz, 1H), 4.79 (m, 2H).
1-(2-C-r-Difluoromethyl-r-D-arabinofuranosyl)cyto-
sine (20a). In a pressure tube 19a (0.25 g, 0.41 mmol) was
dissolved in methanol (20 mL). This solution was cooled to 0
°C and saturated with ammonia. The pressure tube was sealed
and kept at 4 °C with stirring for 2 days. When TLC showed
that the reaction was complete, the mixture was evaporated
under reduced pressure, and the residue was purified by silica
gel chromatography, eluting with 10% methanol in chloroform,
to afford 20a (0.10 g, 84%) as a white solid. 1H NMR (CD3OD)
δ 7.90 (d, J ) 7.6 Hz, 1H), 6.20 (s, 1H), 5.91 (d, J ) 7.7 Hz,
1H), 5.90 (t, J ) 54.0 Hz, 1H), 4.37 (m, 1H), 4.26 (d, J ) 3.4
Hz, 1H), 3.75 (d, J ) 5.2 Hz, 2H); 13C NMR (CD3OD) δ 167.6,
158.7, 143.5, 116.2 (t, J ) 243.9 Hz), 95.8, 93.5, 90.4, 83.3 (t,
J ) 20.6 Hz), 77.9, 63.1; 19F NMR (CD3OD) δ -136.9, -137.01;
HRMS calcd for C10H14F2N3O5 [M + H]+ 294.0902, found
294.0901.
colorless solution was concentrated under vacuum, maintain-
ing the temperature below 30 °C. The residue was coevapo-
rated three times with 10 mL of 50% methanol in water,
washed with chloroform (3 × 5 mL), dried under vacuum, and
redissolved in 50 mL of water. The solution was adjusted to
pH 7 with triethylamine, filtered, and chromatographed on a
DEAE cellulose column (2.5 × 25 cm) using a linear gradient
of 0.15-0.5 M TEAB, pH 8.0 (700 mL). The fractions (peaked
around 0.35 M TEAB) containing uridine bisphosphate were
pooled, evaporated to dryness, and coevaporated several times
with 10 mL of 50% methanol in water to remove TEAB buffer,
1
giving 21 (38 mg) in 50% yield. H NMR (D2O) δ 7.85 (d, J )
8.0 Hz, 1H), 6.04 (s, 1H), 5.93 (t, J ) 53.5 Hz, 1H), 5.82 (d, J
) 8.0 Hz, 1H), 4.80 (t, J ) 9.3 Hz, 1H), 4.15 (m, 2H), 3.77 (t,
J ) 5.3 Hz, 1H); 31P NMR (D2O; peak position changes with
pH) δ 4.35 (triplet in nondecoupled 31P NMR), 3.47 (doublet
in nondecoupled 31P NMR); 19F NMR (D2O) δ -129.07 (d, J )
301.2 Hz), -134.90 (d, J ) 301.2 Hz); HRMS calcd for
C10H14N2O12P2F2 [M + H]+ 455.0068, found 455.0068.
Incorporation of Uridine Bisphosphate (21) into an
Oligonucleotide. In a total volume of 10 µL, 3 mM uridine
bisphosphate 22, 0.3 mM 5′ DNA fragment d(CTGTCAC-
CGAAA), 0.4 mM ATP, 8 mM spermine, 1 mM creatine
phosphate, 10 mM magnesium chloride, 10 mg/mL bovine
serum albumin, 1 mM hexaaminecobalt(III) chloride, 20 mM
DTT, 170 units/mL myokinase, 175 units/mL phosphocreatine
kinase, and 2000 units/mL T4 RNA ligase were incubated in
50 mM Tris-HCl, pH 8.0, at 25 °C for 3 days. After the ligase
was deactivated by incubating at 100 °C for 5 min, 1 µL of
10× alkaline phosphatase buffer (500 mM Tris-HCl, pH 9.0,
10 mM MgCl2) and 1 µL of 30 units/µL calf intestine phos-
phatase (CIP) were added to the solution and incubated at 37
°C for 30 min to remove the 3′-phosphate. The phosphatase
was deactivated in the same manner as above, and the reaction
mixture was purified by 20% denaturing polyacrylamide gel
electrophoresis (DPAGE). The correct band was visualized by
UV shadowing, excised with a clean razor blade, and eluted
in TE buffer (10 mM Tris, pH 8.0; 0.1 mM EDTA) at 4 °C
overnight. The eluant was extracted by phenol/chloroform/
isoamyl alcohol (25/24/1), precipitated three times by n-
butanol, dried under vacuum (speed vac), and redissolved in
water. About 90% of the starting DNA fragment became
ligated based on 32P labeling of the mixture before gel purifica-
tion. To prepare the full-length chimeric oligonucleotide, the
13mer [d(CTGTCACCGAAA)rU2′CF H, final concentration 20
2
µM], phosphorylated 3′ fragment pd(ACACGCAAGATG) and
splint DNA template (dCTTGCGTGTATTTCGGTG) were pre-
annealed in T4 DNA ligase buffer (1X containing 50 mM Tris-
HCl, 10 mM magnesium chloride, 10 mM DTT, 1 mM ATP,
25 g/mL bovine serum albumin) by incubating at 90 °C for 1
min followed by slow cooling to 4 °C. Hexaaminecobalt(III)
chloride (1 mM) and 8 pmol/µL T4 DNA ligase were added,
and the mixture was incubated at 16 °C overnight. The full-
length chimeric product was purified and precipitated by the
same procedure as described above for the 5′ fragment.
1-(2-C-r-Difluoromethyl-r-D-arabinofuranosyl)uracil
(20b). Using the similar procedure as described for 20a,
compound 20b was obtained in 90% yield as a white solid. 1H
NMR (CD3OD) δ 8.00 (d, J ) 8.2 Hz, 1H), 6.21 (s, 1H), 5.95 (t,
J ) 53.2 Hz, 1H), 5.71 (d, J ) 8.2 Hz, 1H), 4.43 (t, J ) 5.3 Hz,
1H), 4.19 (s, 1H), 3.77 (dd, J ) 6.5, 11.6 Hz, 1H), 3.70 (dd, J
) 5.3, 11.6 Hz, 1H); 19F NMR (CD3OD) δ -135.9 (d, J ) 296.4
Hz), -138.8 (d, J ) 296.4 Hz); HRMS calcd for C10H13F2N2O6
[M + H]+ 295.0742, found 295.0741.
2′-â-Difluoromethyluridine 3′,5′-Bisphosphate (21). Uri-
dine 7 (50 mg, 0.17 mmol) was stirred in anhydrous diphos-
phoryl chloride (0.24 mL, 1.7 mmol) under argon in an ice bath.
The reaction was allowed to warm to room temperature over
∼4 h. After TLC (n-PrOH:30% aqueous NH3:H2O, 6:3:1, v:v:v)
showed that the reaction was complete, ice-chilled water (5
mL) was added, and the mixture was kept at 0 °C for 10 min
before addition of an ice-cooled 0.5 M triethylammonium
bicarbonate (TEAB) solution (20 mL, pH 8.0). The resulting
Acknowledgment. J.D.Y. is a Research Associate
and J.A.P. is an Investigator at Howard Hughes Medical
Institute.
Supporting Information Available: 1H NMR spectra of
3, 4, 6-8, 9a-c, 10a-c, 15-18, 19a,b, 20a,b, and 21; 13C
NMR spectra of 3, 4, 6, 7, 9a,b, 10a, 15, 18, 19a, and 20a;
19F-1H NOE spectra of 3 and 6; NOESY spectra of 6, 8, 9a,b,
and 19a; 31P spectra of 21; MALDI mass spectroscopy of
rCCGAAAU2′CF H; P1 nuclease digestion of the oligonucleotides.
2
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO0507542
7910 J. Org. Chem., Vol. 70, No. 20, 2005