Rigid 2′,4′-difluororibonucleosides: Synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase

Article abstract of DOI:10.1021/jo500794v

We report on the synthesis and conformational properties of 2′-deoxy-2′,4′-difluorouridine (2′,4′-diF-rU) and cytidine (2′,4′-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially ?loc? the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2′,4′-diF-rU 5′-triphosphate (2′,4′-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2′,4′-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC₅₀ > 50 μM). 2′,4′- diF-rU/C represents rare examples of ?locked? ribonucleoside mimics that lack a bicyclic ring structure.

Full text of DOI:10.1021/jo500794v

Article
pubs.acs.org/joc
Rigid 2′,4′-Difluororibonucleosides: Synthesis, Conformational
Analysis, and Incorporation into Nascent RNA by HCV Polymerase
Saul Martínez-Montero,† Glen F. Deleavey,† Anupriya Kulkarni,‡ Nerea Martín-Pintado,§
́
Petra Lindovska,† Michael Thomson,† Carlos Gonzalez,§ Matthias Gotte,‡ and Masad J. Damha*,†
́
̈
†Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC Canada H3A 0B8
‡Department of Microbiology and Immunology, McGill University, 3775 University, Montreal, QC Canada H3A 2B4
§Instituto de Química Física Rocasolano, CSIC, C/. Serrano 119, 28006 Madrid, Spain
S
* Supporting Information
ABSTRACT: We report on the synthesis and conformational properties of 2′-deoxy-2′,4′-difluorouridine (2′,4′-diF-rU) and
cytidine (2′,4′-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic
effects induced by the two fluorines essentially “lock” the conformation of the sugar in the North region of the pseudorotational
cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2′,4′-diF-rU 5′-triphosphate
(2′,4′-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2′,4′-diF-rUTP inhibited RNA synthesis in
dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 μM). 2′,4′-diF-
rU/C represents rare examples of “locked” ribonucleoside mimics that lack a bicyclic ring structure.
structural and biological dependence on nucleoside conforma-
tion. In the examples provided in Figure 2A, conformational
rigidity is conferred by the bicyclic nature of the nucleoside
sugar moiety. Marquez and co-workers have studied the
antiherpes activity of N-methanocarbathymidine and S-
methanocarbathymidine.6 The North isomer exhibited high
antiherpes activity; meanwhile, the South isomer was found to
be inactive, proving the important role of the sugar
conformation on the biological activity of nucleoside
analogues.6,7 On the other hand, LNA (3, locked nucleic
acid), features a methylene bridge joining the 2′-OH to C4′,
restricting the sugar in the North region of the pseudorotational
cycle. The rigid conformation shown by LNA and related
derivatives is widely applied to increase thermal stability of
duplexes and to improve gene-silencing properties of
oligonucleotide-based therapeutics.8−12
INTRODUCTION
■
Considerable research efforts have been concentrated to
prepare chemically modified nucleoside derivatives as effective
anticancer1 and antiviral agents.2,3 In this aspect, the conforma-
tional preferences of nucleosides can have a profound impact
on their biological activity. The furanose ring of the nucleosides
in solution can adopt two major antipodal conformations
classified as North (N, C-3′endo, phase ≈ 0°) and South (S, C-
2′endo, phase ≈ 180°) described in the pseudorotation cycle
(Figure 1).4 In the case of nucleoside antiviral compounds, the
sugar conformation plays a crucial role in determining
biological activity, which depends on kinase phosphorylation
and polymerase-catalyzed incorporation steps.5 Nucleosides
with a sugar pucker in the South range of the pseudorotation
cycle are preferentially phosphorylated by kinases, while North-
type nucleosides are preferentially incorporated by poly-
merases.6,7
A sugar modification that improves the biological properties
of some nucleoside analogues is the introduction of fluorine, a
common tool used in drug discovery efforts.13,14 Our research
The level of understanding and control over nucleoside
conformation has a significant impact on the ability to
successfully develop therapeutic nucleosides and oligonucleo-
tides. A variety of conformationally rigid “locked” nucleoside
analogues have been used to expand the understanding of the
Received: April 8, 2014
© XXXX American Chemical Society
A
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interactions from parallel aligned C−F bonds in these
analogues,20 the 4′-fluorine should introduce a strong anomeric
effect favoring the North pucker and an additional gauche effect
that reinforces such conformation (Figure 2B).
Since the discovery of Nucleocidin, a potent and toxic
antibiotic 4′-fluorinated nucleoside derivative found in
nature,21,22 some other 4′-fluorinated nucleoside analogues
have been prepared.23−27 However, in nearly all cases, isolating
the free 4′-fluorinated nucleoside was either not achieved or the
isolated compound was very unstable to cleavage of the
glycosidic linkage in water. This feature was deemed problem-
atic for nucleoside-based antiviral applications. Since 2′-
fluorination is known to enhance the stability of the glycosidic
linkage, we hypothesized that 4′-fluorinated derivatives of 2′-F-
rU nucleosides would be less prone to degradation in aqueous
solution.28 Additionally, the anticipated North conformation of
these analogues featuring minimal modification to the natural
ribose sugar of RNA makes them excellent candidates for
incorporation into siRNAs and biological study. Herein we
describe the synthesis, characterization, and conformational
analysis of two novel 2′-deoxy-2′,4′-difluororibonucleosides,
namely, 2′,4′-diF-rU and 2′,4′-diF-rC. We also report on
preliminary incorporation studies of 2′,4′-diF-rU nucleotide by
NS5B RNA polymerase.
Figure 1. Pseudorotational cycle describing the sugar conformations of
nucleosides; E = envelope, T = twist. Superscripts and subscripts
indicate the specific atoms in the ribose ring that project away from the
plane defined by the remaining ring atoms. Natural nucleosides have
characteristic minima in the North (0−36°) and South regions (144−
180°).
RESULTS AND DISCUSSION
■
Our investigation of 2′,4′-difluorinated nucleoside analogues
began with the synthesis of 2′-deoxy-2′,4′-difluorouridine
(2′,4′-diF-rU) shown in Scheme 1. Our chosen synthetic
route, inspired by reported syntheses of 4′-substituted nucleo-
sides,22,24−27 began with commercially available 2′-deoxy-2′-
fluorouridine [2′-F-rU (4)]. Reaction of 4 with iodine and
triphenylphosphine yielded the 5′-iodo derivative, 5. This was
followed by an elimination reaction in the presence of sodium
methoxide to produce the 4′,5′-unsaturated nucleoside 6. Both
steps proceeded in high yields.
With compound 6 in hand, the next step required installation
of the desired 4′-fluorine. The syntheses of all other reported
examples of 4′-fluorinated nucleosides effect fluorination
through addition of iodine fluoride across the 4′,5′-
olefin.22,24−27 The stereochemical outcome of this addition is
highly dependent on the nucleobase identity and functionalities
at the 2′ and 3′ positions. The uracil nucleobase likely
participates in this reaction, with the C2 carbonyl of uracil
opening the iodonium intermediate 13 to form the 4′-anhydro
nucleoside 14 and subsequently yielding the right stereo-
chemistry at C4′ upon attack by fluoride (Scheme 2A).26 We
have observed that addition of iodine monofluoride (I-F) to
alkene 6 can produce unidentified reaction products. The best
conditions we have found provide the 5′-iodo-4′-fluoro
nucleoside 7 in 64% yield, obtained through the gradual
addition of iodine/AgF in acetonitrile at 0 °C. The stereo-
Figure 2. (A) Some examples of conformationally bicyclic “locked”
nucleosides. (B) Anomeric effect (left) due to the overlap of a lone-
pair orbital of O4′ (p-type) with the σ*C4′−F4′ antibonding orbital and
gauche effect (right) due to the interaction between the σC3′−H3′
bonding orbital and the σC*4′−F4′ antibonding orbital. The combined
effects stabilize the North sugar pucker in 2,′4′-diF-rU.
group has focused on the development of fluorinated
nucleoside derivatives for biological studies and oligonucleotide
modification encouraged by the conformational changes that
fluorine imparts in the ribose and arabinose moiety.15−18 In the
present study, we explore the utility of two fluorines as a tool
for creating rigidified nucleoside structures similar to those in
Figure 2A. The fluorine of 2′-deoxy-2′-fluorouridine (2′-F-rU)
imparts a preference for the North pucker through gauche
effects but does not provide the sufficient rigidity to compare
with true locked analogues.19 We reasoned that replacing the
H4′ of 2′-F-rU with a fluorine, creating a 2′,4′-difluorinated
analogue, would impart additional bias toward the North
conformation without the need of a bicyclic scaffold. Although
an energy penalty may be expected from dipole−dipole
1
chemistry at C4′ of nucleoside 7 was assessed by 2D H−1H
NOESY NMR experiments and later by extensive analysis of
the 1H NMR coupling constants of 4 and 10. For example, the
NOESY spectrum of 7 (and the parent compound 4) shows a
strong correlation between H5′ and H3′ as in naturally
occurring ribonucleosides.
With the installation of the 4′-fluorine, substitution of the 5′-
iodine was required to furnish the desired nucleoside analogue.
However, displacement of the 5′-iodine from 4′-fluorinated
compounds has proven surprisingly difficult in many
instances.22,24−27 A variety of oxygenated nucleophiles were
B
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Scheme 1. Stereoselective Synthesis of 2′,4′-diF-rU (10) and 2′,4′-diF-rC (12)
Scheme 2. Postulated Mechanisms To Explain the Formation of Compounds 7 and 9
unsuccessful, and the presence of the 4′-fluorine has been
estimated to decrease the ease of displacement of the 5′-iodine
by about 1000-fold.26 Activation of the iodine as hypoiodate
was found to be effective,24 especially when a 3′-benzoyl group
is present, which may assist in the displacement through
migration from the 3′ to the 5′ position (Scheme 2B). Thus,
benzoylation of 7 using benzoyl chloride afforded 8 in 83%
yield as a precursor for the substitution reaction. Indeed,
displacement of the 5′-iodine was achieved with mCPBA in
water-saturated dichloromethane with concomitant migration
of the benzoyl group to the 5′ position to afford protected
nucleoside 9 (Scheme 2B). A simple deprotection step to
remove the 5′-O-benzoyl group produced the desired final
product 2′,4′-diF-rU (10) in quantitative yield (Scheme 1).
To expand our collection of difluorinated nucleoside
analogues, we next sought to prepare 2′,4′-diF-rC (12). We
took advantage of 5′-protected intermediate 9, which was used
for the conversion of uracil to cytosine following benzoyl
protection of the 3′-hydroxyl group, to form compound 11.
The triazole intermediate method29 followed by full depro-
tection with ammonia was used to obtain 2′,4′-diF-rC (12)
(Scheme 1). The NOESY NMR spectra of 12 show clear
C
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1
1
Table 1. H−1H and H−19F Coupling Constant Values for Nucleosides 4 and 10 at 25 °C in D2O and MeCN-d3 (500 MHz)
2′,4′-diF-rU (10)
J (Hz) ( 0.2) (D2O)
J (Hz) ( 0.2) (MeCN-d3)
J (Hz) ( 0.2) (MeCN-d3)
J (Hz) ( 0.2) (D2O)
2′-F-rU (4)
H1′−H2′
H1′−F2′
H2′−H3′
H3′−F2′
H3′−F4′
F4′−H5′
F4′−H5″
H5′−H5″
0
0
1.9
17.2
4.4
1.4
19.6
4.8
H1′−H2′
H1′−F2′
H2′−H3′
H3′−F2′
H3′−H4′
H4′−H5′
H4′−H5′
H5′−H5″
21.5
5.8
20.0
5.5
22.1
20.7
6.5
25.6
21.0
4.3
20.7
7.5
21.2
8.4
2.1
2.4
10.0
12.8
4.3
2.8
4.4
12.4
12.8
13.2
Figure 3. (A) JH1′H2′ vs sugar pseudorotation angle. Sugar pucker curves of both nucleosides overlap. (B) Energy profiles of 2′-F-rU and 2′,4′-diF-rU.
correlations between H6 of the base and the H5′ protons as
well as between H5′ protons and H3′, again supporting the
right configuration at C4′ (see Supporting Information). No
decomposition of these compounds was observed in aqueous
solution after several days.
pure North conformation) (Figure 1). However, the coupling
constant of 1.4 Hz obtained for 2′-F-rU (4) in aqueous solution
corresponds to either a single conformation with a higher
pseudorotation phase angle of approximately 50° or a pure
North sugar pucker (P ∼ 0°) in a conformational equilibrium
with a South pucker with a relative population of around 10%.
The experimental J coupling data indicate that this minor
conformer does not exist in the case of 2′,4′-diF-rU.
The percentage of North and South conformers in solution
was calculated by applying the empirical equation S(%) = 10 ×
1
J
H1′H2′, where JH1′H2′ is the H−1H coupling constant between
the ribose ring H1′ and H2′ protons.30,31 For 2′,4′-diF-rU (10),
H1′H2′ is 0 Hz (Table 1), indicating a pure North conformation
To further explore the impact of 4′-fluorination on the sugar
puckering preference, quantum mechanical calculations were
carried out using Gaussian 03 at the M062x/6-31+G(d,p)
level.39 The pseudorotation energetic profiles of 2′-F-rU and
2′,4′-diF-rU were calculated by means of a constrained energy
optimization (see Supporting Information). These quantum
mechanical calculations indicate a clear preference for the
North pucker and, most importantly, that the energy of the
minor South conformation (pseudorotation angle 175°) is
significantly higher for 2′,4′-diF-rU than it is for 2′-F-rU. This
result is in complete agreement with the experimental NMR
data and further supports the notion that these monocyclic
nucleoside analogues are true mimics of previously reported
bicyclic nucleosides (Figure 3B).
The rigid RNA-like conformation of 2′,4′-diF-rU (10) led us
to prepare the corresponding triphosphate and evaluate it as a
substrate for an RNA polymerase. Among the polymerases
available, we chose HCV NS5B RNA polymerase given the
potent anti-HCV activity found in 4′-substituted nucleo-
sides.40−47 HCV NS5B is capable of initiating RNA synthesis
either de novo or in the presence of short RNA primers. In this
assay, we measured enzymatic activity from the extension of a
radio-labeled dinucleotide (rGG) primer complementary to the
3′ end of a model 20 nt oligo-RNA template. Single nucleotide
incorporation events were assessed during the elongation stage
J
in aqueous solution. Furthermore, JF4′H3′ is 20.7 Hz (Table 1),
consistent with the pseudo-trans-diaxial orientation of H3′ and
F4′ of a North puckered nucleoside (see Figure 2B). In the
South conformer, these two atoms are pseudoequatorial, and
hence a much smaller coupling constant value (JH,F = 4−10 Hz)
would be expected.22,32−34 In the case of 2′-F-rU, JH1′H2′ is 1.4
Hz, consistent with the existence of both North (86%) and
South (14%) conformers in aqueous solution. In MeCN, the
fraction of the South conformer of 2′-F-rU increases to 19%
(JH1′H2′ = 1.9 Hz); meanwhile, 2′,4′-diF-rU adopts exclusively
the North conformation (JH1′H2′ = 0 Hz).
Since the presence of the electronegative 4′-fluorine can
affect the JH1′H2′ and mislead the comparison of coupling
constant values,35 we calculated JH1′H2′ for 2′-F-rU and 2′,4′-
diF-rU by applying a generalized Karplus equation that takes
into account the inductive effect of the 4′-fluorine (see
Supporting Information).36−38 Figure 3A shows the depend-
ence of JH1′H2′ on the pseudorotation angle for these
nucleosides. Both curves overlap, suggesting that the 4′-fluorine
does not have a significant effect on JH1′H2′. According to the
Karplus equation, a JH1′H2′ constant less than 1 Hz (as is the
case for 2′,4′-diF-rU), is only consistent with a single sugar
conformation with a pseudorotation phase angle of 0−10° (a
D
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and possible inhibition by 2′,4′-diF-rUTP and 3′-deoxyuridine
triphosphate (3′-dUTP, an obligate chain terminator). The
oligo template/primer duplex utilized in these studies is shown
in Figure 4. Extension of the 2 nt primer by rGTP and rATP
as a chain terminator under these conditions (Figure S4,
Supporting Information).
CONCLUSIONS
■
A new approach to “lock” the ribofuranose sugar without
resorting to a bicyclic framework has been developed through
the design and synthesis of 2′,4′-difluororibonucleosides.
Placing a 4′-fluorine at C4′ of 2′-fluorouridine introduces
strong stereoelectronic effects that lock the conformational
equilibrium toward a pure North conformation as assessed by
NMR and computational studies. 2′,4′-diF-rU nucleotide was
incorporated into RNA by the NS5B HCV polymerase and the
primer subsequently extended to the corresponding full-length
product. Furthermore, 2′,4′-diF-rUTP inhibited RNA synthesis
at early stages in dinucleotide-primed reactions, although with
relatively high half-maximal inhibitory concentrations (IC50
>
50 μM).
EXPERIMENTAL SECTION
■
Procedures and Experimental Data. 1-(2-Deoxy-2-fluoro-5-
iodo-β-D-furanosyl)uracil (5). Iodine (4.03 g, 15.85 mmol) and
triphenylphoshpine (4.47 g, 17.08 mmol) were added to a suspension
of 4 (3.0 g, 12.2 mmol) in pyridine (12.8 mL) and anhydrous CH3CN
(240 mL). After being stirred at room temperature for 48 h, solvents
were concentrated under vacuum and the residue was purified by
column chromatography (1−4% MeOH in CH2Cl2) to give 5 as a pale
Figure 4. Incorporation assay of 3′-dU and 2′,4′-diF-rU nucleotides in
the absence of competing rUTP and the next triphosphate, rCTP,
leading to chain termination. Underlined A indicates the site for the
incorporation of the modified nucleotide.
1
yellow solid (3.2 g, 74%): Rf (10% MeOH/CH2Cl2) 0.41; H NMR
(MeOH-d4, 300 MHz) δ 3.44 (dd, 1H, H-5′, JHH = 6.1 Hz, JHH = 11.2
Hz), 3.63 (dd, 1H, H-5″, JHH = 3.6 Hz, JHH = 11.2 Hz), 3.81 (m, 1H,
H-4′), 4.16 (ddd, 1H, H-3′, JHH = 5.0 Hz, JHH = 8.0 Hz, JHF = 19.9
Hz), 5.17 (ddd, 1H, H-2′, JHH = 1.8 Hz, JHH = 5.0 Hz, JHF = 53.3 Hz),
5.72 (d, 1H, H-5, JHH = 8.1 Hz), 5.89 (dd, 1H, H-1′, JHH = 1.8 Hz, JHF
= 21.0 Hz), 7.71 (d, 1H, JHH = 8.1 Hz); 13C NMR (MeOH-d4, 75.5
MHz) δ 3.5 (C-5′), 72.7 (d, C-3′, JCF = 16.6 Hz), 80.9 (C-4′), 90.0 (d,
C-1′, JCF = 37.0 Hz), 93.4 (d, C-2′, JCF = 187.2 Hz), 101.6 (C-5),
142.2 (C-6), 150.3 (C-2), 164.6 (C-4); HRMS (ESI+) m/z calcd for
C9H10FIN2NaO4 [M + Na]+ 378.9562, found 378.9550.
yields a stalled elongation complex with an extended 15 nt
primer.48,49 Incorporation of these analogues in the absence of
competing rUTP and the next nucleoside triphosphate, rCTP,
is demonstrated by a concentration-dependent increase in the
formation of a 16 nt product (Figure 4; Supporting
Information).
Next, chain elongation was assessed in the presence of
multiple nucleotide incorporation events that permit competi-
tion with the inhibitors (Figure 5). In this experiment, 5 μM
rNTPs and increasing concentrations (0−1000 μM) of 2′,4′-
diF-rUTP (or 3′-dUTP) were added as the elongating
nucleotides. Under these conditions, RNA synthesis was
completely blocked at concentrations of 2′,4′-diF-rUTP of
approximately 500 μM (IC50 54.7, Figure S3, Supporting
Information). The pattern of products observed in these assays
suggests that this compound acts predominantly at the level of
initiation (Figure 5). Chain termination at position +16 is not
evident. In contrast, 3′-dUTP was able to inhibit RNA
synthesis, as a chain terminator with increasing accumulation
of the +16 oligonucleotide (IC50 15.4, Figure S3, Supporting
Information) and no significant effect during initiation.49
Additional nucleotide incorporation experiments were
applied to assess whether 2′,4′-diF-rU may act as a chain
terminator under conditions that ignore the initiation stage.
Previous studies on nucleoside analogues with a 3′-hydroxyl
group show that they may act as pseudo-obligate chain
terminator if the 3′-OH group is sterically hindered to enter
into a phosphodiester linkage with the incoming rNTP.50,51 For
evaluation of 2′,4′-diF-rUTP as a chain terminator, rATP and
rGTP were added as the elongating nucleotides followed by
addition of 10 μM 2′,4′-diF-rUTP (or 3′-dUTP) at their 3′-
termini. Only the primer with the terminal 2′,4′-diF-rU
underwent further extension to afford the full-length product
(+20), demonstrating that 2′,4′-diF-rU nucleotide does not act
1-(2,5-Dideoxy-2-fluoroerythro-β-D-4-enofuranosyl)uracil (6). A
commercially available solution of 25% sodium methoxide in MeOH
(7.6 mL, 35 mmol) was added to a suspension of compound 5 (2.5 g,
7.02 mmol) in anhydrous MeOH (66 mL). The reaction mixture was
stirred at reflux for 24 h. MeOH was evaporated, and the residue was
filtered over a small bed of silica gel using 10% MeOH/CH2Cl2 as
eluent to remove the salts. The obtained solid was then purified by
column chromatography (4% MeOH/CH2Cl2) to afford 6 as a white
solid (1.20 g, 75%): Rf (10% MeOH/CH2Cl2) 0.38; 1H NMR
(MeOH-d4, 300 MHz) δ 4.38 (s, 1H, H5′), 4.60 (s, 1H, H5″), 4.90
(m, 1H, H-3′), 5.17 (dd, 1H, H-2′, JHH = 4.9 Hz, JHF = 52.5 Hz), 5.71
(d, 1H, H-5, JHH = 8.0 Hz), 6.02 (dd, 1H, H-1′, JHH = 1.7 Hz, JHF
=
17.4 Hz), 7.48 (d, 1H, H-6, JHH = 8.1 Hz); 13C NMR (MeOH-d4, 75.5
MHz) δ 68.4 (d, C-3′, JCF = 16.6 Hz), 84.4 (C-5′), 90.4 (d, C-1′, JCF
=
35.5 Hz), 91.1 (d, C-2′, JCF = 188.8 Hz), 101.9 (C-5), 141.1 (C-6),
150.3 (C-4′), 161.0 (C-2), 164.5 (C-4); HRMS (ESI+) m/z calcd for
C9H9FN2NaO4 [M + Na]+ 251.0439, found 251.0439.
1-(2-Deoxy-2,4-difluoro-5-iodo-β-D-ribofuranosyl)uracil (7). A
suspension of alkene 6 (350 mg, 1.53 mmol) and silver fluoride
(6.12 mmol, 777 mg) in MeCN (20.4 mL) was vigorously stirred at 0
°C while a solution of iodine (777 mg, 3.06 mmol) in MeCN (12.4
mL) was added over 15 min. After completion, the reaction mixture
was directly filtered over a small bed of silica gel using 50% MeOH/
CH2Cl2 as eluent to remove the Ag salts. Fractions containing the
product were collected; solvents were evaporated, and the resulting
residue was purified by column chromatography (2% MeOH/CH2Cl2)
to afford 7 as a white solid (371 mg, 64%): Rf (10% MeOH/CH2Cl2)
1
0.44; H NMR (DMSO-d6, 500 MHz) δ 3.49 (dd, 1H, H-5′, JHH
=
11.7 Hz, JHF = 23.4 Hz), 3.58 (dd, 1H, H-5″, JHF = 4.3 Hz, JHH = 11.7
Hz), 4.63 (dddd, 1H, H-3′, JHH = 5.7 Hz, JHH = 8.2 Hz, JHF = 20.9 Hz,
E
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Figure 5. Primer extension assay from the dinucleotide primer [32P]-rGG for 3′-dUTP (left) and 2′,4′-diF-rUTP (right). Underlined A indicates the
site for the incorporation of the modified nucleotide.
JHF = 23.8 Hz), 5.38 (ddd, 1H, H-2′, JHH = 1.1 Hz, JHH = 5.9 Hz, JHF
=
(ESI+) m/z calcd for C16H13F2IN2NaO5 [M + Na]+ 500.9729, found
500.9714.
53.7 Hz), 5.72 (d, 1H, H-5, JHH = 8.0 Hz), 5.89 (m, 2H, H-1′ + OH),
7.70 (d, 1H, JHH = 8.0 Hz), 11.6 (br s, 1H, NH); 13C NMR (MeCN-
1-(5-O-Benzoyl-2-deoxy-2,4-difluoro-β-D-ribofuranosyl)uracil (9).
3-Chloroperoxybenzoic acid (mCPBA) (77% purity, 449 mg, 2.00
mmol of mCPBA) was added to a suspension of 8 (248 mg, 0.52
mmol) in CH2Cl2 (14 mL) and H2O (0.8 mL). The mixture was
heated at 40 °C for 5 h, after which time solvents were concentrated
and the residue was purified by flash column chromatography (40%
AcOEt/hexanes) to afford 9 (126 mg, 66% yield) as a white solid: Rf
d3, 75.5 MHz) δ 1.9 (d, C-5′, JCF = 33.2 Hz), 71.3 (dd, C-3′, JCF
=
17.4, JCF = 23.4 Hz), 91.4 (d, C-2′, JCF = 186.5 Hz), 93.3 (d, C-1′, JCF
= 38.5 Hz), 102.4 (C-5), 113.2 (d, C-4′, JCF = 258.2 Hz), 143.0 (C-6),
149.9 (C-2), 162.6 (C-4); 19F NMR (DMSO-d6, 470.35 MHz) δ
−109.5 (dddd, F-4′, JHF = 4.2 Hz, JFF = 10.0 Hz, JHF = 20.8 Hz, JHF
=
25.0 Hz), −193.7 (ddd, F-2′, JFF 9.9 Hz, JHF = 23.4 Hz, JHF = 53.5 Hz);
HRMS (ESI+) m/z calcd for C9H9F2IN2NaO4 [M + Na]+ 396.9467,
found 396.9467.
1
(70% AcOEt/hexanes) 0.40; H NMR (MeCN-d3, 300 MHz) δ 3.91
(d, 1H, OH, JHH = 10.7 Hz), 4.55 (dd, 1H, H-5′, JHF = 6.9 Hz, JHH
=
12.3 Hz), 4.64 (dd, 1H, H-5″, JHF = 7.5 Hz, JHH = 12.3 Hz), 4.90 (m,
1H, H-3′), 5.35 (dd, 1H, H-2′, JHH = 6.0 Hz, JHF = 53.7 Hz), 5.58 (dd,
1H, H-5, JHH = 1.9 Hz, JHH = 8.0 Hz), 5.96 (d, 1H, H-1′, JHF = 22.2
Hz), 7.40 (d, 1H, H-6, JHH = 8.1 Hz), 7.55 (t, 2H, Bz, JHH = 8.0 Hz),
7.68 (t, 1H, Bz, JHH = 7.7 Hz), 8.07 (m, 2H, Bz), 9.16 (br s, 1H, NH);
13C NMR (acetone-d6, 75.5 MHz) δ 61.2 (d, C-5′, JCF = 43.0 Hz), 70.5
(dd, C-3′, JCF = 16.9, JCF = 22.0 Hz), 90.5 (d, C-2′, JCF = 185.7 Hz),
94.2 (d, C-1′, JCF = 38.5 Hz), 102.4 (C-5), 115.7 (d, C-4′, JCF = 229.5
Hz), 128.7 (Bz), 129.4 (Bz), 129.7 (Bz), 133.4 (Bz), 143.2 (C-6),
150.0 (C-2), 162.5 (C-4), 165.1 (Bz); HRMS (ESI+) m/z calcd for
C16H14F2N2NaO6 [M + Na]+ 391.0712, found 391.0696.
1-(3-O-Benzoyl-2-deoxy-2,4-difluoro-5-iodo-β-D-ribofuranosyl)-
uracil (8). Benzoyl chloride (28 μL, 0.241 mmol) was added dropwise
to a solution of compound 7 (90 mg, 0.241 mmol), triethylamine (169
μL, 1.212 mmol), and DMAP (0.5 mg, 0.004 mmol) in THF (6 mL).
The reaction was stirred for 15 min at room temperature and was then
quenched by addition of 3 mL of MeOH. Solvents were evaporated,
and the residue obtained was purified by column chromatography
(40% AcOEt/hexanes) to give benzoylated compound 8 (96 mg, 83%)
as a white solid: Rf (70% AcOEt/hexanes) 0.57; 1H NMR (MeCN-d3,
300 MHz) δ 3.59 (dd, 1H, H-5′, JHH = 11.9 Hz, JHF = 21.9 Hz), 3.75
(dd, 1H, H-5″, JHF = 8.2 Hz, JHH = 11.9 Hz), 5.52−6.13 (m, 4H, H-1′
+ H-2′ + H-3′ + H-5), 7,47 (d, 1H, H-6, JHH = 8.1 Hz), 7.58 (t, 2H,
Bz, JHH = 8.0 Hz), 7.71 (t, 1H, Bz, JHH = 7.7 Hz), 8.12 (m, 2H, Bz),
9.24 (br s, 1H, NH); 13C NMR (MeCN-d3, 75.5 MHz) δ 1.6 (d, C-5′,
JCF = 32.4 Hz), 71.5 (dd, C-3′, JCF = 15.9, JCF = 21.1 Hz), 89.9 (d, C-
2′, JCF = 190.2 Hz), 94.7 (d, C-1′, JCF = 39.2 Hz), 102.6 (C-5), 114.4
(d, C-4′, JCF = 234.1 Hz), 128.7 (Bz), 128.9 (Bz), 129.8 (Bz), 134.0
(Bz), 143.8 (C-6), 150.0 (C-2), 162.7 (C-4), 165.0 (Bz); HRMS
1-(2-Deoxy-2,4-difluoro-β-D-ribofuranosyl)uracil (10). Protected
nucleoside 9 (47 mg, 0.128 mmol) was treated with 2 M NH3 in
MeOH, and the mixture was stirred overnight at room temperature
and then evaporated to dryness under reduced pressure. Purification
by column chromatography (1−10% MeOH/CH2Cl2) gave 10 (33.7
1
mg, 99%) as a white solid: Rf (10% MeOH/CH2Cl2) 0.22; H NMR
(D2O, 50 °C, 500 MHz) δ 4.00 (dd, 1H, H-5′, JHF = 7.1 Hz, JHF = 12.8
Hz), 4.06 (dd, 1H, H-5″, JHF = 10.1 Hz, JHH = 12.8 Hz), 4.91 (ddd,
F
dx.doi.org/10.1021/jo500794v | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1H, H-3′, JHH = 5.8 Hz, JHF = 21.2 Hz, JHF = 23.2 Hz), 5.53 (dd, 1H,
H-2′, JHH = 5.8 Hz, JHF = 53.2 Hz), 6.02 (d, 1H, H-5, JHH = 8.0 Hz),
6.30 (d, 1H, H-1′, JHF = 21.5 Hz), 7.79 (d, 1H, H-6, JHH = 8.0 Hz);
13C NMR (MeOH-d4, 75.5 MHz) δ 59.2 (d, C-5′, JCF = 41.5 Hz), 68.7
(dd, C-3′, JCF = 16.6, JCF = 21.1 Hz), 91.2 (d, C-2′, JCF = 188.0 Hz),
91.7 (d, C-1′, JCF = 37.0 Hz), 101.6 (C-5), 117.1 (d, C-4′, JCF = 232.5
Hz), 142.0 (C-6), 150.2 (C-2), 164.6 (C-4); 19F NMR (D2O, 470.35
MHz) δ −109.5 (ddt, F-4′, JHF = 7.5 Hz, JFF = 10.3 Hz, JHF = 20.7 Hz),
μM of HCV NS5B, 0.2 μM radio-labeled GG primer, and 5 μM of
rGTP and rATP. Reactions were carried out in a buffer containing 40
mM HEPES (pH 8), 15 mM NaCl, 1 mM dithiothreitol, and 0.5 mM
EDTA. All reactions were started using 6 mM MgCl2 and allowed to
proceed at room temperature for 40 min up to position +15. To these
reactions were added increasing concentrations of 3′-dUTP or 2′,4′-
diF-rUTP analogue to look at their incorporation against A at position
+16. The compounds were allowed to incorporate for 5 min, and the
reaction was then stopped using EDTA and formamide. The samples
were heat-denatured at 95 °C, and the products were resolved on a
20% polyacrylamide gel and visualized using a Bio-Rad phosphor-
imager.
Primer Extension Assay in the Presence of 3′-dUTP and 2′,4′-diF-
rUTP from the Dinucleotide Primer (Figure 5). The standard reaction
mixture contained 500 nM of RNA template, 1 μM of HCV NS5B, 0.2
μM radio-labeled GG primer, and 5 μM of rGTP, rATP, rCTP, and
0.1 μM of UTP as the competing nucleotide. Reactions were carried
out in a buffer containing 40 mM HEPES (pH 8), 15 mM NaCl, 1
mM dithiothreitol, and 0.5 mM EDTA. Increasing concentrations of
3′-dUTP or 2′,4′-diF-rUTP were added, and the reactions were started
using 6 mM MgCl2 and allowed to proceed at room temperature for
40 min. The reactions were then stopped using EDTA and formamide,
samples heat-denatured at 95 °C, and the products resolved on a 20%
polyacrylamide gel and visualized using a Bio-Rad phosphorimager.
Primer Extension Reaction to Full-Length Product. The standard
reaction mixture contained 500 nM of RNA template, 1 μM of HCV
NS5B, 0.2 μM radio-labeled rGG primer, and 5 μM of rGTP and
rATP. Reactions were carried out in a buffer containing 40 mM
HEPES (pH 8), 15 mM NaCl, 1 mM dithiothreitol, and 0.5 mM
EDTA. All reactions were started using 6 mM MgCl2 and allowed to
proceed at room temperature for 40 min up to position +15. 3′-dUTP
or 2′,4′-diF-rUTP was then added to the reactions at a concentration
of 10 μM and allowed to incorporate for 10 min to confirm full
incorporation of the analogue. To these reactions were added
increasing concentrations of the next rNTP and rCTP, and the
reactions were allowed to proceed for an additional 10 min and then
stopped using EDTA and formamide. The samples were heat-
denatured at 95 °C, and the products were resolved on a 20%
polyacrylamide gel and visualized using a Bio-Rad phosphorimager
(see Figure S4, Supporting Information).
−193.7 (dddd, F-2′, JFF = 9.9 Hz, JHF = 21.1 Hz, JHF = 22.5 Hz, JHF
=
52.2 Hz); HRMS (ESI+) m/z calcd for C9H10F2N2NaO5 [M + Na]+
287.0450, found 287.0448.
1-(3,5-Di-O-benzoyl-2-deoxy-2,4-difluoro-β-D-ribofuranosyl)-
uracil (11). Benzoyl chloride (58 μL, 0.489 mmol) was added
dropwise to a solution of compound 9 (180 mg, 0.489 mmol), Et3N
(346 μL, 2.48 mmol), and DMAP (1 mg, 0.008 mmol) in dry THF
(13 mL). After 15 min, the crude reaction mixture was evaporated to
dryness under reduce pressure. The resulting residue was purified by
column chromatography (30% AcOEt/hexanes) to afford 11 (174 mg,
75%) as a white solid: Rf (70% AcOEt/hexanes) 0.68; 1H NMR
(acetone-d6, 300 MHz) δ 4.70 (d, 1H, H-5′, JHF = 1.6 Hz), 4.73 (s, 1H,
H5″), 5.72 (d, 1H, H-5, JHH = 8.1 Hz), 5.98 (ddd, 1H, H-2′, JHH = 1.1
Hz, JHH = 5.8 Hz, JHF = 53.2 Hz), 6.13−6.34 (m, 2H, H-1′ + H-3′),
7.41 (t, 2H, Bz, JHH = 7.8 Hz), 7.38−7.63 (m, 4H, Bz), 7.70 (t, 1H, JHH
= 7.5 Hz, Bz), 7.87 (d, 1H, H-6, JHH = 8.1 Hz), 8.00−8.08 (m, 4H,
Bz); 13C NMR (acetone-d6, 75.5 MHz) δ 62.3 (d, C-5′, JCF = 39.3
Hz), 71.3 (dd, C-3′, JCF = 15.1, JCF = 18.9 Hz), 89.1 (d, C-2′, JCF
=
189.5 Hz), 95.8 (d, C-1′, JCF = 40.0 Hz), 102.5 (C-5), 115.1 (d, C-4′,
JCF = 234.1 Hz), 128.5−133.8 (12C, Bz), 144.1 (C-6), 150.2 (C-2),
162.5 (C-4), 164.7 (Bz), 165.0 (Bz); HRMS (ESI+) m/z calcd for
C23H18F2N2NaO7 [M + Na]+ 495.0974, found 495.0964.
1-(4-Fluoro-2-deoxy-2-fluoro-β-D-ribofuranosyl)cytosine (12).
Phosphoryl chloride (129.6 μL, 1.39 mmol) was added dropwise to
a solution, cooled on a ice−water bath, of 11 (164 mg, 0.348 mmol),
dry Et3N (967 μL, 6.95 mmol), and 1,2,4-triazole (360 mg, 5.21
mmol) in dry MeCN (7 mL). After 2 h, the reaction mixture was
evaporated to dryness and the residue was filtered through a small bed
of silica gel (50% AcOEt/hexanes). The residue was then treated with
aqueous NH3 (33%, 5 mL), and the solution was stirred at room
temperature for 30 min. After concentration under reduced pressure,
the residue was dissolved in 2 M NH3 in MeOH (5 mL) and stirred
for 6 h at room temperature. Purification by silica gel chromatography
(2−10% MeOH/CH2Cl2) affords the cytosine derivative 12 (16 mg,
18% overall yield): Rf (20% MeOH/CH2Cl2) 0.25; 1H NMR (MeOH-
d4, 400 MHz) δ 3.78 (d, 2H, H-5′, JHF = 4.8 Hz), 4.61 (ddd, 1H, H-3′,
ASSOCIATED CONTENT
■
JHH = 5.4 Hz, JHF = 21.2 Hz, JHF = 24.7 Hz), 5.08 (dd, 1H, H-2′, JHF
53.7 Hz, JHH = 5.4 Hz), δ 5.89 (br s, 1H, H-5), 6.12 (d, 1H, H-1′, JHF
=
=
S
* Supporting Information
1H, 13C, some 19F, and 2D NMR spectra for compounds 4−12.
Additional computational data. Full gel images for HCV NS5B
polymerase-catalyzed incorporation assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
19.6 Hz), 7.81 (d, 1H, H-6, JHH = 7.5 Hz); 13C NMR (MeOH-d4, 75.5
MHz) δ 59.4 (d, C-5′, JCF = 41.9 Hz), 68.7 (dd, C-3′, JCF = 16.8 Hz,
JCF 21.8 Hz), 92.3 (d, C-1′, JCF = 36.2 Hz), 91.5 (d, C-2′, JCF = 189.3
Hz), 94.9 (C-5), 117.1 (d, C-4′, JCF = 231.9 Hz), 142.1 (C-6), 156.2
(C-2), 166.6 (C-4); 19F NMR (MeOH-d4, 282 MHz) δ −125.30
(dddd, F-4′, JHF = 4.8 Hz, JHF = 4.8 Hz, JFF = 11.4 Hz, JHF = 21.1 Hz),
−197.62 (dddd, F-2′, JHF = 53.7 Hz, JHF = 24.7 Hz, JHF = 19.6 Hz, JFF
= 11.4 Hz); HRMS (ESI+) m/z calcd for C9H11F2N3O4Na [M + Na]+
286.0610, found 286.0609.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: masad.damha@mcgill.ca.
2′-Deoxy-2′,4′-difluorouridine triphosphate was synthesized by
custom-made laboratory (ChemGenes): 31P NMR (D2O, 80 MHz)
δ −7.4 (γ-P), −13.59 (α-P), −22,15 (β-P); HRMS (ESI+) m/z calcd
for C9H9F2Li3N2)14P3 [M + 3Li]+ 514.9639, found 514.9638.
HCV Polymerase Incorporation Reaction. The RNA sequence
used for this study to mediate RNA synthesis was as follows: 5′-
CUCGAUUUCCUUUUCUCUCC-3′ (Trilink). The sequence con-
tained a single site for incorporation of the uridine analogue at position
+16 and was PAGE-purified. A 5′-GG-3′ dinucleotide primer (Trilink)
was used for primer extension. The 5′-end of the dinucleotide primer
was labeled with [γ-32P] ATP using the T4 polynucleotide kinase
(Thermo Scientific). Ribonucleoside triphosphates were purchased
from Thermo Scientific, and the 3′-dUTP was purchased from Trilink.
Incorporation Assay of 3′-dUTP and 2′,4′-diF-rUTP (Figure 4).
The standard reaction mixture contained 500 nM of RNA template, 1
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support was provided by Natural Sciences and
Engineering Research Council of Canada (M.J.D.), the McGill
CIHR Drug Development Training Program (S.M.M.), a
Vanier NSERC Scholarship (G.F.D), the Canadian Institutes of
́
Health Research and Fonds du Recherche du Quebec (M.G.),
the National CIHR Research Training Program in Hepatitis C
(A.K.). N.M.P. and C.G. thank Spanish MICINN (CTQ2010-
21567-C02-02).
G
dx.doi.org/10.1021/jo500794v | J. Org. Chem. XXXX, XXX, XXX−XXX

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(28) Marquez, V. E.; Tseng, C. K. H.; Mitsuya, H.; Aoki, S.; Kelley, J.
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Med. Chem. 1990, 33, 978−985.
DEDICATION
■
This paper is dedicated to John A. (“Jack”) Secrist on the
occasion of his retirement, after 34 years of seminal
contributions and service at the Southern Research Institute,
Alabama.
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