Adjusting the Structure of 2′-Modified Nucleosides and Oligonucleotides via C4′-α-F or C4′-α-OMe Substitution: Synthesis and Conformational Analysis

Article abstract of DOI:10.1021/acs.joc.8b01329

We report the first syntheses of three nucleoside analogues, namely, 2′,4′-diOMe-rU, 2′-OMe,4′-F-rU, and 2′-F,4′-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4′-α-position of a 4′,5′-olefinic intermediate. Conf

Full text of DOI:10.1021/acs.joc.8b01329

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Article
Adjusting the Structure of 2#-Modified Nucleosides and Oligonucleotides via
C4#-#-F or C4#-#-OMe Substitution: Synthesis and Conformational Analysis
Elise Malek-Adamian, Mihai Burai Patrascu, Sunit Kumar Jana,
Saul Martinez-Montero, Nicolas Moitessier, and Masad J. Damha
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01329 • Publication Date (Web): 02 Jul 2018
Downloaded from http://pubs.acs.org on July 5, 2018
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Adjusting the Structure of 2ʹꢀModified Nucleosides and
Oligonucleotides via C4ʹꢀαꢀF or C4ʹꢀαꢀOMe Substitution:
Synthesis and Conformational Analysis
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Elise MalekꢀAdamian, Mihai Burai Patrascu, Sunit Kumar Jana, Saúl MartínezꢀMontero,
Nicolas Moitessier, and Masad J. Damha*
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC,
Canada H3A 0B8.
* Correspondence to: Email address masad.damha@mcgill.ca
ABSTRACT
We report the first syntheses of three nucleoside analogs, namely 2′,4′ꢀdiOMeꢀrU, 2′ꢀOMe,4′ꢀFꢀ
rU, and 2′ꢀF,4′ꢀOMeꢀaraU via stereoselective introduction of fluorine or methoxy functionalities
at the C4ʹ alpha position of a 4ʹ,5ʹꢀolefinic intermediate. Conformational analyses of these
nucleosides and comparison to other previously reported 2′,4′ꢀdisubstituted nucleoside analogs
make it possible to evaluate the effect of fluorine and methoxy substitution on sugar pucker, as
assessed by NMR, Xꢀray diffraction and computational methods. We found that that C4ʹꢀαꢀ
F/OMe substituents reinforce the C3ʹꢀendo (North) conformation of 2ʹꢀOMeꢀrU. Furthermore,
the predominant C2ʹꢀendo (South/East) conformation of 2ʹꢀFꢀaraU switches to C3ʹꢀendo upon
introduction of these substituents at C4ʹ. The nucleoside analogs were incorporated into DNA
and RNA oligonucleotides via standard phosphoramidite chemistry and their effects on thermal
stability of homoꢀ and heteroduplexes were assessed via UV thermal melting experiments. We
found that 4ʹ substituents can modulate the binding affinity of the parent 2ʹꢀmodified oligomers,
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inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study
expands the spectrum of oligonucleotide modifications available for rational design of
oligonucleotides therapeutics.
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INTRODUCTION
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Beyond their applications as smallꢀmolecule therapeutic agents, nucleoside analogs are
extensively used for oligonucleotide modification in gene silencing1,2ꢀ4 and, more recently, gene
editing applications.5ꢀ7,8 In this context, the conformation of the nucleotide components is a main
factor in defining binding affinity towards the target RNA or ssDNA which determines, to a
large extent, the therapeutic and offꢀtarget effects. Many different modifications in the
carbohydrate moiety have been explored in order to modulate the conformation of the sugar
ring.9 Electronegative substituents at C2ʹ in the ribo (α) configuration10 such as methoxy
(OMe),11,12 methoxyethyl (MOE),13 and fluoro (F)14,15 tailor the sugar pucker towards the C3′ꢀ
endo (North) conformation, whereas electronegative C2ʹꢀβꢀsubstituents (e.g., 2ʹꢀF) drive the
sugar towards the South/East conformation. Nucleosides that adopt the C3′ꢀendo conformation
(i.e. 2ʹꢀF, 2ʹꢀOMe, 2ʹꢀMOE ribonucleotides) are widely used in RNA targeting applications (e.g.,
siRNAs, AONs).16,17ꢀ19
We and others have recently shown that modification at the 4ʹꢀposition can also have a strong
effect on sugar pucker preference.20ꢀ26 We first designed 2′,4′ꢀdifluorinated ribonucleosides with
the rationale that a C4′ꢀαꢀfluorine would reinforce the North conformation of 2′ꢀFꢀ
ribonucleosides.
Indeed, as shown by NMR and molecular modeling, 2′,4′ꢀdiFꢀrU adopts
exclusively the North conformation without requiring a bridge linking the 2ʹ and 4ʹ positions as
seen in LNA, cEt and their derivatives.21,27 In the case of 2′ꢀFꢀaraU, introduction of a C4′ꢀαꢀ
fluorine caused a switch from South to mainly North pucker (~8:2% North:South ratio), pointing
to a dominant FꢀC4ʹꢀO4ʹ anomeric effect.20 In a more recent study we showed that 2′ꢀF,4′ꢀOMeꢀ
rU also adopts a Northern conformation (~ 9:1 N/S),23,28 and that siRNAs containing several 2′ꢀ
F,4′ꢀOMeꢀrU units in the sense or antisense strands triggered RNAiꢀmediated gene silencing
with efficiencies comparable to that of 2ʹꢀFꢀrU.20,22,23 Furthermore, the C4′ꢀαꢀOMe moiety
conferred increased nuclease resistance due to the close proximity between 4ʹꢀOMe substituent
and the vicinal 5ʹꢀ and 3ʹꢀphosphate group.23
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Guided by these results, we report herein the synthesis of several new 2ʹ,4ʹꢀmodified arabino and
ribonucleoside analogs and study their conformation via NMR, Xꢀray crystallography and
computational methods (Figure 1). When appropriate, comparisons are drawn with the
conformation of the previously reported 2ʹ,4ʹꢀmodified analogs (Figure 1). All new 2ʹ,4ʹꢀ
modified arabino and ribonucleoside analogs were converted into their respective
phosphoramidite derivatives for incorporation into DNA and RNA strands by solidꢀphase
methods, and the resulting modified strands mixed with complementary DNA or RNA strands to
study their binding properties.
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Figure 1. Structures of nucleoside analogues studied in this work. A) 2′ꢀOMeꢀmodified
ribonucleosides. B) 2′ꢀFꢀmodified ribonucleosides. C) 2′ꢀFꢀmodified arabinonucleosides. The
synthesis of those coloured in blue are reported herein. Ura is uracil nucleobase.
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RESULTS AND DISCUSSION
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Synthesis. We first focused on installing a OMe group at C4ʹ of 2′ꢀOMeꢀrU and 2′ꢀFꢀaraU,
following our previously published protocols,23 producing two new compounds 2ʹ,4ʹꢀdiOMeꢀrU
(11) and 2ʹꢀF,4ʹꢀOMeꢀaraU (12), respectively. Briefly, 2′ꢀOMeꢀrU and 2′ꢀFꢀaraU were first
converted to the 5ʹꢀiodine derivative to allow for elimination reaction with DBU in THF to
provide the desired alkene derivatives, 3 and 4, respectively (Scheme 1).
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Scheme 1. Synthesis of 2′,4′ꢀdiOMeꢀrU (11) and 2′ꢀF,4′ꢀOMeꢀaraU (12) and their
phosphoramidite derivatives (15) and (16), respectively.a
aReagents and conditions: (i) I2, PPh3/ACN, pyridine, rt, 48 h, 1: 91%, 2: 93% ; (ii) DBU/THF,
rt, overnight, 3: 86%, 4: 78% (iii) PbCO3, I2/MeOH, 0 °C, overnight, 5: 82%, 6: 77% (iv)
o
BzCl/pyridine, rt, overnight, 7: 77%, 8: 78% (v) mCPBA/DCM, H2O, 40 C, 6 h, 9: 62%, 10:
o
85% (vi) 2M NH3/MeOH, rt, overnight, 11: 81%. 12: 86% (vii) DMTrCl, pyridine, 45 C, 2 h,
13: 57%, 14: 78%, (viii 15) 2ꢀCyanoethyl N,Nꢀdiisopropylchlorophosphoramidite, DIPEA/THF,
rt, 7 h, 15: 42%, 16: 86%.
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Subsequent reaction with PbCO3 and I2 in methanol yielded the corresponding 5ʹꢀiodoꢀC4ʹꢀ
alphaꢀOMe in 82 and 77% yield for 5 and 6, respectively, with high stereoselectivity. PbCO3 is
essential for the stereoselectivity achieved in this reaction.23,29 In its absence, a mixture of alpha
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and beta anomers together with other uncharacterized products were observed in the HꢀNMR of
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the reaction crude mixture (Figure S3). We propose a mechanism in which PbCO3 coordinates to
the ring oxygen effectively blocking the beta face of the nucleoside and forcing the MeOH to
attack from the 4ʹꢀalpha face exclusively (Scheme 2). When Pb2+ is replaced by a smaller cation
such as Ag2+, we observed a twoꢀfold decrease in stereoselectivity (Supporting Information,
Figures S1 and S2), supporting the mechanism shown in Scheme 2. This observation is
corroborated by the finding that protection of the N3 position with the benzyloxymethyl acetal
(BOM) protecting group led to the same αꢀstereoselectivity at C4ʹ (Scheme 2; Supporting
Information, Figure S4).
Scheme 2. Proposed mechanism and role of PbCO3 in the reaction of a 2ʹꢀsubstituted 4ʹ,5ʹꢀolefin
with PbCO3/I2 in MeOH. R1 = OMe, F; R2 = H or BOM group.
Benzoylation of the 3ʹꢀOH with concomitant migration of the Bz group followed by treatment
with mCPBA in water saturated dichloromethane and ammonia deprotection, afforded the target
nucleosides 11 and 12. Their corresponding phosphoramidites 15 and 16 were obtained using
conventional methods by first treatment with DMTrCl in pyridine to obtain 13 and 14 followed
by treatment with ClP(OCEt)N(iPr)2 in the presence in DIPEA and THF to afford
phosphoramidites 15 and 16 in 42% and 86% yield respectively.
We further exploited the synthetic utility of the 4ʹ,5ʹꢀolefin of the 2ʹꢀOMeꢀrU intermediate (3), to
prepare the 4ʹꢀfluorinated analog of 2ʹꢀOMeꢀrU (Scheme 3).21 The 4ʹꢀfluorination was
accomplished by slow addition of I2 to a suspension of AgF and 3 in acetonitrile at 0 oC to obtain
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17 in 65% yield. Unprotected nucleoside 20 was obtained in good overall yield after
benzoylation of the 3ʹꢀOH, oxidative iodine substitution reaction with mCPBA in water/DCM,
and final ammonia deprotection. Dimethoxytritylation followed by treatment with 2ꢀcyanoethyl
N,N,N,Nꢀtetraisopropylphosphorodiamidite in the presence of 4,5ꢀdicyanoimidazole in ACN and
DCM30 produced the target phosphoramidite 22 in 64% yield.
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Scheme 3. Synthesis of 2′ꢀOMe,4′ꢀFꢀrU, (20) and its phosphoramidite derivative (22).
aReagents and conditions: (iii) AgF, I2/ACN, 0 °C, 30 min, 17: 65% (iv) BzCl/pyridine, rt,
o
overnight, 18: 83% (v) mCPBA/DCM, H2O, 40 C, 6 h, 19: 83 % (vi) 2M NH3/MeOH, rt,
o
overnight, 20: 95 %, (vii) DMTrCl, pyridine, 45 C, 2 h, 21: 60%, (viii) 2ꢀcyanoethyl N,N,N,Nꢀ
tetraisopropyldiphosphoramidite, DCI/ACN solution, DCM, rt, 30 min, 22: 64%.
NMR Conformational Analysis. With our set of nucleosides in hand, we proceeded to analyse
their sugar conformations by NMR spectroscopy
.
The percentage of North and South
conformers of each ribonucleoside in solution was calculated by applying the equation S(%) = 10
21,31
1
× JH1′ꢀH2′
,
where JH1′ꢀH2′ is the H−1H coupling constant between the ribose ring H1′ and H2′
hydrogens (Table 1 and Table S1). Table 1 summarizes, in descending order, the percentage of
North conformation for all the nucleosides under study in three different solvents. Substitution of
the 4′ꢀH for electronegative substituents 4′ꢀF and 4′ꢀOMe resulted in a larger North bias in all
2′,4′ꢀsubstituted nucleosides relative to their corresponding 2′ꢀsubstituted nucleosides. This
effect was more pronounced in the case of 4ʹꢀfluoro substitution, in agreement with a fluorine
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being a better acceptor of hyperconjugation than a methoxy group,32 thus providing stronger 4ʹꢀ
anomeric and gauche effects.
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Table 1: Percentage of different nucleoside in the North conformation in various solvents at
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298K.
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% North in Solutiona,b
Entry
Nucleoside
Solvent
D2O
CD3OD
100
CD3CN
100
1
2
2′,4′ꢀdiFꢀrUc
2′ꢀOMe,4′ꢀFꢀrU
2′ꢀF, 4′ꢀOMeꢀrUd
2′ꢀFꢀrUd
100
87.0
87.0
85.0
80.0
80.0
72.0
60.0
55.5
40.0
86.0
86.0
81.0
80.5
ꢀ
93.0
87.0
80.0
78.0
ꢀ
3
4
5
2′ꢀF,4′ꢀOMeꢀaraU
2′,4′ꢀdiFꢀaraUe
2′,4′ꢀdiOMeꢀrU
2′ꢀOMeꢀrU
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7
70.0
64.0
54.0
38.0
74.0
60.0
54.0
44.0
8
9
rUd
10
2′ꢀFꢀaraU
a Obtained from equation: S(%) = 10 × JH1′ꢀH2′
b
Sugar pucker populations for 2′ꢀFꢀaraU and 2′ꢀF,4′ꢀOMeꢀaraU were estimated according to
MartínezꢀMontero et al.20
c Previously acquired data from MartínezꢀMontero et al.21
d Previously acquired data from MalekꢀAdamian, E. et al.23
e Previously acquired data from MartínezꢀMontero et al.20
To assess changes in sugar pucker equilibria as a function of temperature, JH1′ꢀH2′ coupling
constants were measured in 10 K intervals over the range of 323ꢀ193 K in CD3OD (Supporting
Information), and the %North at each temperature was calculated (Table S2). Assuming that a
twoꢀstate N/S equilibrium is maintained, all nucleosides under study progressively became more
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North as the temperature was reduced, consistent with the notion that the North conformation is
thermodynamically more favoured. In contrast, 2ʹ,4ʹꢀdiFꢀrU adopted exclusively
the North conformation when heated to 323 K, confirming the conformational rigidity imposed
by the two fluorine atoms. In order to quantify electronic interactions that favor the North
conformation, we then turned to computational methods.
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Computational Analysis. Recently, we developed and validated a molecular dynamicsꢀbased
protocol for analyzing the conformation of nucleosides in solution.28 We applied this protocol to
the abovementioned set of 4ʹꢀmodified nucleosides to get more insight into the origin of their
conformational preferences. For each stable conformation we determined the pseudorotational
phase angle P, the puckering amplitude φmax (the maximum degree of pucker) and the
appropriate dihedral angles (Supporting Information, Section S5). In addition, relevant
hyperconjugation and anomeric effects and possible intramolecular hydrogen bonding were also
computed (Supporting Information). Predicted N/S ratios determined by either Boltzmann
population distribution analysis or energy differences between conformations were in close
agreement with the experimentally determined N/S ratios (Supporting Information). This further
confirmed the accuracy of the method and the generated conformations could now be further
analyzed.
Lowest Energy Conformations and Electronic Effects. Anomeric and hyperconjugation
effects through the means of natural bond orbital (NBO) analysis were evaluated as described in
our recent study.28 The anomeric effect nO4ʹ → σ*C4ʹOMe/F (Figure 2A) favours the North
conformation except for 2ʹꢀF,4ʹꢀOMeꢀaraU; the strength of the anomeric effect followed the
order: 2ʹꢀOMe,4ʹꢀFꢀrU (P = 55.6°) > 2ʹ,4ʹꢀdiOMeꢀrU (P = 60.5°) > 2ʹꢀF,4ʹꢀOMeꢀaraU (P = 47.2
°).
Several factors seem to influence the strength of the anomeric effect: a) the puckering amplitude
of the sugar ring, with a weaker effect observed for lower amplitudes for both North and South
conformations, b) the hyperconjugation accepting ability of the 4ʹ substituent, and c) the P angle,
with deviations from ideal envelope conformations associated with weaker anomeric effects
(Supporting Information).
In the case of 2ʹꢀF,4ʹꢀOMeꢀaraU, a plausible explanation for the South conformation exhibiting a
slightly larger anomeric effect is that the North conformation (P = 47.2°, φmax = 22.1°) deviates
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from an ideal envelope conformation (P = 54°, φmax = 25ꢀ45°).33 This leads to a poor overlap
between the lone pair on the anomeric oxygen and σ*C4ʹOMe due to orbital misalignment.
However, 2ʹꢀF,4ʹꢀOMeꢀaraU was shown to be predominantly North, and as such another
electronic effect apart from the anomeric effect should be the driving force behind the observed
conformation. Indeed, the data in Table S40 suggests that the σC3’H3’ – σ*C4’OMe hyperconjugation
effect (Figure 2C) is the most pronounced computed electronic effect (with an energy difference
of ~1.9 kcal/mol in favour of North), similar in strength to 2ʹ,4ʹꢀdiOMeꢀrU (~2.0 kcal/mol) and
2ʹꢀOMe,4ʹꢀFꢀrU (~3.1 kcal/mol).
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Interestingly, this effect is exhibited in conjunction with a σC3’C4’ – σ*C2’H hyperconjugation
effect (Figure 2B) (~0.8 kcal/mol in favour of the South conformer) that is significantly lower
than that observed for 2ʹꢀOMe,4ʹꢀFꢀrU (~3.5 kcal/mol) and 2ʹ,4ʹꢀdiOMeꢀrU (~2.2 kcal/mol), most
likely afforded by orientation of the fluorine in the arabino configuration. As such, the loss of an
important hyperconjugation effect favouring the South pucker (σC3’C4’ – σ*C2’H), coupled with a
strong hyperconjugation effect favouring the North pucker (σC3’H3’ – σ*C4’OMe) contribute to the
observed North conformation of 2ʹꢀF,4ʹꢀOMeꢀaraU.
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A.
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B.
C.
South
North
Figure 2. Stereoelectronic effects in 2ʹꢀF,4ʹꢀOMeꢀaraU : A) Depiction of the nO4ʹ → σ*C4ʹOMe
anomeric effect. B) Depiction of the σC3ʹC4ʹ → σ*C2ʹH hyperconjugation effect. C) Depiction of
the σC3ʹH3ʹ → σ*C4ʹOMe hyperconjugation effect.
A Comparison between Crystal and Predicted Structures. The structures of 2ʹ,4ʹꢀdiOMeꢀrU
and 2ʹꢀOMe,4ʹꢀFꢀrU, first synthesized in this study, were unambiguously confirmed by Xꢀray
crystallography. Additionally, we obtained the crystal structures of previously reported 2ʹ,4ʹꢀdiFꢀ
rU and 2ʹꢀF,4ʹꢀOMeꢀrU .28 These were compared to their predicted conformations by
computational analysis and the superposition of these are shown in Figure 3.
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B.
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C.
D.
Figure 3. Superposition between the predicted conformation (green) and the crystal structure
(pink). A) 2ʹ,4ʹꢀdiOMeꢀrU, heavy atom RMSD = 0.47Å. B) 2ʹꢀOMe,4ʹꢀFꢀrU left) unit 1, heavy
atom RMSD = 0.82Å and right) unit 2, heavy atom RMSD = 0.82Å. C) 2ʹꢀF,4ʹꢀOMeꢀrU left) unit
1, heavy atom RMSD = 0.51 Å and right) unit 2, heavy atom RMSD = 0.59 Å. D) 2ʹ,4ʹꢀdiFꢀrU,
heavy atom RMSD = 0.59Å.
2′ꢀOMe,4′ꢀFꢀrU was crystalized in its most stable conformation (P1 = 22.4°, φ1ꢀmax = 34.1°, P2 =
21.2°, φ2ꢀmax = 33.8°).
In the case of 2ʹꢀF,4ʹꢀOMeꢀrU (4ʹꢀexo)., two slightly different
conformations appeared in the asymmetric unit crystal (P1 = 62.3°, φ1ꢀmax = 36.6°, and P2 = 60.5°,
φ2ꢀmax = 37.1°). However, the orientation of the base and the 3ʹ and 5ʹ hydroxyls differ from the
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computational data. In contrast, 2ʹ,4ʹꢀdiOMeꢀrU and 2ʹ,4ʹꢀdiFꢀrU were crystallized in the South
conformation (P = 163.2°, φmax = 39.5°) and the East conformation (P = 74.7o, φmax = 35.4o28),
respectively. Clearly, these results do not depict how nucleosides behave in solution, and hence
crystal packing and intermolecular hydrogen bonding interactions between molecules in different
unit cells likely affect the conformation of the nucleoside in the crystal.34
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Oligonucleotide Synthesis. To systematically compare the effect of the structure of the three
novel 2ʹ,4ʹꢀmodified nucleotides discussed above with the parent 2ʹꢀmodified nucleotides and
other 2ʹ,4ʹꢀmodifications previously described in our laboratory, we performed single
incorporations in a central location of a previously used oligonucleotide sequence.22 To achieve
this, phosphoramidite 15, 16, and 22 were incorporated into DNA and RNA oligonucleotides via
standard phosphoramidite chemistry (see Experimental Section) with coupling efficiencies
similar to 2ʹꢀFꢀaraU and 2ʹꢀOMeꢀrU amidites. Oligonucleotides were purified by ion exchange
HPLC using 1 M lithium perchlorate buffer. All 2′,4′ꢀmodified oligonucleotides were examined
by ESI mass spectrometry and confirmed to possess the correct mass (Table S35, Supporting
Information)
Melting Temperature of Modified Duplexes. Thermal stability was assessed by determining
the Tm values of 5′ꢀd(CCA TXA TAG C) and 5′ꢀr(CCA UXA UAG C) hybridized with
complementary DNA or RNA (Table 2). Incorporation of 2ʹ,4ʹꢀdiOMeꢀrU in DNA resulted in a
o
destabilization effect when paired with either complementary DNA or RNA (ꢁTm = ꢀ1.5 C). In
contrast, its incorporation into RNA had a positive impact on thermal stability whether the target
o
was a complementary DNA or RNA strand (ꢁTm = + 1.5 C). This suggests that the geometrical
constrains imposed by the two bulky OMe groups are wellꢀaccommodated in the Aꢀform
structure adopted by RNA:RNA duplexes. Similarly, the 2ʹꢀOMe,4ʹꢀFꢀrU analog had a
destabilizing effect when incorporated into a DNA:DNA duplex. However, the effect was neutral
or slightly stabilizing when incorporated in either DNA:RNA hybrids (ꢁTm = neutral; +0.5 oC) or
o
RNA:RNA duplex (ꢁTm = +1.5 C). Comparison with the isomeric 2ʹꢀF,4ʹꢀOMeꢀrU analog
previously reported indicates that permutation of the fluorine and methoxy substituents has little
effect on the thermal stability of the corresponding modified duplexes (Table 2).23
Comparison of 2ʹ,4ʹꢀdiFꢀrU with 2ʹꢀF,4ʹꢀOMeꢀrU suggest that a 4ʹꢀOMe provides a significant
improvement over a 4ʹꢀF in stability of hybrid duplexes and slight improvement in RNA
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duplexes (Table 2).22 The origins for this behavior remains unclear; however, we hypothesize
that the greater inductive effect of F versus OMe (at C4ʹ) diminishes internucleotide 2′C−H···O4′
hydrogen bonds that contribute to the stabilization of Aꢀlike duplex structures.35 This interaction
is enhanced in 2′ꢀFꢀrU by the polarization of the 2′C−H bond by the geminal fluorine at C2′.22
While this effect should also operate in 2′,4′ꢀdiFꢀrU and 2′ꢀF,4′ꢀOMeꢀrU, the electronegative
substituents at C4′ is expected to affect the charge distribution on the furanose sugar ring (e.g., F
at C2′). The consequence of this would be a reduction in both the acceptor capability of O4′ and
the polarization of the 2′C−H bond in the order 4′ꢀF > 4′ꢀOMe > 4′ꢀH, consistent with the Tm
changes in DNA/RNA and RNA/RNA duplexes by 2ʹꢀFꢀrU (stabilizing), 2ʹꢀF,4ʹꢀOMeꢀrU
(stabilizingꢀneutral), and 2ʹ,4ʹꢀdiFꢀrU (destabilizingꢀneutral). Clearly, highꢀfield NMR analysis is
needed to gain a better understanding of these subtle structural effects.
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Incorporation of 2ʹꢀF,4ʹꢀOMeꢀaraU into DNA:DNA duplexes is destabilizing (ꢁTm = ꢀ2.5 C), an
expected result if the predominant North conformation of the nucleoside is maintained in the
duplex. By the same token, incorporation of 2ʹꢀF,4ʹꢀOMeꢀaraU into an RNA duplex is slightly
stabilizing, in contrast to 2ʹꢀFꢀaraU which adopts mainly a South/East conformation and hence
causes destabilization of the same duplexes. In the context of DNA:RNA hybrids, 2ʹꢀFꢀaraU
adopts a South/East pucker, allowing the formation of internucleosidic CꢀH···FꢀC
pseudohydrogen bonds at pyrimidineꢀpurine steps and resulting in the stabilizing effect observed
when it is incorporated in the DNA strand of DNAꢀRNA hybrids.36,37 This is consistent with the
o
observed stabilization (ꢁTm = +1.5 C) when a single dT is replaced with 2ʹꢀFꢀaraU at a
pyrimidineꢀpurine step. Such pseudohydrogen bonding is unlikely to form in a North conformer
(2ʹꢀF,4ʹꢀOMeꢀaraU) hence losing the stabilization typical of 2ʹꢀFꢀaraU nucleotides when placed
in DNA (ꢁTm = ꢀ0.0 oC; Table 2).
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Table 2. Comparison of the relative melting temperatures of duplexes containing a single
incorporation of modified uridine nucleotide.a
ꢀTm/mod. (oC)
2ʹꢀF ribo modifications
2ʹꢀOMe ribo modifications
2ʹꢀF arabino modifications
Duplex Sequence
(5ʹ to 3ʹ)
(3ʹ to 5ʹ)
2ʹꢀF
2ʹ,4ʹꢀdiFb
2ʹF,4ʹOMeb
2ʹꢀOMe
2ʹ,4ʹꢀdiOMe
2ʹOMe,4ʹF
2ʹꢀaraF
2ʹaraF,4ʹOMe
1
d
4
(CCATXATAGC)
ꢀ0.5
+2.0
+1.5
+1.5
n.d.
ꢀ9.4
ꢀ1.5
ꢀ0.5
ꢀ1.0
+1.5
+1.5
0.0
ꢀ1.0
+1.5
+1.5
+1.0
ꢀ1.5
ꢀ1.5
+1.5
+1.5
ꢀ1.5
+0.5
0.0
ꢀ0.5
+1.5
ꢀ4.5
ꢀ1.5
ꢀ2.5
0.0
d(GGTAATATCG)
15
d(CCATXATAGC)
16
r(GGUAAUAUCG)
17
r(CCAUXAUAGC)
18
0.0
d(GGTAATATCG)
19
r(CCAUXAUAGC)
20
+1.5
+0.5
r(GGUAAUAUCG)
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a Tm values were measured in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM
NaCl and 0.1 mM EDTA with a strand concentration of 1.5 µM where ꢁTm = (Tm of modified
duplex – Tm of corresponding unmodified duplex). X indicates the modified uridine nucleotide:
2′ꢀFꢀrU, 2′,4′ꢀdiFꢀrU, 2′ꢀFꢀ4′ꢀOMeꢀrU, 2′ꢀOMeꢀrU, 2′,4′ꢀdiOMeꢀrU, 2′ꢀOMeꢀ4′ꢀFꢀrU, 2ʹꢀaraF, or
2′ꢀFꢀ4′ꢀOMeꢀaraU. b Values obtained from previous report.22,23
CONCLUSIONS
Three new 2ʹ,4ʹꢀmodified nucleosides, 2ʹꢀF,4ʹꢀOMeꢀaraU, 2ʹꢀOMe,4ʹꢀFꢀrU, and 2ʹ,4ʹꢀdiOMeꢀrU,
were synthesized with high stereoselectivity from 4ʹ,5ʹꢀolefinic nucleoside precursors using
PbCO3 as the catalyst. Computational studies suggest that both electronic and steric effects
influence their sugar pucker, favoring in all cases the North form. A combination of two
hyperconjugation effects (σC3’H3’ – σ*C4’OMe and σC3’C4’ – σ*C2’H) drives the conformation of 2ʹꢀ
F,4ʹꢀOMeꢀaraU towards North. Evaluation of the duplex stabilizing properties of these new
monomers and comparison with the parent 2ʹꢀmodified nucleotide analogs shows that the 4ʹ
modification clearly impacts duplex stability. The North 2ʹ,4ʹꢀdisubsituted RNA analogues 2ʹꢀ
OMe,4ʹꢀFꢀrU, and 2ʹ,4ʹꢀdiOMeꢀrU and the ANA analogue 2ʹꢀF,4ʹꢀOMeꢀaraU are well tolerated in
RNA:RNA duplexes, stabilizing or nearly neutral with respect to effects of duplex thermal
stability (+0.5 to +1.5 oC/nt). This outcome suggests that these analogues (and those previously
reported by our group) may be very well suited for siRNA and guide RNAs, allowing retention
of an Aꢀform structure and negligible effects on thermal stability, while introducing modified
nucleotides foreign to serum nucleases and immunostimulatory receptors. The neutral or
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minimally destabilizing character of 2ʹꢀOMe,4ʹꢀFꢀrU; 2ʹ,4ʹꢀdiFꢀrU; 2ʹꢀF,4ʹꢀOMeꢀaraU in
DNA:RNA contexts could be useful for CRISPR/Cas9 technologies where slightly destabilizing
chemical modifications in the sgRNA have been shown to reduce offꢀtarget effects while
keeping gene editing efficiency.7 This study expands the “chemist’s toolbox” of nucleoside (and
RNA/DNA) modifications available for a wide range of applications. Further investigations are
currently in progress in our laboratory to assess the full potential of the oligonucleotide
modifications described herein.
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EXPERIMENTAL SECTION
Procedures and Experimental Data. HRMS was obtained by ESIꢀMS using a Thermo
Scientific Exactive Plus Orbitrap mass spectrometer or a Bruker Daltonics Maxis Impact
quardupole timeꢀofꢀflight (QTOF) mass spectrometer.
Synthesis of 2′,4′ꢁdiOMeꢁrU. 5′ꢀiodoꢀ2′ꢀdeoxyꢀ2′ꢀmethoxyuridine (1). A suspension of 2′ꢀOMe
uridine (2.0 g, 7.752 mmol) was prepared in anhydrous acetonitrile (160 mL) and pyridine (8
mL). Iodine (2.56 g, 10.08 mmol) and then triphenylphosphine (2.85 g, 10.85 mmol) were added
under inert atmosphere to the reaction mixture. The reaction was left to stir at room temperature
for 48 hours, after which the solvents were removed by vacuum and the resulting residue was
purified by column chromatography (1ꢀ10% MeOH in CH2Cl2) to give 1 as a yellow foam (2.59
g, 91%): Rf (10% MeOH/CH2Cl2) 0.45; 1H NMR (500 MHz, CD3OD) δ 7.74 (d, J = 8.1 Hz, 1H),
5.91 (d, J = 3.9 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 4.07 (t, J = 5.8 Hz, 1H), 3.95 (dd, J = 5.6, 4.0
Hz, 1H), 3.84 (td, J = 5.9, 4.6 Hz, 1H), 3.59 (dd, J = 11.0, 4.6 Hz, 1H), 3.50 (s, 3H), 3.45 (dd, J =
13
11.0, 5.9 Hz, 1H). C NMR (126 MHz, CD3OD) δ 166.2, 152.1, 142.8, 103.2, 89.9, 84.2, 84.1,
74.1, 59.2, 5.8. HRMS (ESI+) m/z calcd for C10H13IN2NaO5 [M + Na]+ 390.9761, found
390.9760.
4′,5′ꢀDidehydroꢀ2′,5′ꢀdideoxyꢀ2′ꢀmethoxyuridine (3). A solution of 1 (2.58 g, 7.030 mmol) was
prepared in anhydrous THF (66 mL) under inert atmosphere. 1,8ꢀDiazabicyclo[5.4.0]undecꢀ7ꢀ
ene (DBU) was added dropwise to the solution (6.31 mL, 42.18 mmol) over a period of 5
minutes. The reaction was left to stir at room temperature overnight. The reaction mixture was
then evaporated and purified by column chromatography (1ꢀ12% MeOH/CH2Cl2) to afford the
1
alkene as a white foam (1.45 g, 86%): Rf (10% MeOH/CH2Cl2) 0.42; H NMR (500 MHz,
CD3OD) δ 7.48 (d, J = 8.1 Hz, 1H), 6.11 (d, J = 3.9 Hz, 1H), 5.74 (dd, J = 8.1, 1.0 Hz, 1H), 4.67
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(dt, J = 5.4, 1.5 Hz, 1H), 4.53 (t, J = 1.8 Hz, 1H), 4.35 (dd, J = 2.2, 1.3 Hz, 1H), 4.00 (dd, J =
5.2, 3.9 Hz, 1H), 3.51 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 165.9, 163.0, 151.9, 141.4, 103.4,
89.7, 86.2, 83.0, 69.7, 58.7. HRMS (ESI+) m/z calcd for C10H12N2NaO5 [M + Na]+ 263.0644,
found 263.0636.
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2′ꢀDeoxyꢀ2′,4ʹꢀdimethoxyꢀ5′ꢀiodo uridine (5). A suspension of alkene 3 (1.45 g, 6.046 mmol) and
PbCO3 (3.23 g, 12.09 mmol) was prepared in anhydrous methanol (41 mL). A solution of iodine
(3.07 g, 12.09 mmol) in methanol (11 mL) was added dropwise to the rapidly stirring solution at
0 oC. The reaction was left to stir overnight at room temperature, the solvents were concentrated
under vacuum, and the resulting residue was purified by column chromatography (2ꢀ5%
MeOH/CH2Cl2) to afford the product as an orange foam (1.96 g, 82%): Rf (10% MeOH/CH2Cl2)
0.73; 1H NMR (500 MHz, CD3OD) δ 7.66 (d, J = 8.1 Hz, 1H), 5.89 (d, J = 3.8 Hz, 1H), 5.75 (d,
J = 8.1 Hz, 1H), 4.51 (d, J = 7.2 Hz, 1H), 3.95 (dd, J = 7.2, 3.8 Hz, 1H), 3.66 (d, J = 11.4 Hz,
1H), 3.51 (d, J = 11.4 Hz, 1H), 3.47 (s, 3H), 3.38 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 165.7,
151.6, 142.9, 104.7, 103.3, 90.6, 82.8, 74.2, 59.5, 3.3. HRMS (ESI+) m/z calcd for
C11H15IN2NaO6 [M + Na]+ 420.9872, found 420.9861.
3ʹꢀOꢀBenzoylꢀ2′ꢀdeoxyꢀ2′,4ʹꢀdimethoxyꢀ5′ꢀiodo uridine (7). A suspension of the nucleoside 5
(1.45 g, 3.643 mmol) was prepared in pyridine (5 mL). Benzoyl chloride (508 ꢂL, 4.372 mmol)
was slowly added dropwise to the mixture, and the reaction was left to stir at room temperature
overnight, following which the solvents were concentrated under vacuum. The resulting residue
was purified by column chromatography (2ꢀ5% MeOH/CH2Cl2) to afford the product as an
orange solid (1.41 g, 77%): Rf (10% MeOH/CH2Cl2) 0.78; 1H NMR (500 MHz, CD3OD) δ 8.55
(dt, J = 4.6, 1.7 Hz, 1H), 8.14 – 8.11 (m, 2H), 7.75 (d, J = 8.0 Hz, 1H), 7.67 – 7.62 (m, 1H), 7.53
– 7.49 (m, 2H), 5.98 (d, J = 4.0 Hz, 1H), 5.78 (d, J = 8.1 Hz, 1H), 5.64 (d, J = 7.3 Hz, 1H), 4.41
13
(dd, J = 7.3, 4.0 Hz, 1H), 3.67 (d, J = 2.7 Hz, 2H), 3.44 (s, 3H), 3.37 (s, 3H). C NMR (126
MHz, CD3OD) δ 166.9, 151.9, 149.6, 143.5, 139.0, 134.7, 134.0, 131.0, 130.7, 129.6, 125.7,
105.6, 103.6, 92.3, 82.0, 75.3, 59.7, 4.2. HRMS (ESI+) m/z calcd for C18H19IN2NaO7 [M + Na]+
525.0135, found 525.0132.
5ʹꢀOꢀBenzoylꢀ2′ꢀdeoxyꢀ2′,4ʹꢀdimethoxy uridine (9). A solution of nucleoside 7 (1.41 g, 2.805
mmol) was prepared in CH2Cl2 (79 mL) and H2O (4.5 mL). 3ꢀChloroperoxybenzoic acid
(mCPBA) (77% purity, 2.52 g, 11.22 mmol) was added slowly to the stirring solution. The
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reaction was heated to 40 C and allowed to react for 6 hours, after which the solvents are
concentrated and the resulting residue was purified by column chromatography (30ꢀ70% EtOAc
in hexanes) to afford the product as a white foam (683 mg, 62%): Rf (50% EtOAc/hexanes) 0.56;
1H NMR (500 MHz, CD3CN) δ 7.95 (dd, J = 8.5, 1.3 Hz, 2H), 7.74 (d, J = 8.2 Hz, 1H), 7.68 (t, J
= 7.5 Hz, 1H), 7.54 – 7.50 (m, 2H), 5.86 (d, J = 2.4 Hz, 1H), 5.66 (d, J = 8.1 Hz, 1H), 4.68 (d, J
= 12.0 Hz, 1H), 4.60 (d, J = 7.0 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.10 – 4.03 (m, 1H), 3.50 (s,
3H), 3.47 (s, 3H). 13C NMR (126 MHz, CD3CN) δ 169.9, 167.3, 150.4, 143.6, 136.4, 134.5,
132.8, 130.8, 129.6, 105.9, 102.9, 93.2, 82.9, 72.7, 62.3, 60.0, 20.8, 14.4. HRMS (ESI+) m/z
calcd for C18H20N2NaO8 [M + Na]+ 415.1117, found 415.1122.
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2′ꢀDeoxyꢀ2′,4ʹꢀdimethoxy uridine (11). A solution of the nucleoside 9 (683 mg, 1.742 mmol) was
prepared in methanolic ammonia (2M, 10 mL) and stirred overnight at room temperature. The
resulting mixture was evaporated to remove solvents and then purified by column
chromatography (2ꢀ20% MeOH/CH2Cl2) to afford the product as a white foam (406 mg, 81%):
Rf (10% MeOH/CH2Cl2) 0.54; 1H NMR (500 MHz, CD3OD) δ 7.88 (d, J = 8.1 Hz, 1H), 6.01 (d,
J = 3.0 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 4.43 (d, J = 6.7 Hz, 1H), 3.85 – 3.83 (dd, J = 3.5, 6.7
13
Hz, 1H), 3.84 – 3.82 (d, J = 12.0 1H), 3.63 (d, J = 11.8 Hz, 1H), 3.51 (s, 3H), 3.39 (s, 3H). C
NMR (126 MHz, CD3OD) δ 166.0, 151.9, 142.5, 107.7, 103.0, 90.1, 84.1, 71.1, 61.0, 59.7, 50.1.
HRMS (ESI+) m/z calcd for C11H16N2NaO7 [M + Na]+ 311.0855, found 311.0852.
5′ꢀOꢀ[Bis(4ꢀmethoxyphenyl)phenylmethyl]ꢀ2′ꢀdeoxyꢀ2′,4′ꢀdimethoxy uridine (13). A solution of
nucleoside 11 (300 mg, 1.164 mmol) was prepared in pyridine (15 mL) under argon. 4,4′ꢀ
dimethoxytrityl chloride (552 mg, 1.628 mmol) was added, and the reaction was allowed to stir
for 2 h at 45 °C. Pyridine was subsequently evaporated under vacuum, and the resulting residue
purified by column chromatography using deꢀacidified silica gel by treatment with 10%
trimethylamine in CH2Cl2 and MeOH/CH2Cl2 washing (1−3% MeOH/CH2Cl2). This afforded
dimethoxytritylated nucleoside 13 as a white foam (391 mg, 57%). Rf (10% MeOH/CH2Cl2)
0.64; 1H NMR (500 MHz, CD3OD) δ 7.89 (d, J = 8.1 Hz, 1H), 7.43 – 7.40 (m, 2H), 7.33 – 7.28
(m, 6H), 7.27 – 7.23 (m, 1H), 6.90 – 6.86 (m, 4H), 5.97 (d, J = 2.0 Hz, 1H), 5.24 (d, J = 8.1 Hz,
1H), 4.66 (d, J = 6.8 Hz, 1H), 3.85 (dd, J = 6.8, 2.0 Hz, 1H), 3.78 (s, 6H), 3.53 (s, 3H), 3.49 (d, J
= 10.0 Hz, 1H), 3.34 (d, J = 10.0 Hz, 1H), 3.19 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 166.00,
160.36, 151.69, 145.82, 142.51, 136.62, 136.42, 132.91, 131.54, 131.48, 131.45, 129.50, 129.46,
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128.96, 128.65, 128.63, 128.13, 114.24, 113.99, 113.94, 107.34, 102.80, 90.66, 88.58, 84.25,
71.91, 62.42, 59.94, 58.44, 55.74, 55.69, 49.94. HRMS (ESI+) m/z calcd for C32H34N2NaO9 [M
+ Na]+ 613.2162, found 613.2160.
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5′ꢀOꢀ[Bis(4ꢀmethoxyphenyl)phenylmethyl]ꢀ2′ꢀdeoxyꢀ2′,4′ꢀdimethoxy uridine, 3'ꢀ[2ꢀcyanoethyl
N,Nꢀbis(1ꢀmethylethyl)phosphoramidite] (15). Dimethoxytritylated nucleoside 13 (391 mg,
0.663 mmol), was dried under vacuum overnight and coevaporated with MeCN four times. Dry
THF (7 mL) was added under argon. To the solution was added EtN(iPr)2 (603 ꢂL, 3.315 mmol)
and then ClPOCEtN(iPr)2 (163 ꢂL, 0.729 mmol). The reaction mixture was stirred for 7 hours at
room temperature. After the reaction reached completion, the mixture was extracted using DCM
and saturated sodium bicarbonate, the organic phases were collected and dried using MgSO4 to
finally be filtered and concentrated for direct loading on a deacidified silica column (eluent: 30ꢀ
70% EtOAc in hexanes) to afford phosphoramidite 15 as a white foam (220 mg, 42%). Rf (50%
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EtOAc/hexanes) 0.38. H NMR (500 MHz, CD3CN) δ 9.03 (s, 1H), 7.83 – 7.62 (m, 1H), 7.45 –
7.39 (m, 2H), 7.35 – 7.22 (m, 7H), 6.88 (dd, J = 8.2, 4.4 Hz, 4H), 6.00 (dd, J = 13.9, 2.7 Hz, 1H),
5.17 (dd, J = 8.2, 4.7 Hz, 1H), 4.74 (ddd, J = 47.1, 11.2, 6.9 Hz, 1H), 3.93 (td, J = 7.7, 7.1, 2.7
Hz, 1H), 3.77 (d, J = 2.3 Hz, 6H), 3.66 – 3.58 (m, 2H), 3.46 (d, J = 13.7 Hz, 3H), 3.43 – 3.36 (m,
1H), 3.31 – 3.23 (m, 1H), 3.20 (d, J = 18.8 Hz, 3H), 2.68 (td, J = 6.4, 5.3 Hz, 1H), 2.55 (ddd, J =
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15.2, 6.6, 5.4 Hz, 1H), 1.27 – 1.09 (m, 11H), 1.07 (d, J = 6.7 Hz, 2H). C NMR (126 MHz,
CD3CN) δ 163.56, 159.70, 150.86, 145.40, 141.28, 135.96, 132.37, 131.19, 131.10, 129.22,
129.04, 128.76, 128.19, 127.92, 119.36, 113.95, 107.58, 102.81, 102.70, 90.24, 89.93, 87.90,
83.24, 82.97, 72.33, 72.22, 71.54, 71.42, 63.18, 62.55, 59.55, 59.37, 59.33, 55.78, 55.76, 50.69,
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50.41, 43.96, 43.86, 24.96, 24.90, 24.80, 24.67, 20.73. P NMR (203 MHz, CD3CN) δ 150.70,
150.26. HRMS (ESI+) m/z calcd for C41H51N4NaO10P [M + Na]+ 813.3241, found 813.3243.
Synthesis of 2ʹꢁF,4ʹꢁOMeꢁaraU. 5′ꢀiodoꢀ2′ꢀdeoxyꢀ2′ꢀfluoro arabino uridine (2). The same
procedure was followed as previously described, and the results matched experimental findings.4
4′,5′ꢀDidehydroꢀ2′,5′ꢀdideoxyꢀ2′ꢀfluoro arabino uridine (4). A solution of 2 (2.4 g, 6.761 mmol)
was prepared in anhydrous THF (61 mL) under inert atmosphere. 1,8ꢀDiazabicyclo[5.4.0]undecꢀ
7ꢀene (DBU) was added dropwise to the solution (6.1 mL, 40.56 mmol) over a period of 5
minutes. The reaction was left to stir at room temperature overnight. The reaction mixture was
then evaporated and purified by column chromatography (1ꢀ12% MeOH/CH2Cl2) to afford the
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alkene as a white foam (1.2 g, 78%). Rf (10% MeOH/CH2Cl2) 0.59; H NMR (500 MHz,
CD3OD) δ 7.47 (dd, J = 8.2, 2.1 Hz, 1H), 6.51 (dd, J = 19.4, 3.3 Hz, 1H), 5.72 (d, J = 8.1 Hz,
1H), 5.11 – 4.95 (m, 1H), 4.69 – 4.68 (m, 1H), 4.67 – 4.65 (m, 1H), 4.47 (dd, J = 2.3, 0.8 Hz,
1H). 13C NMR (126 MHz, CD3OD) δ 166.0, 161.8, 142.6, 102.7, 95.6, 94.1, 88.4, 86.9, 74.5. 19F
NMR (471 MHz, CD3OD) δ ꢀ204.41, ꢀ204.43, ꢀ204.44, ꢀ204.45, ꢀ204.45, ꢀ204.48, ꢀ204.52, ꢀ
204.52, ꢀ204.55, ꢀ204.56, ꢀ204.58. HRMS (ESI+) m/z calcd for C9H9FN2NaO4 [M + Na]+
251.0444, found 251.0436.
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2′,5′ꢀDideoxyꢀ2′ꢀfluoroꢀ5′ꢀiodoꢀ4′ꢀmethoxy arabino uridine (6). A suspension of alkene 4 (2 g,
8.772 mmol) and PbCO3 (4.69 g, 17.54 mmol) was prepared in anhydrous methanol (56 mL). A
solution of iodine (4.45 g, 17.54 mmol) in methanol (16 mL) was added dropwise to the rapidly
stirring solution at 0 oC. The reaction was left to stir overnight at room temperature, the solvents
were concentrated under vacuum, and the resulting residue was purified by column
chromatography (2ꢀ10% MeOH/CH2Cl2) to afford the product as a yellow foam (2.6 g, 77%): Rf
(10% MeOH/CH2Cl2) 0.62; 1H NMR (500 MHz, CD3OD) δ 7.72 (dd, J = 8.1, 1.7 Hz, 1H), 6.26
(dd, J = 12.0, 5.3 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 5.13 (ddd, J = 54.4, 5.3, 4.5 Hz, 1H), 4.55
(dd, J = 24.6, 4.5 Hz, 1H), 3.80 (dd, J = 11.6, 0.9 Hz, 1H), 3.54 (d, J = 11.6 Hz, 1H), 3.42 (s,
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3H). C NMR (126 MHz, CD3OD) δ 165.7, 151.8, 142.8, 103.5, 102.5, 97.7, 96.1, 82.9, 79.7,
1.7. 19F NMR (471 MHz, CD3OD) δ ꢀ201.20, ꢀ201.23, ꢀ201.26, ꢀ201.28, ꢀ201.32, ꢀ201.35, ꢀ
201.37, ꢀ201.40. HRMS (ESI+) m/z calcd for C10H12FIN2NaO5 [M + Na]+ 408.9673, found
408.9655.
3ʹꢀOꢀBenzoylꢀ2′ꢀdeoxyꢀ2′ꢀfluoroꢀ5′ꢀiodoꢀ4′ꢀmethoxy arabino uridine (8). A suspension of the
nucleoside 6 (3.4 g, 8.854 mmol) was prepared in pyridine (13 mL). Benzoyl chloride (1.2 mL,
10.63 mmol) was slowly added dropwise to the mixture, and the reaction was left to stir at room
temperature overnight, following which the solvents were concentrated under vacuum. The
resulting residue was purified by column chromatography (2ꢀ5% MeOH/CH2Cl2) to afford the
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product as a yellow foam (3.4 g, 78%): Rf (10% MeOH/CH2Cl2) 0.78; H NMR (500 MHz,
DMSOꢀd6) δ 11.59 (s, 1H), 8.11 – 7.98 (m, 2H), 7.84 (dd, J = 8.1, 1.9 Hz, 1H), 7.74 – 7.66 (m,
1H), 7.62 – 7.50 (m, 2H), 6.33 (dd, J = 10.8, 5.9 Hz, 1H), 5.99 (dd, J = 24.1, 4.8 Hz, 1H), 5.75 (t,
J = 4.1 Hz, 2H), 3.89 (d, J = 11.5 Hz, 1H), 3.71 (d, J = 11.4 Hz, 1H), 3.35 (s, 3H). 13C NMR (126
MHz, DMSOꢀd6) δ 163.9, 161.9, 149.2, 141.0, 133.0, 128.8, 127.9, 101.0, 100.2, 93.7, 92.2,
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77.6, 48.2, 2.1. 19F NMR (471 MHz, DMSOꢀd6) δ ꢀ198.48, ꢀ198.51, ꢀ198.56, ꢀ198.59, ꢀ198.61, ꢀ
198.64, ꢀ198.67. HRMS (ESI+) m/z calcd for C17H16FIN2NaO6 [M + Na]+ 512.9935, found
512.9922.
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5ʹꢀOꢀBenzoylꢀ2′ꢀdeoxyꢀ2′ꢀfluoroꢀ4′ꢀmethoxy arabino uridine (10). A solution of nucleoside 8 (1
g, 2.041 mmol) was prepared in CH2Cl2 (28 mL) and H2O (2.3 mL). 3ꢀChloroperoxybenzoic acid
(mCPBA) (77% purity, 1.8 g, 8.163 mmol) was added slowly to the stirring solution. The
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reaction was heated to 40 C and allowed to react for 6 hours, after which the solvents are
concentrated and the resulting residue was purified by column chromatography (30ꢀ80% EtOAc
in hexanes) to afford the product as a white foam (840 mg, 85%): Rf (50% EtOAc/hexanes) 0.47;
1H NMR (500 MHz, CD3CN) δ 9.16 (s, 1H), 8.05 (dt, J = 8.4, 1.8 Hz, 2H), 7.69 – 7.64 (m, 1H),
7.55 – 7.50 (m, 2H), 7.40 (dd, J = 8.1, 1.7 Hz, 1H), 6.30 (dd, J = 11.4, 5.3 Hz, 1H), 5.46 (d, J =
8.2 Hz, 1H), 5.17 (dt, J = 53.9, 5.1 Hz, 1H), 4.67 (dd, J = 12.2, 1.0 Hz, 1H), 4.55 (d, J = 8.9 Hz,
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1H), 3.78 (s, 1H), 3.47 (s, 3H), 3.45 – 3.31 (m, 1H). C NMR (126 MHz, CD3CN) δ 167.0,
165.6, 151.8, 142.3, 134.7, 130.7, 129.8, 103.8, 102.5, 97.4, 95.8, 82.7, 77.9, 62.0, 50.7. 19F
NMR (471 MHz, CD3CN) δ ꢀ202.02, ꢀ202.05, ꢀ202.07, ꢀ202.10, ꢀ202.14, ꢀ202.16, ꢀ202.19, ꢀ
202.21. HRMS (ESI+) m/z calcd for C17H17FN2NaO7 [M + Na]+ 403.0917, found 403.0899.
2′ꢀdeoxyꢀ2′ꢀfluoroꢀ4′ꢀmethoxy arabino uridine (12). A solution of the nucleoside 10 (290 mg,
0.7513 mmol) was prepared in methanolic ammonia (2M, 10 mL) and stirred overnight at room
temperature. The resulting mixture was evaporated to remove solvents and then purified by
column chromatography (2ꢀ20% MeOH/CH2Cl2) to afford the product as a white foam (180 mg,
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86%): Rf (10% MeOH/DCM) 0.31; H NMR (500 MHz, CD3OD) δ 7.84 (dd, J = 8.1, 1.2 Hz,
1H), 6.32 (dd, J = 8.1, 5.8 Hz, 1H), 5.71 (d, J = 8.1 Hz, 1H), 5.20 (dt, J = 54.8, 5.8 Hz, 1H), 4.50
(dd, J = 24.7, 5.8 Hz, 1H), 3.90 (dd, J = 12.0, 1.6 Hz, 1H), 3.68 (d, J = 12.0 Hz, 1H), 3.40 (s,
3H). 13C NMR (126 MHz, CD3OD) δ 165.9, 152.0, 142.6, 105.8, 102.4, 97.6, 96.0, 82.3, 75.4,
59.7, 50.3. 19F NMR (471 MHz, CD3OD) δ ꢀ202.82, ꢀ202.84, ꢀ202.88, ꢀ202.89, ꢀ202.94, ꢀ202.96,
ꢀ202.99, ꢀ203.01. HRMS (ESI+) m/z calcd for C10H13FN2NaO6 [M + Na]+ 299.0655, found
299.0641.
5′ꢀOꢀ[Bis(4ꢀmethoxyphenyl)phenylmethyl]ꢀ2′ꢀdeoxyꢀ2′ꢀfluoroꢀ4′ꢀmethoxy arabino uridine (14). A
solution of nucleoside 12 (240 mg, 0.869 mmol) was prepared in pyridine (12 mL) under argon.
4,4′ꢀdimethoxytrityl chloride (412 mg, 1.217 mmol) was added, and the reaction was allowed to
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stir for 2 h at 45 °C. Pyridine was subsequently evaporated under vacuum, and the resulting
residue purified by column chromatography using deꢀacidified silica gel by treatment with 10%
trimethylamine in CH2Cl2 and MeOH/CH2Cl2 washing (1−3% MeOH/CH2Cl2). This afforded
dimethoxytritylated nucleoside 14 as a white foam (390 mg, 78%). Rf (10% MeOH/CH2Cl2)
0.57; 1H NMR (500 MHz, CD3CN) δ 9.16 (s, 1H), 7.64 (dd, J = 8.2, 1.3 Hz, 1H), 7.47 – 7.41 (m,
2H), 7.38 – 7.23 (m, 8H), 6.90 (d, J = 8.8 Hz, 4H), 6.27 (dd, J = 8.5, 5.7 Hz, 1H), 5.34 (d, J = 8.2
Hz, 1H), 5.18 (dt, J = 54.4, 5.7 Hz, 1H), 4.62 (dd, J = 23.5, 5.6 Hz, 1H), 3.78 (s, 6H), 3.68 (s,
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1H), 3.45 (d, J = 10.3 Hz, 1H), 3.30 (d, J = 10.3 Hz, 1H), 3.17 (s, 3H), 1.97 (s, 1H). C NMR
(126 MHz, CD3CN) δ 163.6, 159.8, 151.1, 145.5, 141.6, 136.2, 136.2, 131.1, 129.0, 128.1,
114.2, 104.4, 104.3, 102.5, 97.18, 95.65, 87.91, 81.91, 81.77, 76.65, 76.46, 61.50, 55.95, 50.47,
34.75, 23.06, 15.05. 19F NMR (471 MHz, CD3CN) δ ꢀ202.56, ꢀ202.58, ꢀ202.61, ꢀ202.63, ꢀ202.67,
ꢀ202.69, ꢀ202.72, ꢀ202.74. HRMS (ESI+) m/z calcd for C31H31FN2NaO8 [M + Na]+ 601.1962,
found 601.1955.
5′ꢀOꢀ[Bis(4ꢀmethoxyphenyl)phenylmethyl]ꢀ2′ꢀdeoxyꢀ2′ꢀfluoroꢀ4′ꢀmethoxy arabino uridine, 3'ꢀ[2ꢀ
cyanoethyl N,Nꢀbis(1ꢀmethylethyl)phosphoramidite] (16). Dimethoxytritylated nucleoside 14
(250 mg, 0.432 mmol), was dried under vacuum overnight and coevaporated with MeCN four
times to remove water. Dry THF (4.5 mL) was added under argon. To the solution was added
EtN(iPr)2 (385 ꢂL, 2.162 mmol) and then ClPOCEtN(iPr)2 (106 ꢂL, 0.476 mmol). The reaction
was allowed to stir for 7 hours at room temperature. After the reaction reached completion, the
mixture was extracted using DCM and saturated sodium bicarbonate, the organic phases were
collected and dried using MgSO4 to finally be filtered and concentrated for direct loading on a
deacidified silica column (eluent: 30ꢀ70% EtOAc/hexanes) to afford phosphoramidite 16 as a
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white foam (290 mg, 86%). Rf (3:2 EtOAc/hexanes) 0.66. H NMR (500 MHz, CD3CN) δ 9.01
(s, 1H), 7.76 (td, J = 8.0, 1.3 Hz, 1H), 7.47 – 7.38 (m, 2H), 7.37 – 7.22 (m, 7H), 6.92 – 6.85 (m,
4H), 6.38 (ddd, J = 20.4, 7.3, 6.1 Hz, 1H), 5.47 – 5.30 (m, 1H), 5.19 (dd, J = 23.9, 8.2 Hz, 1H),
5.04 – 4.89 (m, 1H), 4.06 (q, J = 7.1 Hz, 1H), 3.78 (d, J = 2.6 Hz, 6H), 3.67 – 3.50 (m, 4H), 3.29
(t, J = 9.8 Hz, 1H), 3.19 (d, J = 8.4 Hz, 3H), 2.63 (t, J = 6.0 Hz, 1H), 2.58 – 2.50 (m, 1H), 1.23 –
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1.09 (m, 10H), 1.04 (d, J = 6.7 Hz, 2H). C NMR (126 MHz, CD3CN) δ 163.4, 159.9, 159.9,
145.4, 141.6, 141.3, 136.0, 131.3, 131.2, 129.2, 129.0, 128.2, 128.2, 114.2, 102.5, 88.20, 61.60,
60.26, 60.12, 59.55, 55.97, 55.94, 50.98, 50.77, 44.35, 44.25, 44.19, 44.09, 24.98, 24.92, 24.87,
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24.78, 24.73, 24.68, 20.74, 14.51. F NMR (471 MHz, CD3CN) δ ꢀ199.10, ꢀ199.11, ꢀ199.13, ꢀ
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199.15, ꢀ199.16, ꢀ199.18, ꢀ199.21, ꢀ199.23, ꢀ199.25, ꢀ199.26, ꢀ199.28, ꢀ199.30, ꢀ199.46, ꢀ199.47,
ꢀ199.49, ꢀ199.51, ꢀ199.53, ꢀ199.54, ꢀ199.56, ꢀ199.58, ꢀ199.59, ꢀ199.63, ꢀ199.64, ꢀ199.66. HRMS
(ESI+) m/z calcd for C40H48FN4NaO9P [M + Na]+ 801.3041, found 801.3036.
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Synthesis of 2′ꢁOMe,4′ꢁFꢁrU. 2′,5′ꢀDideoxyꢀ2′ꢀmethoxyꢀ5′ꢀiodoꢀ4′ꢀfluorouridine (17): A
suspension of the nucleoside 3 (100 mg, 0.417 mmol) and AgF (211 mg, 1.667 mmol) was
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prepared in dry acetonitrile (6 mL). The suspension was brought to 0 C followed by dropwise
addition of a solution of I2 (211 mg, 0.833 mmol) in dry acetonitrile (3.4 mL) over a period of 10
minutes. The reaction mixture was then allowed to stir for 30 minutes and then extracted with
ethyl acetate and brine to afford the product as a yellow foam (104 mg, 65%): Rf (10%
MeOH/DCM) 0.45; 1H NMR (500 MHz, CD3OD) δ 7.60 (d, J = 8.0 Hz, 1H), 5.93 (d, J = 2.1 Hz,
1H), 5.73 (d, J = 8.1 Hz, 1H), 4.62 (dd, J = 19.4, 7.2 Hz, 1H), 4.11 – 4.07 (m, 1H), 3.63 (dd, J =
11.5, 5.6 Hz, 1H), 3.55 (d, J = 11.6 Hz, 1H), 3.52 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 165.9,
151.6, 143.7, 115.2, 103.5, 94.07, 82.55, 72.80, 59.86, 2.49. 19F NMR (471 MHz, CD3OD) δ ꢀ
112.56, ꢀ112.57, ꢀ112.60, ꢀ112.61, ꢀ112.64, ꢀ112.65. HRMS (ESI+) m/z calcd for
C10H12FIN2NaO5 [M + Na]+ 408.9673, found 408.9652.
3ʹꢀOꢀBenzoylꢀ2′ꢀdeoxyꢀ2′ꢀmethoxyꢀ5′ꢀiodoꢀ4′ꢀfluorouridine (18): A suspension of the nucleoside
17 (400 mg, 1.042 mmol) was prepared in pyridine (1.5 mL). Benzoyl chloride (145 ꢂL, 1.250
mmol) was slowly added dropwise to the mixture, and the reaction was left to stir at room
temperature overnight, following which the solvents were concentrated under vacuum. The
resulting residue was purified by column chromatography (2ꢀ5% MeOH/DCM) to afford the
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product as a yellow foam (420 mg, 83%): Rf (50% EtOAc/hexanes) 0.43; H NMR (500 MHz,
CD3CN) δ 9.13 (s, 1H), 8.09 (dd, J = 8.3, 1.4 Hz, 2H), 7.69 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.8
Hz, 2H), 7.44 (d, J = 8.1 Hz, 1H), 5.89 (d, J = 2.5 Hz, 1H), 5.71 – 5.68 (m, 1H), 5.67 (d, J = 7.3
Hz, 1H), 4.49 (dd, J = 7.2, 2.6 Hz, 1H), 3.70 (dd, J = 11.8, 8.1 Hz, 1H), 3.57 (dd, J = 21.6, 11.8
Hz, 1H), 3.35 (s, 3H), 2.11 (s, 1H). 13C NMR (126 MHz, CD3CN) δ 167.8, 166.2, 163.7, 151.0,
143.4, 134.8, 134.1, 130.7, 129.5, 103.7, 95.33, 80.78, 73.12, 59.87, 3.42. 19F NMR (471 MHz,
CD3CN) δ ꢀ107.29, ꢀ107.31, ꢀ107.33, ꢀ107.33, ꢀ107.34, ꢀ107.35, ꢀ107.37, ꢀ107.39. HRMS (ESI+)
m/z calcd for C17H16FIN2NaO6 [M + Na]+ 512.9935, found 512.9929.
5ʹꢀOꢀBenzoylꢀ2′ꢀdeoxyꢀ2′ꢀmethoxyꢀ4′ꢀfluorouridine (19): A solution of nucleoside 18 (560 mg,
1.143 mmol) was prepared in CH2Cl2 (32 mL) and H2O (1.8 mL). 3ꢀChloroperoxybenzoic acid
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(mCPBA) (77% purity, 1.03 g, 4.571 mmol) was added slowly to the stirring solution. The
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reaction was heated to 40 C and allowed to react for 6 hours, after which the solvents are
concentrated and the resulting residue was purified by column chromatography (30ꢀ70% EtOAc
in hexanes) to afford the product as a white foam (360 mg, 83%): Rf (50% EtOAc/hexanes) 0.53;
1H NMR (500 MHz, CD3CN) δ 9.10 (s, 1H), 8.06 – 8.02 (m, 2H), 7.68 – 7.64 (m, 1H), 7.54 –
7.50 (m, 2H), 7.33 (d, J = 8.2 Hz, 1H), 5.92 (d, J = 1.4 Hz, 1H), 5.49 (dd, J = 8.2, 1.4 Hz, 1H),
4.62 – 4.60 (m, 1H), 4.58 (d, J = 7.5 Hz, 1H), 4.54 (d, J = 7.5 Hz, 1H), 4.04 (dd, J = 6.9, 1.5 Hz,
1H), 3.61 (s, 1H), 3.51 (s, 3H). 13C NMR (126 MHz, CD3CN) δ 166.3, 163.7, 150.8, 142.3,
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134.6, 130.6, 129.7, 103.3, 93.89, 81.07, 71.61, 71.43, 62.70, 62.36, 60.02. F NMR (471 MHz,
CD3CN) δ ꢀ121.94, ꢀ121.96, ꢀ121.97, ꢀ121.99, ꢀ122.00, ꢀ122.02. HRMS (ESI+) m/z calcd for
C17H17FN2NaO7 [M + Na]+ 403.0917, found 403.0911.
2′ꢀdeoxyꢀ2′ꢀmethoxyꢀ4′ꢀfluorouridine (20). A solution of the nucleoside 19 (140 mg, 0.3646
mmol) was prepared in methanolic ammonia (2M, 10 mL) and stirred overnight at room
temperature. The resulting mixture was evaporated to remove solvents and then purified by
column chromatography (2ꢀ20% MeOH/CH2Cl2) to afford the product as a white solid (85 mg,
1
95%): Rf (10% MeOH/DCM) 0.38. H NMR (500 MHz, CD3OD) δ 7.84 (d, J = 8.1 Hz, 1H),
6.11 (d, J = 1.4 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 4.48 (dd, J = 20.2, 6.6 Hz, 1H), 3.90 (dd, J =
6.6, 1.5 Hz, 1H), 3.77 (dd, J = 11.9, 3.5 Hz, 1H), 3.73 (dd, J = 11.9, 3.8 Hz, 1H), 3.57 (s, 3H).
13C NMR (126 MHz, CD3OD) δ 166.0, 151.7, 142.2, 119.4, 117.6, 91.85, 83.01, 70.14, 60.91,
59.74. 19F NMR (471 MHz, CD3OD) δ ꢀ124.29, ꢀ124.30, ꢀ124.31, ꢀ124.34, ꢀ124.34, ꢀ124.35.
HRMS (ESI+) m/z calcd for C10H13FN2NaO6 [M + Na]+ 299.0655, found 299.0639.
5′ꢀOꢀ[Bis(4ꢀmethoxyphenyl)phenylmethyl]ꢀ2′ꢀdeoxyꢀ2′ꢀmethoxyꢀ4′ꢀfluoro uridine (21).
A
solution of nucleoside 20 (80 mg, 0.290 mmol) was prepared in pyridine (4 mL) under argon.
4,4′ꢀdimethoxytrityl chloride (137 mg, 0.406 mmol) was added, and the reaction was allowed to
stir for 2 h at 45 °C. Pyridine was subsequently evaporated under vacuum, and the resulting
residue purified by column chromatography using deꢀacidified silica gel by treatment with 10%
trimethylamine in CH2Cl2 and MeOH/CH2Cl2 washing (1−3% MeOH/CH2Cl2). This afforded
dimethoxytritylated nucleoside 21 as a white foam (100 mg, 60%). Rf (10% MeOH/CH2Cl2)
0.62; 1H NMR (500 MHz, CD3OD) δ 7.87 (d, J = 8.1 Hz, 1H), 7.42 – 7.38 (m, 2H), 7.35 – 7.31
(m, 2H), 7.30 – 7.26 (m, 5H), 6.90 – 6.86 (m, 4H), 6.10 (d, J = 1.0 Hz, 1H), 5.27 (d, J = 8.1 Hz,
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1H), 4.73 (dd, J = 21.2, 6.5 Hz, 1H), 3.96 (dd, J = 6.5, 1.0 Hz, 1H), 3.79 (s, 6H), 3.58 (d, J = 1.8
13
Hz, 3H), 3.51 (dd, J = 10.0, 3.2 Hz, 1H), 3.37 – 3.33 (m, 1H). C NMR (126 MHz, CD3OD) δ
166.0, 160.4, 151.6, 145.5, 142.2, 136.4, 136.1, 131.4, 129.4, 129.0, 128.2, 119.0, 117.2, 114.3,
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102.9, 92.14, 88.73, 82.93, 70.83, 62.30, 61.93, 59.84, 55.74. F NMR (471 MHz, CD3OD) δ ꢀ
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120.41, ꢀ120.45, ꢀ121.06, ꢀ121.11. HRMS (ESI+) m/z calcd for C31H31FN2NaO8 [M + Na]+
601.1962, found 601.1932 .
5′ꢀOꢀ[Bis(4ꢀmethoxyphenyl)phenylmethyl]ꢀ2′ꢀdeoxyꢀ2′ꢀfluoroꢀ4′ꢀmethoxy arabino uridine, 3'ꢀ[2ꢀ
cyanoethyl N,Nꢀbis(1ꢀmethylethyl)phosphoramidite] (22). Dimethoxytritylated nucleoside 21
(140 mg, 0.242 mmol), was coevaporated with MeCN four times to remove water and dried
under vacuum overnight. Dry DCM (0.540 mL) was added under argon. 2ꢀCyanoethyl N,N,N,Nꢀ
tetraisopropyldiphosphoramidite (77 ꢂL, 0.242 mmol) was added to the solution followed by
dropwise addition of a solution of 4,5ꢀdicyanoimidazole (DCI) in MeCN (26 mg, 0.243 mL). The
reaction mixture was stirred for 30 minutes at room temperature. The reaction progress was
monitored by TLC (1:1 EtOAc, Hexanes). After the reaction reached completion, the mixture
was extracted using DCM and saturated sodium bicarbonate, the organic phases were collected
and dried using Na2SO4 to finally be filtered and concentrated for direct loading on a deacidified
silica column (eluent: 30ꢀ70% EtOAc in hexanes) to afford phosphoramidite 22 as a white foam
1
(120 mg, 64%). Rf (50% EtOAc/hexanes) 0.48. H NMR (500 MHz, CD3CN) δ 9.03 (s, 1H),
7.64 (dd, J = 29.1, 8.1 Hz, 1H), 7.46 – 7.38 (m, 2H), 7.36 – 7.23 (m, 7H), 6.92 – 6.83 (m, 4H),
6.06 (d, J = 4.6 Hz, 1H), 5.25 (dd, J = 8.1, 2.5 Hz, 1H), 4.84 – 4.67 (m, 1H), 4.09 – 4.00 (m, 1H),
3.78 (d, J = 3.2 Hz, 6H), 3.74 – 3.68 (m, 1H), 3.62 (dq, J = 10.5, 6.7 Hz, 2H), 3.50 (s, 3H), 3.44
– 3.32 (m, 2H), 3.12 (dd, J = 7.3, 5.6 Hz, 1H), 2.71 – 2.49 (m, 2H), 1.21 – 1.10 (m, 9H), 1.06 –
13
0.96 (m, 3H). C NMR (126 MHz, CD3CN) δ 163.7, 159.9, 159.9, 150.9, 145.3, 141.5, 141.3,
135.9, 131.3, 131.2, 131.2, 129.1, 129.0, 128.2, 114.2, 103.1, 103.0, 92.77, 92.57, 88.10, 82.10,
81.54, 70.70, 59.75, 59.71, 59.59, 59.51, 55.96, 55.93, 44.24, 44.13, 44.03, 25.07, 25.01, 24.98,
24.93, 24.80, 24.75, 21.03, 20.97, 15.07. 19F NMR (471 MHz, CD3CN) δ ꢀ117.92, ꢀ117.96, ꢀ
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118.65, ꢀ118.69. P NMR (203 MHz, CD3CN) δ 151.33, 150.22. HRMS (ESI+) m/z calcd for
C40H48FN4NaO9P [M + Na]+ 801.3041, found 801.3034. Note: When ClPOCEtN(iPr)2 was used
as the phosphitylating reagent for this reaction, it produced significantly lower yield of product..
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Computational Methods. All computations were performed according to our previously
established protocol.28
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Solid Phase Synthesis. All oligonucleotides (ONs) were synthesized on an Applied Biosystems
(ABI) 3400 DNA synthesizer at 1 ꢁmol scale using Unylinker CPG as solid support, mesh 500A.
DNA phosphoramidites and 2′,4ʹꢀmodified phosphoramidites were prepared as 0.1 M solutions
in acetonitrile, RNA phosphoramidites as 0.15 M solutions in acetonitrile. 5ꢀEthylthiotetrazole
was used as activator, 3% trichloroacetic acid in dichloromethane was used to deblock DMTr
groups, acetic anhydride in tetrahydrofuran (THF) and 16% Nꢀmethylimidazole in THF was used
to cap unreacted 5ʹꢀOH groups, and 0.1 M I2 in 1:2:10 pyridine/water/THF was used for
oxidation of phosphite triester linkages. DNA monomers were coupled for 200 s (300 s for dG);
all other phosphoramidites were coupled for 600 s (900 s for rG). All 2′,4ʹꢀmodified
phosphoramidites were coupled for 1200 s. Deprotection and cleavage from the solid support for
DNA oligonucleotides was achieved with concentrated aqueous ammonia for 48 h at room
temperature. After decanting to remove the CPG, the deprotection solution was removed under
vacuum in a SpeedVac lyophilizer. For RNAꢀcontaining oligonucleotides, deprotection and
cleavage from the solid support was achieved with 3:1 aqueous ammonia/EtOH for 48 h at room
temperature, and desilylation was achieved in neat TREATꢀHF (150 ꢂL) with shaking at room
temperature for 48 h. Purifications were performed by HPLC, using a Protein Pak DEAE 5PW
analytical anionexchange column. A stationary phase of MilliꢀQ water and a mobile phase of 1
M LiClO4 in water was used for analysis and purification using a gradient of 0−60% over 37
min. Following purification, excess LiClO4 salts were removed using NAPꢀ25 sephadex sizeꢀ
exclusion columns. Oligonucleotides were quantitated by UV (extinction coefficients were
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determined
using
the
IDT
OligoAnalyzer
tool
(www.idtdna.com/analyzer/Applications/OligoAnalyzer). Extinction coefficients for RNA were
used for oligonucleotides containing 2′,4ʹꢀmodifications.
Thermal Denaturation Studies. Equimolar amounts of complementary sequences were
combined, dried, and rediluted in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM
NaCl and 0.1 mM EDTA (1 mL). They were then transferred into cuvettes in a Varian UV
spectrophotometer. The samples were heated at 90 °C and the cooled to 5 °C. The change in
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absorbance at 260 nm was then monitored upon heating from 5 to 90 °C. Melting temperatures
were determined using the hyperchromicity method.
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ASSOCIATED CONTENT
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SUPPORTING INFORMATION
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1H, C, F, and P NMR spectra for compounds 1ꢀ22, some crystal structures, and additional
computational results such as relevant electronic effects, PMFs and puckering distributions for
selected nucleosides. Biological activity of nucleosides 11, 12, 20, and 23 against 60 cancer cell
lines from the Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute is included. Additional Tm data and mass analysis of
reported oligonucleotides. Available free of charge from the ACS website.
ACKNOWLEDGEMENTS
Financial support was provided by Natural Sciences and Engineering Research Council of
Canada (M.J.D. and N.M.), NSERC CHRP (M.J.D.), the McGill CIHR Drug Development
Training Program (E.M.A and S.M.M) CGS Master’s NSERC Scholarship (E.M.A.), as well as
Compute Canada for their generous CPU time allocations. We would also like to acknowledge
Dr. Francine Bélanger at the University of Montréal for performing the Xꢀray crystallography of
nucleosides.
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