Synthesis and Structural Characterization of 2'-Fluoro-α-L-RNA-Modified Oligonucleotides

Article abstract of DOI:10.1002/cbic.201100161

We describe the synthesis and binding properties of oligonucleotides that contain one or more 2'-fluoro-α-L-RNA thymine monomer(s). Incorporation of 2'-fluoro-α-L-RNA thymine into oligodeoxynucleotides decreased thermal binding stability slightly upon hybridization with complementary DNA and RNA with the smallest destabilization towards RNA. Thermodynamic data show that the duplex formation with 2'-fluoro-α-L-RNA nucleotides is enthalpically disfavored but entropically favored. 2'-Fluoro-α-L-RNA nucleotides exhibit very good base pairing specificity following Watson-Crick rules. The 2'-fluoro-α-L-RNA monomer was designed as a monocyclic mimic of the bicyclic α-L-LNA, and molecular modeling showed that this indeed is the case as the 2'-fluoro monomer adopts a C3'-endo/C2'-exo sugar pucker. Molecular modeling of modified duplexes show that the 2'-fluoro-α-L-RNA nucleotides partake in Watson-Crick base pairing and nucleobase stacking when incorporated in duplexes while the unnatural α-L-ribo configured geometry of the sugar is absorbed by changes in the sugar-phosphate backbone torsion angles. The duplex behavior of our new nucleotide follows that of α-L-LNA, by and large.

Full text of DOI:10.1002/cbic.201100161

DOI: 10.1002/cbic.201100161
Synthesis and Structural Characterization of 2’-Fluoro-a-l-
RNA-Modified Oligonucleotides
Troels Bundgaard Jensen, Anna Pasternak, Andreas Stahl Madsen, Michael Petersen, and
Jesper Wengel*[a]
We describe the synthesis and binding properties of oligonu-
cleotides that contain one or more 2’-fluoro-a-l-RNA thymine
monomer(s). Incorporation of 2’-fluoro-a-l-RNA thymine into
oligodeoxynucleotides decreased thermal binding stability
slightly upon hybridization with complementary DNA and RNA
with the smallest destabilization towards RNA. Thermodynamic
data show that the duplex formation with 2’-fluoro-a-l-RNA
nucleotides is enthalpically disfavored but entropically favored.
2’-Fluoro-a-l-RNA nucleotides exhibit very good base pairing
specificity following Watson–Crick rules. The 2’-fluoro-a-l-RNA
monomer was designed as a monocyclic mimic of the bicyclic
a-l-LNA, and molecular modeling showed that this indeed is
the case as the 2’-fluoro monomer adopts a C3’-endo/C2’-exo
sugar pucker. Molecular modeling of modified duplexes show
that the 2’-fluoro-a-l-RNA nucleotides partake in Watson–Crick
base pairing and nucleobase stacking when incorporated in
duplexes while the unnatural a-l-ribo configured geometry of
the sugar is absorbed by changes in the sugar–phosphate
backbone torsion angles. The duplex behavior of our new nu-
cleotide follows that of a-l-LNA, by and large.
Introduction
Chemically modified oligonucleotides (ONs) are broadly ap-
plied within the field of nucleic acid chemical biology to im-
prove properties, like resistance against enzymatic degradation
and binding affinity toward DNA and RNA.[1–3] Modification at
the 2’-position of the furanose ring has been a preferred strat-
egy towards increasing the RNA-binding affinity and nucleolyt-
ic resistance. Introducing a 2’-fluorine substituent instead of
the 2’-hydroxy group has been intensively studied as fluorine
and the hydroxy group are similar in size while their electrone-
gativity and hydrogen bond behavior are different.[4–6] Intro-
duction of 2’-deoxy-2’-fluoro-b-d-ribonucleic acid (2’-F-RNA)
monomers[7,8] into DNA ONs induces increased thermal stability
of duplexes formed with RNA complements; this is explained
by the fact that 2’-F-RNA is an RNA mimic and its furanose ring
adopts a C3’-endo pucker.[9,10] The use of 2’-F-RNA within anti-
sense mediated gene silencing has been limited by the fact
that 2’-F-RNA-modified antisense ONs, except the so-called
gapmers, are unable to induce RNase H-mediated cleavage of
an RNA target.[4,11] Inversion of the stereochemistry at the C2’-
position of 2’-F-RNA results in 2’-deoxy-2’-fluoro-b-d-arabino-
nucleic acid (2’-F-ANA; Figure 1).[6,7,12,13] Introduction of 2’-F-
ANA monomers into DNA ONs likewise induces increased ther-
mal stability of duplexes formed with DNA and RNA comple-
ments.[14] 2’-F-ANA is a DNA mimic as the furanose moiety
adopts an O4’-endo pucker;[13] this has stimulated its use in the
antisense field as 2’-F-ANA-modified antisense ONs induce
RNase H-mediated cleavage of a complementary RNA strand,
although cleavage rates are generally lower than with unmodi-
fied DNA ONs.[15,16]
Figure 1. Selected DNA-mimicking nucleotides. In Tables 1 and 2, the nota-
tions aLTL (a-l-LNA), aLT (a-l-RNA) and X (2’-F-a-l-RNA) are used for the three
different a-l-ribo configured monomers; T: thymin-1-yl.
has the same constitution as an LNA monomer with a 2’-O-4’-
C-methylene-linked furanose ring but inversed configuration at
three of its stereocenters. ONs containing a-l-LNA show ap-
pealing hybridization properties towards RNA comple-
[a] Dr. T. Bundgaard Jensen, Dr. A. Pasternak, Dr. A. Stahl Madsen,
Dr. M. Petersen, Prof. J. Wengel
Nucleic Acid Center, Department of Physics and Chemistry
University of Southern Denmark, Campusvej 55
5230 Odense M (Denmark)
Previously we have reported ONs containing the DNA-mim-
icking monomers a-l-LNA[17,18] and a-l-RNA[19,20] (Figure 1). An
a-l-LNA (a-l-ribo-configured locked nucleic acid) monomer
E-mail: jwe@ifk.sdu.dk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100161.
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2’-Fluoro-a-l-RNA-Modified Oligonucleotides
ments.[21,22] The furanose conformation of an a-l-LNA
monomer is N-type (C3’-endo, 3E) but it is a DNA
mimic as a consequence of the unnatural l configura-
tion.[23] NMR spectroscopic studies as well as molecu-
lar modeling simulations of partially and fully modi-
fied a-l-LNA:RNA duplexes have shown very similar
overall duplex geometry to the corresponding un-
modified DNA:RNA duplex, although some local rear-
rangements of the phosphate backbone were ob-
served to accommodate the unnatural a-l-LNA nu-
cleotides.[23–25] Despite the DNA-mimicking nature of
a-l-LNA, antisense ONs containing such monomers
induce only limited RNase H-mediated cleavage of
complementary RNA.[24] RNase H recognizes
a
DNA:RNA duplex by interacting within the minor
groove, and an NMR structure of a duplex between
an a-l-LNA containing DNA ON with the RNA com-
plement has revealed that the phosphate groups ad-
joining a-l-LNA nucleotides are rotated into the
minor groove, which likely results in suboptimal geo-
metries for RNA cleavage.[24] a-l-RNA monomers[19,20]
have the same constitution as a-l-LNA monomers
but lack the methylene group between the O2’ and
C4’ atoms. Thus, being more structurally flexible than
a-l-LNA, it was not surprising to observe that incor-
poration of a-l-RNA monomers into ONs induces de-
creased thermal binding affinity toward DNA and
RNA complements, although with a preferential bind-
ing towards complementary RNA as also observed
for a-l-LNA.[19,20] Interestingly, it was demonstrated
that DNA ONs containing a single a-l-RNA monomer
retain the ability to elicit RNase H activity.[19]
Scheme 1. Synthesis of 2’-F-a-l-RNA phosphoramidite derivative 8. Reagents and condi-
tions (yield): a) BSA, TMSOTf, thymine, MeCN, 808C (92%); b) BOMCl, DBU, DMF, room
temperature (94%); c) sat. methanolic ammonia, CH2Cl2, room temperature (99%); d) pyr-
idine, bis(methoxyethyl)aminosulfur trifluoride, toluene, room temperature (50%); e) i:
BCl3, CH2Cl2, À788C; ii: H2O, room temperature (77%); f) DMTrCl, pyridine, room temper-
ature (88%); g) diisopropylammonium tetrazolide, bis(N,N-diisopropylamino)-2-cyanoe-
thoxyphosphine, CH2Cl2, room temperature (94%); h) automated ON synthesis.
Inspired by the excellent RNA targeting potential of a-l-
LNA-modified ONs and their interesting behavior as outlined
above, we have now synthesized the 2’-F-a-l-RNA monomer
(Figure 1). This allows us to compare the effect of a monocyclic
a-l-configured sugar with the bicyclic a-l-LNA sugar and to
compare the effect of the 2’-fluorine substituent with a 2’-hy-
droxy group as found in a-l-RNA. To this end we have evaluat-
ed the thermostability of 2’-F-a-l-RNA-modified duplexes, de-
rived thermodynamic data for duplex formation and per-
formed molecular modeling at both the monomer and duplex
level.
troducing a BOM (benzyloxymethyl) group to give nucleoside
3. Cleavage of the O2’ ester was carried out by using saturated
methanolic ammonia to afford nucleoside 4, which was then
treated with deoxofluor [bis(2-methoxyethyl)aminosulfur tri-
fluoride] to give the fluorinated nucleoside 5 with the C2’ ste-
reocenter inverted. Removal of the protection groups was ach-
ieved by using BCl3 to give nucleoside 6, which was selectively
protected at the O5’-position by treatment with DMTrCl (4,4’-
dimethoxytritylchloride). The thus obtained nucleoside 7 was
eventually subjected to standard phosphitylation to afford the
desired phosphoramidite 8 in 27% overall yield calculated
from starting sugar 1. The configuration of compound 6 was
verified by a NOESY experiment (Figure S11 in the Supporting
Information). Cross-correlations were observed between H1’
and H2’ as well as H2’ and H3’ showing close proximity of
these atoms. As the stereoconfiguration at the 3’-position was
retained during the synthesis, this experiment verifies the a-l-
ribo configuration of compounds 6–8 and monomer X.
Results and Discussion
Synthesis
Phosphoramidite building block 8, suitable for incorporation of
2’-F-a-l-RNA monomer X into ONs, was synthesized
(Scheme 1). The known coupling sugar 1 was synthesized by
using a previously published procedure,[26] and subsequent
standard Vorbrꢁggen glycosylation afforded intermediate 2.
Fluorination was envisioned to be performed in a two-step in-
version reaction, and as pyrimidine nucleosides are known to
form 2,2’-anhydro intermediates, it was necessary to protect
the N3-position of the thymine unit. This was achieved by in-
Synthesis of ONs containing monomer X was performed on
the 0.2 mmol scale with an automated DNA synthesizer. Stan-
dard cycle protocols were used for unmodified phosphorami-
dites (2 min coupling time, 1H-tetrazole as activator) whereas
extended coupling time (15 min) was used for phosphorami-
dite 8, resulting in stepwise coupling yields above 95%. All
synthesized ONs were, if necessary, purified by RP-HPLC, and
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J. Wengel et al.
their composition and purity (>
80%) were confirmed by MALDI-
MS (Table S3 in the Supporting
Information) and ion-exchange
HPLC, respectively.
Table 1. Thermal denaturation temperatures (Tm values) of matched and singly mismatched duplexes.[a]
Tm [8C]
DNA: 3’-CACTBTACG
RNA: 3’-CACUBUACG
ON
Sequence
B=
A
T
G
C
A
U
G
C
ON1
ON2
5’-GTGATATGC
5’-GTGAXATGC
28.5
26.0
13.5
<5
20.0
14.5
13.5
<5
26.5
25.5
13.5
<5
22.0
18.0
<5
11.5
Thermal denaturation and ther-
modynamic studies
[a] The Tm values were measured as the maximum of the first derivatives of the melting curves (A260 vs. temper-
ature) recorded in a medium salt buffer (100 mm NaCl, 0.1 mm EDTA, 10 mm NaH2PO4, 5 mm Na2HPO4, pH 7.0)
by using 1.0 mm of each of the two complementary strands. The Tm values are averages of at least two meas-
urements.
Initially, the effect of a single in-
corporation of monomer X on
duplex stability was evaluated
by thermal denaturation studies
by using a medium salt buffer
(Table 1). A slight decrease in
thermostability was observed
against both DNA and RNA com-
plementary strands with mono-
mer X being tolerated better in
the RNA context. Furthermore,
mismatch discrimination studies
were carried out to investigate if
monomer X participates in base
pairing with the opposing nu-
cleotide (Table 1). Improved mis-
match discrimination relative to
the unmodified oligonucleotide
(ON1) was observed against
both DNA and RNA targets,
which shows that monomer X
partakes in Watson–Crick base
pairing.
Table 2. Thermal denaturation temperatures (Tm values)[a] and thermodynamic data.[b]
Tm [oC]
ÀDG837 [kcalmolÀ1
]
ON
Sequence
DNA
RNA
DNA
RNA
ON1
5’-GTGATATGC
28.5
30[c]
26.0
26[c]
34.5
<5
<5[c]
17.5
20[c]
<5
26.5
28[c]
25.5
28[c]
34.0
14.5
12[c]
13.5
19[c]
<5
6.82Æ0.16
6.29Æ0.15
ON2
ON3
ON4
ON5
ON6
ON7
5’-GTGAXATGC
5’-GTGA(aLT)ATGC
5’-GTGA(aLTL)ATGC
5’-GXGAXAXGC
5’-G(aLT)GA(aLT)A(aLT)GC
5’-T10
6.31Æ0.06
6.22Æ0.14
–
–
8.14Æ0.09
8.30Æ0.14
–
–
–
–
–
–
ON8
ON9
ON10
ON11
ON12
5’-X9T
–
–
–
–
–
–
–
–
–
–
5’-(aLT)9T
<5[c]
63[c]
<5
<5[c]
66[c]
<5
5’-(aLTL)9T
5’-[(X)(aLTL)]4(X)T
5’-[(aLT)(aLTL)]4(aLT)T
<5[c]
27[c]
[a] The Tm values were measured as the maximum of the first derivatives of the melting curves (A260 vs temper-
ature) recorded in a medium salt buffer (100 mm NaCl, 0.1 mm EDTA, 10 mm NaH2PO4, 5 mm Na2HPO4, pH 7.0)
by using 1.0 mm of each of the two complementary strands. The Tm values are averages of at least two meas-
urements. [b] For the thermodynamic data of helix formation, see Table S5 in the Supporting Information.
[c] From ref. [19]. aLTL: a-l-LNA; aLT: a-l-RNA; X: 2’-F-a-l-RNA.
In Table 2 is included a direct
comparison of the effect on
thermal denaturation tempera-
ture of monomers X and a-l-RNA (ON2, ON3, ON5 and ON6).
It is clear that substitution of the 2’-hydroxy group of an a-l-
RNA monomer with a 2’-fluoro substituent has no major influ-
ence on the thermal stability of the resulting duplexes.
X-modified duplex formation might be due to the constrained
sugar moiety but changes in solvation will also contribute to
this term. The less favorable enthalpic term is difficult to ex-
plain readily in terms of structure as our modeling of X-modi-
fied DNA duplexes (see below) show no large structural differ-
ences between modified and unmodified duplexes. The
change in enthalpy might be due to an unfavourable back-
bone geometry imposed by the a-l-configured sugar or small
changes in stacking interactions.
We next determined the thermodynamic parameters for the
9-mer ON1 and its variants modified with either X (ON2) or
a-l-LNA (ON4) hybridized with DNA and RNA complements
(Table 2 and Table S5 in the Supporting Information). It should
be noted that melting temperatures for the duplexes follow
the trend of Table 1, and in accordance with the literature a
single a-l-LNA modification yields a significant elevation in
melting temperature. Introduction of monomer X into the
DNA:DNA duplex decreased the thermodynamic stability by
0.51 kcalmolÀ1 relative to the unmodified duplex, whereas an
a-l-LNA monomer made the duplex more stable by 1.32 kcal
molÀ1. When hybridized with DNA, the X-modified ON2 result-
ed in a less enthalpically favored and less entropically disfa-
vored duplex relative to the unmodified all-DNA duplex
(Table S5 in the Supporting Information). A similar trend seems
to be observed for ON4 hybridized with DNA, however, here
the limits of errors for DH8 and DS8 values restrict further con-
clusions to be drawn. The less unfavorable entropy term of the
The thermodynamic picture is slightly different when the X
and a-l-LNA-modified oligonucleotides are hybridized with
RNA. The presence of monomer X within a DNA:RNA hetero-
duplex did not induce any significant change of Gibbs free
energy, whereas introduction of a-l-LNA within the DNA
strand of the DNA:RNA heteroduplex considerably improved
thermodynamic stability. Our modeling in this study and previ-
ous NMR structures show that the a-l-LNA nucleobases
appear to partake in efficient Watson–Crick pairing and stack-
ing without much change from unmodified duplexes. How-
ever, a quantitative energetic description would require high
level quantum mechanical calculations on base stacking and
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2’-Fluoro-a-l-RNA-Modified Oligonucleotides
backbone geometry, which is outside the scope of the present
study.
Finally, different combinations of the monomers were evalu-
ated in a homothymine 10-mer context. As reported earlier,
the almost fully modified a-l-LNA (ON10) displayed high ther-
mostability towards both DNA and RNA complements relative
to the unmodified reference (ON7).[19] In contrast, the corre-
sponding ONs containing 2’-F-a-l-RNA (ON8) or a-l-RNA
(ON9) monomers melted below 58C when hybridized with
DNA and RNA complements under medium salt conditions,
whereas duplexes of low stability were observed for ON8 hy-
bridized with DNA and RNA complements at high salt condi-
tions, again with preferential binding towards RNA (Table S4 in
the Supporting Information). ON12, containing alternating a-l-
RNA and a-l-LNA monomers displayed efficient hybridization
with RNA but no melting was observed with its DNA comple-
ment. No melting was observed for the corresponding ON11,
which contains 2’-F-a-l-RNA monomers, X, instead of a-l-RNA
monomers, not even with RNA or at high salt concentration
(Table S4 in the Supporting Information).
Figure 2. CD spectra of duplexes of ON2 hybridized with complementary
DNA and RNA as well as of unmodified reference duplexes.
with ON2 hybridized with DNA and RNA complements and
with ONs containing either an a-l-RNA or a-l-LNA monomer.
We used starting structures with standard B- and A-type
duplex geometries for the simulations of dsDNA duplexes and
DNA:RNA hybrids, respectively, as justified by CD spectroscopy.
All duplexes were subjected to molecular dynamics followed
by energy minimization of snapshots by using the AMBER
force field[28,29] and the GB/SA solvent model[30] as implement-
ed in MacroModel V.9.1.[31]
Modeling of monomers
In an attempt to explain the results from the thermal denatura-
tion and thermodynamic studies described above, we analyzed
the sugar conformations of the X monomer using quantum
mechanical calculations and compared it with the sugar con-
formations of a-l-RNA and a-l-LNA. Each of the three a-l-con-
figured nucleosides have a deep energy minimum for confor-
mations with a pseudorotational angle close to zero with a-l-
LNA showing the most shallow energy curve (data not shown).
Thus, all these nucleosides are constrained in the N-range of
the pseudorotation cycle. We also scanned the rotation profile
of the glycosidic angle c for the three a-l-configured nucleo-
sides and again the results were similar with a global minimum
near c=À1508 and a local high-energy minimum, DEꢀ5 kcal
molÀ1, near c=508. Having established conformational similari-
ties at the monomeric level we proceeded towards evaluating
the structural characteristics at the duplex level using CD for
overall conformational features and molecular modeling for
detailed atomic information (vide infra).
In agreement with the thermal denaturation experiments, all
simulations showed that the modified nucleotides are accom-
modated in the duplexes and partake in Watson–Crick base
pairing and nucleobase stacking. As expected from the CD
measurements, the global geometry of the modified duplexes
is close to the geometries obtained in the simulations of the
unmodified duplexes. The local geometry is, however, changed
as a consequence of the configuration of the sugars (Figure 3).
Circular dichroism curves
CD spectroscopy was employed to obtain insight into the heli-
cal structure of the 2’-F-a-l-RNA-modified duplexes (Figure 2).
ON2 hybridized with complementary DNA and RNA gave spec-
tra displaying the characteristic features of B-type and inter-
mediate A:B-type duplexes, respectively.[27] The unmodified
duplexes gave virtually identical spectra; this indicates that the
2’-F-a-l-RNA monomer does not perturb the global helical
structures despite its unnatural sugar configuration.
Figure 3. A) Duplexes formed with the DNA complement. Overlays of a
three-nucleotide excerpt containing 2’-F-a-l-RNA monomer X (multicolored)
with DNA (yellow), a-l-RNA (purple) and a-l-LNA (green). B) Corresponding
duplexes formed with the RNA complement. Overlays of a three-nucleotide
excerpt containing 2’-F-a-l-RNA monomer X (multicolored) with DNA
(yellow), a-l-RNA (purple) and a-l-LNA (green). For clarity, all hydrogen
atoms and sodium ions are omitted.
Molecular modeling of duplexes
To obtain an atomic insight into the features of the a-l-modi-
fied duplexes, we performed molecular dynamic simulations
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J. Wengel et al.
There is limited change in the positions of the nucleobases of
the modified nucleotides. This demonstrates that base pairing
and stacking govern the position of the nucleobases while the
malleable sugar–phosphate backbone is left to alter its geome-
try so as to present the bases in optimum positions for base
pairing and stacking. This is similar to what has been observed
in NMR structures of a-l-LNA-modified duplexes.[24,25] First of
all, the glycosidic angles of the modified nucleotides change
from approximately À1408 in the unmodified duplexes to
values near À1808 in the modified nucleotides with smaller
changes observed for 2’-F-a-l-RNA and a-l-RNA monomers in
a dsDNA context. This trend follows the results obtained by
modeling at nucleoside level albeit the exact values differ.
The sugar puckers of the l nucleosides are of the N type.
The 2’-F-a-l-RNA and a-l-RNA monomers show identical puck-
ering (C3’-endo/C2’-exo) whereas the a-l-LNA monomer display
a C3’-endo puckering as reported earlier.[23] Again this follows
the trend of our quantum mechanical nucleoside calculations
described above, and validates the simulation as carried out in
the MacroModel environment.
Conclusions
We have synthesized the 2’-fluoro-a-l-ribofuranose thymine
phosphoramidite building block 8 in 27% overall yield and
used it for incorporation of the 2’-F-a-l-RNA monomer X into
oligonucleotides. A single incorporation of X is well tolerated
in duplexes, although comparison with unmodified reference
duplexes revealed a slight decrease in stability when hybrid-
ized with complementary DNA and RNA. Substitution of the 2-
hydroxy group of an a-l-RNA monomer to a 2’-fluorine sub-
stituent did not induce a stabilizing effect in a duplex context.
Three incorporations of monomer X induce a large decrease in
duplex stability, with a preferential binding towards an RNA
complementary strand. Molecular modeling showed that the
conformation of the furanose ring of monomer X and a-l-RNA
thymine adopt a C3’-endo/C2’-exo pucker whereas the a-l-LNA
thymine adopts a C3’-endo pucker. Moreover, in order to com-
pensate for the unusual configuration of monomer X, the
phosphate backbone must undergo structural changes. In
summary, the novel 2’-F-a-l-RNA monomer displays many of
the same characteristics as the corresponding a-l-RNA and a-
l-LNA monomers, but because of its duplex destabilizing
effect it cannot be considered a monocyclic version of an a-l-
LNA monomer.
As seen in Figure 3, the conformation of the sugar–phos-
phate backbone is dramatically altered in the modified duplex-
es as compared to the unmodified duplexes. Common
changes are in the g-, d- and z-torsion angles from (gauche+,
gauche+ and gaucheÀ) to (trans, gaucheÀ and gauche+),
respectively. Similar changes were observed in NMR structures
of a-l-LNA-modified duplexes.[24,25] Moreover, changes in the
a- and b-torsion angles were observed for the 2’-F-a-l-RNA nu-
cleotide, monomer X and the a-l-RNA monomer when hybrid-
ized with DNA. Furthermore, changes in the backbone torsion
angles of the 3’-flanking nucleotides were also observed. In
the unmodified duplexes, the a- and g-torsion angles adopt a
(gaucheÀ, gauche+) conformation, which is the conformation
with the lowest energy and consequently the most populat-
ed.[32] However, in the duplexes containing the three a-l-ribo
configured monomers, the a- and g-torsion angles adopt a
(gauche+, trans) conformation. Such changes have previously
been observed for a-l-LNA-modified duplexes.[24,25]
Experimental Section
1-(2-O-Acetyl-3,5-di-O-benzyl-a-l-arabinofuranosyl)thymine (2):
Compound 1[26] (1.85 g, 4.47 mmol) and thymine (1.13 mg,
8.94 mmol) were coevaporated with anhyd. MeCN (2ꢂ10 mL) and
resuspended in anhyd. MeCN (25 mL) followed by addition of N,O-
bis-(trimethylsilyl)acetamide (BSA, 3.9 mL, 15.6 mmol) while being
stirred at room temperature. The resulting suspension was refluxed
until it became a clear solution (>45 min) and was then cooled to
room temperature. Trimethylsilyl triflate (TMSOTf, 2.0 mL,
11.2 mmol) was added and the mixture was heated to 808C for
25 h. The reaction mixture was cooled to room temperature and
poured into sat. aq. NaHCO3 (30 mL) and the resulting mixture was
extracted with CH2Cl2 (4ꢂ100 mL). The combined organic phase
was dried (Na2SO4), filtered, and evaporated to dryness under re-
duced pressure. The residue was purified by silica gel column chro-
matography by using as eluent EtOAc (0–100%) in PE to afford
To summarize, all l nucleotides incorporated into ONs dis-
played a pronounced N-type sugar conformation as well as an
anti conformation of the glycosidic torsion angle. Furthermore,
rearrangements of the phosphate backbone were observed to
accommodate the unnatural l nucleotides, as well as rear-
rangements of the backbone torsion angles the 3’-flanking nu-
cleotide.
1
compound 2 as a white foam (1.97 mg; 92%). H NMR (400 MHz,
CDCl3): d=8.53 (s, 1H; NH), 7.36–7.05 (m, 11H; Ph and H6), 6.05 (d,
J=2.3 Hz, 1H; H1’), 5.22 (t, J=1.9 Hz, 1H; H2’), 4.67–4.36 (m, 5H;
2ꢂOCH2Ph and H4’), 4.02 (t, J=2.1 Hz, 1H; H3’), 3.51 (dd, J=10.1,
5.7 Hz, 1H; H5a’), 3.44 (dd, J=10.1, 6.5 Hz, 1H; H5’b), 1.92 (s, 3H;
Me), 1.73 (d, J=1.2 Hz, 3H; Me); 13C NMR (101 MHz, CDCl3): d=
168.6, 162.5, 149.2, 136.5, 135.8, 134.9, 127.5, 127.5, 127.2, 126.9,
126.8, 126.8, 109.9 (C5), 88.6 (C1’), 84.1 C4’), 81.2 (C3’), 79.5 (C2’),
72.5 (CH2), 71.1 (CH2), 68.1 (C5’), 19.7 (Me), 11.4 (Me); elemental
analysis calcd (%) for C26H28N2O7: C 64.99, H 5.87, N 5.83; found: C
For the corresponding 2’-F-ANA derivative, the presence of a
weak interaction between the 2’-F substituent and a nucleo-
base has been reported to be one of the causes of stabilization
of the duplexes observed by incorporation of 2’-F-ANA nucleo-
tides.[6] To what extent a similar interaction takes place with
the 2’-F-a-l-RNA nucleotide X is unclear but the similar duplex
stabilities obtained for monomer X and the corresponding a-l-
RNA nucleotide (Table 2) suggest such an interaction to be
very weak at best.
64.79,
H 5.80, N
5.70; ESI-HRMS: m/z 503.1798 ([M+Na]+,
C26H28N2O7·Na calcd 503.1789).
1-(2-O-Acetyl-3,5-di-O-benzyl-a-l-arabinofuranosyl)-3-(benzylox-
ymethyl)thymine (3): Compound 2 (1.50 g, 3.12 mmol) was dis-
solved in anhyd. DMF (30 mL) and the resulting mixture was stirred
at
08C.
1,8-Diazabicyclo[5.4.0]undec-7-ene
(DBU,
467 mL,
3.12 mmol) was added followed by dropwise addition of benzylox-
ymethyl chloride (BOMCl, 520 mL, 3.74 mmol). The reaction mixture
1908
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1904 – 1911

2’-Fluoro-a-l-RNA-Modified Oligonucleotides
was stirred for 2 h whereupon sat. aq. NaHCO3 (50 mL) was added.
The reaction mixture was stirred for additional 10 min followed by
extraction with EtOAc (150 mL). The organic phase was washed
with H2O (5ꢂ100 mL). The organic phase was dried (Na2SO4), fil-
tered and evaporated to dryness under reduced pressure. The resi-
due was purified by silica gel column chromatography by using as
eluent EtOAc (0–50%) in PE to afford compound 3 as a colorless
CDCl3): d=À185.5; elemental analysis calcd (%) for
C32H33FN2O6·1= EtOAc: C 68.13, H 6.03, N 4.84; found: C 67.77, H
5
5.65, N 4.69; ESI-HRMS: m/z 583.2219 ([M+Na]+, C32H33FN2O6·Na
calcd 583.2215).
1-(2-Deoxy-2-fluoro-a-l-ribofuranosyl)thymine (6): Compound 5
(720 mg, 1.28 mmol) was dissolved in anhyd. CH2Cl2 (50 mL) and
the solution was stirred and cooled to À788C by using dry ice and
acetone. Boron trichloride (1.0m solution in CH2Cl2, 10.2 mL,
10.2 mmol) was added dropwise and the reaction mixture was
stirred for 30 min at À788C. Afterwards, MeOH (25 mL) was added
and the resulting mixture was allow to warm to room temperature.
The reaction mixture was evaporated to dryness under reduced
pressure and the residue was redissolved in H2O (50 mL) and the
resulting mixture was stirred for 22 h at room temperature. The
mixture was then evaporated to dryness under reduced pressure
and the residue was coevaporated with anhyd. MeCN (3ꢂ20 mL).
The residue was purified by silica gel column chromatography by
using as eluent MeOH (10%) in CH2Cl2 to afford nucleoside 6 as a
white foam (258 mg; 77%). 1H NMR (400 MHz, CD3CN): d=9.21
(brs, 1H; NH), 7.37 (dd, J=2.3, 1.1 Hz, 1H; H6), 5.95 (dd, J=16.7,
2.6 Hz, 1H; H1’), 5.15 (dt, J=51.4, 2.8 Hz, 1H; H2’), 4.40–4.26 (m,
2H; H3’ and H4’), 3.85 (d, J=4.1 Hz, 1H; 3’-OH), 3.65–3.54 (m, 2H;
H5’), 3.06 (t, J=5.9 Hz, 1H; 5’-OH), 1.83 (d, J=1.2 Hz, 3H; Me);
13C NMR (101 MHz, CD3CN): d=164.8 (C=O), 151.6 (C=O), 137.7
(C6), 111.1 (C5), 101.0 (d, J=184.9 Hz, C2’), 90.8 (d, J=37.0 Hz, C1’),
88.6 (d, J=3.8 Hz, C4’), 74.9 (d, J=24.8 Hz, C3’), 62.1 (d, J=1.8 Hz,
C5’), 12.5 (Me); 19F NMR (376 MHz, CD3CN): d=À189.33; elemental
analysis calcd (%) for C10H13FN2O5: C 46.16, H 5.04, N 10.77; found:
C 45.78, H 4.95, N 10.50; ESI-HRMS: m/z 283.0700 ([M+Na]+,
C10H13FN2O5·Na calcd 283.0701).
1
oil (1.78; 94%). H NMR (400 MHz, CDCl3): d=7.39–7.21 (m, 16H;
Ph and H6), 6.13 (d, J=1.9 Hz, 1H; H1’), 5.46 (q, J=9.7 Hz, 2H;
NCH2), 5.29 (d, J=8.7 Hz, 1H; H2’), 4.70–4.48 (m, 7H; H4’ and 3ꢂ
OCH2), 4.07 (s, 1H; H3’), 3.58 (dd, J=10.0, 5.7 Hz, 1H; H5’a), 3.51
(dd, J=10.0, 6.6 Hz, 1H; H5’b), 1.99 (s, 3H; Me), 1.82 (s, 3H; Me);
13C NMR (101 MHz, CDCl3): d=169.6 (C=O), 163.4 (C=O), 151.0 (C=
O), 138.0 (Ph), 137.6 (Ph), 136.9 (Ph), 134.6 (C6), 128.5 (Ph), 128.3
(Ph), 128.1 (Ph), 128.0 (Ph), 127.8 (Ph), 127.7 (Ph), 127.6 (Ph), 110.2
(C5), 90.5 (C1’), 85.4 (C4’), 82.3 (C3’), 80.5 (C2’), 73.5(CH2), 72.1(CH2),
72.0 (CH2), 70.5 (CH2), 69.2 (C5’), 20.7 (Me), 13.2 (Me); elemental
analysis calcd (%) for C34H36N2O8·1= H2O: C 66.98, H 6.12, N 4.59;
2
found: C 66.61, H 5.80, N 4.29; ESI-HRMS: m/z 623.2350 ([M+Na]+,
C34H38N2O8·Na calcd 623.2364).
1-(3,5-Di-O-benzyl-a-l-arabinofuranosyl)-3-(benzyloxymethyl)-
thymine (4): Compound 3 (1.70 g, 2.83 mmol) was dissolved in
CH2Cl2 (20 mL) and sat. methanolic ammonia (60 mL) was added
and the reaction mixture was stirred at room temperature for 4 h.
The solution was evaporated to dryness under reduced pressure.
The residue was purified by silica gel column chromatography by
using as eluent EtOAc (0–50%) in PE to give compound 4 as a
1
white foam (1.56 g; 99%). H NMR (400 MHz, CDCl3): d=7.42–7.17
(m, 16H; Ph and H6), 5.94 (d, J=1.5 Hz, 1H; H1’), 5.49 (q, J=
9.7 Hz, 2H; NCH2), 4.72–4.48 (m, 7H; H4’ and 3ꢂOCH2), 4.29 (d, J=
8.6 Hz, 1H; H2’), 4.18 (d, J=8.6 Hz, 1H; 2’-OH), 4.05–4.02 (m, 1H;
H3’), 3.75 (dd, J=10.6, 2.9 Hz, 1H; H5’a), 3.65 (dd, J=10.6, 3.3 Hz,
1H; H5’b), 1.82 (s, 1H; Me); 13C NMR (101 MHz, CDCl3): d=163.5
(C=O), 151.4 (C=O), 138.0, 136.9, 136.6, 134.3 (C6), 128.7, 128.6,
128.3, 128.3, 128.1, 128.0, 127.7, 127.7, 127.6, 109.4 (C5), 94.6 (C1’),
85.7 (C4’), 84.8 (C3’), 80.4 (C2’), 74.0 (CH2), 72.3 (CH2), 72.0 (CH2),
70.5, 70.3 (C5’), 13.2 (Me); elemental analysis calcd (%) for
C32H34N2O7: C 68.80, H 6.13, N 4.69; found: C 68.50, H 5.76, N 4.69;
ESI-HRMS: m/z 581.2279 ([M+Na]+, C32H34N2O7·Na calcd 581.2259).
1-(2-Deoxy-5-O-(4,4’-dimethoxytrityl)-2-fluoro-a-l-ribofurano-
syl)thymine (7): Compound 6 (229 mg, 0.88 mmol) was coevapo-
rated with anhyd. pyridine (2ꢂ5 mL) and redissolved in anhyd.
pyridine (5 mL). 4,4’-Dimethoxytrityl chloride (DMTrCl, 386 mg,
1.14 mmol) was added and the resulting mixture was stirred for
3 h at room temperature. EtOH (0.5 mL) was added and stirring
was continued for additional 10 min followed by evaporation to
dryness under reduced pressure. The residue was redissolved in
CH2Cl2 (100 mL) and washed successively with sat. aq. NaHCO3
(25 mL) and brine (25 mL). The organic phase was dried (Na2SO4),
filtered and evaporated to dryness under reduced pressure. The
residue was purified by silica gel column chromatography by using
as eluent EtOAc (50–100%) in PE to afford nucleoside 7 as a white
1-(2-Deoxy-3,5-di-O-benzyl-2-fluoro-a-l-ribofuranosyl)-3-(benzyl-
oxymethyl)thymine (5): Compound 4 (1.50 g, 2.69 mmol) was co-
evaporated with anhyd. toluene (2ꢂ20 mL) and redissolved in
anhyd. toluene (25 mL) and the resulting mixture was stirred at
room temperature. Anhyd. pyridine (1.1 mL, 13.5 mmol) and
deoxo-fluor (50% in toluene, 3.9 mL, 10.8 mmol) were added and
the reaction was stirred for 20 h at room temperature. The reaction
mixture was diluted with EtOAc (100 mL) and washed successively
with sat. aq. NaHCO3 (50 mL) and brine (50 mL). The organic phase
was dried (Na2SO4), filtered and evaporated to dryness under re-
duced pressure. The residue was purified by silica gel column chro-
matography by using as eluent EtOAc (0–50%) in PE to afford
1
foam (448 mg; 88%). H NMR (400 MHz, CDCl3): d=9.53 (brs, 1H;
NH), 7.49–7.41 (m, 2H), 7.37–7.17 (m, 8H), 6.86–6.78 (m, 4H), 5.80
(d, J=17.8 Hz, 1H; H1’), 5.32 (d, J=50.2 Hz, 1H; H2’), 4.53–4.38 (m,
2H; H3’ and H4’), 3.78 (s, 6H; 2ꢂOMe), 3.73 (d, J=5.7 Hz, 1H;
3’OH), 3.31 (dd, J=9.0, 6.2 Hz, 1H; H5’a), 3.24 (dd, J=10.2, 5.3 Hz,
1H; H5’b), 1.89 (d, J=1.1 Hz, 3H; Me); 19F NMR (376 MHz, CDCl3):
d=À181.4; elemental analysis calcd (%) for C31H31FN2O7·1= EtOAc:
4
C 65.74, H 5.69, N 4.79; found: C 65.38, H 5.63, N 4.75; ESI-HRMS:
m/z 585.2010 ([M+Na]+, C31H31FN2O7·Na calcd 585.2008).
1
compound 5 as a colorless oil (775 mg; 50%). H NMR (400 MHz,
CDCl3): d=7.39–7.18 (m, 15H; Ph), 7.15 (d, J=1.2 Hz, 1H; H6), 6.11
(dd, J=16.2, 1.3 Hz, 1H; H1’), 5.48 (q, J=9.7 Hz, 2H; NCH2), 5.17
(dt, J=49.8, 1.4 Hz, 1H; H2’), 4.71–4.54 (m, 7H; H4’ and 3ꢂOCH3),
4.26–4.18 (m, 1H; H3’), 3.62 (ddd, J=10.1, 6.0, 1.8 Hz, 1H; H5’a),
3.54 (ddd, J=10.1, 6.4, 1.2 Hz, 1H; H5’b), 1.83 (d, J=1.2 Hz, 3H;
Me); 13C NMR (101 MHz, CDCl3): d=163.3 (C=O), 150.9 (C=O),
138.0, 137.6, 136.4, 134.6 (C6), 128.6, 128.5, 128.4, 128.3, 128.3,
127.9, 127.8, 127.8, 127.7, 109.8 (C5), 97.7 (d, J=187.6 Hz, C2’), 91.1
(d, J=37.1 Hz, C1’), 85.5 (C4’), 81.6 (d, J=25.5 Hz, C3’), 73.6, 72.3,
72.3, 70.5, 69.2 (d, J=2.6 Hz, H5’), 13.1 (Me); 19F NMR (376 MHz,
1-(3-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-2-deoxy-5-
O-(4,4’-dimethoxytrityl)-2-fluoro-a-l-ribofuranosyl)thymine (8):
Compound 7 (104 mg, 0.18 mmol) was coevaporated with 1,2-DCE
and redissolved in anhyd. CH2Cl2 (5 mL) and the resulting mixture
was stirred under argon at room temperature. Diisopropyl ammo-
nium tetrazolide (46 mg, 0.27 mmol) and bis(N,N-diisopropylami-
no)-2-cyanoethoxyphosphine (85 mL, 0.27 mmol) were added and
stirring was continued for 18 h at room temperature. The resulting
mixture was diluted with sat. aq. NaHCO3 (25 mL) and extracted
with CH2Cl2 (2ꢂ50 mL). The combined organic phase was dried
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1909

J. Wengel et al.
(Na2SO4), filtered and evaporated to dryness under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy by using as eluent EtOAc (30–40%) in PE to afford phosphora-
midite 8 as a white foam (129 mg; 94%); 31P NMR (162 MHz,
CDCl3): d=152.2, 151.3; elemental analysis calcd (%) for
Quantum mechanical calculations: Ab initio quantum mechanical
calculations were carried out by using the Gaussian 03 program.[33]
To determine the potential energy of pseudorotation, sugar torsion
angles n2 and n4 were calculated by using the theory of Altona and
Sundaralingam with a maximal puckering amplitude of 368.[34] For
each point along the pseudorotation pathway, full geometry opti-
mizations (MP2/6-31G) were carried out while maintaining the
desired n2 and n4 angles and constraining the e torsion angle to a
value consistent with duplex geometry. Single point energies were
determined by using second-order Møller–Plesset theory (MP2)
with the cc-pVTZ basis set.
C40H48FN4O8P·1= EtOAc: C 62.93, H 6.36, N 7.30; found: C 62.93, H
19
6.43, N 6.91; ESI-HRMS: m/z 785.3088 ([M+Na]+, C40H48FN4O8P·Na
calcd 785.3086).
General procedure for synthesis and purification of oligonucleo-
tides: Synthesis of ONs was performed on the 0.2 mmol scale by
using an automated DNA synthesizer. Standard cycle procedures
were applied for unmodified phosphoramidites by using a solution
of 1H-tetrazole (0.45m) as activator. Stepwise coupling yields, as
determined by a spectrophotometric DMTr+ assay, were >99% for
standard phosphoramidites and >95% (15 min coupling time) for
phosphoramidite 8. Removal of the nucleobase protection groups
and cleavage from the solid support was effected by using stan-
dard conditions (32% aqueous ammonia for 12 h at 558C). Un-
modified DNA and RNA strands were obtained from commercial
suppliers and, if necessary, further purified as described below. Pu-
rification of ONs was performed by DMTr-ON RP-HPLC by using
the Waters system 600 equipped with Xterra MS C18 column
(5 mm, 150ꢂ7.8 mm) and a precolumn Xterra MS C18 column
(5 mm, 10ꢂ7.8 mm) by using the representative gradient protocol
depicted in Table S1 in the Supporting Information. Fractions con-
taining pure ONs were collected and evaporated, followed by de-
tritylation (80% aq. AcOH, 20 min), precipitation (acetone, À188C,
12 h) and washing with acetone. The composition of ONs was veri-
fied by MALDI-MS analysis (Table S3 in the Supporting Informa-
tion), whereas the purity (>80%) was verified by ion-exchange
HPLC by using a LaChrome L-7000 system (VWR International)
equipped with a Dionex DNAPac Pa-100 (250ꢂ4.0 mm) by using
the representative gradient protocol shown in Table S2 in the Sup-
porting Information.
Molecular dynamics calculations: The unmodified DNA:DNA
duplex was constructed with a standard B-type helical geometry
and the DNA:RNA duplex was constructed with a standard A-type
helical geometry. These duplexes were subsequently modified to
the duplexes of interest by using the MacroModel V9.1 suite of
programs.[31] Furthermore, the negative charge of the phospho-
diester was neutralized with sodium ions, which were placed 3.0 ꢃ
from the two none-bridging oxygen atoms. The model structures
were energy minimized, followed by 5 ns stochastic dynamics sim-
ulations during which 1000 structures were collected (300 K, 2 fs
time step, SHAKE constraints on all bonds to hydrogen) and these
were subsequently energy minimized. The simulations were carried
out by using the AMBER force field[28,29] and the GB/SA solvent
model.[30] The collected structures were used to calculate the sugar
pucker, P, the glycosidic torsion angle and the backbone torsion
angles. In all calculations, the first 100 structures generated were
discarded, as the respective duplexes were not in equilibrium.
Acknowledgements
We gratefully thank The Danish National Research Foundation
for funding.
General procedure for thermal denaturation studies: Concentra-
tions of ONs were calculated by using the following extinction co-
efficients (OD260 [mmol]): DNA: A (15.2), G (12.0), T (8.4), and C (7.1);
RNA: A (15.4), G (13.7), U (10.0), and C (9.0). Concentrations of
modified ONs were calculated as for unmodified DNA strands. ONs
(1.0 mmol of each strand) were thoroughly mixed in a Tm buffer
(medium salt buffer: 100 mm NaCl, 0.1 mm EDTA, pH 7.0 adjusted
with 10 mm NaH2PO4/5 mm Na2HPO4; high salt buffer: 700 mm
NaCl, 0.1 mm EDTA, pH 7.0 adjusted with 10 mm NaH2PO4/5 mm
Na2HPO4), denatured by being heated to 708C and subsequently
cooled to the starting temperature of the experiment. Quartz opti-
cal cells with a path-length of 1.0 cm were used. Thermal denatura-
tion curves (A260 vs. temperature) were obtained on a Perkin–Elmer
Lambda 35 UV/Vis spectrometer equipped with a PTP-6 Peltier
temperature programmer. Thermal denaturation temperatures (Tm
values) were determined as the maximum of the first derivative of
the melting curve. A temperature ramp of 1.08C was used in all
experiments. Reported thermal denaturation temperatures are an
average of two measurements within Æ1.08C.
Keywords: ab initio calculations · conformation analysis · l-
nucleosides · l-nucleotides · molecular modeling
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Received: March 11, 2011
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Published online on July 4, 2011
ChemBioChem 2011, 12, 1904 – 1911
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