Synthesis, Duplex Stabilization and Structural Properties of a Fluorinated Carbocyclic LNA Analogue

Full text of DOI:10.1002/cbic.201200669

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DOI: 10.1002/cbic.201200669
Synthesis, Duplex Stabilization and Structural Properties
of a Fluorinated Carbocyclic LNA Analogue
Punit P. Seth,*[a] Pradeep S. Pallan,[b] Eric E. Swayze,[a] and Martin Egli[b]
Fluorine enjoys a privileged status in medicinal
chemistry by virtue of its “polar hydrophobic”
nature.[1] Fluorine has roughly the same atomic
radius as hydrogen but it is highly electronegative.
In the context of oligonucleotide medicinal chemis-
try, fluorine has been used as an isostere for the 2’-
oxygen atom in RNA and also as an isostere for one
of the 2’-hydrogen atoms in DNA, as exemplified by
2’-fluoro RNA (FRNA, 1) and 2’-fluoro arabino nucleic
acid (FANA, 2) respectively (Scheme 1). DNA oligonu-
cleotides modified with FRNA and FANA, show im-
proved duplex thermal stability when paired with
RNA complements relative to unmodified DNA.
However, while FRNA increases oligonucleotide
duplex thermal stability by increasing the strength
of the Watson–Crick base-pairing and nucleobase-
stacking interactions,[2] improved thermal stability of
FANA-modified duplexes results from the formation
of pseudo H···F bonds at pyrimidine–purine steps.[3]
Recent reports have unequivocally shown the
beneficial effects of FRNA substitution on the biolog-
ical properties of modified oligonucleotides for sev-
eral antisense mechanisms.[4] We showed that intro-
ducing fluorine in an axial or equatorial orientation
at the 3’-position of hexitol nucleic acid (HNA) pro-
vided analogues FHNA (3) and Ara-FHNA (4) respec-
tively.[5] In thermal denaturation experiments, 3 had
Scheme 1. Structures and relative duplex stabilization properties of DNA oligonucleo-
tides modified with various fluorinated and cLNA analogues and paired with comple-
mentary RNA.
a stabilizing effect upon duplex thermostability
whereas 4 was destabilizing. Using crystal structure
data, we showed that the axial fluorine atom in 3
proved activity in animals in comparison to a sequence-
matched 2’-O-methoxyethyl ASO with comparable RNA affini-
ty.[6] In both cases, the improved activity with the fluorinated
ASOs was attributed to improved functional uptake of the
modified ASOs in liver tissue.
projects into the minor groove where it is well tolerated. In
contrast, the equatorial fluorine in 4 experiences a steric clash
with the electronegative 4’-oxygen atom of the 3’-adjacent nu-
cleotide resulting in duplex instability. In animal experiments,
FHNA-modified antisense oligonucleotides (ASOs) showed ex-
cellent activity for downregulating gene expression in mouse
liver despite lower RNA affinity as compared to a benchmark
locked nucleic acid (LNA, 6) ASO control. We also showed that
2’-fluorocyclohexenyl nucleic acid (F-CeNA, 5) behaves like
a nuclease-stable mimic of 1 and exhibits significantly im-
Given the excellent antisense activity observed with ASOs
containing fluorinated nucleoside building blocks, we wanted
to study the impact of introducing fluorine on the [2.2.1]dioxa-
bicyclo ring scaffold of locked nucleic acid (LNA, 6).[7] We envis-
aged that ASOs modified with such building blocks could have
the dual advantage of high RNA affinity afforded by the LNA
scaffold combined with the beneficial effects of fluorine substi-
tution for facilitating tissue uptake. We further anticipated that
the 2’,4’-bridge in LNA was an ideal position for incorporating
fluorine as this would position the appended fluorine atom in
the same general orientation (into the minor groove) as the 3’-
fluorine atom in 3. However, it is not feasible to introduce a flu-
orine atom directly on the 2’,4’-bridging group in LNA due to
the synthetic difficulties associated with assembling a bicyclic
geminal fluorinated ether. To circumvent these issues, we
[a] Dr. P. P. Seth, Dr. E. E. Swayze
Department of Medicinal Chemistry, Isis Pharmaceuticals
2855 Gazelle Court, Carlsbad, CA 92010 (USA)
E-mail: pseth@isisph.com
[b] Dr. P. S. Pallan, Prof. M. Egli
Department of Biochemistry, Vanderbilt University School of Medicine
Nashville, TN 37232-0146 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200669.
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examined the feasibility of introducing fluorine on the 2’,4’-
bridge of carbocyclic LNA (cLNA, 7) where the 2’-oxygen atom
in LNA is replaced with a carbon atom (Scheme 1).
cLNA represents an interesting class of nucleic acid ana-
logues of which several members have been synthesized by
the Chattopadhyaya group.[8] The majority of cLNA analogues
reported in the literature show slight to significant decrease in
binding affinity for complementary nucleic acids relative to
LNA. The RNA affinity of cLNA-modified oligonucleotides can
be partially restored by introducing polar functionality on the
2’,4’-bridge of these analogues.[9] The polar functionality helps
to restore the water of hydration network in the minor groove
which is important for maintaining the stability of oligonucleo-
tide duplexes.[10] Interestingly, the cLNA analogue 8, in which
the 2’-oxygen atom was replaced with a exocyclic methylene
group, showed LNA-like duplex stabilization properties while
the corresponding saturated analogues (9 and 10) were less
stabilizing.[11] Using crystal structure data, we showed that the
exocyclic methylene group retains net negative electrostatic
potential at the brim of the minor groove and participates in
CH···O type interactions with acceptor molecules in the minor
groove of the modified duplex. In contrast, the saturated cLNA
analogues 9 and 10 were unable to extend the water of hydra-
tion network around the backbone phosphates and the nucle-
obases resulting in diminished duplex stabilizing properties.
Given this background, we hypothesized that a fluorinated
cLNA analogue such as 11 (Scheme 1) could exhibit improved
duplex stabilizing properties as a consequence of introducing
a highly electronegative fluorine atom in the vicinity of the 2’-
carbon atom of the nucleoside furanose ring.[2a] In addition, oli-
gonucleotides modified with such building blocks could bene-
fit from fluorine substitution for potential therapeutic applica-
tions. In this report, we describe the synthesis, biophysical and
structural evaluation of oligonucleotides modified with a fluori-
nated cLNA analogue 11.
Synthesis of the fluorinated cLNA phosphoramidite 19 was
initiated from the known nucleoside intermediate 11
(Scheme 2).[11] Attempts to cleave the exocyclic double bond in
11 using osmium tetroxide and sodium periodate gave poor
conversions to the intermediate ketone. Presumably, the bulky
tert-butyldiphenylsilyl (TBDPS) and 2-naphthylmethyl (Nap)
protecting groups on the 5’- and the 3’-hydroxyl groups create
steric crowding and limit access of the catalyst to the exocyclic
methylene group in 11. To address this, the TBDPS group was
removed using tetra-n-butylammonium fluoride (TBAF) and
the 5’-hydroxyl group was re-protected as the acetate ester to
provide 12. In the absence of the 5’-O-TBDPS protecting
group, oxidative cleavage of the double bond was accom-
plished using osmium tetroxide and sodium periodate in mod-
erate yield (~50% for two steps). Attempts to improve the
yield by optimizing the reaction conditions were unsuccessful
because of oxidative decomposition caused by reaction of
osmium tetroxide with the C5=C6 double bond in the uracil
nucleobase. The intermediate ketone was typically not isolated
but directly reduced with sodium borohydride in methanol at
ꢀ788C to yield the alcohol 13 as a single diastereoisomer. Pre-
sumably, attack of the hydride nucleophile occurs from the
Scheme 2. Synthesis of (R)-F-cLNA Uracil phosphoramidite 19. a) TBAF, THF,
RT, 16 h, 63%; b) Ac2O, pyridine, RT, 16 h, 95%; c) OsO4, NaIO4, 1,4-dioxane,
H2O, RT, 16 h; d) NaBH4, MeOH, -788C to RT, 16 h, 46% (2 steps); e) CF3-
(CF2)3SO2F, DBU, THF; f) CsF, tBuOH, 508C, 4 h; g) DAST, CH2Cl2, 08C, 3 h, 10%;
h) DDQ, CH2Cl2, RT, 16 h, 65%; i) NH3, MeOH, RT, 3 h; j) DMTrCl, pyridine, RT,
16 h, 70% (2 steps); k) 2-Cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiami-
dite, DMF, NMI, 1H-tetrazole, RT, 8 h, 73%.
less hindered re face of the intermediate ketone away from the
bulky 3’-O-Nap protecting group.
Fluorination of the secondary hydroxyl group in 13 was very
problematic. Use of nonafluorobutanesulfonyl fluoride[12] pro-
vided the nonaflate intermediate 14 which was recalcitrant to
further displacement with fluoride. Triflation of the secondary
alcohol in 13 followed by reaction with cesium fluoride in tert-
butanol[13] at elevated temperatures did not yield the desired
fluorinated nucleoside and only decomposition prevailed. Suc-
cessful fluorination was finally achieved by reacting 13 with di-
ethylaminosulfur trifluoride (DAST) in dichloromethane at 08C
to provide 15 as a single diastereoisomer, presumably through
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an SN2 displacement of the secondary hydroxyl group in 13.
However, attempts to scale up this reaction on a gram scale
lead to significant decomposition and only trace amounts of
the fluorinated nucleoside 15 could be isolated after purifica-
tion by silica gel chromatography. The Nap group was next re-
moved using 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in
dichloromethane[14] to provide nucleoside 16. Further removal
of the 5’-acetyl group to provide 17, followed by selective pro-
tection of the 5’-hydroxyl group with 4,4’-dimethoxytrityl chlo-
ride (DMTrCl) provided nucleoside 18. A phosphitylation re-
action provided the desired phosphoramidite 19.
Table 1. Sequence, analytical data and duplex stabilizing properties of
modified oligonucleotides evaluated in thermal denaturation experi-
ments.
ODN Modification
Sequence (5’!3’)[a] Tm [8C] DTm/mod. [8C][b]
A1
A2
A3
A4
A5
A6
A7
DNA
F-cLNA
d(GCGTTTTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUTTTGCT)
45.6
48.5
48.5
47.6
46.2
50.1
50.4
control
+2.9
+2.9
+2.0
+0.6
+4.5
+4.8
(R)-Me-cLNA[c]
(S)-Me-cLNA[c]
FHNA
LNA[c]
methylene-cLNA[c] d(GCGTTUTTTGCT)
[a] Underlined letter indicates modified nucleotide. [b] Tm values were
measured in sodium phosphate buffer (10 mm, pH 7.2) containing NaCl
(100 mm) and EDTA (0.1 mm). Sequence of RNA complement: 5’-r(AG-
CAAAAAACGC)-3’. [c] Ref. [11].
The precise orientation of the fluorine atom in 17 was deter-
mined by examination of the fluorine–proton coupling con-
stants (Scheme 2). The H1’ and H3’ protons in 17 appear as
singlets, which is consistent with the nucleoside furanose ring
being locked in the C3’-endo conformation (dihedral angles
~908). H2’ shows a small coupling (H2’–F dihedral angle ~908)
to the neighboring fluorine atom and appears as a narrow
doublet (J=4.0 Hz). In contrast, fluorine shows a large geminal
coupling to H7’ (J=56.4 Hz), a large coupling to H6’ (J=
27.5 Hz, dihedral angle ~08) and a smaller coupling to H6’’ (J=
14.2 Hz, dihedral angle ~1208) which is consistent with an axial
orientation for the fluorine atom.
at position 6 (nucleotides in strands 1 and 2 of the duplex are
numbered 1–10 and 11–20, respectively). We previously ob-
served that this decamer adopts an A-form conformation if at
least one of the 2’-deoxynucleotides is replaced with a 2’-sub-
stituted residue.[11,15] In addition, we relied on the same se-
quence in our studies of the conformational properties of 3
and 4[5a] as well as of 5.[6] The decamer was crystallized and its
structure determined at 1.80 ꢁ resolution. Final electron densi-
ty is shown in Figure 1A and selected crystal data and final re-
finement parameters are listed in Table S1 in the Supporting
Information (PDB ID: 4HQH).
Synthesis of the modified oligonucleotides was carried out
on polystyrene resin using standard conditions for incorpora-
tion of the DNA monomers and an extended coupling time for
introducing the modified nucleoside phosphoramidite 18
under conditions described previously.[14b] After the synthesis
was complete, the cyanoethyl protecting groups on the back-
bone phosphodiesters were removed by using triethylamine.
Removal of the heterocyclic protecting groups and cleavage
from the solid support was accomplished by heating with
aqueous ammonia at 558C. Purification of the oligonucleotide
using ion-exchange chromatography followed by desalting on
C18 cartridges provided the desired oligonucleotide for evalua-
tion in thermal denaturation (Tm) experiments.
As expected, the duplex adopts an A-form conformation
with average values for helical rise and twist of 2.9 ꢁ and 318,
respectively. All sugars except for those of residues G11 and
G13 adopt a C3’-endo pucker; both G11 and G13 exhibit a C2’-
exo pucker. The backbone torsion angles of both nucleotides
U*6 and U*16 fall into the standard scꢀ/ap/sc+/sc+/ap/scꢀ
ranges for a to z. P···P distances of 5.64 ꢁ (U*6pA7 step) and
5.61 ꢁ (U*16pA17 step) are consistent with the A-form charac-
ter of the backbone. Consistent with previous crystal structures
of DNA duplexes modified with cLNA analogues 8, 9 and 10,[11]
A5 is the only residue in the duplex that displays an extended
variant of the backbone with torsion angles a and g both in
the ap range rather than the standard scꢀ and sc+ conforma-
tions, respectively.
Biophysical evaluation of the (R)-F-cLNA 11 modified oligo-
nucleotide (A2, DTm +2.98C per modification) showed that
this modification exhibited similar duplex stabilizing properties
as the (R)-Me-cLNA, 9 (A3, DTm +2.98C/mod.) and the (S)-Me-
cLNA 10 (A4, DTm +2.08C/mod.) but improved properties rela-
tive to the 3-modified oligonucleotide (A5, DTm +0.68C/mod.;
Table 1). The improved hybridization of 11 relative to 3 is a con-
sequence of locking the furanose ring in the RNA-like C3’-endo
sugar pucker. The hexitol ring system in 3 mimics the RNA-like
C3’-endo sugar pucker but this scaffold is conformationally
more dynamic as compared to the locked sugar moieties in
LNA and related analogues. However, the duplex stabilizing
property of analogue 11 was clearly reduced as compared to
LNA 6 (A6, DTm +4.58C/mod.) and methylene-cLNA 8 (A7, DTm
+4.88C/mod.).
Fluorine atoms jut into the minor groove but do not engage
in lattice interactions with atoms from neighboring duplexes
(Figure 1B). Water molecules in the minor groove are hydrogen
bonded to acceptors from the base and sugar moieties where-
as distances between water and fluorine are inconsistent with
hydrogen bond formation (Figure 1B). This observation is con-
sistent with the conclusion based on crystal structures of FRNA
duplexes that showed fluorine to be a poor hydrogen-bond
acceptor in the minor groove.[2b] Superimposition of the modi-
fied base-pair steps from the decamer containing LNA-Ts[16]
(Figure 1C) and the corresponding decamer with (R)-F-cLNA-Us
indicates nearly identical conformations (Figure 1D).
We next examined the structural properties of a self-comple-
mentary DNA duplex modified with the F-cLNA analogue 11 to
ascertain if the fluorine atom can participate in interactions
with acceptor molecules in the minor groove. For the crystallo-
graphic studies, we chose the DNA decamer of sequence 5’-
d(GCGTAU*ACGC)-3’ with (R)-F-cLNA (U*) replacing thymidine
In conclusion, we report the synthesis, biophysical and struc-
tural evaluation of oligonucleotides modified with a fluorinated
cLNA analogue. We show that fluorine substitution does not
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Figure 1. A) The central A5pU*6:A15pU*16 base-pair step (U*=(R)-F-cLNA-U) viewed into the minor groove. The green meshwork represents Fourier 2Fo-Fc
sum electron density drawn at the 1.2s level. Carbon, oxygen, nitrogen and phosphorus atoms are colored in pink, red, blue and orange, respectively, and
C6’, C7’ and F7’ in the modified uridines are highlighted as spheres. B) Solvent environment of fluorine atoms in the minor groove. Hydrogen bonds between
water molecules (small red spheres) and minor-groove acceptors are indicated with thin lines. Residue G1 from an adjacent duplex is shown with carbon
atoms colored in green. Relatively long distances of >3.3 ꢁ between fluorine and water are inconsistent with hydrogen bond formation. C) The central
A5pT*6:A15pT*16 (T*=LNA-T) in the crystal structure of the LNA-modified decamer viewed into the minor groove (PDB ID: 1I5W).[16] Carbon, oxygen, nitro-
gen and phosphorus atoms are colored in light blue, red, blue and orange, respectively. D) Superimposition of the (R)-F-cLNA-modified A5pU*6:A15pU*16
and LNA-modified A5pT*6:A15pT*16 base-pair steps. The root-mean-square deviation between the two fragments amounts to 0.63 ꢁ.
confer added duplex stabilization beyond that imparted by
locking the conformation of the furanose ring in the C3’-endo
conformation using the 2’,4’-carbocyclic bridging group. A
recent study of FRNA-modified duplexes using NMR and ther-
modynamic data showed that the axial fluorine atom at the 2’-
position polarizes the nucleobase and increases RNA affinity by
favorably affecting Watson–Crick base-pairing and stacking
interactions.[2a] Taken together with our results, these observa-
tions collectively suggest that increased electronegativity in
Figure 2. Overlay of A) FHNA (green carbons) with LNA (grey carbons) B) F-
cLNA (pink carbons) and LNA (grey carbons) C) FHNA (green carbons) and F-
cLNA (pink carbons) showing that the fluorine atom in F-cLNA, unlike the 3’-
proximity to the 2’-position on the furanose ring alone is not
sufficient for polarization of the nucleobase and underscore
the importance of the anti-periplanar orientation of the nucle-
obase and the 2’-electron-withdrawing-group for optimal
duplex stabilization, even for nucleic acid analogues with con-
formationally locked furanose rings (Figure 2).
F atom in FHNA or the 2’-oxygen atom in LNA, is not anti-periplanar to the
nucleobase.
In the context of cLNA analogues, replacing the electron
withdrawing 2’-oxygen atom of LNA with a saturated carbon
atom provides analogues that are less stabilizing than LNA.
This suggests that the slight electron donating nature of the
saturated carbon substituent at the 2’-position doubly handi-
caps these analogues as they lack the nucleobase polarization
imparted by the 2’-oxygen atom and also suffer from reduced
duplex hydration in the minor groove.[11] Nonetheless, fluori-
nated cLNA analogues such as 11, present the opportunity to
investigate the effect of combining fluorine in the minor
groove of LNA duplexes with increased duplex stability (rela-
tive to FHNA and F-CeNA) on the antisense properties of modi-
fied oligonucleotides. However, a more efficient synthesis of
the fluorinated nucleoside phosphoramidite is required before
a complete investigation of the biological properties of fluori-
nated cLNA analogues can be completed. These efforts are on-
going and results will be reported in due course.
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Acknowledgements
We thank Dr. Charles R. Allerson for assistance with oligonucleo-
tide synthesis.
Keywords: carbocyclic LNA · duplex stability · fluorinated
nucleosides · nucleic acids · X-ray crystallography
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Received: October 24, 2012
Published online on November 28, 2012
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