Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3′-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides

Article abstract of DOI:10.1021/ja207086x

The synthesis, biophysical, structural, and biological properties of both isomers of 3′-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently acc

Full text of DOI:10.1021/ja207086x

ARTICLE
pubs.acs.org/JACS
Synthesis, Improved Antisense Activity and Structural Rationale for
the Divergent RNA Affinities of 30-Fluoro Hexitol Nucleic Acid (FHNA
and Ara-FHNA) Modified Oligonucleotides
Martin Egli,*,† Pradeep S. Pallan,† Charles R. Allerson,‡,§ Thazha P. Prakash,‡ Andres Berdeja,‡ Jinghua Yu,‡
Sam Lee,‡ Andrew Watt,‡ Hans Gaus,‡ Balkrishen Bhat,‡,§ Eric E. Swayze,‡ and Punit P. Seth*,‡
†Department of Biochemistry, School of Medicine, Vanderbilt University, 607 Light Hall, Nashville, Tennessee 37232, United States
‡Isis Pharmaceuticals Inc., 1891 Rutherford Road, Carlsbad, California 92008, United States
S Supporting Information
b
ABSTRACT: The synthesis, biophysical, structural, and biolo-
gical properties of both isomers of 30-fluoro hexitol nucleic acid
(FHNA and Ara-FHNA) modified oligonucleotides are re-
ported. Synthesis of the FHNA and Ara-FHNA thymine
phosphoramidites was efficiently accomplished starting from
known sugar precursors. Optimal RNA affinities were observed
with a 30-fluorine atom and nucleobase in a trans-diaxial
orientation. The Ara-FHNA analog with an equatorial fluorine
was found to be destabilizing. However, the magnitude of
destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at
pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA
duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the
destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an
internucleotide CꢀF HꢀC pseudo hydrogen bond between F30 of Ara-FHNA and C8ꢀH of the nucleobase from the 30-
3 3 3
adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in
saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish
FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py) HꢀC(Pu) pseudo
3 3 3
hydrogen bonds in nucleic acid structures.
’ INTRODUCTION
RNA.9 In the hexitol series, the furanose ring found in DNA 1
and RNA 2 is replaced with a six-membered pyranose ring and
the position of the nucleobase is moved from the anomeric
carbon to the 20-position (Figure 1). This arrangement positions
the nucleobase in an axial orientation and mimics the N-type
sugar pucker of furanose nucleosides.
The Herdewijn group described the synthesis, biophysical,
structural, and preliminary biological characterization of HNA
(hexitol nucleic acid, 7), ANA (altritol nucleic acid, 8), 30-O-Me
ANA 9, and MNA (mannitol nucleic acid, 10) modified
oligonucleotides.9 However and much to our surprise, the 30-
fluoro hexitol derivative (FHNA, 11) and its “arabino” analog
(Ara-FHNA, 12) have not been described to date. Given the use-
fulness of the 20-fluoro modification (FRNA, 5) in siRNA-,10,11
miRNA-,12 and aptamer-13 based oligonucleotide therapeutic
applications and the general importance of fluorine as a “polar
hydrophobic” substituent in medicinal chemistry,14 we were
intrigued by the potential of FHNA as a modification for anti-
sense therapeutics. As well we were keen on assessing Ara-FHNA
Antisense oligonucleotides (ASOs) bind to their cognate
mRNA by WatsonꢀCrick base pairing and modulate mRNA
function.1 Over the past decade, interest in antisense technology
has grown exponentially with the discovery of siRNA and
miRNA based mechanisms for modulating gene expression2
and by the realization that RNA plays a far more important role
in cell biology than serving just as a conduit for the transfer of
genetic information.3 In addition, second generation ASOs that
downregulate gene expression by employing an RNase H
mechanism have shown robust antisense pharmacology in hu-
mans and established nucleic acid based therapeutics as a viable
drug discovery platform.4
As part of a comprehensive effort aimed at understanding the
structureꢀactivity relationship (SAR) of ASOs containing high
affinity modifications, with the aim of advancing a new oligonu-
cleotide modification toward human trials, we recently compared
the antisense properties of locked nucleic acid (LNA, 13) and
related analogs such as 20,40-constrained 20-O-Et (cEt, 14)
(Figure 1).5ꢀ8 As an extension of that work, we evaluated the
in vivo properties of the hexitol class of oligonucleotide mod-
ifications that are also known to increase affinity for cognate
Received: July 28, 2011
Published: September 14, 2011
r
2011 American Chemical Society
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Scheme 2. Synthesis of Ara-FHNA Thymine
Phosphoramidite
Figure 1. Structural relationships between 20-modified RNA, hexitol,
and 20,40-bridged nucleic acids.
Scheme 1. Synthesis of FHNA Thymine Phosphoramidite
(Ara-FHNA) results in a significant destabilization relative to
both HNA and DNA. Thus, the consequences of 30-fluoro
substitution of HNA differ distinctly from those of 20-fluoro
substitution of DNA (FRNA and FANA), in that both latter
analogs exhibit increased RNA affinity relative to the parent oligomer.
Subsequently, we conducted a detailed crystallographic analysis
of FHNA-, Ara-FHNA-, FRNA- and FANA-modified oligo-20-
deoxynucleotides that provides insight into the structural origins
of the initially surprising observations with regard to the relative
pairing affinities.
’ RESULTS
Synthesis of FHNA and Ara-FHNA Pyrimidine Nucleoside
Phosphoramidites. Synthesis of FHNA pyrimidine nucleoside
phosphoramidites was accomplished as shown in Scheme 1.
Treatment of known epoxide 15 with thymine in the presence of
DBU provided the corresponding hexitol nucleoside 16.16,17
Mesylation of the secondary hydroxyl group provided mesylate
17 which was treated with aqueous sodium hydroxide to pro-
vide the “ara” nucleoside 18 via formation and opening of the
anhydro-nucleoside intermediate as described previously.18
Treatment of nucleoside 18 with nonafluorobutanesulfonyl
fluoride and DBU provided the fluorinated nucleoside 19 via
SN2 inversion of the 30-hydroxyl group,19,20 which was typically
contaminated with 5ꢀ10% of a byproduct arising from elimina-
tion of the nonaflate intermediate. It was difficult to completely
remove the elimination byproduct from the fluorinated nucleo-
sides at this stage by silica gel chromatography. Instead the
partially purified mixture was hydrogenated using palladium
because the 20-fluoroarabino modification (FANA, 6) displayed
increased RNA affinity relative to DNA and was found to be one
of only a handful of analogs that elicits cleavage of the targeted
RNA strand by RNase H.15
In this manuscript, we describe the synthesis, biophysical,
structural, and biological properties of oligo-20-deoxyribonucleo-
tides modified with one or more FHNA and Ara-FHNA mono-
mers. Whereas axial 30-fluoro substitution (FHNA) boosts
the RNA affinity of HNA, fluorine in the equatorial orientation
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Scheme 3. Synthesis of FHNA 5-Methylcytosine
Phosphoramidite
Figure 2. Structural determination of FHNA and Ara-FHNA nucleo-
sides showing key NOESY crosspeaks.
displacement of the triflate by N3 of thymine. The formation of
28 was also accompanied with the formation of a separable
byproduct (∼20%) arising from displacement of the sugar triflate
by O-2 of N3-Bom thymine. As our motivation at this point
was to prepare enough material to complete our initial evaluation
of Ara-FHNA in Tm studies, we did not attempt to optimize
the nucleoside formation reaction any further. Removal of the
N3-Bom group and the 4,6-O-benzylidene was accomplished by
catalytic hydrogenation using 20% palladium hydroxide on
carbon to provide the Ara-FHNA thymine nucleoside 29, which
was not purified but directly converted to the 60-O-DMTr ether
30. A phosphitylation reaction provided the desired phosphor-
amidite 31.
The 5-methylcytosine FHNA phosphoramidite 35 was
synthesized according to the procedure outlined in Scheme 3.
The 40-hydroxyl group in 21 was first protected as the tert-
butyldimethylsilyl ether to provide 32 in excellent yield. The
thymine nucleobase was next converted to the 4-N-benzoyl
protected 5-Methylcytosine nucleobase by a three-step se-
quence.25 Reaction of 32 with POCl3 followed by displace-
ment of the 4-chloro intermediate with 1,2,4-triazole provided
the corresponding triazolide which was further displaced with
aqueous ammonia to provide the 5-methylcytosine nucleoside.
The crude nucleoside was dried and the exocyclic amino group
on the heterocycle was reacted with benzoic anhydride to
provide the 4-N-benzoyl protected nucleoside 33 in good yield
(68% over three steps). The 40-O-TBS group in 33 was removed
using TBAF to provide 34 followed by a phosphitylation reaction
to provide the desired phosphoramidite 35. All the hexitol
phosphoramidites were incorporated into oligonucleotides using
standard automated phosphoramidite chemistry using proce-
dures described previously.25
hydroxide on carbon to provide the deprotected nucleoside 20,
which could now be conveniently purified by chromatography.
While the overall yield for the fluorination/benzylidene removal
was ∼50% for the two step process, this procedure could be used
conveniently for the preparation of 20 on a multigram scale. The
60-hydroxyl group was then protected with dimethoxytrityl
chloride to provide nucleoside 21, which was converted to the
desired phosphoramidite 22 by means of a phosphitylation
reaction.
For synthesis of Ara-FHNA phosphoramidite 31, all methods
to carry out direct fluorination of nucleoside 16 were unsuccess-
ful due to competing formation of the anhydro-nucleoside via
attack of O-2 of the pyrimidine ring on the leaving group
generated by activation of the 30-hydroxyl group. To circumvent
these problems, we carried out the fluorination on diacetone
allofuranose 23 to install the problematic fluorine atom early in
the synthesis (Scheme 2). Fluorination was accomplished by first
conversion of the 30-hydroxyl group in 23 to the correspond-
ing triflate followed by displacement with cesium fluoride in
t-BuOH21 to provide 24 in essentially quantitative yield. Treat-
ment of 24 with an acidic resin under aqueous conditions
resulted in hydrolysis of both acetonide protecting groups and
rearrangement to the more stable pyranose ring structure which
was per-acetylated to provide fluoro sugar 25 as an anomeric
mixture.22,23 Conversion of the anomeric acetates to the anome-
ric bromide using HBr/acetic acid followed by reduction using
nBu3SnH/AIBN provided the fluoro hexitol sugar 26.24 Removal
of the acetate protecting groups followed by reprotection of the
40- and 60-hydroxyl groups as the cyclic benzylidene furnished the
protected hexitol intermediate 27. Synthesis of the N3-Bom
protected thymine nucleoside 28 was accomplished by first
conversion of the 20-hydroxyl group in 27 to the triflate followed
by displacement with N3-Bom protected thymine. It was neces-
sary to use the protected nucleobase to prevent competing
The relative orientation of the fluorine atom in FHNA and
Ara-FHNA nucleosides was assigned by examination of the
fluorine-proton coupling constants in nucleosides 19 and 28
and by NOESY spectroscopy (Figure 2). In the case of FHNA
nucleoside 19, the axial fluorine atom showed a large geminal
coupling (JF,H3 = 47.8 Hz) to H30, another large trans-diaxial
0
coupling to H40 (JF,H4 = 29.8 Hz), and a smaller (axial/
equatorial) coupling to H20 (JF,H2 = 8.0 Hz). In turn, H30
0
0
showed a large coupling to the geminal fluorine (JH3 ,F = 48.2 Hz)
and small couplings (<3 Hz) to H20 and H40. In contrast, in
nucleoside 28, the equatorial fluorine showed a large geminal
coupling to H30 (JF,H3 = 48.2 Hz), intermediate (equatorial/
0
axial) coupling to H40 (JF,H4 = 10.3 Hz), an0d a long-range
0
coupling to H1e0 (JF,H1e = 5 Hz). In addition, H3 showed a large
0
0
geminal coupling to fluorine (JH3 ,F = 48.2 Hz), a large trans-
diaxial coupling to H40 (JH3 ,H4 = 9.8 Hz), and a smaller (axial/
0
0
equatorial) coupling to H20 (JH3 ,H2 = 6.2 Hz). NOESY cross-
0
0
peaks were visible between H40 and the pyrimidine H6 for both
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Table 1. Characterization of FHNA, Ara-FHNA, HNA, and
LNA Analogs Based on Tm Values for DNA Sequences
Containing One, Two, or Three Modified Nucleotides
Table 3. 30-Exonuclease Stability of the Phosphosphodiester
Linkage in DNA, MOE, LNA, FHNA, and Ara-FHNA Mod-
ified Oligonucleotides
Sequence (50ꢀ30)
Modificationa
T1/2 (min)b
ΔTm/modification (°C) b
TTTTTTTTTTTT
TTTTTTTTTTtt
TTTTTTTTTTtt
TTTTTTTTTTtt
TTTTTTTTTTtt
a Uppercase letters indicate 20-deoxynucleotides, bold letters indicate
modified nucleotides, and MOE indicates 20-O-methoxyethyl ribose
modification. b Each oligonucleotide was prepared as a 500 μL mixture
containing the following: 12.5 μL of 200 μM oligomer, 50 μL of SVDP at
0.005 Units/mL in SVPD buffer (50 mM Tris-HCl, pH 7.5, 8 mM
MgCl2) final concentration 0.0005 Units/mL, 438.5 μL of SVP buffer.
Samples were incubated at 37 °C in a thermoblock. Aliquots (50 μL) were
taken at different intervals. EDTA was added to aliquots immediately after
removal to quench enzyme activity, and the samples were analyzed on IP
HPLC/MS. The results are expressed as half-life (T1/2) in minutes.
DNA
0.23
3
Sequencea
(50 to 30)
MOE
DNA
FHNA
Ara-FHNA
HNA
LNA
LNA
3
GCGTTtTTTGCT
GCGTTttTTGCT
CCAGtGAtAtGC
a Uppercase letters indicate 20-deoxynucleotides; bold lower case letters
indicate modified nucleotides. b Tm values were measured at 4 μM oligo
concentration in 10 mM sodium phosphate buffer (pH 7.2) containing
100 mM NaCl and 0.1 mM EDTA; the sequences of the RNA
complements were 50-r(AGCAAAAAACGC)-30 and 50-r(GCAUAU-
CACUGG)-30; Tm values reflect the average of at least three individual
measurements with a standard deviation of e0.27 °C.
(45.6)
(45.6)
(42.6)
+0.6
+1.7
+2.6
ꢀ2.8
ꢀ4.1
ꢀ1.2
+0.5
+0.9
+1.4
+5.2
+4.7
+6.7
FHNA
Ara-FHNA
160
101
Table 2. Mismatch Discrimination Properties of FHNA
Tm (ΔTm = Tm(mismatch) ꢀ Tm(match)) (oC)a,b
characterized the mismatch discrimination and nuclease resis-
tance properties of FHNA oligonucleotides. In the mismatch
experiments, FHNA showed substantially better discrimination
for the G-T wobble base pair but diminished recognition of the
T-T mismatched pair relative to DNA and LNA (Table 2). Both
the FHNA- and the Ara-FHNA-modified DNAs also exhibited
much higher resistance to exonuclease digestion as compared to
LNA- and MOE-modified oligonucleotides (Table 3).
Modification
X = A
X = G
X = C
X = T
DNA
FHNA
LNA
45.6
46.2
50.8
41.5 (ꢀ4.1)
36.3 (ꢀ9.9)
45.6 (ꢀ5.2)
32.4 (ꢀ13.4)
34.3 (ꢀ11.9)
34.1 (ꢀ16.7)
31.9 (ꢀ13.7)
38.9 (ꢀ7.3)
37.2 (ꢀ13.6)
a Sequence used for evaluation of 50-d(GCGTTtTTTGCT)-30 where
the bold letter indicates the modified nucleotide. b Sequence of RNA
complement 50-r(AGCAAAAXAACGC)-30 where X indicates the
mismatch site.
X-ray Crystallography of Oligonucleotides Containing
FHNA-T or Ara-FHNA-T Modifications. To gain insight into
the origins of the divergent hybridization behaviors of the FHNA
and Ara-FHNA modifications, we determined crystal structures
of DNA decamer duplexes [d(GCGTAtACGC)]2 containing
either FHNA-T or Ara-FHNA-T (t6 and t16; nucleotides in the
paired strands are numbered 1ꢀ10 and 11ꢀ20) at resolutions of
1.56 and 1.67 Å, respectively. Selected crystal data, data collec-
tion, and refinement parameters are listed in the Supporting
Information (Table S2) along with examples of the quality of
the final electron density (Figure S1). The decamers adopt
A-form geometry, and their overall conformations are similar
(Supporting Information, Figure S2). Visual inspection of the
structures and the analysis of helical parameters28 and the sugar-
backbone geometry demonstrate that FHNA and Ara-FHNA
residues fit well into the oligo-20-deoxynucleotide duplex. Thus,
there are no kinks associated with the sites of incorporation of
modified residues as judged by the curvature of the helical axis
and local rolling of base pairs. The average values for helical rise
and twist are 2.93 Å and 31.8°, respectively, in the FHNA-
modified duplex, and they are 2.86 Å and 32.1°, respectively, in
the Ara-FHNA-modified duplex. Among the global base-axis
parameters, both X-displacement (ꢀ4.3 Å, FHNA; ꢀ4.6 Å, Ara-
FHNA) and inclination (5.6°, FHNA; 8.6°, Ara-FHNA) are
slightly higher in the Ara-FHNA-modified duplex. The sugars of
all 20-deoxynucleotides adopt the C30-endo pucker. In both
duplexes, residue A5 displays a so-called extended variant of
the backbone with torsion angles α and γ both in the ap range
instead of the standard scꢀ and sc+ conformations respectively.
In the FHNA-modified duplex, G13 also adopts an extended
backbone. However, the six backbone torsion angles and the
glycosidic angles of all FHNA and Ara- FHNA nucleotides t6 and
t16 fall into the standard ranges.
19 and 28 establishing the axial orientation of the nucleobase. In
addition, for 28, NOESY crosspeaks were visible between H30,
H10, and H50, while NOESY crosspeaks were only visible between
H10 and H50 for 19.
FHNA and Ara-FHNA Show Opposite Trends in Affinity for
Complementary RNA. We first evaluated the thermal stability
(Tm) of FHNA and Ara-FHNA modified duplexes using single,
double, and triple incorporations of the modified nucleotide and
compared them to LNA and HNA modified duplexes (Table 1).
FHNA exhibited slightly improved Tm as compared to HNA, but
the magnitude of stabilization was significantly less than that
observed for LNA modified duplexes. Interestingly, we observed
higher duplex stabilization when FHNA T was incorporated in
tandem or between A or G as compared to incorporation in a
stretch of dTs. In contrast, Ara-FHNA was destabilizing. The
duplex destabilization observed with Ara-FHNA is somewhat
unexpected as the corresponding furanose analog, FANA 6,
improves the thermal stability of oligonucleotide duplexes by
formation of a pseudo H-bond at pyrimidine/purine steps.26
However, the result is consistent with the binding properties
reported for MNA 10 which has an equatorial hydroxyl group at
the 30-position.27 Remarkably, when three Ara-FHNA-Ts were
incorporated before A or G, the loss in stability was considerably
diminished compared to sequences with either one or two
modifications in a stretch of dTs (Table 1). For now we note
that there appears to be a sequence-dependent effect on the
stability as a result of the Ara-FHNA modification.
Mismatch Discrimination and Nuclease Resistance
Properties of FHNA Modified Oligonucleotides. We also
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Figure 4. Comparison between the conformations of dinucleotides
containing the Ara-FHNA (afh; gray carbons) or FANA (fa; magenta
carbons) modifications. Superimposition of afh(T)pd(A) and fa(T)pd-
(A),29 (A) viewed into the major groove and (B) rotated by 90° and
viewed perpendicularly to the thymine plane. Please see Figure 3 for
color codes of individual atoms.
In this hypothetical model, the shortest among these distances is
between F30(t6) and O40(dA7): 2.19 Å. Even considering a van
der Waals radius for fluorine of 1.2 Å (matching that of H), such a
contact would be strongly destabilizing. Therefore, in order to
accommodate the Ara-FHNA residue, one would expect the
dinucleotide step to undergo a conformational change.
Figure 3. Comparison between the conformations of dinucleotides
containing the FHNA (fh; pink carbons), Ara-FHNA (afh; gray
carbons), or FRNA (fr; green carbons) modifications. Superimposition
of fh(T)pd(A) and fr(C)pfr(G),11 (A) viewed into the major groove and
(B) rotated by 90° and viewed perpendicularly to the thymine plane.
Superimposition of fh(T)pd(A) and afh(T)pd(A), (C) viewed into the
major groove, revealing slight unstacking of bases in the case of Ara-
FHNA (double arrow), and (D) rotated by 90° and viewed perpendi-
cularly to the thymine plane. Nitrogen, oxygen, and phosphorus atoms
are colored blue, red, and orange, respectively, and axial 20- and axial or
equatorial 30-fluorine atoms are highlighted in light green and are
labeled.
Indeed, the superimposition of dinucleotides containing either
FHNA-T or Ara-FHNA-T followed by 20-deoxy-A reveals that
the latter residue is pushed away in the Ara-FHNA-modified
decamer duplex (Figure 3A,C). The resulting internucleotide
distances between the equatorial fluorine and the 40-oxygen in
the two strands of 2.75 Å (F30[t6] O40[dA7]) and 2.82 Å
3 3 3
(F30[t16] O40[dA17]) correspond to the sum of the van der
3 3 3
Waals radii. However, avoiding a steric clash causes partial
unstacking of the adenine from Ara-FHNA-T (Figure 3C,D),
which likely contributes to the unfavorable RNA affinity ob-
served for Ara-FHNA relative to FHNA, HNA, and DNA
(Table 1).
We take the similar overall geometries of the two duplexes as
evidence that putative deviations at the local level between the
two structures, at the sites of either FHNA or Ara-FHNA
incorporation, can be attributed to the particular steric and
stereoelectronic characteristics of the two modifications. In the
following paragraphs we analyze the conformational features of
the base-pair steps containing FHNA and Ara-FHNA modifica-
tions. We compare them to base-pair steps in A-form duplexes
featuring the corresponding furanose analogs, FRNA and FANA,
respectively, which we have previously analyzed in considerable
detail in terms of their conformational preferences,11,29 and corr-
elate the stability data (Table 1) with the structural observations.
The axial fluorine atom in FHNA juts out into the minor
groove in a direction that is virtually perpendicular to the
trajectory of the backbone, and this precludes short contacts
between the 30-substituent and backbone and/or base atoms of
the 30-adjacent nucleotide (dA7 in strand 1 and dA17 in strand 2;
Figure 3A,B). The spacing between adjacent bases in FHNA and
FRNA are very similar, and the only difference is a slight shift of
adenine into the major groove in the FHNA-modified structure
relative to the corresponding FRNA base. We used FRNA as a
reference here because FRNA and RNA adopt virtually identical
structures.11 Moreover, rather than a difference between the TpA
(FHNA) and CpG (FRNA) steps in the A-form settings
compared here, the slight offset in the positions of the nucleo-
bases is most likely a consequence of the geometric constraints of
the furanose and hexose sugars.
Unlike in the case of HNA, addition of a fluorine in the arabino
configuration to 20-deoxyribose (FANA 6, Figure 1) has a
positive effect on the RNA affinity, whereby the stability increase
is highest at pyrimidine-purine steps due to formation of an
F20(Py) HꢀC8(Pu) pseudo hydrogen bond.26 FANA-T (t)
3 3 3
incorporated into the A-form decamer d(GCGTAtACGC) was
found to be unable to adopt a pure C30-endo pucker.29 Moreover,
it forces the 30-adjacent deoxyribose into a C20-endo pucker
(Supporting Information, Figure S4). The resulting dinucleotide
step exhibits a considerable roll and F20(FANA-T) C8(dA)
3 3 3
contacts as short as 2.55 Å.29 A comparison between the con-
formation of dinucleotides containing either a 50-Ara-FHNA-T
or a -FANA-T demonstrates different degrees of rolling and a
larger slide of the adenine moiety following the Ara-FHNA
nucleotide (Figure 4). The latter is a consequence of the steric
constraints of the HNA framework and the equatorial 30-fluorine
that pushes away the adjacent sugar. These constraints preclude
tight F30(Ara-FHNA-T) C8(dA) contacts such as those in
3 3 3
the FANA-modified duplex (Figure 4 and Supporting Informa-
tion, Figure S4). Nevertheless, the resultant distances of 3.09 and
3.12 Å in strands 1 and 2, respectively, of the Ara-FHNA-modi-
fied duplex appear still sufficient to offset the loss of stability
due to diminished stacking (Figure 3C), so as to merely reduce
the Tm by 1.2 °C/modification for multiple incorporations at
pyrimidine-purine steps (Table 1).
In contrast to the axial fluorine in FHNA, modeling an
equatorial 30-fluorine into the structure of the FHNA-modified
duplex leads to several short contacts between fluorine and atoms
from the 30-adjacent nucleotide (Supporting Information, Figure S3).
Biological Evaluation of FHNA Modified Antisense Oligo-
nucleotides. We next evaluated FHNA A1, HNA A2, and LNA
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A3 ASOs in Tm, cell culture, and a single dose escalation
experiment in mice using a 14-mer oligonucleotide sequence
targeting mouse phosphatase and tensin homologue (PTEN)
mRNA (Table 4, Figure 5A,B). In Tm measurements we mea-
sured duplex thermal stability versus the length matched 14-mer
and a longer 20-mer RNA complement. We used the 20-mer
complement since the biological receptor (i.e., mRNA) for these
ASOs is not length matched to the ASO. When the Tm was
measured versus the 14-mer RNA complement, both the hexitol
ASOs A1 and A2 exhibited similar duplex thermal stabilities
which were substantially lower than that of LNA ASO A3.
However, all the ASOs exhibited improved duplex thermal
stabilities when the Tm was measured using the longer 20-mer
complement. Presumably, the overhanging nucleotides improve
duplex thermostability by providing additional stacking interac-
tions with the modified nucleotides in the flanks of the ASO.30
However, these interactions appear to be somewhat more
important for FHNA (ΔTm 20-mer-14-mer +6.2 °C) as com-
pared to HNA (ΔTm +3.1 °C) and LNA (ΔTm +4.3 °C)
oligonucleotides.
In cell culture, FHNA ASO A1 and HNA ASO A2 exhibited
similar potency for downregulating PTEN mRNA but both
ASOs were slightly less potent than LNA ASO A3 (Table 4
and Figure S6). In the animal experiment, mice were injected i.p.
with a single dose of 3.2, 10, 32, and 100 mg/kg of ASOs A1, A2,
and A3 in saline and liver PTEN mRNA and plasma alanine
amino transferase (ALT) levels were measured postsacrifice. In
this experiment, the FHNA ASO A1 exhibited near identical
potency as the LNA ASO A3 while the HNA ASO A2 was 2-fold
less potent. However, there was a dramatic difference in the
markers for hepatotoxicity for these ASOs. The LNA ASO A3
showed ALT increases for the high dose group treated animals
while the hexitol ASOs did not. To determine if the improved
activity of FHNA was because of improved biodistribution,
we measured tissue levels of all three ASOs in mouse liver
(Supporting Information, Figure S5). Both FHNA and LNA
ASOs showed comparable tissue accumulation at all doses except
for the high dose group where the LNA ASO levels were higher.
The HNA ASO showed slightly lower tissue accumulation at all
the doses tested. In a subsequent experiment, we evaluated the
Table 4. Tm, in Vitro and in Vivo activity of FHNA, HNA, and
LNA gapmer ASOs targeting mouse PTEN mRNA
Tm (°C)b vs Tm (°C)c vs
14-mer RNA 20-mer RNA (μM)d (mg/kg)e (mg/kg)f
IC50
ED50
ED50
ASOa
FHNA A1
HNA A2
LNA A3
53.9
54.9
61.1
60.1
58.0
65.4
2.3
2.4
1.5
7.8
13.8
7.2
2.2
NT
2.1
a Sequence used for evaluation of mCTTAGCACTGGCmCT; bold
letters indicate modified residues in flanks, and uppercase letters indicate
20-deoxynucleotides in the gap; all linkages are phosphorothioate
b
modified. Sequence of 14-mer RNA complement 50-r(AGGCCAG-
UGCUAAG)-30. c Sequence of 20-mer RNA complement 50-r(UCAA-
GGCCAGUGCUAAGAGU)-30. d IC50 for PTEN mRNA reduction in
e
mouse bEND cells with electroporation. Potency for PTEN mRNA
f
reduction in mouse liver from single dose study. Potency for PTEN
mRNA reduction in mouse liver from 3-week study.
Figure 5. Biological evaluation of of FHNA A1, HNA A2, and LNA A3 ASOs in mice. (A) Mice (n = 4/dose group) were injected i.p. with a single dose
of 3.2, 10, 32, and 100 mg/kg of ASO in saline, and animals were sacrificed 72 h after the last dose. Liver PTEN mRNA was measured using quanti-
tive RT-PCR. (B) Plasma ALT levels after sacrifice. (C) Mice (n = 4/dose group) were injected i.p. twice a week for three weeks with 0.5, 1.5, 4.5, and
15 mg/kg of FHNA A1 and LNA A3 ASOs in saline, and animals were sacrificed 72 h after the last dose. Liver PTEN mRNA was measured using
quantitative RT-PCR, and plasma ALT levels were recorded postsacrifice. (D) Tissue levels of FHNA and LNA ASOs from 3-week study.
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Journal of the American Chemical Society
ARTICLE
FHNA A1 and LNA A3 ASOs in a three week study to investigate
the effect on potency and toxicity in a subchronic dosing
schedule. Mice were injected i.p. with 0.5, 1.5, 4.5, and 15 mg/
kg of each ASO, and liver PTEN mRNA and plasma ALT levels
were measured postsacrifice. Once again, the FHNA ASO A1
(ED50 = 2.2 mg/kg) exhibited identical potency as LNA ASO A3
(ED50 = 2.1 mg/kg). However, the high dose group LNA ASO
A3 treated animals showed an increase in ALT levels but the
FHNA ASOs did not show increased ALT levels at all the doses
evaluated (Figure 5C). As in the case of the single dose study, we
measured the tissue levels of FHNA ASO A1 and LNA ASO A3
in mouse liver but did not observe any meaningful differences in
accumulation (Figure 5D).
’ ASSOCIATED CONTENT
Supporting Information. 1H, 13C, and 19F NMR spectra
S
b
and analytical data for all new compounds; 31P NMR spectra for
all phosphoramidites; analytical data for oligonucleotides and
dose response curves for cell culture experiments are provided.
Atomic coordinates and structure factor data for the three crystal
structures have been deposited in the Protein Data Bank (http://
www.rcsb.org; 3Q61, FHNA-modified decamer and 3SD8, Ara-
FHNA-modified decamer). This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
martin.egli@vanderbilt.edu; pseth@isisph.com
’ DISCUSSION
Present Addresses
§Regulus Therapeutics, 3545 John Hopkins Ct., San Diego,
California 92121, United States
Our biophysical and structural characterization of the FHNA
and Ara-FHNA modifications reveals properties that are distinct
from those of the corresponding fluoro-modified analogs of
DNA, FRNA, and FANA. FHNA compares favorably with
HNA in terms of RNA affinity, whereas Ara-FHNA causes a
stability loss relative to HNA, FANA, and DNA that appears to be
sequence dependent. Crystal structures at high resolution pro-
vide a qualitative rationalization of the thermodynamic data and
reveal that the equatorial 30-fluorine substituent in Ara-FHNA
pushes away the 30-flanking nucleotide, thus disrupting stack-
ing of nucleobases. Evaluation of FHNA-modified ASOs in
animal experiments revealed that these ASOs show comparable
potency to LNA without producing hepatotoxicity. The excellent
in vivo activity was obtained without the aid of complicated
cationic lipids based formulations to deliver the ASO and is
comparable to that observed with chemically modified and for-
mulated siRNAs disclosed recently.31 Interestingly, while both
FHNA and HNA modified ASOs showed similar activity in
cell culture, the improved activity seen with FHNA in animal
experiments suggests a positive role of fluorination for improv-
ing ASO uptake into tissues for therapeutic applications, an area
of high interest.
’ ACKNOWLEDGMENT
We thank Dr. Karsten Schmidt for HRMS measurements and
Ms. Merry Daniel for help with the crystallization experiments.
This work was supported in part by NIH Grant R01 GM55237
(to M.E.). Vanderbilt University is a member institution of LS-
CAT at the Advanced Photon Source (Argonne, IL). Use of the
APS was supported by the U.S. Department of Energy, Basic
Energy Sciences, Office of Science, under Contract No. W-31-
109-Eng-38.
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