Structural requirements for hybridization at the 5′-position are different in α-l-LNA as compared to β-d-LNA

Article abstract of DOI:10.1016/j.bmcl.2011.11.012

The synthesis and biophysical evaluation of R and S-5′-Me-α-l- LNA nucleoside phosphoramidites and modified oligo-2′-deoxyribonucleotides is reported. Synthesis of the nucleoside phosphoramidites was accomplished in multi-gram quantities starting from dia

Full text of DOI:10.1016/j.bmcl.2011.11.012

Bioorganic & Medicinal Chemistry Letters 22 (2012) 296–299
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural requirements for hybridization at the 50-position
are different in
a-L-LNA as compared to b-D-LNA
Punit P. Seth a, , Charles R. Allerson b, , Michael E. Østergaard a, Eric E. Swayze a
⇑
a Department of Medicinal Chemistry, Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA
b Regulus Therapeutics, 3545 John Hopkins Court, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
The synthesis and biophysical evaluation of R and S-50-Me-
a-L-LNA nucleoside phosphoramidites and
Article history:
modified oligo-20-deoxyribonucleotides is reported. Synthesis of the nucleoside phosphoramidites was
accomplished in multi-gram quantities starting from diacetone glucose. The 50-methyl group in the S
configuration was introduced by reacting the sugar 50-aldehyde with MeMgBr. Synthesis of the R-50-Me
isomer was accomplished from the S-50-Me nucleoside by a late stage inversion using Mitsunobu condi-
tions. Evaluation of the modified oligonucleotides in thermal denaturation experiments revealed that
Received 26 September 2011
Revised 2 November 2011
Accepted 3 November 2011
Available online 11 November 2011
Keywords:
LNA
R-50-Me-
This result is in contrast to the b-
the R-50-Me group completely reversed the stabilization effect on duplex thermostability.
Ó 2011 Elsevier Ltd. All rights reserved.
a-
L
-LNA showed similar RNA affinity as
a-L
-LNA while the S-50-Me analog was less stabilizing.
D
-series where the S-50-Me isomer showed LNA-like affinity for RNA while
a-
L
-LNA
50-Me-
Duplex thermal stability
a-L-LNA
a
-
L
-LNA (4) represents the alpha anomer of enantio-LNA and
exhibits LNA-like high affinity recognition of complementary nu-
cleic acids (Fig. 1).1,2 As a result,
-LNA modified oligonucleotides
have been used as antisense agents3 and for oligonucleotide based
diagnostic applications.4 To further explore the utility of
-LNA
modified oligonucleotides, a number of analogs have been reported
recently. For example, replacing the 20-oxygen in
-LNA with a
indicative of reduced immune stimulation. To ascertain if introduc-
ing a methyl group at the 50-position of
-LNA could have a
a-L
a
-L
similar impact on the biophysical and biological properties of mod-
ified oligonucleotides, we prepared the corresponding building
block phosphoramidites for incorporation into oligonucleotides.
In this letter, we report the synthesis and biophysical evaluation of
a
-L
a
-L
S- and R-50-Me-
a
-
L
-LNA (5 and 6, respectively, Fig. 1) modified
substituted nitrogen or carbon atom resulted in moderate to severe
reductions in duplex thermostability except when the 20-nitrogen
was substituted with pyrenyl moieties in which case large in-
creases in duplex thermostability were observed due to intercala-
tion of the pyrenyl ring within the oligonucleotide duplex.5–9 In
contrast, introducing a methyl group at the 30- or the 60-position
oligo-20-deoxyribonucleotides and show that the structural requi-
rements for hybridization in the
a
-L
-series are opposite from those
in the b-
D
-series.
of a-L
-LNA was well tolerated.10,11 As part of a comprehensive ef-
fort aimed at elucidating the SAR of antisense oligonucleotides
(ASOs) containing high affinity modifications,12–15 we recently
showed that the configuration of a 50-Me group can modulate the
Me
Me
O
O
O
O
O
O
Ur
Ur
Ur
O
O
O
O
O
O
biophysical and biological properties of b-D-LNA 1 (referred to as
β-D-LNA, 1
Δ Tm +5 C/mod.
LNA henceforth) ASOs.16 In that study, high affinity recognition
of complementary RNA was only observed when the 50-methyl
group was present in the (S) configuration (S-50-Me-LNA, 2). In
contrast, introducing a methyl group in the (R) configuration
(R-50-Me-LNA, 3) completely reversed the stabilizing effect on du-
plex thermostability. In addition, mice treated with S-50-Me-LNA
ASOs showed lower drug induced increases in spleen weight
S-5'-Me-β-D-LNA, 2
R-5'-Me-β-D-LNA, 3
Δ Tm 0 °C/mod.
°
Δ Tm +5
°
C/mod.
O
O
O
Ur
Ur
Ur
O
O
O
O
O
O
Me
O
Me
O
O
α-L-LNA, 4
Δ Tm +5 C/mod.
S-5'-Me-α-L-LNA, 5
Δ Tm +2 C/mod.
R-5'-Me-α-L-LNA, 6
Δ Tm +5 C/mod.
°
°
°
⇑
Corresponding author. Tel.: +1 760 603 2587.
E-mail address: pseth@isisph.com (P.P. Seth).
Figure 1. Structures of 50-Me-b- -LNA and 50-Me-
a-L-LNA.
D
Present address.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.012

P. P. Seth et al. / Bioorg. Med. Chem. Lett. 22 (2012) 296–299
297
Synthesis of the 50-Me-
23 was initiated from diacetone glucose 7 (Scheme 1). We chose to
start from a sugar precursor as opposed to the -LNA nucleoside
a
-L
-LNA uracil phosphoramidites 20 and
hydroxyl groups in 15 followed by treatment of the crude product
with aqueous sodium hydroxide in 1,4-dioxane provided the pro-
a
-L
tected 50-Me- -LNA nucleoside 16 by a 20-inversion/cyclization
a-L
itself as addition of MeMgBr or related organometallic reagents to
nucleosidic aldehydes is low yielding due to decomposition of the
nucleobase and other competing side-reactions. Reaction of the so-
dium salt of 7 with 2-bromonaphthalene in DMF provided the
30-O-Nap protected sugar following chromatography on silica gel.
Selective cleavage of the 5,6-acetonide using aqueous acetic acid,
cleavage of the resulting diol with periodate followed by an
Aldol/Cannizzaro sequence provided diol 8 as a solid. Reaction of
diol 8 with tert-butyldiphenylsilyl chloride and triethylamine in
dichloromethane provided a 1:1 mixture of the regioisomeric
mono-TBDPS protected sugars 9 and 10 which had to be separated
by tedious chromatography. Interestingly, the mono-TBDPS pro-
tected sugar derivative analogous to 9 in the allofuranose series
is a solid which can be crystallized out directly from the crude
reaction mixture.17 Swern oxidation of the primary alcohol in 9 fol-
lowed by treatment of the intermediate aldehyde with MeMgBr in
THF provided a 1:1 mixture of the R-Me and S-Me sugars 11 and
12, respectively. Once again, the sterochemical outcome of the
Grignard addition was very different from that observed in the
allofuranose series where addition of the nucleophile takes place
predominantly from the si-face of carbonyl group.17 The next step
involved reaction of the secondary hydroxyl in 12 with sodium hy-
dride and benzyl bromide in anhydrous DMF to provide the benzyl
ether 13 in excellent yield. Gratifyingly, no deprotection or migra-
tion of the nearby TBDPS protecting group was seen during the
reaction. The choice of benzyl as an orthogonal protecting group
was crucial since an acyl group was not expected to survive the
strongly basic conditions required for cyclization to the [2.2.1] ring
system later in the synthesis. Next, acetolysis of the 1,2-acetonide
cascade described previously.10,18 The 30-O-Nap protecting group
was next removed with DDQ and replaced with a tert-butyldimeth-
ylsilyl protecting group (17) before removal of the 50-O-benzyl
group. This protecting group swap was required because we were
unable to selectively protect the secondary 50-alcohol as the DMTr
ether in the presence of an unprotected secondary 30-hydroxyl
group and removal of the 30-O-Nap group with DDQ or catalytic
hydrogenation resulted in cleavage of the 50-O-DMTr ether.16 Ef-
forts to cleave the 50-O-Bn ether in 17 using catalytic hydrogena-
tion were unsuccessful. Instead, the 50-O-Bn ether was cleanly
deprotected using boron trichloride in dichloromethane to provide
18. Now, protection of the liberated 50-hydroxyl as the DMTr ether
followed by removal of the 30-O-TBS group with buffered triethyl-
amine trihydrofluoride provided the 50-O-DMTr nucleoside 19 in
good yield.
S-50-Me-
-LNA phosphoramidite 20.
Synthesis of the R-50-Me-
-LNA uracil phosphoramidite was
A phosphitylation reaction provided the desired
a-L
a
-L
accomplished as outlined in Scheme 2. Inversion of configuration
of the secondary 50-hydroxyl group in nucleoside 18 by means of
a Mitsunobu reaction followed by deprotection of the p-nitro-
benzoate ester using methanolic ammonia provided the R-50-Me-
a-L
-LNA nucleoside 21.16 Protection of the 50-hydroxyl group as
the DMTr ether followed by deprotection of the 30-O-TBS group
using buffered triethylamine trihydrofluoride provided the
50-O-DMTr protected nucleoside 22. A phosphitylation reaction
provided the desired R-50-Me-
a-L-LNA phosphoramidite 23.
The structure and the absolute stereochemistry of the 50-methyl
group was unambiguously assigned by X-ray crystallography using
the fully deprotected nucleoside 24 which was prepared from 21
by treatment with triethylamine trihydrofluoride (Fig. 2). Since
the stereochemistry of the 50-methyl group in 24 and therefore
in 21 is (R), the stereochemistry of the methyl group in 18 has to
be (S).
followed by
a Vorbruggen reaction with persilylated uracil
provided the nucleoside 14 in good yield. Removal 20-O-acetyl
group followed by deprotection of the TBDPS protecting group pro-
vided the partially deprotected nucleoside 15. Mesylation of both
O
O
1. NaH, NapBr
DMF, 98%
TBDPSCl
Et3N, CH2Cl2
TBDPSO
HO
HO
TBDPSO
HO
HO
H
+
O
O
O
O
O
O
O
O
2. AcOH, H2O
3. NaIO4
4. NaOH, HCHO
(67%, 3 steps)
(98%, 1:1)
O
O
O
O
O
O
HO
O
7
Nap
9
Nap
10
Nap
8
1. Swern Oxi.
2. MeMgBr
TBDPSO
TBDPSO
1. AcOH, Ac2O
cat. H2SO4
TBDPSO
BnO
NaH, BnBr
DMF (95%)
O
O
O
HO
O
HO
+
O
9
O
2. Uracil, BSA
TMSOTf
(65%, 2 steps)
(95%, 2 steps)
1:1 mix.
Me
O
Me
Me
O
O
O
O
O
Nap
Nap
Nap
12
11
13
1. NH3/MeOH
2. TBAF, THF
TBDPSO
BnO
HO
BnO
Me
1. DDQ, CH2Cl2
1. MsCl, pyr
O
O
O
O
Ur
Ur
Ur
OH
Ur
BnO
Me
O
BnO
O
2. TBSCl, DMF
Imidazole
(75%, 4 steps)
2. Aq. NaOH
1,4-dioxane
(85%, 2 steps)
Me
Me
O
O
OAc
O
O
TBS
17
Nap
14
Nap
15
Nap
16
O
N(iPr)2
NC
P
1. DMTrCl, pyr
(80%)
O
O
BCl3, CH2Cl2
(83%)
O
N(iPr)2
Ur
Ur
Ur
HO
Me
DMTrO
Me
DMTrO
Me
O
O
O
NMI, 1H-tetrazole
DMF (68%)
2. Et3N.3HF
Et3N, THF
(87%)
O
HO
O
P
TBS
18
19
NC
O
N(iPr)2
20
Scheme 1. Synthesis of S-50-Me-
a-
L-LNA uracil phosphoramidite.

298
P. P. Seth et al. / Bioorg. Med. Chem. Lett. 22 (2012) 296–299
as the activator and standard oxidizing and capping reagents. An ex-
1. Ph3P, DIAD
4-NO2-BzOH
1. DMTrCl, pyr
(84%)
O
O
tended coupling time of 8 min was used for incorporation of the mod-
ified nucleosides with an efficiency of 85–90% per incorporation. In
Ur
Ur
HO
Me
HO
Me
O
O
2. Et3N.3HF
Et3N, THF
(82%)
2. MeOH/NH3
(95%, 2 steps)
O
this sequence either a single or tandem incorporations of R-50-Me-
a-
O
TBS
21
TBS
18
L
-LNA (A2 and A3,
duplex thermostability which was comparable to that observed with
-LNA modified oligonucleotides A6 and A7 ( Tm + 4.6 °C/mod.). In
contrast, single or tandem incorporations of S-50-Me-
-LNA (A4
and A5, Tm + 1.7–2.5 °C/mod.) were less stabilizing. We also mea-
sured the mismatch stabilizing properties of both R- and S-50-Me-
-LNA and found them to be comparable to that of DNA and -LNA
modified oligonucleotides. Interestingly, tandem incorporations of
R-50-Me-
-LNA resulted in a slight increase in discrimination for
the GT wobble base-pair (
Tm ꢀ7.4 °C).
To help explain the divergent behavior of the 50-Me group on
hybridization in the -series relative to the b- -series, we at-
tempted to understand the conformation around the C40–C50 bond
(torsion angle ) at the nucleoside level using NOESY spectroscopy.
In natural nucleic acids such as DNA and RNA, there is restricted
rotation around
with three main orientations (Fig. 3).19 Conforma-
tion III is least preferred as it positions the 40 and 50-oxygen atoms in
DTm + 4.3–4.8 °C/mod.) provided an increase in
a
-L
D
O
N(iPr)2
a-L
NC
P
O
O
D
N(iPr)2
Ur
Ur
DMTrO
Me
DMTrO
Me
O
O
a-
NMI, 1H-tetrazole
DMF (98%)
L
a-L
HO
O
P
22
NC
O
N(iPr)2
a-L
23
D
Scheme 2. Synthesis of R-50-Me-
a-L-LNA uracil phosphoramidite.
a
-L
D
c
Et3N.3HF, THF
95%
O
O
Ur
Ur
HO
Me
HO
Me
O
O
c
O
HO
an anti orientation (
c
in ꢀsc range). Conformation I (
c in +sc range)
24
TBS
21
is most preferred as it places the 40- and 50-oxygen atoms in a gauche
orientation and this conformation is further stabilized by a CHꢁ ꢁ ꢁO
type H-bond between the 50-oxygen and H6 in pyrimidine (or H8
in purine) nucleosides.20 In the LNA series, NOESY studies on the
protected nucleosides showed that
ferred conformation I (
in +sc range) for the S-50-Me analog while
was in the ap range for the R-50-Me isomer.16 In the
-series, there
c was in the canonical and pre-
c
c
a-L
is no possibility of a CHꢁ ꢁ ꢁO type interaction between the H6/H8 on
the nucleobase with the 50-oxygen since the interatomic distance
between H6 on the nucleobase and the 50-oxygen is 3.84 Å in the
R-50-Me-
a
-L
-LNA nucleoside crystal structure as compared to
2.60 Å in LNA.21,22 As a result, conformations analogous to I and II
in the -series are most likely equally preferred at the nucleoside
level. This is reflected in the NOESY spectra of both the R- and S-50-
Me- -LNA nucleosides where crosspeaks were observed between
the 50-Me and the 50-H with both the protons at the 60-position
indicative of conformational mobility around . Interestingly, the
conformation around observed in the crystal structure of nucleo-
a
-L
Figure 2. Crystal structure of R-50-Me-
a-L-LNA uracil nucleoside 24.
a
-L
We evaluated the hybridization properties of both R- and S-50-
Me- -LNA using single and tandem incorporations of the modified
nucleotides (Table 1). Oligonucleotides with full phosphodiester back-
bone were synthesized at 2 mol scale using T-CPG support, 0.l M
c
c
a
-L
side 24 (R-50-Me) is identical to that of the
a-L-LNA nucleotide in a
modified duplex with RNA.21 However, in the absence of a crystal
l
structure for the corresponding S-50-Me nucleoside, it is not possible
solutions of all phosphoramidites in acetonitrile, 0.5 M 1H-tetrazole
Table 1
Duplex thermal stability measurements of R-50-Me-
a
-L-LNA, S-50-Me-
a
-L-LNA, R-60-Me-
a
-L-LNA, 30-Me-
a
-L-LNA and
a-L-LNA modified oligonucleotides versus RNA complements
Oligo
Mass calcd
Mass found
% UV purity
Modification
Sequence (50-30)a
D
Tm/mod.b (°C)
Mismatch discrimination
(Tm(mismatch)–Tm(match)
versus RNA
)
X = A (match)
X = G
X = C
X = U
A1
A2
3633.4
3661.4
3632.9
3660.8
—
98.4
DNAc
d(GCGTTTTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
(control)
+4.8
ꢀ4.1
ꢀ5.8
ꢀ13.3
ꢀ16.0
ꢀ13.8
ꢀ16.1
R-50-Me-
R-50-Me-
a
a
-
-
L
-LNA
-LNA
A3
3689.4
3661.4
3689.4
3661.4
3689.4
3661.4
3689.4
3647.4
3661.3
3689.0
3660.9
3688.9
3660.8
3689.0
3660.9
3688.9
3646.5
3660.8
96.3
98.6
97.9
98.4
96.3
98.6
97.9
97.8
97.9
L
+4.3
+2.5
+1.7
+4.1
+4.0
+5.5
+4.8
+4.6
+4.6
ꢀ7.4
ꢀ5.5
ꢀ5.1
ꢀ6.7
ꢀ6.3
ꢀ5.3
ꢀ5.3
ꢀ4.7
nd
ꢀ15.1
ꢀ14.2
ꢀ15.1
ꢀ14.6
ꢀ14.6
ꢀ15.5
ꢀ15.0
ꢀ15.4
nd
ꢀ16.4
ꢀ12.9
ꢀ14.1
ꢀ13.3
ꢀ15.1
ꢀ13.9
ꢀ15.4
ꢀ13.8
nd
A4
S-50-Me-
S-50-Me-
30-Me-
30-Me-
60-Me-
60-Me-
a
a
-
L
-LNA
A5
-
L
-LNA
A6
a
a
a
a
-
-
-
-
L
L
L
L
-LNAc
-LNAc
-LNAc
-LNAc
A7
A8
A9
A10
A11
a
a
-
-
L
-LNAc
-LNAc
L
a
Bold and underlined alphabet indicates modified residue.
b
Tm values were measured in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM EDTA, Sequence of RNA complement 50-r(AGCAAAXAACGC)-
30; Mismatch discrimination values were calculated by subtracting the Tm measured versus the mis-matched RNA complement (X = G, C or U) from the Tm versus the matched
RNA complement (X = A) for each modification.
c
Ref. 10 and.11.

P. P. Seth et al. / Bioorg. Med. Chem. Lett. 22 (2012) 296–299
299
structure of R-50-Me-LNA with the preferred conformation of the
S-50-Me-LNA nucleoside monomer but the relative positions of the
methyl groups were found to be distinctly different (Fig. 4).
O
O
O
O
γ
Bx
Bx
Bx
5'
O
1'
2'
OH
OH
OH
4'
3'
Bx
Ur
In conclusion, synthesis of both R- and S-50-Me-
a-L-LNA uracil
O
H
O
H
O
H
O
H
H
H
H
O
O
nucleoside building block phosphoramidites was accomplished
OH
H
O
H
starting from diacetone glucose. Evaluation of the modified oligonu-
RNA
I (γ in +sc)
II (γ in ap)
III (γ in -sc)
cleotides in thermal stability experiments showed that R-50-Me-a-
L-
LNA improved duplex thermostability while the S-50-Me isomer was
less stabilizing. This result is in contrast to the b- -series where the
O
O
O
O
Ur
Ur
D
O
O
O
O
S-50-Me isomer showed LNA-like affinity for RNA while the R-50-Me
group completely reversed the stabilization effect on duplex ther-
mostability. Efforts to understand the divergent hybridization prop-
erties using structural information and evaluation of the modified
oligonucleotides in biological experiments are underway and these
results will be reported in due course.
Me
O
O
O
H
Me
H
Me
O
preferred in
preferred in
S-5'-Me-LNA
5'-Me-β-D-LNA
R-5'-Me-LNA
6'
O
H
O
H
O
H
O
O
O
Ur
Ur
Ur
H
H
H
R2
R1
Ur
O
O
O
O
O
O
References and notes
O
O
R1
R2
R1
R2
O
R2
O
R1
R-5'-Me-α-L-LNA
1. Rajwanshi, V. K.; Hakansson, A. E.; Sorensen, M. D.; Pitsch, S.; Singh, S. K.;
Kumar, R.; Nielsen, P.; Wengel, J. Angew. Chem., Int. Ed. 2000, 39, 1656.
2. Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am. Chem. Soc. 2002,
124, 5974.
3. Fluiter, K.; Frieden, M.; Vreijling, J.; Rosenbohm, C.; De Wissel, M. B.;
Christensen, S. M.; Koch, T.; Orum, H.; Baas, F. Chem. Biol. Chem. 2005, 6, 1104.
4. Østergaard, M. E.; Hrdlicka, P. J. Chem. Soc. Rev. 2011. doi:10.1039/C1CS15014F.
5. Kumar, T. S.; Madsen, A. S.; Østergaard, M. E.; Wengel, J.; Hrdlicka, P. J. J. Org.
Chem. 2008, 73, 7060.
(R1 = Me, R2 = H)
observed in NMR of
α-L-LNA/RNA duplex and
crystal structure of 24
S-5'-Me-α-L-LNA
(R1 = H, R2 = Me)
Figure 3. Conformational analysis around the C40-C50-bond in b-
D-LNA and a-L-LNA
nucleosides.
6. Kumar, T. S.; Wengel, J.; Hrdlicka, P. J. Chem. Biol. Chem. 2007, 8, 1122.
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J. J. Org. Chem. 2009, 74, 1070.
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Wancewicz, E. V.; Witchell, D.; Swayze, E. E. J. Med. Chem. 2009, 52, 10.
13. Prakash, T. P.; Siwkowski, A.; Allerson, C. R.; Migawa, M. T.; Lee, S.; Gaus, H. J.;
Black, C.; Seth, P. P.; Swayze, E. E.; Bhat, B. J. Med. Chem. 2010, 53, 1636.
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Prakash, T. P.; Watt, A. T.; Egli, M.; Swayze, E. E. J. Am. Chem. Soc. 2010, 132,
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Watt, A. T.; Gaus, H.; Bhat, B.; Swayze, E. E.; Seth, P. P. J. Am. Chem. Soc. 2011,
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Figure 4. Structural overlay of the X-ray crystal structure of R-50-Me-LNA nucle-
oside (light blue) over the preferred conformation of S-50-Me-LNA nucleoside
monomer (marroon, created and minimized in Chem3D Ultra 7.0).
16. Seth, P. P.; Allerson, C. R.; Siwkowski, A.; Vasquez, G.; Berdeja, A.; Migawa, M.
T.; Gaus, H.; Prakash, T. P.; Bhat, B.; Swayze, E. E. J. Med. Chem. 2010, 53, 8309.
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Prakash, T. P.; Migawa, M. T.; Bhat, B.; Swayze, E. E. J. Org. Chem. 2010, 75, 1569.
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to make any further conclusions at this point. It is also difficult to
predict what type of structural distortions in backbone torsion an-
gles
a
and b occur in the oligonucleotide duplex upon introduction
-LNA nucleosides. This information
of R- and S-50-Me substituted
a
-L
can only be ascertained when high resolution structures of the mod-
ified duplexes become available. Lastly, we also overlaid the crystal