Synthesis and biophysical evaluation of 3′-Me-α-l-LNA - Substitution in the minor groove of α-l-LNA duplexes

Article abstract of DOI:10.1016/j.bmcl.2011.06.104

The synthesis and biophysical evaluation of 3′-Me-α-l-LNA is reported. The synthesis of the nucleoside building block phosphoramidite was accomplished starting from diacetone glucose. The 3′-Me group was introduced in the desired configuration by hydride

Full text of DOI:10.1016/j.bmcl.2011.06.104

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4690–4694
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biophysical evaluation of 30-Me-
in the minor groove of -L-LNA duplexes
a-L-LNA – Substitution
a
Punit P. Seth a, , Charles A. Allerson b, Michael E. Østergaard a, Eric E. Swayze a
⇑
a Department of Medicinal Chemistry, Isis Pharmaceuticals, 1891 Rutherford Road, Carlsbad, CA 92008, United States
b Regulus Therapeutics, 3545 John Hopkins Ct., San Diego, CA 92121-1121, United States
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 30-Me-
a-L-LNA is reported. The synthesis of the nucleoside
Article history:
building block phosphoramidite was accomplished starting from diacetone glucose. The 30-Me group
was introduced in the desired configuration by hydride mediated opening of an exocyclic epoxide. Inver-
sion of the 20-hydroxyl group was achieved by means of an oxidation/reduction sequence followed by
cyclization onto a 50-leaving group to assemble the [2.2.1] ring system. Biophysical evaluation of 30-
Received 6 May 2011
Revised 22 June 2011
Accepted 24 June 2011
Available online 30 June 2011
Me-
similar to
a
-
L
-LNA modified oligonucleotides showed good duplex thermal stabilizing properties which were
Keywords:
a
-
L
-LNA. Mismatch discrimination experiments revealed that 30-Me-
a-L
-LNA possess slightly
a
-
L
-LNA
enhanced discrimination properties for the GU wobble base-pair as compared to related nucleic acid
analogs.
30-Me-
a-L-LNA
Minor groove
Thermal stability
Ó 2011 Elsevier Ltd. All rights reserved.
Locked nucleic acid (b-
D
-LNA or LNA, 1) modified oligonucleo-
(DTm/unit +33 °C) were observed due to stacking of the pyrenyl
tides provide unprecedented increases in the thermal stability of
oligonucleotide duplexes.1 As a result, LNA and related analogs
have been extensively used for RNase H antisense,2–7 splice modu-
lation,8 siRNA and microRNA9 based therapeutic antisense applica-
tions. In addition, the high affinity recognition of cognate nucleic
acids seen with LNA has resulted in the widespread use of this
modification for oligonucleotide based diagnostic applications.10
moiety at the abasic site within the duplex structure.
O
major
groove
Y
O
5'
O
3'
2'
O
O
O
Bx
Wengel showed that the alpha anomer of enantio-LNA (a-L-LNA,
6'
X
4'
5) also exhibits LNA-like high affinity recognition of complemen-
tary nucleic acids despite the structural differences between the
nucleoside monomers.11 Subsequent structural studies showed
that the 20,40-bridge in LNA duplexes lies in the minor groove while
Bx
Z
O
1'
minor
groove
β-D-series (ΔTm /mod.)
β-D-LNA 1
1, X = Y = H, Z = O (+5 oC)
2, X = Me, Y = H, Z = O (+5 oC)
3, X = Y = H, Z = CCH2 (+5 oC)
4, X = H, Y = alkyl, Z = O (~0 oC)
this bridge is located inside the major groove for a-L-LNA modified
duplexes.12,13 As a result of these differences, introducing substitu-
tion along the 20,40-bridge in LNA orients them into the minor
groove of the duplex, where they are generally well tolerated
(Fig. 1).4,14 On the other hand, introducing substitution at the
30-position of LNA projects the appended group into the major
groove where it negatively impacts duplex stability.15 In contrast
major
groove
O
5'
6'
X
O
O
Bx
O
Z
O
4'
3'
Y
O
to the b-
in -LNA generally disrupts hybridization. In a series of elegant
studies,16–18 Hrdlicka showed that alkyl groups on the 20-nitrogen
D
-LNA series, introducing substitution on the 20,40-bridge
Bx
O
2'
1'
a-L
minor
α-L-LNA 5
groove
atom of 20-amino-
a-L-LNA were poorly tolerated except when the
α-L-series (ΔTm/mod.)
5, X = Y = H, Z = O (+5 oC)
20-nitrogen had a pyrenyl moiety attached. In that case, very large
increases in binding towards RNA strands containing abasic sites
6, X = Me, Y = H, Z = O (+5 oC)
7, X = Y = H, Z = CCH2 (-10 oC)
8, X = H, Y = Me, Z = O this study
⇑
Corresponding author. Tel.: +1 760 603 2587.
E-mail address: pseth@isisph.com (P.P. Seth).
Figure 1. Structures of b-D-LNA, a-L-LNA and related analogs.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.104

P. P. Seth et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4690–4694
4691
1. Oxalyl chloride,
DMSO, Et3N
2. PPh3CH3Br
nBuLi
LiBEt3H
CH2Cl2
MCPBA
CH2Cl2
O
O
O
O
O
O
O
O
H
H
H
H
O
O
O
O
O
O
O
O
(67%)
(96%, 2 steps)
~10:1
diastereoselectivity
O
HO
O
O
O
O
HO
Me
9
10
11 (87%)
12
1. AcOH, H2O
2. NaIO4
3. HCHO, NaOH
NaH, BnBr
DMF
O
O
MsO
MsO
BnO
HO
HO
H
MsCl
O
O
O
O
OHC
Me
O
O
O
O
(98%)
(20%, 3 steps)
BnO
BnO
O
O
O
O
Me
Me
Me
14
+ hydrate
13
16
15
1. AcOH, Ac2O
H2SO4 (cat.)
2. Uracil, BSA
TMSOTf
1. Oxalyl chloride
DMSO, Et3N
2. NaBH4, MeOH
2',4'
cyclize
MsO
MsO
MsO
BnO
MsO
MsO
BnO
O
Hydride
O
O
O
Ur
OH
Ur
Ur
Ur
MsO
BnO
MsO
BnO
O
3. aq. NaOH
1,4-dioxane
(61%, 3 steps)
3. MeOH/NH3
(65%, 3 steps)
OH
Me
17
O
Me
19
Me
18
Me
20
Cl
P
KOAC
18-crown-6
1,4-dioxane
1. Pd(OH)2/H2
MeOH
2. MeOH/NH3
NC
O
N(iPr)2
O
O
O
Ur
Ur
Ur
DMTrO
O
AcO
DMTrO
O
O
O
(76%)
3. DMTrCl, pyr
(73%, 3 steps)
NMI, DCM (97%)
O
BnO
HO
P
Me
N(iPr)2
Me
21
Me
22
NC
23
Scheme 1. Synthesis of 30-Me-
a-L-LNA uridine phosphoramidite.
We recently showed that that replacing the 20-oxygen atom in
Aq. NaOH, 1,4-dioxane, rt, 16h
a
-
L
-LNA with an exocyclic methylene group (7) was detrimental
to duplex thermal stability.19 As a continuation of that exercise,
Aq. NaOH
MsO
OBn
we also showed strong duplex stabilization properties with 60-
Me-
Me
O
MsCl
pyridine
50 oC, 8 days
-LNA (6) where the methyl group points towards the 30-
Ur
O
a-L
OMs
Ur
MsO
BnO
17
O
phosphodiester linkage and is situated towards the edge of the ma-
jor groove of the modified duplex.20 While the above SAR was
informative with regard to the steric requirements for hybridiza-
tion within the major groove, we also wanted to evaluate the effect
OMs
Me
26
24
3'-Me-β-D-xylo-LNA
hydrolyze
2' mesylate
of introducing steric bulk in the minor groove of a-L-LNA duplexes.
X
2',4'-
cyclize
The minor groove of oligonucleotide duplexes serves as an impor-
tant recognition element for many nucleic acid binding proteins
such as RNase H21,22 and introducing substitution could have a ma-
jor impact on the biological properties of ASOs containing such
O
N
OBn
Me
OMs
OMs
MsO
OMs
Ur
O
N
modifications. Moreover, identifying sites on the
a-L-LNA scaffold
O
O
for site-specific introduction of conjugates or fluorescent tags in
the minor groove of the modified duplexes could be useful for oli-
gonucleotide based diagnostic applications. In this letter, we pres-
O
O
Me
25
O
Ms
eclipsed
ent the synthesis and hybridization properties of 30-Me-
a
-
L
-LNA
(8) and show that introducing steric bulk in the minor groove of
-LNA modified duplexes maintains the high affinity recognition
of complementary nucleic acids observed with -LNA.
Synthesis of the 30-Me-
-LNA uracil phosphoramidite 23 was
27
anhydro
formation
a-L
a-L
2',4'-
MsO
MsO
BnO
MsO
a
-L
cyclize
OH
O
O
Ur
OH
20
initiated from diacetone glucose 9 (Scheme 1). In diacetone glu-
cose, oxidation of the 30-hydroxyl followed by addition of MeMgBr
is known to provide almost exclusively the undesired epimeric (R)
30-methyl sugar due to attack of the organometallic reagent from
the less hindered concave face of the cis-fused bicyclic 1,2-aceto-
nide.23 To circumvent this, we first prepared the exocyclic olefin
10 from diacetone glucose by a Swern/Wittig sequence. Reaction
of 10 with MCPBA resulted in conversion of the exocyclic olefin
to a mixture of epoxide diastereomers (ꢀ10:1) from which the
major and desired product 11 could be cleanly isolated after chro-
MsO
BnO
N
O
N
O
Me
Me
28
Scheme 2. Rationale for formation of 30-Me-b-
D-xylo-LNA.
matography. Reduction of the epoxide 11 using super hydride pro-
vided the tertiary alcohol 12 with the methyl group now in the

4692
P. P. Seth et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4690–4694
desired (S) configuration.24 The tertiary alcohol in 12 was
protected as the benzyl ether to provide 13 in excellent yield.
Selective hydrolysis of the 5,6-acetonide, followed by oxidative
cleavage of the alcohol with periodate and treatment with excess
formaldehyde and sodium hydroxide provided diol 14 in low yield.
The major product appeared to be compound 15 which is formed
by beta-elimination of the 30-benzyl ether upon treatment of the
intermediate aldehyde with a strong base such as sodium hydrox-
ide. Interestingly, no beta-elimination of the 30-ether is reported
with analogous sugar intermediates which do not have a 30-methyl
group of when the 30-alkyl group is in the opposite configura-
tion.15,25 Efforts to suppress the beta-elimination reaction by cool-
ing the reaction prior to adding sodium hydroxide, adding a large
excess of formaldehyde to trap the anion prior to beta-elimination
or slow addition of base were not successful. However, we were
still able to prepare multi-gram quantities of diol 14 which were
adequate to provide gram quantities of the final phosphoramidite
23, due to the relative ease of preparing tertiary alcohol 13
(ꢀ50 g) early in the synthetic scheme. Next, diol 14 was reacted
with methanesulfonyl chloride to provide the bis-mesylate 16.
Acetolysis of the 1,2-acetonide followed by a Vorbruggen reaction
with per-silylated uracil to install the nucleobase provided nucleo-
side 17 after removal of the 20-acetyl group with methanolic
ammonia.
Bn
O
"H6
H6'
3'
Me
H6'
H6
N
H6
R
O
R
O
6
O
4'
4'
BnO
O
N
O
O
O
2'
H6"
2'
1'
H
3'
Me
1'
H
NH
NH
O
26 (R = CH2OMs)
20 (R = CH2OMs)
Figure 2. Stereochemical assignment for nucleosides 20 and 26 showing relevant
NOESY crosspeaks.
ꢀ85% for single and ꢀ75% for the tandem incorporations. In this se-
quence, 30-Me-
a-L-LNA showed good hybridization properties (A2,
D
Tm +4.0 °C/mod.), which were slightly lower than those observed
for -LNA (A6, Tm +4.6 °C/mod.) and LNA (A8, Tm +4.6 °C/
mod.) modified oligonucleotides. Incorporation of two tandem 30-
Me- -LNA (A3) or -LNA (A7) nucleotides provided an almost
a
-
L
D
D
a
-
L
a-L
identical increase in Tm per modification as compared to a single
incorporation of the high affinity nucleotide. We also measured
the mismatch discrimination properties of 30-Me-
a-
L
-LNA and
-LNA (A4 and A5) using mismatched RNA
complements. For both, the single and tandem incorporation of
30-Me-
-LNA, we observed excellent discrimination for the UC
Tm ꢁ14.6 °C/mod.) and the UU Tm ꢁ15.1 °C/mod.) mis-
matched pairs which were similar to the discrimination observed
with R-60-
-LNA, -LNA, LNA and DNA for the same mis-
matches. Interestingly, we observed slightly enhanced selectivity
for the GU wobble base pair using 30-Me-
-LNA (
Tm ꢁ6.7 °C/
mod.) as compared to R-60-Me-
-LNA (
Tm ꢁ5.3 °C/mod.),
LNA ( Tm ꢁ5.1 °C/mod.) or DNA (
Tm ꢁ4.7 °C/mod.), LNA (
ꢁ4.1 °C/mod.).
Lastly, we constructed a structural model of a modified duplex
the related 60-Me-
a-L
Attempts to invert the 20-hydroxyl group by means of an anhy-
dro-formation/ring-opening sequence were unsuccessful due to
very slow formation of the anhydro-nucleoside intermediate 28
(Scheme 2). Instead, under the reaction conditions employed, the
20-mesylate was slowly hydrolyzed to generate the 20-hydroxyl
group (25) which cyclized into the 50-mesylate to provide the 30-
Me-xylo nucleoside 26. Presumably, formation of the anhydro-
nucleoside has to proceed through a transition state where the
30-methyl group eclipses the adjacent 20-mesylate (27) and this
disfavors SN2 displacement of the 20-mesylate by the 2-oxygen
atom of the pyrimidine nucleobase. To further confirm the identity
a-L
(D
(D
a-
L
a-L
a
-L
D
a
D
-L
D
a-L-
DTm
D
containing 30-Me-
a-L-LNA using a published structure of an a-L-
of the cyclized b-
D
-xylo nucleoside 26, nucleoside 17 was treated
LNA/RNA duplex as described previously (Fig. 3).19 The model
clearly shows that the 30-Me group lies in the minor groove of
the oligonucleotide duplex and does not experience any steric
interactions with the sugar-phosphate backbone. The model also
with aqueous sodium hydroxide to provide 26 by direct displace-
ment of the 50-mesylate by the 20-hydroxyl group. To avoid this
problem, the 20-hydroxyl group was inverted by first oxidation to
the 20-ketone 18, followed by reduction with sodium borohydride
to provide 19 almost exclusively. Presumably, attack of the hydride
takes place from the relatively less hindered re face of the ketone in
18 to provide alcohol 19. Further treatment of 19 with aqueous so-
dium hydroxide provided the 20,40-cyclized nucleoside 20. The 50-
mesylate in 20 was next displaced by heating with potassium ace-
tate and 18-crown-6 in 1,4-dioxane to provide the 50-acetate 21.
For solubility reasons, we chose to remove the benzyl group first
using catalytic hydrogenation, followed by removal of the 50-ace-
tate and reprotection of the 50-hydroxyl group as the DMTr ether
to provide nucleoside 22 in good yield. Finally, a phosphitylation
reaction provided the desired phosphoramidite 23.
indicates that the 30-position in
a-L-LNA is a suitable site for
appending additional substitution in the form of conjugates or re-
porter molecules which could be very useful for oligonucleotide
based diagnostic applications.
In conclusion, we present that synthesis and biophysical char-
acterization of 30-Me-
a-L-LNA modified oligonucleotides. The syn-
thesis was accomplished starting from diacetone glucose and
utilized hydride opening of an exocyclic epoxide to install the 30-
methyl group in the desired configuration. The 30-protected ether
was found to be extremely susceptible to beta-elimination during
installation of the 40-hydroxymethyl functionality. Interestingly,
presence of the 30-Me group almost completely abolished forma-
tion of the anhydro nucleoside, an extremely facile reaction of thy-
midine nucleosides. Instead, inversion of the 20-hydroxyl group
was accomplished by means of an oxidation reduction sequence
with reduction of the 20-ketone taking place away from the bulky
The stereochemistry of the cyclized nucleosides 20 and 26 was
confirmed by NMR spectroscopy (Fig. 2).26 In nucleosides 20 and
26, H10 and H20 appear as singlets indicative of locked [2.2.1] ring
system. For nucleoside 20, NOESY crosspeaks were visible between
the 30-Me group and the H10 and between H60 and H6 on the
pyrimidine nucleobase. For nucleoside 26, NOESY crosspeaks were
visible between the 30-Me group and H60 and between H10 and H600.
nucleobase. Biophysical evaluation revealed that 30-Me-
shows similar hybridization properties to -LNA and LNA itself.
Examination of 30-Me-
-LNA in mismatch discrimination exper-
iments indicated that this modification possesses slightly better
discrimination for the GU wobble base-pair as compared to 60-
-LNA, -LNA, LNA and DNA. Lastly, a structural model showed
a-L-LNA
a
-L
a
-L
The effect of 30-Me-
a-L-LNA modification on duplex thermal
stability was measured using a single and two tandem incorpora-
tion of the modified nucleoside (Table 1). Oligonucleotides were
a-
L
a-L
synthesized on a 2
lmol scale using T-CPG support, 0.l M solutions
that the 30-Me-group resides in the minor groove of the modified
duplex and could also serve as a site for introduction of other func-
tional groups such as conjugates and reporter molecules. Further
evaluation of this modification in biological experiments is under-
way and the results will be reported in due course.
of all phosphoramidites in acetonitrile, 0.5 M 1H-tetrazole as the
activator and standard oxidizing and capping reagents. An ex-
tended coupling time of 8 min was used for incorporation of the
modified nucleosides and the efficiency of incorporation was

P. P. Seth et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4690–4694
4693
Table 1
Thermal stability measurements of 30-Me-
a
-L-LNA, R-60-Me-
a
-L-LNA, a-L-LNA and LNA modified oligonucleotides versus RNA complements
O
O
O
O
O
O
O
O
Me
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
NH
NH
NH
NH
Me
O
O
O
O
LNA
α-L-LNA
3'-Me-α-L-LNA
6'-Me-α-L-LNA
b
Oligomer
Mass
% UV purity
Modification
DNA
Sequence (50–30)a
DTm (°C) versus RNA
Calcd
Found
X = A
X = G
X = C
X = U
A1
A2
3633.4
3661.4
3632.9
3660.8
–
98.4
d(GCGTTTTTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
d(GCGTTUUTTGCT)
d(GCGTTUTTTGCT)
(45.6)
+4.1
ꢁ4.1
ꢁ6.7
ꢁ6.3
ꢁ5.3
ꢁ5.3
ꢁ4.7
nd
ꢁ13.3
ꢁ14.6
ꢁ14.6
ꢁ15.5
ꢁ15.0
ꢁ15.4
nd
ꢁ13.8
ꢁ13.3
ꢁ15.1
ꢁ13.9
ꢁ15.4
ꢁ13.8
nd
30-Me-
30-Me-
60-Me-
60-Me-
a-
a-
a-
a-
L
L
L
L
-LNA
-LNA
-LNA
-LNA
A3
A4
A5
A6
A7
A8
3689.4
3661.4
3689.4
3647.4
3661.3
3647.4
3689.0
3660.9
3688.9
3646.5
3660.8
3646.8
96.3
98.6
97.9
97.8
97.9
98.1
+4.0
+5.5
+4.8
+4.6
+4.6
+4.6
a
a
-
-
L
-LNA
-LNA
L
LNA
ꢁ5.1
ꢁ14.9
ꢁ12.9
Sequence of RNA complement 50-r(AGCAAAXAACGC)-30, X = A represents the perfectly matched complement, while X = G, C or U represent the mis-matched complements.
a
Bold and underlined alphabet indicates modified residue.
Tm values were measured in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM EDTA,
b
DTm values were calculated by subtracting the Tm
measured for the duplexes of modified oligomers versus matched and mis-matched RNA complements from the Tm of the perfectly matched A1/RNA (45.6 °C) duplex.
Figure 3. Hypothetical model of a 30-Me-
minor groove of the modified duplex. The
Letter is uracil.
a
a
-
-
L
-LNA modified duplex with RNA showing (a) the 30-Methyl group highlighted in green and (b) the 30-Me group located in the
-LNA nucleobase in the published report (Ref.13) is thymine while the nucleobase for 30-Me-
-LNA described in the present
L
a-L
6. Gupta, N.; Fisker, N.; Asselin, M. C.; Lindholm, M.; Rosenbohm, C.; Orum, H.;
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26. Compound 20: 1H NMR (300 MHz, CDCl3) d: 1.64 (s, 3H), 3.06 (s, 3H), 4.06 (d,
J = 8.5 Hz, 1H), 4.41 (d, J = 8.5 Hz, 1H), 4.46–4.65 (m, 4H), 4.69 (s, 1H), 5.80 (d,
J = 8.1 Hz, 1H), 5.94 (s, 1H), 7.24–7.47 (m, 5H), 7.75 (d, J = 8.1 Hz, 1H), 9.29 (br s,
1H). Compound 26: 1H NMR (300 MHz, CDCl3) d: 1.57 (s, 3H), 3.11 (s, 3H), 3.96
(d, J = 11.1 Hz, 1H), 4.04 (d, J = 9.0 Hz, 1H), 4.11 (d, J = 8.9 Hz, 1H), 4.58 (m, 2H),
4.62 (s, 1H), 4.72 (d, J = 11.9 Hz, 1H), 5.21 (d, J = 8.1 Hz, 1H), 5.63 (s, 1H), 7.11
(m, 2H), 7.22–7.38 (m, 3H), 7.47 (d, J = 8.1 Hz, 1H), 9.15 (br s, 1H).
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