Structure-based design of a highly constrained nucleic acid analogue: Improved duplex stabilization by restricting sugar pucker and torsion angle γ

Article abstract of DOI:10.1002/anie.201203680

Dual conformational restriction: A new, highly constrained modification of the α-L-locked nucleic acid (α-L-LNA) scaffold that locks the sugar furanose ring in an N-type configuration and also restricts rotation around torsion angle γ was synthesized (see scheme). This new modification increases the thermostability of an oligonucleotide duplex compared to using a single mode of constraint alone. Copyright

Full text of DOI:10.1002/anie.201203680

Angewandte
Chemie
DOI: 10.1002/anie.201203680
Modified Nucleotides
Structure-Based Design of a Highly Constrained Nucleic Acid
Analogue: Improved Duplex Stabilization by Restricting Sugar Pucker
and Torsion Angle g**
Stephen Hanessian,* Benjamin R. Schroeder, Robert D. Giacometti, Bradley L. Merner,
Michael Østergaard, Eric E. Swayze, and Punit P. Seth*
Oligonucleotide modifications, which enhance affinity for
cognate RNA, have been used extensively as antisense
agents[1] and for oligonucleotide-based diagnostic applica-
tions.[2] In general, modifications that improve affinity by
conformational restriction of the sugar–phosphate backbone
have been the most successful, because they do not interfere
with the specificity of Watson–Crick base pairing and can be
used across a wide range of oligonucleotide sequences with
predictable results.[1,3] Over the past two decades, at least
three distinct successful strategies for incorporating covalent
conformational constraints into natural nucleic acids (NAs)
have been described. The first strategy restricts rotation
around torsion angles g and d in 2’-deoxynucleotide subunits
(1), as exemplified by the tricyclo-DNA (tcDNA 2) scaffold
reported by Leumann.[4] A second strategy reported by
Escudier[5] improves affinity for RNA and DNA complements
by restricting backbone torsion angles a and b into the
canonical ap and -sc range found in A-form DNA duplexes
(a,b-constrained nucleic acid or a,b-CNA 3). The third, and
perhaps the most successful strategy, involves locking the
nucleoside furanose ring in an N-type (northern) sugar
pucker, as exemplified by 2’,4’-bridged (locked) nucleic
acids 4.[6,7]
While each of the above approaches have been explored
individually, incorporating more than one type of conforma-
tional constraint into a single modification could provide
incremental increases in the binding affinity of a modified
oligonucleotide that may be unattainable through a single
mode of constraint. In this Communication, we report a new,
highly constrained oligonucleotide modification analogous to
that represented by the generic formula of 5 (Scheme 1),
which improves oligonucleotide duplex thermostability by
Scheme 1. Rationale for the design of a-l-tricyclic nucleic acids.
[*] Prof. Dr. S. Hanessian, Dr. B. R. Schroeder, R. D. Giacometti,
Dr. B. L. Merner
Department of Chemistry, Universitꢀ de Montrꢀal
C.P. 6128, Succ. Centre-ville, Montrꢀal, QC H3C 3J7 (Canada)
E-mail: stephen.hanessian@umontreal.ca
locking the furanose sugar pucker, as well as restricting
rotation around torsion angle g.
We chose a-l-ribo-configured locked nucleic acid (a-l-
LNA) 6 as the starting point to evaluate our strategy of dual
conformational restriction (Scheme 1). a-l-LNA (6) exhibits
LNA-like high-affinity recognition of complementary nucleic
acids when incorporated in appropriate oligonucleotide
sequences.[7] The increased affinity of a-l-LNA-modified
oligonucleotides for RNA stems from a combination of
locking the sugar furanose ring in an N-type conformation
along with the a-l-configuration of the nucleoside mono-
mer.[8] Structural studies of an a-l-LNA-modified DNA/RNA
M. Østergaard, E. E. Swayze, P. P. Seth
Department of Medicinal Chemistry, Isis Pharmaceuticals
2855 Gazelle Court, Carlsbad, CA 92010 (USA)
E-mail: pseth@isisph.com
[**] R.D.G. is grateful to NSERC (CGS D) for a postgraduate scholar-
ship. We thank Benoꢁt DeschÞnes-Simard for X-ray crystallographic
analyses.
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201203680.
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duplex showed that the 2’,4’-bridge of a-l-LNA lies inside the
major groove and also mapped the orientation of torsion
angle g for the modified nucleotide within the oligonucleotide
duplex.[9] Using the NMR structure of the modified duplex as
a starting point, we previously introduced (R)-configured
methyl groups at the 5’- and 6’-positions on the a-l-LNA
scaffold to give analogues 7 and 8, respectively.[10] Evaluation
of oligonucleotides modified with 7 or 8 in thermal denatura-
tion experiments revealed that both of these analogues
display a-l-LNA-like affinity for RNA. Accordingly, we
hypothesized that tethering these methyl groups together,
forming a six-membered ring between the 5’- and 6’-positions
of the a-l-LNA nucleoside monomer (that is 2’,4’-5’,6’-bis-
constrained-a-l-tricyclic nucleic acid (9) or 2’,4’-5’,6’-bc-a-l-
TriNA), would further improve affinity for RNA by restrict-
ing rotation around angle g.
While considering various approaches to 9, we were aware
that stereocontrolled formation of the 2’,4’-anhydro bridge
(that is the 1,4-dioxa[2.2.1]heptane motif) in 9 would present
a major synthetic challenge, and thus planned the route
accordingly. Oxidation of 10, readily available from diace-
tone-d-glucose,[11] afforded aldehyde 11, which was subjected
to a Sakurai allylation reaction[12] to give 12 after pivaloyla-
tion and removal of the TBS protecting group (Scheme 2).
Oxidation of the a-configured hydroxymethyl group to the
corresponding aldehyde and subsequent treatment with
vinylmagnesium bromide afforded a 1:1 mixture of allylic
alcohols 13, which were converted into the spirocyclic cyclo-
hexene using a ring-closing metathesis reaction in the
presence of Grubbsꢀ second-generation catalyst and in
excellent overall yield. An oxidation–reduction sequence
afforded enantiopure spirocycle 14, which was subjected to
catalytic hydrogenation and protected as the 2-naphthyl-
methyl (Nap) ether to give 15. A three-step sequence
culminating with a Vorbrꢁggen glycosylation afforded orthog-
onally O-protected spirocyclic thymidyl nucleoside 16 in 80%
overall yield.[13] Chemoselective ester hydrolysis, followed by
mesylation of the 2’-OH group gave mesylate 17.
In the original synthetic proposal for 9, we envisaged that
exposing the 2’,6’-dimesylate analogue of 17 to hydroxide
would facilitate formation of the 2’,4’-anhydro bridge in
a single synthetic step, however, formation of the 2,6’-
anhydronucleoside prevailed instead. These initial attempts
at constructing the 2’,4’-anhydro bridge of 9 indicated that it
was essential to protect the free nitrogen atom of 17 prior to
cycloetherification.
Accordingly,
1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU)-induced formation of the 2,2’-
Scheme 2. Synthesis of thymidyl spironucleoside 23.[14] Reagents and
conditions: a) pyridinium chlorochromate (PCC), NaOAc, 4 ꢂ molec-
ular sieves (MS), CH2Cl2, 08C to RT, 5 h; b) BF3·OEt2, allyltrimethylsi-
lane, CH2Cl2, À788C, 3 h, 78% over two steps; c) pivaloyl chloride
(PivCl), 4-dimethylaminopyridine (DMAP), pyridine, 1008C, 24 h, 92%;
d) tetra-n-butylammonium fluoride (TBAF), THF, RT, 3 h, 88%; e) PCC,
NaOAc, 4 ꢂ MS, CH2Cl2, 08C to RT, 3 h, 81%; f) vinylmagnesium
bromide, Et2O, 08C, 30 min., 95% (dr=1:1); g) Grubbs II (1 mol%),
CH2Cl2, 408C, 1 h, 93%; h) PCC, NaOAc, 4 ꢂ MS, CH2Cl2, 08C to RT,
4 h; i) CeCl3·7H2O, NaBH4, MeOH, 08C, 20 min., 85% over two steps;
j) H2, 10% Pd/C, THF, RT, 4 h, 96%; k) NapBr, NaH, tetra-n-butylam-
monium iodide (TBAI), tetrahydrofuran/N,N-dimethylformamide
(THF/DMF; 1:1), 08C to RT, 2.5 h, 83%; l) 80% AcOH, 808C, 24 h;
m) Ac2O, DMAP, pyridine, 08C to RT, 15 h, 95% over two steps;
n) thymine, N,O-bis(trimethylsilyl)acetamide (BSA), 1,2-dichloro-
ethane, 808C, 1 h; then trimethylsilyl trifluoromethanesulfonate
(TMSOTf), 08C to 508C, 19 h, 84%; o) K2CO3, MeOH, RT, 15 h;
p) methanesulfonyl chloride (MsCl), pyridine, 08C to RT, 12 h, 86%
over two steps. q) DBU, MeCN, 808C, 12 h; r) NaOH, EtOH/H2O
(1:1), 908C, 2 h; s) DBU, BOMCl, DMF, 08C, 1 h, 65% over three
steps; t) Tf2O, pyridine, CH2Cl2, 08C, 30 min.; u) NaNH2, DMF, 558C,
15 min., 52% over two steps; v) 2,3-dichloro-5,6-dicyanobenzoquinone
(DDQ), CH2Cl2/H2O (9:1), RT, 1 h, 92%; w) levulinic acid (LevOH), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC·HCl), iPr2NEt,
DMAP, CH2Cl2., RT, 10 h, 84%; x) H2, Pd(OH)2/C, MeOH/EtOAc (1:1),
RT, 3 d; then iPr2NEt, RT, 1 h, 95%; y) NC(CH2)2OP(NiPr2)2, 1H-
tetrazole, N-methylimidazole, DMF, RT, 6 h, 55%.
2
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Chemie
anhydronucleoside intermediate 18, followed by basic hydrol-
ysis, and benzyl chloromethyl ether (BOM) protection of the
imide nitrogen atom gave the C2’-inverted nucleoside 19.
Conversion of 19 to the corresponding triflate gave 20, which
upon exposure to NaNH2 in N,N-dimethylformamide (DMF)
gave 21, containing the tricyclic core of 9, in 52% yield over
two steps. Cleavage of the O-Nap group, conversion to the
levulinate ester, and hydrogenolysis, followed by phosphity-
lation led to 23, the crucial monomer in the synthesis of the
oligonucleotides for melting temperature (Tm) studies.
Confirmation of the structure of the tricyclic spironucleo-
side 21 was found through an X-ray crystal structure of
a corresponding p-nitrobenzoate ester.[14] Considering the
possibility for b-elimination of the triflate ester to occur in 20,
it is remarkable that intramolecular SN2 displacement pre-
vailed to give 21 as the major product, even on gram scale.
Nevertheless, the formation of tricyclic nucleoside 21 was
accompanied by a benzyl enol ether by-product, likely
resulting from triflyl migration to the 2’-OH group and
subsequent elimination.[14]
duplex stabilization versus complementary DNA as com-
pared to LNA (A2, DTm + 1.78C/mod.) and a-l-LNA (A3,
DTm + 1.48C/mod.), although the magnitude of stabilization
was smaller given the higher baseline Tm of the parent
sequence A1 for its DNA complement.[16] In addition, 9
showed excellent mismatch discrimination properties, which
were comparable or improved relative to DNA, LNA, and a-
l-LNA modifications at the same position.[14] The tricyclic
analogue 9 also showed excellent duplex-stabilizing proper-
ties versus RNA and DNA complements in a second sequence
(A7, A9, and A11, DTm + 4.7 to + 8.38C/mod. for RNA and
+ 3.0 to + 7.48C/mod. for DNA) as compared to a-l-LNA
(A6, A8, and A10, DTm + 4.5 to + 6.38C/mod. for RNA and
+ 3.8 to + 6.58C/mod. for DNA).
In conclusion, we show that a strategy of dual conforma-
tional restriction can indeed be useful for stabilizing oligonu-
cleotide duplexes. The duplex-stabilizing properties of 9 are
impressive because several previous attempts to increase
duplex thermostability by appending six-membered rings to
restrict conformational freedom of the nucleoside furanose
ring were unsuccessful.[17] It is conceivable that the duplex-
stabilizing properties of 9 could be further improved by
introducing heteroatoms or other polar functional groups to
the six-membered carbocyclic ring to preserve the water of
hydration network around the sugar–phosphate backbone.[18]
Further attempts to apply this strategy of dual conformational
restriction to other classes of nucleic acid analogues, and to
investigate its potential for the antisense approach, are
currently in progress and the results of these experiments
will be reported later.
We measured the ability of tricyclic nucleoside motif 9 to
stabilize oligonucleotide duplexes versus complementary
DNA and RNA using two oligonucleotide sequences
(Table 1). Oligonucleotide syntheses were carried out on
Table 1: Duplex thermal-stability measurements of LNA-, a-l-LNA-, and
2’,4’-5’,6’-bc-a-l-TriNA-modified oligonucleotides versus DNA and RNA
complements.[a]
No. Modification Sequence (5’- 3’)
DTm 8C vs.
DTm 8C vs.
DNA
RNA
A1 DNA
A2 LNA
A3 a-l-LNA
d(GCGTTTTTTGCG) (49.1)
d(GCGTTTTTTGCG) +1.7
(46.0)
+5.2
+5.7
+7.1
(43.6)
+5.6
+5.3
+6.3
+8.3
+4.5
+4.7
Received: May 11, 2012
Revised: July 6, 2012
Published online: && &&, &&&&
d(GCGTTTTTTGCG) +1.4
d(GCGTTTTTTGCG) +2.6
d(CCAGTGATATGC) (47.3)
d(CCAGTGATATGC) +3.8
d(CCAGTGATATGC) +3.0
d(CCAGTGATATGC) +6.5
d(CCAGTGATATGC) +7.4
d(CCAGTGATATGC) +4.4
d(CCAGTGATATGC) +4.4
A4
9
A5 DNA
A6 a-l-LNA
Keywords: antisense agents · conformational restriction ·
.
nucleosides · oligonucleotides · structure-based design
A7
A8 a-l-LNA
A9
A10 a-l-LNA
A11
9
9
9
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1 mmol scale with universal UnyLinker polystyrene support.
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(A4, DTm + 7.18C/modification) showed excellent duplex-
stabilizing properties versus complementary RNA relative to
LNA (A2, DTm + 5.28C/mod.) and a-l-LNA (A3, DTm
+ 5.78C/mod.) when inserted in a stretch of dT residues.
Analogue 9 (A4, DTm + 2.68C/mod.) also showed good
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4
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Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Modified Nucleotides
S. Hanessian,* B. R. Schroeder,
R. D. Giacometti, B. L. Merner,
M. Østergaard, E. E. Swayze,
P. P. Seth*
&&&& — &&&&
Structure-Based Design of a Highly
Constrained Nucleic Acid Analogue:
Improved Duplex Stabilization by
Restricting Sugar Pucker and Torsion
Angle g
Dual conformational restriction: A new,
highly constrained modification of the a-
l-locked nucleic acid (a-l-LNA) scaffold
that locks the sugar furanose ring in an N-
type configuration and also restricts
rotation around torsion angle g was
synthesized (see scheme). This new
modification increases the thermostabil-
ity of an oligonucleotide duplex com-
pared to using a single mode of con-
straint alone.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!