Synthesis and biophysical properties of constrained d -altritol nucleic acids (cANA)

Article abstract of DOI:10.1021/ol401730d

The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how conformationally restricting the d-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found that the thermal stability was reduced (cANA/RNA -1.1 C/modification) compared to DNA/RNA, suggesting the constrained system results in a small destabilizing perturbation in the duplex structure.

Full text of DOI:10.1021/ol401730d

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Synthesis and Biophysical Properties
of Constrained D‑Altritol Nucleic
Acids (cANA)
Michael T. Migawa,* Thazha P. Prakash, Guillermo Vasquez, Punit P. Seth, and
Eric E. Swayze
Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc., 2855 Gazelle Court,
Carlsbad, California 92010, United States
mmigawa@isisph.com
Received June 19, 2013
ABSTRACT
The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how
conformationally restricting the D-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found
that the thermal stability was reduced (cANA/RNA ꢀ1.1 °C/modification) compared to DNA/RNA, suggesting the constrained system results in a
small destabilizing perturbation in the duplex structure.
Some antisense oligonucleotides (ASOs)1 modified with
monomers having a conformationally constrained pento-
furanosyl sugar show an increase in binding affinity with
their complementary mRNA; this has been well documen-
ted in the case of LNA (1, Figure 1).2ꢀ6 LNA can be
thought of as a conformationally restricted 20-OMe RNA,
where the methyl group is constrained to the 40-position,
thereby locking it into its ‘northern’ conformation. When
incorporating this conformational restricted 20-OMe RNA,
i.e. LNA, into an ASO, a large increase in affinity is
observed when paired with complementary RNA. This
observation also holds for a variety of other conformation-
ally restricted analogs, such as cEt-BNA (2),7 R-LNA,8 and
ENA.9
Herdewijn et al. have been studying anhydrohexitol-
based oligonucleotides, where the pyranosyl ring system is
replaced with a hexitol ring system.10ꢀ16 The parent in this
series is hexitol nucleic acid (3, HNA), which was incorpo-
rated intoanASO and reported to possess a strong binding
affinity when duplexed with RNA, in addition to many
(9) Morita, K.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.;
Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. Lett. 2002,
12, 73.
(1) Antisense Drug Technology: Principles, Strategies, and Applica-
tions, 2nd ed.; Crooke, S. T., Ed.; CRC Press: Boca Raton, FL, 2007.
(2) Lindholm, M. W.; Elmen, J.; Fisker, N.; Hansen, H. F.; Persson,
R.; Moller, M. R.; Rosenbohm, C.; Orum, H.; Straarup, E. M.; Koch, T.
Mol. Ther. 2011, 376.
(10) D’Alonzo, D.; Van Aerschot, A.; Guaragna, A.; Palumbo, G.;
Schepers, G.; Capone, S.; Rozenski, J.; Herdewijn, P. Chem.;Eur. J.
2009, 15, 10121.
(11) Maier, T.; Przylas, I.; Strater, N.; Herdewijn, P.; Saenger, W.
J. Am. Chem. Soc. 2005, 127, 2937.
(3) Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P.
J. Am. Chem. Soc. 2002, 124, 5974.
(12) Allart, B.; Van Aerschot, A.; Herdewijn, P. Nucleosides Nucleo-
tides 1998, 17, 1523.
(4) Nielsen, C. B.; Singh, S. K.; Wengel, J.; Jacobsen, J. P. J. Biomol.
Struct. Dyn. 1999, 17, 175.
(13) Allart, B.; Khan, K.; Rosemeyer, H.; Schepers, G.; Hendrix, C.;
Rothenbacher, K.; Seela, F.; Aerschot, A. V.; Herdewijn, P. Chem.;
Eur. J. 1999, 5, 2424.
(14) Van Aerschot, A.;Maurinsh, Y.;Allart, B.;Boudou, V.; Herdewijn,
P. Collect. Symp. Ser. 1999, 2, 100.
(5) Braasch, D. A.; Corey, D. R. Chem. Biol. 2001, 8, 1.
(6) Petersen, M.; Wengel, J. Trends Biotechnol. 2003, 21, 74.
(7) Seth, P. P.; Vasquez, G.; Allerson, C. A.; Berdeja, A.; Gaus, H.;
Kinberger, G. A.; Prakash, T. P.; Migawa, M. T.; Bhat, B.; Swayze, E. E.
J. Org. Chem. 2010, 75, 1569.
(15) Wouters, J.; Herdewijn, P. Bioorg. Med. Chem. Lett. 1999, 9,
1563.
(8) Hakansson, A. E.; Wengel, J. Bioorg. Med. Chem. Lett. 2001, 11,
935.
(16) Froeyen, M.; Wroblowski, B.; Esnouf, R.; De Winter, H.; Allart,
B.; Lescrinier, E.; Herdewijn, P. Helv. Chim. Acta 2000, 83, 2153.
r
10.1021/ol401730d
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interesting biological properties.12 An improvement in hy-
bridization was also demonstrated for D-altritol nucleic acids
(4, ANAs) and its methylated analog (5, Me-ANA).13,17
Due to the reported strong affinity that altritol nucleic acids
have toward RNA,14 we sought to discover if we could
conformationally restrict the sugar moiety in an analogous
manner to the pentofuranosyl sugars to further increase the
affinity in the altritol class of nucleoside analogs. Toward
this end, we prepared an ASO containing a constrained
ANA monomer (6, cANA) and ascertained its biophysical
properties.
Scheme 1
Scheme 2
Figure 1. Several modified nucleic acid monomers.
The synthesis of amidites suitable for ASO incorporation,
18 and 19, began with the widely available and inexpensive
diacetone glucose 7 (Scheme 1). Conversion to the protected
hexitol (8) was carried out by a slight modification of known
methods.18,19 A subsequent deoxygenation of compound 8
at the anomeric position was achieved by converting the
anomeric acetate to its corresponding chloride with HCl
followed by a reduction of the R-chloride under radical
conditions, deacetylation with methanolic ammonia, and
4,6-benzylidine formation to give the monoprotected diol
(9) in 30% yield over four steps.20 The 2-methylnapthyl
group (Nap) was removed with DDQ in aqueous DCM in
81% yield, followed by bis-tosylation, and then selective
hydrolysis gave the monotosylated alcohol 11 in 61% yield
over two steps.21
Treatment of compound 11 with sodium hydride rapidly
gave epoxide 12 in a 91% yield, which was converted into
the alcohol 13 by an ozonolysisꢀborohydride reduction
sequence. Our key intermediate, 13, could then be coupled
(17) Van Aerschot, A.; Meldgaard, M.; Schepers, G.; Volders, F.;
Rozenski, J.; Busson, R.; Herdewijn, P. Nucleic Acids Res. 2001, 29,
4187.
(18) Bleriot, Y.; Vadivel, S. K.; Herrera, A. J.; Greig, I. R.; Kirby,
A. J.; Sinay, P. Tetrahedron 2004, 60, 6813.
(19) Rao, A. V. R.; Gurjar, M. K.; Devi, T. R.; Kumar, K. R.
Tetrahedron Lett. 1993, 34, 1653.
to uracil or thymine by an epoxide ring-opening reaction
under sodium salt conditions to give the corresponding
nucleoside, 14a and 14b, in 52% and 69% yields,
(20) Auge, J.; David, S. Carbohydr. Res. 1977, 59, 255.
(21) Brockway, C.; Kocienski, P.; Pant, C. J. Chem. Soc., Perkin
Trans. 1 1984, 875.
B
Org. Lett., Vol. XX, No. XX, XXXX

respectively (Scheme 2). Tosylation of the primary hydro-
xyl followed by ring closure gave the corresponding pro-
tected, constrained nucleosides 15a and 15b.
were stabilizing, resulting in 1.6 °C/mod and 0.1°/mod,
respectively.
To explain the destabilizing behavior of the cANA
incorporation, we speculated that the conformation of
the cANA monomer in the duplex differs somewhat from
that of Me-ANA. Recently, it has been suggested that very
slight perturbations in the lean of the nucleobase or the
rotation angle of the glycosidic bond axis can result in a
large destabilizing effect.22 While the crystal structure of
15a suggests that a nearly perfect overlay with Me-ANA
or HNA is possible, it is also likely that the addition of
the 30,50-CH2O- bridge may introduce additional steric
effects modifying the preferred conformation. As shown
in Figure 3a, the Me-ANA monomer prefers to adopt
a conformation that situates the nucleobase in an axial
orientation which is necessary for WatsonꢀCrick base
pairing in the duplex. However, as seen in Figure 3b, an
additional 1,6 steric interaction could render this confor-
mation undesirable, with the six-membered ring adopting
the preferred chair conformation, while the outer seven-
membered prefers a chair or twist-chair conformation. This
would situate the nucleobase in an equatorial position,
preferentially, resulting in a destabilizing, rather than sta-
bilizing, effect in the context of the ASO/RNA duplex.
Additionally, in the case of Me-ANA, the methyl group lies
‘outside’ the main pyranosyl ring, whereas in the con-
strained system the methyl group lies ‘underneath’ the ring.
This gives rise to additional steric bulk being added under-
neath the pyranosyl ring which may be unfavorable within
the duplex structure.
It is known that the 5-methyl analog of ANA possesses
a higher Tm than the 5-unsubstituted analog; therefore,
we sought to further transform the thymidine derivative
(i.e., 15b) into an amidite suitable for ASO synthesis for Tm
studies while using 15a for X-ray crystallography studies
(Figure 2). While X-ray analysis unambiguously confirmed
the structure of the uracil derivative, the thymine derivative
was assigned by its nearly identical NMR spectra. With
the structure of 15b verified, we removed the benzylidine to
give the unprotected nucleoside 16 in quantitative yield,
installed a DMTr group at the 50-position in 78% yield,
and followed this by conversion into the T and MeC
amidites using well-known and previously reported
methodology.7 We were able to obtain both phosphorami-
dites of the pyrimidine analogs in quantities sufficient for
Tm studies.
Figure 2. Structure of compound 15a from crystal structure
coordinates.
The synthesis of the ASO was carried out (mCTTAG-
CACTGGCmCT), and the Tm of this ASO duplex with
complementary RNA was determined using previously
reported procedures.7 As shown in Table 1, the cANA
ASO (20) Tm was reduced by ꢀ1.1 °C/modification com-
pared toDNA (23), while the HNA (22) and Me-ANA(21)
Figure 3. Conformational analysis of Me-ANA (a) and cANA (b).
Table 1. Biophysical Data of ASOs Containing Me-ANA,
HNA, and cANA Modificationsa
In conclusion, we were able to report the first synthesis
of a constrained altritol nucleoside and incorporate it into
an ASO to determine that it is a destabilizing modification
as compared to the unconstrained hexitol nucleic acids.
Additional work will need to be carried out to determine
the precise nature of the destabilizing effect of cANA
and determine if alternative modes of conformational
Tm
no.
sequences (50-30)
chemistry
(°C)
20
21
22
23
d(mCB1TB1TAGCACTGGCmCB1TB1
d(CAUATAGCACTGGCCAUA)
d(mCHTHTAGCACTGGCmCHTH)
d(CTTAGCACTGGCCT)
)
cANA
44
Me-ANA23
HNA23
DNA
48.9
54.9
48.5
a All internucleosidic linkages are phosphorothioate, mCB1 = cANA-
5-methyl-cytidine, TB1 = cANA thymidine, CA = 30-O-methyl-ANA-
cytidine, UA = 30-O-methyl-ANA-cytidine uridine, mCH = HNA-5-
methylcytidine, TH = HNA-5-methylcytidine.
(22) Mori, K.; Kodama, T.; Obika, S. Org. Lett. 2011, 13, 6050.
(23) Egli, M.; Pallan, P. S.; Allerson, C. R.; Prakash, T. P.; Berdeja,
A.; Yu, J.; Lee, S.; Watt, A.; Gaus, H.; Bhat, B.; Swayze, E. E.; Seth, P. P.
J. Am. Chem. Soc. 2011, 133, 16642.
Org. Lett., Vol. XX, No. XX, XXXX
C

constraint will result in a stabilizing effect when paired
with RNA.
Supporting Information Available. Experimental pro-
cedures, spectroscopic data for new compounds, and
crystallographic data for compound 15a are available.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. The authors thankArnoldL. Rheingold
at the University of California San Diego for obtaining
the crystal structure and Tracy Reigle for assistance in the
preparation of this manuscript.
The authors declare no competing financial interest.
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Org. Lett., Vol. XX, No. XX, XXXX