N-Methyl substituted 2′,4′-BNANC: A highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization

Article abstract of DOI:10.1039/b707352f

Oligonucleotides modified with a novel BNA analogue, 2′, 4′-BNA^NC[N-Me], were synthesized, and in comparison to 2′,4′-BNA (LNA), have similarly high RNA affinity, better RNA selectivity and much higher resistance to nuclease degradation, sugges

Full text of DOI:10.1039/b707352f

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N-Methyl substituted 29,49-BNANC: a highly nuclease-resistant nucleic
acid analogue with high-affinity RNA selective hybridization{
Kazuyuki Miyashita,a S. M. Abdur Rahman,{a Sayori Seki,a Satoshi Obikaab and Takeshi Imanishi*a
Received (in Cambridge, UK) 16th May 2007, Accepted 28th June 2007
First published as an Advance Article on the web 9th July 2007
DOI: 10.1039/b707352f
Several years ago, our group and Wengel and co-workers
independently discovered 29-O,49-C-methylene bridged nucleic
acids (29,49-BNA11 or LNA12) (Fig. 1), which possesses higher
nuclease resistance than natural DNA and exhibit unprecedented
ability to hybridize with complementary RNA and DNA. This
nucleic acid analogue has applications in therapeutics and
genomics13 and is now commercially available. However, inade-
quate RNA selectivity and nuclease resistance of 29,49-BNA
(LNA) modified oligonucleotides might present obstacles to
developing ideal antisense molecules.14 Development of ethylene
bridged nucleic acid (ENA) resulted in improved nuclease
resistance to some extent but affinity to RNA was lowered slightly
and RNA selectivity of ENA is similar to that of 29,49-BNA
(LNA).15 Therefore, we focused our attention on fine-tuning the
BNA structure. As a result of our continued investigation, we
report here the development of another bridged nucleic acid
analogue; namely, N-methyl substituted 29,49-BNANC or 29,49-
BNANC[N–Me]),16 which displays high-affinity RNA selective
hybridizing ability17 and excellent nuclease resistance.
As shown in Scheme 1, 29,49-BNANC[N–Me] monomer 5,
thymine phosphoroamidite 6 and oligonucleotides 7–10 were
synthesized from nucleoside derivative 1.16 The compound 1 was
treated with hydrazine and the resulting free amino group18 was
reacted with benzyl chloroformate to give 2. Base mediated
cyclization of 2 proceeded smoothly to afford the tricyclic
compound 3, which was subjected to boron trichloride in
dichloromethane to furnish the desired cyclized product 4 in good
yield. 29,49-BNANC[N–Me] monomer 5 was then obtained by
reductive methylation and subsequent desilylation. The structure
of 5 was confirmed by 1H NMR spectroscopy and X-ray
crystallography.19 In order to synthesize phosphoroamidite, the
primary hydroxyl group of 5 was protected with a 4,49-dimethoxy-
trityl group and the secondary hydroxyl was phosphitylated with
2-cyanoethyl N,N,N9,N9-tetraisopropylphosphordiamidite, form-
ing the 29,49-BNANC[N–Me]-thymine phosphoroamidite 6. The
29,49-BNANC[N–Me]-modified oligonucleotides 7–10 were then
synthesized in an automated DNA synthesizer according to a
Oligonucleotides modified with a novel BNA analogue, 29,
49-BNANC[N–Me], were synthesized, and in comparison to
29,49-BNA (LNA), have similarly high RNA affinity, better
RNA selectivity and much higher resistance to nuclease
degradation, suggesting that the novel BNA analogue may be
particularly useful for antisense approaches.
For practical use in antisense approaches, chemically modified
oligonucleotides should possess: (i) high resistance to nuclease
degradation and (ii) high affinity to the target RNA strand, along
with excellent RNA selectivity.1 Although during the past few
decades several chemically modified oligonucleotides with robust
ability to bind RNA have been developed, most also have an
increased affinity to complementary DNA.2 Conformationally
restricted amide-linked nucleic acids3 and 29,59-linked DNA4 have
been reported to have selective binding affinity for complementary
RNAs. However, in most cases, their hybridizing ability to
complementary RNAs was similar to or less than that observed for
natural DNA, which greatly restricts their usefulness for practical
applications, such as in antisense technology and for diagnostic
purposes. Recently, a-LNA5 and a-L-LNA6 have been reported to
have excellent RNA specific binding affinity via parallel motif
hybridization. Nevertheless, it was concluded that the thermal
stability of parallel duplexes is lower than those of corresponding
antiparallel duplexes. Backbone extended pyrrolidine peptide
nucleic acids (bep-PNA)7 and thioacetamido nucleic acids
(TANA)8 also possessed promising RNA selective binding affinity.
However, their mode of RNA targeting was achieved via triplex
formation, which requires the use of two folds of antisense nucleic
acids. In addition to that, PNA has its own problems that restrict
its usefulness, including limited aqueous solubility, poor cellular
uptake9 and ambiguity in binding complementary DNA/RNA in
both parallel and antiparallel orientations.10 Our aim is to develop
an antisense oligonucleotide that is sufficiently stable in physio-
logical conditions and binds well to RNA complements while at
the same time, showing lower affinity to DNA complements.
Oligonucleotides which meet these criteria are likely to be much
more useful for in vivo antisense applications.
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6
Yamadaoka, Suita, Osaka, 565-0871, Japan.
E-mail: imanishi@phs.osaka-u.ac.jp; Fax: +81 6 6879 8204;
Tel: +81 6 6879 8200
bPRESTO, Japan Science and Technology Agency (JST), 4-1-8
Honcho Kawaguchi, Saitama, 332-0012, Japan
{ Electronic supplementary information (ESI) available: Full experimental
procedures, characterization data of all new compounds, and UV melting
profiles. See DOI: 10.1039/b707352f
{ On leave from the Faculty of Pharmacy, University of Dhaka, Dhaka-
1000, Bangladesh.
Fig. 1 Structures of 29,49-BNA (LNA), ENA and 29,49-BNANC[N–Me].
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Fig. 2 The change in melting temperature (DTm) of modified oligo-
nucleotides (7–9 and 11–13) relative to the reference oligonucleotide
15, 59-d(GCGTTTTTTGCT)-39. The Tm values of the duplexes formed
by 15 with complementary DNA and RNA were 50 and 45 uC,
respectively. Tm values were obtained from the maxima of the first
derivatives of the melting curves (at 260 nm). Conditions: 4 mM strands
solution in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM
NaCl. Target sequences: DNA, 59-d(AGCAAAAAACGC)-39 and RNA,
59-r(AGCAAAAAACGC)-39.
Scheme 1 Reagents and conditions: (i) H2NNH2?H2O, EtOH, rt, 10 min;
(ii) CbzCl, sat. NaHCO3, CH2Cl2, 0 uC; (iii) NaH, THF, rt, 5 h; (iv) BCl3,
CH2Cl2, 0 uC, 1 h; (v) 20% HCHO, NaBH3CN, PPTS, MeOH, 0 uC, 1 h;
(vi) TBAF, THF, rt, 5 min; (vii) DMTrCl, pyridine, rt, 12 h; (viii)
(iPr2N)2PO(CH2)2CN, dicyanoimidazole, MeCN, rt, 4 h; ix) DNA
synthesizer (AB ExpediteTM 8909). T_ = 29,49-BNANC[N–Me] residue.
standard phosphoroamidite protocol. The coupling efficiency of
the modified oligonucleotides was 96–99% and the isolated yields
were 48 to 63%. For comparison, a set of 29,49-BNA (LNA)-
modified oligonucleotides 11–13 (Fig. 2) and 14 (Fig. 3) were
synthesized, accordingly. The oligonucleotides were purified by
HPLC and characterized by MALDI-TOF mass spectra.§
Fig. 3 Nuclease resistance of T8XT oligonucleotides against SVPDE.
X = natural-T (6) (16), 29,49-BNA-T (#) (14), phosphorthioate-T (n)
(17), and 29,49-BNANC[N–Me]-T (&) (10). Hydrolysis of the oligonucleo-
tides (10 mg) was carried out at 37 uC in 320 ml of a buffer containing
50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 and SVPDE (0.2 mg).
The ability of 29,49-BNANC[N–Me] oligonucleotides to hybri-
dize to complementary RNA and DNA strands was evaluated via
UV melting experiments and compared with that of 29,49-BNA
(LNA)-modified oligonucleotides. The change in melting tempera-
tures (DTm) of the duplexes formed by 29,49-BNANC[N–Me]-
and 29,49-BNA (LNA)-modified oligonucleotides (7–9 and 11–13,
respectively) as compared with duplexes formed by natural DNA
are summarized in Fig. 2.20 It was found that the Tm values for
hybrids formed by 29,49-BNANC[N–Me] oligonucleotides with
RNA complements were high and comparable to Tm values for
hybrids formed by 29,49-BNA (LNA)-modified oligonucleotides,
11–13. In contrast, there is a great difference in their abilities to
form a duplex with complementary DNA. Whereas 29,49-BNA
(LNA)-modified oligonucleotides (11–13) exhibited a gradually
increasing affinity for DNA complements upon increase of the
number of modifications, the corresponding 29,49-BNANC[N–Me]-
modified oligonucleotides showed no change or slightly decreased
affinity towards target DNA; that is, Tm values decreased relative
to that exhibited by the natural DNA 15.21 This interesting
property (i.e. decreased affinity to complementary DNA while
retaining a very strong ability to hybridize with complementary
RNA) of 29,49-BNANC[N–Me]-modified oligonucleotides not only
differs from what has been observed for 29,49-BNA (LNA)-
modified oligonucleotides but also contrasted with the property
of N-alkyl 29-amino-LNA molecules having similar structural
features.22 Unfavorable steric interactions between methyl groups
and the DNA strand may be a cause of the decreased affinity.
Resistance of a decamer oligonucleotide with a single 29,49-
BNANC[N–Me] residue (10) to a 39-exonuclease (snake venom
phosphodiesterase, SVPDE) was assayed and the results were
compared with those obtained by the corresponding natural
oligonucleotide 16, and with 29,49-BNA (LNA)-modified (14) and
phosphorthioate-modified (17) oligonucleotides. After incubation
of oligonucleotide solutions with SVPDE, the reaction mixtures
were analyzed at several time points by reversed-phase HPLC to
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9 U. Koppelhus and P. E. Nielsen, Adv. Drug Delivery Rev., 2003, 55, 267.
10 K. N. Ganesh and P. E. Nielsen, Curr. Org. Chem., 2000, 4, 1931.
11 (a) S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida and
T. Imanishi, Tetrahedron Lett., 1997, 38, 8735; (b) S. Obika, D. Nanbu,
Y. Hari, J. Andoh, K. Morio, T. Doi and T. Imanishi, Tetrahedron
Lett., 1998, 39, 5401.
12 S. K. Sing, P. Nielsen, A. A. Koshkin and J. Wengel, Chem. Commun.,
1998, 455.
13 (a) M. Petersen and J. Wengel, Trends Biotechnol., 2003, 21, 74; (b)
J. S. Jespen and J. Wengel, Curr. Opin. Drug Discovery Dev., 2004, 7,
188; (c) J. S. Jespen, M. D. Sørensen and J. Wengel, Oligonucleotides,
2004, 14, 130.
14 In addition to that, very recently, it is reported that antisense
oligonucleotides containing 29,49-BNA (LNA) are hepatotoxic which
demands discovery of new candidates with similar or better potency; see:
E. E. Swayze, A. M. Siwkowski, E. V. Wancewicz, M. T. Migawa,
T. K. Wyrzykiewicz, G. Hung, B. P. Monia and C. F. Bennet, Nucleic
Acids Res., 2007, 35, 687.
15 (a) K. Morita, C. Hasegawa, M. Kaneko, S. Tsutumi, J. Sone,
T. Ishikawa, T. Imanishi and M. Koizumi, Bioorg. Med. Chem. Lett.,
2002, 12, 73; (b) M. Koizumi, Curr. Opin. Mol. Ther., 2006, 8, 144, and
references therein.
16 Recently, we have communicated the highly stable triplex-forming
ability of 29,49-BNANC[N–H] analogue. The triplex-forming ability
was higher than that of 29,49-BNA (LNA) and ENA;15 see:
S. M. A. Rahman, S. Seki, S. Obika, S. Haitani, K. Miyashita and
T. Imanishi, Angew. Chem., Int. Ed., 2007, 46, 4306.
17 Recently, RNA selective hybridization was reported by using three-
carbon 29,49-linkage LNA analogue; see: N. Alback, M. Petersen and
P. Nielsen, J. Org. Chem., 2006, 71, 7731. However, the affinity of the
analogue to hybridize RNA decreased considerably compared to that of
29,49-BNA (LNA). For example, a duplex formed by a 9-mer
oligonucleotide having three modifications furnished melting tempera-
ture (Tm) at 38 uC which is 12 uC lower than that obtained by a
corresponding LNA–RNA duplex (Tm = 50 uC)12.
monitor the percentage of intact oligonucleotides (Fig. 3). Under
experimental conditions, the natural and 29,49-BNA (LNA)-
modified oligothymidylates were completely digested within 5
and 20 min, respectively. In contrast, the 29,49-BNANC[N–Me]-
modified oligonucleotide was remarkably stable; about 85% of
the oligonucleotide survived after 40 min. The nuclease resistance
of 29,49-BNANC[N–Me] was also slightly better than that of the
phosphorthioate-modified oligonucleotide 17.23 The excellent
resistance of 10 to SVPDE might result from steric hindrance
around the phosphodiester linkage exerted by the six-membered
bridged moiety with a methyl substituent.
In conclusion, we have synthesized a novel bridged nucleic acid
29,49-BNANC[N–Me] and shown that it has high-affinity hybridi-
zation similar to that of 29,49-BNA (LNA) against an RNA
complement. Moreover, the nucleic acid analogue displayed RNA
selectivity superior to that of 29,49-BNA (LNA) and other
structural analogues of 29,49-BNA (LNA). Nuclease resistance of
this nucleic acid analogue is abundantly higher than that of
29,49-BNA (LNA) and also slightly higher than that of phos-
phorthioate. Interestingly, the hydrophobic methyl substituent on
the backbone might present an additional advantage in cellular
uptake of the oligonucleotides.24 All of these phenomena are
essential for antisense applications and research in this direction is
currently in progress.
Notes and references
§ MALDI-TOF-MS data: 7 [M 2 H]2 3688.5 (calc. 3689.5); 8 [M 2 H]2
3746.9 (calc. 3746.5); 9 [M 2 H]2 3804.9 (calc. 3803.6); 10 [M 2 H]2
3036.4 (calc. 3036.1); 11 [M
2
H]2 3660.8 (calc. 3660.4);
18 Direct cyclization of the aminoxy derivative employing various bases
12 [M 2 H]2 3688.7 (calc. 3788.4); 13 [M 2 H]2 3716.9 (calc. 3716.4);
14 [M 2 H]2 3007.1 (calc. 3007.0); 15 [M 2 H]2 3632.6 (calc. 3632.4).
resulted in failure.
19 The pseudorotation phase angle (P) was 23u indicating that the sugar
conformation existed in a typical N-form (C39-endo). CCDC 647092 for
compound 5. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/b707352f.
1 J. Kurreck, Eur. J. Biochem., 2003, 270, 1628.
2 (a) S. M. Freier and K.-H. Altmann, Nucleic Acids Res., 1997, 25, 4429;
(b) R. P. Iyer, A. Ronald, W. Zhou and K. Ghosh, Curr. Opin. Mol.
Ther., 1999, 1, 344; (c) J. Wengel, Acc. Chem. Res., 1999, 32, 301; (d)
D. Renneberg and C. J. Leumann, J. Am. Chem. Soc., 2002, 124, 5993;
(e) T. Imanishi and S. Obika, Chem. Commun., 2002, 1653.
3 (a) A. D. Mesmaeker, C. Lesueur, M. O. Be`vie´rre, A. Waldner,
V. Fritsch and R. M. Wolf, Angew. Chem., Int. Ed. Engl., 1996, 35,
2790; (b) A. Lauritsen and J. Wengel, Chem. Commun., 2002, 530.
4 T. L. Shepperd and R. C. Breslow, J. Am. Chem. Soc., 1996, 118, 9810.
5 (a) P. Nielsen and J. K. Dalskov, Chem. Commun., 2000, 1179; (b)
P. Nielsen, N. K. Christensen and J. K. Dalskov, Chem.–Eur. J., 2002,
8, 712.
20 The Tm profiles of the duplexes formed by the oligonucleotides 7–9 and
12, 13 are provided in the ESI{.
21 The Tm values of duplexes containing single-mismatch RNA strands
also decreased significantly indicating that this nucleic acid analogue also
has excellent sequence selectivity. For example, the Tm value of duplex
formed by 7 with 39-r(CGCAAUAAACGA)-59 decreased by 13 uC.
22 In the case of N-alkyl 29-amino-LNA molecules (such as N-benzyl or
N-methyl 29-amino LNA), Tm values against DNA increased by 3 uC
per modification; see: (a) S. K. Singh, R. Kumar and J. Wengel, J. Org.
Chem., 1998, 63, 10035; (b) M. D. Sørensen, M. Petersen and J. Wengel,
Chem. Commun., 2003, 2130.
23 The Sp-isomer of phosphorthioate was used in this study which is
known to be more resistant to degradation by SVPDE than an Rp-
isomer; see: P. M. J. Burgers, B. K. Sathyanarayana, W. Saenger and
F. Eckstein, Eur. J. Biochem., 1979, 100, 585.
24 Y. Ueno, K. Tomino, I. Sugimoto and A. Matsuda, Tetrahedron, 2000,
56, 7903, and references therein.
6 N. K. Christtensen, T. Bryld, M. D. Sørensen, K. Arar, J. Wengel and
P. Nielsen, Chem. Commun., 2004, 282.
7 (a) T. Govindaraju and V. Kumar, Chem. Commun., 2005, 495; (b)
T. Govindaraju and V. Kumar, Tetrahedron, 2006, 62, 495.
8 K. Gogoi, A. D. Gunjal and V. A. Kumar, Chem. Commun., 2006,
2373.
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Chem. Commun., 2007, 3765–3767 | 3767