Crucial role of linker portion in acridine-bearing oligonucleotides for highly efficient site-selective RNA scission

Article abstract of DOI:10.1016/j.tetlet.2004.03.102

Through a series of linkers, 9-amino-2-methoxy-6-nitroacridine and 9-amino-6-chloro-2-methoxyacridine were tethered to the middle of oligonucleotide, and the abilities of these conjugates for site-selective activation of RNA (inducing site-selective sciss

Full text of DOI:10.1016/j.tetlet.2004.03.102

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 3703–3706
Crucial role of linker portion in acridine-bearing oligonucleotides
for highly efficient site-selective RNA scission
Yun Shi, Akinori Kuzuya, Kenzo Machida and Makoto Komiyama*
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan
Received 21 February 2004; revised 17 March 2004; accepted 18 March 2004
Abstract—Through a series of linkers, 9-amino-2-methoxy-6-nitroacridine and 9-amino-6-chloro-2-methoxyacridine were tethered
to the middle of oligonucleotide, and the abilities of these conjugates for site-selective activation of RNA (inducing site-selective
scission by Lu(III)) were compared. The RNA-activating ability was strongly dependent on the structures of both acridine and
linker. By tethering 9-amino-2-methoxy-6-nitroacridine with a rigid and chiral linker, derived from
selective RNA scission was accomplished.
L-threoninol, quite fast site-
ꢀ 2004 Published by Elsevier Ltd.
Development of artificial ribonucleases, which hydro-
lyze RNA at predetermined site, has been a subject of
growing interest. In most cases, chemical scissors were
tethered to DNA oligomers as sequence-recognizing
moiety.1–5 Recently, we proposed a new approach for
site-selective RNA scission utilizing DNA–acridine
conjugates.6–9 When substrate RNA form hetero-
duplexes with these conjugates, its phosphodiester linkage
opposite the acridine is efficiently activated, and thus
selectively hydrolyzed by metal ions such as lantha-
nide(III), Zn(II), and Mn(II). This site-selective activa-
tion is completely ‘noncovalent’, since the molecular
scissors are not linked to any sequence-recognizing
moiety.
also critical. In this study, we use four linkers and attach
either 9-amino-2-methoxy-6-nitroacridine (pKa ¼ 8:8)
or 9-amino-6-chloro-2-methoxyacridine (pKa ¼ 10:5)
into the middle of oligonucleotide. Effects of the linkers
on site-selective RNA activation have been systemati-
cally studied.
The DNA–acridine conjugates (DNA1–X) used in this
study are presented in Figure 1. Linkers 1 and 2 are
obtained from
respectively, and their side chains are less flexible due to
the amide linkage (linker 4 is also from -threoninol but
L- and D-threoninol as starting material,
L
has a shorter side chain).11 The configuration of the
branching point in these linkers is retained throughout
the synthetic procedure. On the other hand, linker 3
consists of a flexible tetramethylene side chain, and has a
prochiral carbon center at the branching point. Thus,
DNA1–3n and DNA1–3c are used as mixtures of dia-
stereomers. Through these linkers, 9-amino-2-methoxy-
6-nitroacridine (1n, 2n, and 3n) or 9-amino-6-chloro-2-
methoxyacridine (1c, 2c, 3c, and 4) is bound to the
middle of 36-mer DNA1. When RNA1 (labeled with
FAM at the 50-end) is hybridized to these DNA1–X
conjugates, only U19 remains unpaired and it is the
target site for site-selective scission.
In order to promote this site-selective RNA scission still
more, the acridine part in the conjugates was modified in
various ways, and it was found that the RNA-activating
ability increases with increasing acidity of acridine (9-
amino-2-methoxy-6-nitroacridine is the most efficient).10
However, the structure of linker portion that connects
acridine and oligonucleotide has never been sufficiently
optimized. Since the present site-selective RNA activa-
tion originates from both the acid catalysis by the acri-
dine and the conformational change of RNA backbone
due to its intercalation, the linker structure should be
The phosphoramidite monomer 9 for the incorporation
of 1n was prepared according to Scheme 1. First,
L
-threoninol12 was coupled with N-Fmoc-protected
Keywords: Acridine; RNA; DNA; Site-selective scission; Acid cataly-
sis; Linker.
4-aminobutyric acid 5, and then the primary alcohol in
the adduct 6 was protected with dimethoxytrityl (DMTr)
group to produce 7. After the Fmoc group was removed,
* Corresponding author. Tel.: +81-3-5452-5200; fax: +81-3-5452-5209;
e-mail: komiyama@mkomi.rcast.u-tokyo.ac.jp
0040-4039/$ - see front matter ꢀ 2004 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2004.03.102

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Y. Shi et al. / Tetrahedron Letters 45 (2004) 3703–3706
Figure 1. Structures of substrate RNA and DNA–acridine conjugates used in the present study.
Scheme 1. Synthesis of the phosphoramidite monomer 9 for the incorporation of 1n. Reagents and conditions: (i) dioxane, NaHCO3, Fmoc–Cl, rt,
2 h; (ii) L-threoninol, 1,3-dicyclohexylcarbodiimide, 1-hydroxybenzotrizole, N,N-dimethylformamide (DMF), rt, overnight; (iii) DMTr–Cl, pyridine,
CH2Cl2, rt, overnight; (iv) 5% piperidine/DMF, rt, 5 min; (v) 9-(p-chlorophenoxy)-2-methoxy-6-nitroacridine, diisopropylethylamine, DMF, 80 ꢁC,
overnight; (vi) (iPr2N)2PO(CH2)2CN, 1H-tetrazole, acetonitrile, rt, 1 h.
the product was reacted with 9-(p-chlorophenoxy)-2-
methoxy-6-nitroacridine.10 Finally, the resulting acri-
dine derivative 8 was reacted with 2-cyanoethyltetra-
isopropylphosphorodiamidite. Other phosphoramidite
monomers for 1c, 2n, 2c, and 4 were synthesized by
similar procedure. The monomer for 3n was prepared
from diethyl malonate and 4-bromobutyronitrile by the
method of Nelson et al.13 The DNA syntheses and
purification were carried out as described in a previous
paper.7 These conjugates were characterized by MAL-
DI-TOF/MS analyses and also by HPLC determination
of base composition on their digests by snake venom
phosphodiesterase and alkaline phosphatase.
Figure 2 shows typical polyacrylamide gel electrophore-
sis patterns for the scission of RNA1 by Lu(III) at
pH 8.0 and 37 ꢁC. In the absence of oligonucleotide
additives, RNA1 was randomly cleaved by Lu(III) (lane
1). When the DNA–acridine conjugates were added to
the system, however, the phosphodiester linkage in the

Y. Shi et al. / Tetrahedron Letters 45 (2004) 3703–3706
3705
6-nitroacridine than 9-amino-6-chloro-2-methoxyacri-
dine (pKa ¼ 8:8 vs 10.5). It is also noteworthy that
DNA1–1n is even more active than DNA1–3n, which
involves a flexible tetramethylene linker. In order to
link highly acidic 9-amino-2-methoxy-6-nitroacridine to
sequence-recognizing DNA1, rather rigid linker 1 is
superior to flexible linker 3, since it firmly fixes the
acridine to a suitable position for the acid catalysis.
When 9-amino-6-chloro-2-methoxyacridine is bound to
DNA1, however, flexible linker 3 is more favorable than
linkers 1 and 2. Thus, DNA1–3c (kobs ¼ 4:4 ꢀ 10ꢁ2 hꢁ1
)
is twice as active as DNA1–1c (2.3 · 10ꢁ2 hꢁ1), and four
times as active as DNA1–2c (0.95 · 10ꢁ2 hꢁ1). Assum-
ably, this acridine is less acidic so that the major factor
for the site-selective RNA activation is the conforma-
tional change of RNA backbone due to the intercala-
tion, rather than its acid catalysis. This ‘conformational
catalysis’ is never optimized when the acridine is placed
appropriately for the acid catalysis. Accordingly, rigid
linkers 1 and 2 are not very effective here.
Figure 2. Site-selective RNA scission by Lu(III) in the presence of
DNA–acridine conjugates. Lane 1, Lu(III) only; lane 2, DNA1–1n;
lane 3, DNA1–1c; lane 4, DNA1–2n; lane 5, DNA1–2c; lane 6, DNA1–3n;
lane 7, DNA1–3c. At pH8.0 and 37ꢁC for 2h; [RNA1]¼ 5lM; [DNA1–
X] ¼ 10 lM; [Lu(III)] ¼ 100 lM; [Tris–HCl] ¼ 10 mM; [NaCl] ¼ 200 mM.
R, RNA1 only; H, alkaline hydrolysis; T1, RNase T1 digestion; B,
control reaction in the buffer solution.
Furthermore, the stereochemistry of the branching car-
bon atom in the linker portions must be precisely con-
trolled. Thus, DNA1–1n (derived from
2.5 times as active as DNA1–2n (from
L
-threoninol) is
-threoninol),
D
50-side of U19 was selectively and efficiently hydrolyzed
(lanes 2–7). For all the conjugates, the scission band is
notably stronger than the corresponding band in the
random cleavage of RNA1 in lane 1. Apparently, RNA1
is selectively activated just in front of the acridine resi-
due in the conjugates.14
while DNA1–1c is also more active than DNA1–2c. The
stereochemistry of this carbon greatly affects the posi-
tion of acridine in the DNA/RNA heteroduplexes, giv-
ing rise to the difference in the RNA-activating ability.
Consistently, the circular dichroism induced on the
acridine in the middle portion of DNA1–1n/RNA1
duplex is notably different from that for the DNA1–2n/
RNA1 duplex (data not shown). As expected, the length
of linker is also crucially important. Thus, DNA1–4,
The pseudo-first-order rate constants (kobs) of the scis-
sion at the 50-side of U19 in the presence of the DNA–
acridine conjugates are presented in Figure 3. The
conjugate DNA1–1n is remarkably active for the site-
selective RNA scission (kobs ¼ 11:0 ꢀ 10ꢁ2 hꢁ1). Here,
9-amino-2-methoxy-6-nitroacridine is bound to DNA1,
using the linker 1 that possesses amide-based and rather
rigid side chain. This conjugate is 4.8 times as active as
DNA1–1c bearing 9-amino-6-chloro-2-methoxyacridine
through the same linker. Similarly, DNA1–2n
(kobs ¼ 4:4 ꢀ 10ꢁ2 hꢁ1) is 4.6-fold more active than
DNA1–2c. These differences in the ability for RNA
activation (and thus for the resultant RNA scission) are
ascribed to the stronger acidity of 9-amino-2-methoxy-
which is derived from L-threoninol but has a shorter
side chain, shows only one-third ability of DNA1–1c.
In conclusion, a quite efficient RNA activator has been
synthesized by attaching 9-amino-2-methoxy-6-nitro-
acridine into the middle of oligonucleotide through
chiral linker derived from L-threoninol. Precise design of
both acridine and linker is crucially important here.
Acknowledgements
This work was partially supported by Bio-oriented
Technology Research Advancement Institution. The
support by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports, Culture and
Technology, Japan is also acknowledged.
References and notes
1. Komiyama, M.; Sumaoka, J.; Kuzuya, A.; Yamamoto, Y.
Methods Enzymol. 2001, 341, 455–468.
2. Cowan, J. A. Curr. Opin. Chem. Biol. 2001, 5, 634–642.
3. Trawick, B. N.; Daniher, A. T.; Bashkin, J. K. Chem. Rev.
1998, 98, 939–960.
Figure 3. Dependence of RNA-activating ability of DNA–acridine
conjugate on the structure of linker.

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Y. Shi et al. / Tetrahedron Letters 45 (2004) 3703–3706
€
4. Oivanen, M.; Kuusela, S.; Lonnberg, H. Chem. Rev. 1998,
98, 961–990.
10. Kuzuya, A.; Machida, K.; Komiyama, M. Tetrahedron
Lett. 2002, 43, 8249–8252.
€
€
€
5. Haner, R.; Hall, J.; Pfutzer, A.; Husken, D. Pure Appl.
11. Shi, Y.; Kuzuya, A.; Komiyama, M. Chem. Lett. 2003,
464–465.
12. Stanfield, C. F.; Parker, J. E.; Kanellis, P. J. Org. Chem.
1981, 46, 4799–4800.
Chem. 1998, 70, 111–116.
6. Kuzuya, A.; Machida, K.; Mizoguchi, R.; Komiyama, M.
Bioconjugate Chem. 2002, 13, 365–369.
7. Kuzuya, A.; Mizoguchi, R.; Morisawa, F.; Machida, K.;
Komiyama, M. J. Am. Chem. Soc. 2002, 124, 6887–
6894.
8. Kuzuya, A.; Komiyama, M. Chem. Commun. 2000, 2019–
2020.
9. Kuzuya, A.; Komiyama, M. Chem. Lett. 2000, 1378–1379.
13. Nelson, P. S.; Kent, M.; Muthini, S. Nucleic Acids Res.
1992, 20, 6253–6259.
14. Minor cleavage was observed at the 30-side of U19.
However, this cleavage also occurs when unmodified 1,3-
propanediol linker is introduced into DNA1, and is not
much activated by the DNA–acridine conjugates.