Active control by external factors of DNA recognition behavior of α-peptide ribonucleic acids containing basic amino acid residues

Article abstract of DOI:10.1246/cl.2010.112

A series of novel α-peptide ribonucleic acid (αPRNA) oligomers, possessing alternative αPRNA-Gly/Lys sequences, were newly designed, synthesized, and evaluated as the second- generation PRNA. As expected, the αPRNA with Lys formed stable sequence-specific complex with the complementary DNA, for which both the hydrogen-bonding interactions between, the complementary nucleobase pairs and the electrostatic interactions between the ammonium cation on the lysine side chain and the DNA phosphate anion on the backbone are jointly responsible.

Full text of DOI:10.1246/cl.2010.112

doi:10.1246/cl.2010.112
112
Published on the web January 16, 2010
Active Control by External Factors of DNA Recognition Behavior of
¡-Peptide Ribonucleic Acids Containing Basic Amino Acid Residues
Takehiko Wada,*1 Nobuya Sawa,2 Hirofumi Sato,2 Mayuko Kikkawa,2 Keiko Onodera,1 Seiji Sakamoto,1 and Yoshihisa Inoue*2
1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai 980-8577
2Department of Applied Chemistry, Osaka University, 2-1 Yamada-oka, Suita 565-0871
(Received November 27, 2009; CL-091055; E-mail: hiko@tagen.tohoku.ac.jp)
A series of novel ¡-peptide ribonucleic acid (¡PRNA)
oligomers, possessing alternative ¡PRNA-Gly/Lys sequences,
were newly designed, synthesized, and evaluated as the second-
generation PRNA. As expected, the ¡PRNA with Lys formed
stable sequence-specific complex with the complementary DNA,
for which both the hydrogen-bonding interactions between the
complementary nucleobase pairs and the electrostatic interactions
between the ammonium cation on the lysine side chain and the
DNA phosphate anion on the backbone are jointly responsible.
γPRNA•RNA
γPRNA
RNA
O
O
HN
HN
O
O
HN
HN
Base
O
HN
HN
Base
O
O
HN
Base
HN
Base
O
O
O
NH
O
NH
HO OH
O
HO OH
NH2
O
HO OH
HO OH
O
γPRNA
αPRNA1
αPRNA2
αPRNA3
Chart 1. Structure of £PRNA and ¡PRNAs.
Recently gene therapeutic methods for selectively down
regulating the expression of the genetic information of cancer
growth and disease related proteins by specific recognition and
binding to the target mRNA through the siRNA and antisense
RNA strategies have become a target of intensive studies.1 In
particular the down regulation of genetic information by siRNA
is considered one of the most powerful tools not only for
molecular biology and genetic engineering but also for
oligonucleotide therapeutics.1 Nevertheless, the currently used
siRNA molecules have some drawbacks, such as poor reprodu-
cibility, resulting probably from intracellular instability, low cell
membrane permeability, and low binding affinity.1 Most of the
other gene therapeutic methods share some of these problems,
and hence these are regarded as the most crucial research targets
to be attacked.2 To remedy these drawbacks, a variety of
modified nucleotides and nucleic acid model compounds have
been proposed for a better resistance to nucleases and an
enhanced hybridization affinity of the oligomer.2
Peptide nucleic acid (PNA) with a peptide-based backbone
structure is one of the most successful nucleic acid models.3
Inspired by the unique backbone structure and properties of
PNA, several attempts to improve and/or functionalize existing
gene therapeutic compounds have been done through the
modification of the oligonucleotide backbone2 as well as the
development of nucleic acid model compounds with different
backbone structures.2 However, these hitherto reported nucleic
acid derivatives and their model compounds have an intrinsic
restriction or disadvantage, lacking the ability to actively control
the function of oligonucleotide therapeutics by internal physio-
logical and/or external physical/chemical stimuli.
the anti-to-syn switching of the nucleobase orientation, which is
attributable to the synergetic effect of the borate ester formation
with the 2¤,3¤-cis diol of the ribose moiety and the intramolecular
hydrogen-bond formation between the nucleobase and the
furanose moiety of PRNA.4 Despite the unique features useful
for gene therapeutics, PRNA has a potential burden; namely, the
relatively slow hybridization, the low cell membrane perme-
ability, and/or the moderate water solubility critically depend on
the nucleobase sequence in the side chain.
In the present study, we propose a new strategy not only
to improve these potential disadvantages of PRNA but also
to add a functionality to PRNA by introducing derivatizable
amino acid residues. Thus, we have designed and synthesized a
series of ¡PRNAs, in which the moiety 1 is appended to the
¡-(L-glutamine) backbone through the 5¤-amino group, and also
several ¡PRNA derivatives, which have a mixed backbone
composed of ¡PRNA and glycine or basic amino acids, such as
lysine (Chart 1). By using this strategy, both the water solubility
and hybridization ability of PRNA are expected to be improved.
In the target ¡PRNA synthesis, monomer 4 was prepared in
four steps starting from N-Boc-L-Glu-OBzl and 1 (Scheme 1).
N-Boc-L-Glu-OBzl and 1 was reacted with BOP reagent and
HOBt in DMF to give 2U. Subsequently, 2U was treated with
TFA to remove Boc, and then the free N-terminus was protected
with Fmoc by reaction with Fmoc-OSu to give Fmoc-Glu(5¤U)-
O
O
N
U
H2N
U
N
H
U
O
O
O
H
H
N
a
b
TFA •H2
N
Boc
HO OH
HO OH
1U
HO OH
O
OBzl
O
OBzl
2U
We have recently proposed a new category of nucleic acid
model compound; named peptide ribonucleic acid (PRNA), in
which a 5¤-amino-5¤-deoxypyrimidine ribonucleoside (1) moiety
is appended to an oligo(£-L-glutamine) backbone through the
ribose 5¤-amino group (Chart 1).4 £PRNAs form stable com-
plexes with complementary DNAs (cDNAs) and RNAs with
high nucleobase sequence specificities. Furthermore, the recog-
nition and complexation behavior of £PRNAs with target RNAs
can be controlled by borax added as an external factor through
O
O
N
H
U
N
H
U
O
O
H
N
H
c
d
Fmoc
Fmoc
N
HO OH
HO OH
O
OBzl
3U
O
OH
4U
Scheme 1. Reagents: (a) N-Boc-L-Glu-OBzl, Bop reagent, HOBt,
DIEA, DMF, 83%; (b) TFA; (c) Fmoc-OSu, DIEA, DMF, 90%;
(d) H2, Pd/C, DMF/methanol, 96%.
Chem. Lett. 2010, 39, 112-113
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

113
The complexation behaviors of ¡PRNA1, ¡PRNA2
(12mers), and ¡PRNA3 (8mer) with cDNA, d(A)12, and d(A)8
were examined in pH 7.2 phosphate buffers at normal 33 mM
and very dilute 0.33 mM (where the electrostatic interactions
are encouraged) concentrations. The structural difference in
the two ¡PRNA 12mers is the Gly backbone introduced to
¡PRNA2, which led to a critical difference in complex stability.
Thus, ¡PRNA2 forms a complex with d(A)12 that melts at
22 °C, which is similar to the Tm (23 °C) obtained for the
corresponding natural DNA pair. In keen contrast, the complex-
ation of ¡PRNA1 with d(A)12 was not observed (Tm < 5 °C).
This clear distinction indicates that the nucleobase repeating
distance plays one of the most crucial and important roles upon
complexation. Interestingly, ¡PRNA3 8mer, in which Lys
residues are introduced to the backbone, gave a stable complex
(Tm = 25 °C) with d(A)8 immediately after mixing, while no
complex formation was observed for natural d(T)8 and d(A)8
under the same conditions, probably due to electrostatic
repulsion. In contrast, in the borax-containing buffer, the hybrid
complex did not exhibit any melting behavior above 0 °C, or
hypochromic changes, and hence the on-off switching of nucleic
acid recognition was achieved indeed. Moreover, no complex
formation was observed for ¡PRNA3 with not only d(T)8 but
also d(A3TTA3), revealing that the introduction of basic amino
acid residues into PRNA backbone can stabilize the PRNA-
DNA complex, without losing the nucleobase sequence selec-
tivity, and further accelerates the rate of complexation, both of
which are thought to be caused by the electrostatic interaction.
Therefore, a promising strategies to overcome the potential
disadvantage of relatively slow hybridization, low cell mem-
brane permeability, and moderate water solubility is to incor-
porate basic amino acid into ¡PRNA backbone. Studies along
this line are currently in progress.
Figure 1. CD spectral changes of ¡PRNA3 with increasing
concentrations of Na2B4O7 in phosphate buffer (0.033 M KH2PO4-
0.033 M Na2HPO3, pH 7.2); [¡PRNA3] = 1.0 © 10¹4 M.
OBzl (3U). Finally, the C-terminus benzyl ester was removed by
hydrogenation over 10% Pd/C in dry DMF/methanol mixed
solvent to give Fmoc-Glu(5¤U)-OH (4U).5 The ¡PRNA 12mers
of homo-uracil sequence with end-capping Lys3 at the N- and C-
termini, i.e., ¡PRNA1, ¡PRNA-glycine (¡PRNA2), and 8mer
of homo-uracil sequenced ¡PRNA-lysine (¡PRNA3), for the
use in a first examination of the recognition behavior with
DNA, were prepared from 4U by a Fmoc solid-phase peptide
synthesis, which was employed also in a PRNA synthesis
reported previously.4,7 The ¡PRNA oligomers thus obtained
were purified by reversed phase preparative HPLC and finally
confirmed by analytical HPLC and MALDI-TOF MS measure-
ments.6 Good yields of up to 98% at each step were obtained
under the coupling condition.
Since the anti-to-syn switching of nucleobase orientation
upon cyclic borate ester formation is an essential factor for
achieving the “on-demand” gene therapeutics, we first examined
the CD spectral behavior of ¡PRNA1-3. The gradual addition
of borax to a phosphate buffer solution of ¡PRNA3 (pH 7.2)
induced significant CD spectral changes, as shown in Figure 1.
The isodichroic points observed at ca. 220 and 240 nm indicate
We gratefully acknowledge the support of this work by the
Grant-in-Aid for Scientific Research from JSPS.
References and Notes
1
a) L. Du, R. Gatti, Curr. Opin. Mol. Ther. 2009, 11, 116. b) D. Grimm,
Adv. Drug Delivery Rev. 2009, 61, 672. c) E. Wiemer, J. RNAi Gene
Silencing 2006, 2, 173.
that a single step is responsible for these changes. In the phos-
¹1
phate buffer, the [ª]ext value at 270 nm (6600 deg cm2 dmol
)
2
3
M. Wood, H. Yin, G. McClorey, PLoS Genet. 2007, 3, e109.
a) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991,
254, 1497. b) M. Egholm, O. Buchardt, L. Christensen, C. Behrens,
S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, P. E.
Nielsen, Nature 1993, 365, 566.
was comparable to that observed for 1 and larger than that for
original £PRNA, which is compatible with the preferred anti
orientation. In contrast, the [ª]ext value of this oligomer was
greatly reduced to 2800 deg cm2 dmol¹1 by increasing the borate
concentration, which is similar to the value observed for the
original £PRNA in a borate buffer. These results unambiguously
reveal the occurrence of the anti-to-syn switching of the
nucleobase orientation upon addition of borax. A quantitative
analysis of the CD spectral changes, using the nonlinear least-
squares fit to the curve for 1:1 stoichiometric complexation,
4
5
a) T. Wada, N. Minamimoto, Y. Inaki, Y. Inoue, J. Am. Chem. Soc.
2000, 122, 6900. b) H. Sato, Y. Hashimoto, T. Wada, Y. Inoue,
Tetrahedron 2003, 59, 7871.
1H NMR (270 MHz, DMSO-d6): ¤ 1.69-2.07 (m, 2H, ¢-CH2), 2.20
(t, 2H, £-CH2, J = 7.8 Hz), 3.11-3.50 (m, 2H, 5¤-CH2,), 3.72-3.87 (m,
2H, 3¤- and 4¤-H), 3.87-4.09 (m, 1H, ¡- and 2¤-H), 4.15-4.32 (m, 3H,
Fuluorenyl-CH-CH2), 5.19 (d, 1H, 3¤-OH), 5.42 (d, 1H, 2¤-OH), 5.62
(d, 1H, 5-H, J = 7.8 Hz), 5.74 (d, 1H, 1¤-H, J = 5.9 Hz), 7.26-7.78 (m,
8H, Fmoc-H, Fmoc-NH, and 6-H), 7.90 (d, 2H, Fmoc-H), 8.05 (t, 1H,
5¤-NH, J = 5.9 Hz), 11.37 (s, 1H, 3-NH); HR-FAB MS. m/z: found
595.2048 (M + 1), calcd 595.1962 (M + H).
¹1
gave the equilibrium constant of 3000 M for the reversible
esterification of ¡PRNA3 with borax. This value is three times
higher than those of the original £PRNAs, indicating that the
electrostatic repulsion of the positively charged ammonium
groups of Lys residues may extend the peptide backbone, and
therefore the steric hindrance between the nucleoside moieties in
the side chain is reduced. Similar borax-induced ant-to-syn
nucleobase orientation switching was also observed with
¡PRNA1 and -2.7
6
7
NH2-Lys3-(Glu(5¤U))12-Lys3-OH (¡PRNA1): MALDI-TOF MS
(¡-CHCA), m/z: found 5038.97 (M + H)+, calcd 5038.83; NH2-
(Glu(5¤U)-Gly)11-Glu(5¤U)-Lys-OH (¡PRNA2): MALDI-TOF MS
(¡-CHCA), m/z: found 5025.22 (M + H)+, calcd 5025.54; NH2-
(Lys-Glu(5¤U))8-Lys-OH (¡PRNA3): MALDI-TOF MS (¡-CHCA),
m/z: found 4008.16 (M + H)+, calcd 4007.09.
Supporting Information is available electronically on the CSJ-Journal
Web site, http://www.csj.jp/journals/chem-lett/index.html.
Chem. Lett. 2010, 39, 112-113
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/