β-PNA: Peptide nucleic acid (PNA) with a chiral center at the β-position of the PNA backbone

Article abstract of DOI:10.1016/j.bmcl.2011.10.017

Peptide nucleic acid (PNA) monomers with a methyl group at the β-position have been synthesized. The modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthesis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable to that of unmodified PNA-DNA duplex, whereas PNA containing the R-form monomers was not.

Full text of DOI:10.1016/j.bmcl.2011.10.017

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7317–7320
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Bioorganic & Medicinal Chemistry Letters
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b-PNA: Peptide nucleic acid (PNA) with a chiral center at the b-position
of the PNA backbone
Toru Sugiyama a, , Yasutada Imamura b, Yosuke Demizu c, Masaaki Kurihara c,
⇑
Masashi Takano d, Atsushi Kittaka d
a Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
b Faculty of Engineering, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015, Japan
c Division of Organic Chemistry, National Institute of Health Sciences, Ministry of Health and Welfare, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
d Faculty of Pharmaceutical Sciences, Teikyo University, Midori-ku, Sagamihara, Kanagawa 252-5195, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptide nucleic acid (PNA) monomers with a methyl group at the b-position have been synthesized. The
modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthe-
sis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the
S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable
to that of unmodified PNA–DNA duplex, whereas PNA containing the R-form monomers was not.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 16 September 2011
Revised 4 October 2011
Accepted 7 October 2011
Available online 13 October 2011
Keywords:
Peptide nucleic acid
Preorganization
Chirality
Helicity
Peptide nucleic acid (PNA) is a synthetic DNA/RNA mimic in
which the sugar–phosphate backbone is replaced by a peptide
backbone consisting of N-(2-aminoethyl)glycine units.1 PNAs
hybridize to complementary sequences by Watson–Crick base
pairing with high affinity and strict sequence specificity.2 PNAs
are resistant to nucleases and proteases3 and have a low affinity
for proteins.4 These properties make PNAs an attractive agent for
biological and medical applications. To improve the antisense
and antigene properties of PNAs, it is reasonable to increase bind-
ing affinity for DNA or RNA by suitable preorganization. A lot of
backbone modifications of PNAs have been explored under the
concept of preorganization.5,6 However, only limited numbers of
modifications improved the hybridization properties.7–10
the effects of cyclic structures are quite substantial. The b-position
of the PNA backbone corresponds to the C40 of the deoxyribose
moiety of DNA and the C40 is a chiral carbon atom. Thus, the intro-
duction of a chiral center at the b-position may significantly affect
the conformation and the DNA binding ability of PNA oligomers.
Here we report the synthesis of PNA monomers with a methyl
group at the b-position and their incorporation into oligomers as
well as their DNA binding properties.
We designed a new PNA monomer possessing a methyl group of
S-configuration at the b-position of the PNA backbone (Fig. 1) be-
cause molecular modeling, based on the crystal structure of a
PNA–DNA duplex,18 suggested this configuration is less disruptive
to hybridization than the R-configuration (Fig. 2).19
A simple strategy for preorganizing a PNA is to add substituents
to the PNA backbone. Several modified PNAs were obtained by
The requisite chiral b-PNA thymine monomer 9 was prepared
as follows (Scheme 1). Treatment of chiral diamine 1 with 2 mol
incorporating substituents at either the
Comparison of the effects of substituents at the
those of the -position revealed that
a
- or the
c
-position.9–13
-position with
c-modification is more
a
c
Base
effective to improve the DNA binding ability.14 On the other hand,
to our knowledge, PNAs with a single substituent only at the
b-position have not been previously reported. Although some cyc-
lic PNA analogs contain a chiral center at the b-position,7,8,15–17
they do not directly reflect the effects of the b-substituents because
Base
Base
O
O
O
O
O
4'
O
γ
β
N
O
P
N
N
N
α
β
H
O
O-
H
β-(S)-Me PNA
DNA
PNA
⇑
Corresponding author. Tel./fax: +81 3 5465 8743.
E-mail address: csugi@mail.ecc.u-tokyo.ac.jp (T. Sugiyama).
Figure 1. Structures of DNA, PNA and b-(S)-Me PNA.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.017

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T. Sugiyama et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7317–7320
Figure 2. Molecular modeling of b-Me PNA/DNA duplex. The methyl group (green) of S-form b-PNA projects away from the duplex (left). The methyl group (green) of R-form
b-PNA sterically clashes with the methylene of the thyminylacetyl moiety (right).
NH2
CbzOPh
EtOH, r.t.
76 %
NH2
NHZ
H2N
ZHN
H2N
+
1
2
3
(92 : 8)
BrCH2CO2Me
K2CO3
CH2Cl2, r.t.
OMe
MeO
NHZ
O
N
H
N
O
O
O
O
+
+
ZHN
OMe
N
ZHN
OMe
O
OMe
4
5
6
H
N
Column chromatography
ZHN
OMe
77 % from (2+3)
4
(84 % based on 2)
T
T-CH2CO2H
EDCI
O
O
NaOH
4
N
DMF, 0 °C
quant.
MeOH-H2O (3:1)
r.t.
ZHN
OMe
7
T
T
1) H2, Pd-C
O
O
O
O
MeOH, rt
N
N
ZHN
OH
FmocHN
OH
2) FmocOSu
NaHCO3
acetone-H2O, rt
8
9
79 % (3 steps from 7)
Scheme 1. Synthesis of b-(S)-Me PNA thymine monomer.
equiv of CbzOPh20 in EtOH overnight gave a mixture of isomers 2
and 3 in a 92:8 ratio in 76% overall yield. The ratio was deter-
mined from the 1H NMR signals for the methyl proton. Since
these isomers could not be separated by usual chromatographic
methods, the mixture of isomers was used directly for alkylation
reaction. Treatment of the isomeric mixture with 4 mol equiv of
methyl bromoacetate gave 4 in 77% yield (84% from 2) as a pure
isomer. This alkylation is noteworthy in that the minor isomer 3
was completely dialkylated under this condition and became
chromatographically separable. Coupling of the thymin-1-ylacetic

T. Sugiyama et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7317–7320
7319
Table 1
1.2
PNA sequences
PNA1-DNA Tm=51.4 °C
PNA2-DNA Tm=51.0 °C
PNA3-DNA Tm= n.d.
PNA
Sequencea
H-GTAGATCACT-LLYS-NH2
H-GtSAGAtSCACtS-LLYS-NH2
H-GtRAGAtRCACtR-DLYS-NH2
1.15
1.1
1.05
1
PNA 1
PNA 2
PNA 3
aeg
b-(S)-Me
b-(R)-Me
tS = b-(S)-Me PNA, tR = b-(R)-Me PNA
a
Written from N to C terminal. PNAs terminate in a lysine amide.
O
N
O
N
HN
HN
O
O
O
O
O
O
20
30
40
50
60
70
80
N
N
N
H
N
H
Temperature (°C)
Figure 3. UV-melting curves of the PNA–DNA hybrid duplexes at 5
l
M strand
tS
tR
concentration each. Samples were prepared in 10 mM sodium phosphate buffer
containing 1 mM EDTA (pH 7.4). Thermal denaturation was monitored at 260 nm
at a rate of 0.2 °C per minute. The melting transitions were determined from the
first derivatives of the UV-melting curves. n.d.: not determined. DNA: 50-AGTG
ATCTAC-30.
acid was accomplished with EDCI in DMF in quantitative yield.
Alkaline hydrolysis of the ester 7 proceeded quantitatively to af-
ford 8. Catalytic hydrogenation of 8 followed by its subsequent
Fmoc protection with FmocOSu gave b-PNA monomer 9 in 79%
yield for 3 steps from 7. For comparison, the enantiomer of 9
was also prepared from (R)-1,2-diaminopropane. These mono-
mers were used directly for the synthesis of PNA oligomers.
Using a standard manual solid-phase peptide synthesis proce-
dure,21 three PNA oligomers were synthesized to probe the effect
of a methyl group at the b-position of the PNA backbone (Table
1). We chose a 10-residue mixed-base PNA sequence that has been
well documented.2a,22 b-PNA monomers could be successfully
incorporated into the PNA sequence without difficulty. After
deprotection and cleavage from the resin, each PNA was purified
by reversed-phase HPLC and characterized by ESI-TOF mass spec-
trometry.23 PNA oligomers synthesized here were readily soluble
2
A
1.5
1
0.5
0
-0.5
-1
PNA1
PNA2
PNA3
-1.5
(more than 500 lM) in water.
To evaluate the effect of b-backbone modification on the DNA
binding affinity of PNA, we measured the melting temperatures
(Tms) of the b-PNAs with the complementary DNA strand in
10 mM sodium phosphate buffer containing 1 mM EDTA (Fig. 3).
b-PNA containing three S-form chiral units (PNA2) and unmodified
PNA (PNA1) have similar Tm values. In contrast, PNA3, the enantio-
mer of PNA2, did not show a well-defined melting transition. Since
the effect of C-terminal lysine on Tm is generally small, the melting
behavior of PNA3-DNA should be dominated by the R-form chiral
units. These results indicate that the stereochemistry of the b-po-
sition is critical to the hybridization stability of PNA and is strictly
-2
200
220
240
260
280
300
320
Wavelength (nm)
B
2
1.5
1
0.5
0
limited to S-configuration. This contrasts to
a-modified PNAs in
which chirality arising from a methyl group incorporated at the
a
-position hardly affects the hybridization properties.10
-0.5
-1
To gain structural insights, we measured the circular dichroism
(CD) spectra of single-stranded PNAs (PNA1-3) and their hybrids
with the complementary DNA in the same buffer used in UV-melt-
ing experiments. A comparison of the CD spectra of single-
stranded PNAs (PNA1-3) is shown in Figure 4A. No exciton cou-
pling pattern was observed for unmodified PNA (PNA1) in the
nucleobase absorption regions (220–300 nm), indicating the lack
of helical base-stacking. On the other hand, the b-PNAs, PNA2
and PNA3, showed distinct CD signals. The shape of the spectrum
of PNA2 was similar to that of right-handed PNAs10,13,19 and the
signs of the CD bands of PNA2 and PNA3 were opposite each other.
Thus it is reasonable to propose that PNA2 adopts a right-handed
helical structure and PNA3 is left-handed. As shown in Figure 4B,
the CD profile of PNA2-DNA hybrid duplex is very similar to that
of PNA1-DNA hybrid, with a positive band at 266 nm and a
PNA1-DNA
PNA2-DNA
PNA3-DNA
-1.5
-2
200
220
240
260
280
300
320
Wavelength (nm)
Figure 4. (A) Circular dichroism (CD) spectra of single-stranded PNA1 (thick solid
curve), PNA2 (thick broken curve), and PNA3 (thin solid curve) at 5 M strand
concentration each in the same buffer used in UV-melting experiments, recorded at
room temperature. (B) CD spectra of the corresponding PNA–DNA hybrids at 5
l
lM
strand concentration each, recorded at room temperature after annealing in the
same buffer. DNA: 50-AGTGATCTAC-30.
low-intensity negative band at 242 nm, suggesting a high degree
of helical identity between PNA1/PNA2-DNA hybrids. In contrast,

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T. Sugiyama et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7317–7320
the CD spectrum of PNA3-DNA hybrid is distinctly different from
those of PNA1/PNA2-DNA hybrids. These results are consistent
with the Tm data. Furthermore, the CD spectrum of single-stranded
PNA2 is similar but somewhat different from that of the
corresponding hybrid duplex, PNA2-DNA (PNA2 in Fig. 4A vs.
PNA2-DNA in Fig. 4B). The most significant differences appeared
in the 200–230 nm region. The positive band at 213 nm in sin-
gle-stranded PNA2 was red-shifted to 222 nm in PNA2-DNA du-
plex and an intense negative band was observed at 200 nm only
in the hybrid duplexes. These differences reflect conformational
changes that occur upon DNA hybridization of PNA2. Since the in-
duced secondary structure of b-Me PNA did not contribute to the
total hybridization stability, the benefit of the induced structure
in terms of DNA binding might be canceled by unfavorable steric
interactions arising from b-methyl groups in the PNA–DNA hybrid
duplex. To minimize the energy loss associated with conforma-
tional changes, further sophisticated design of PNA is required.
In conclusion, we have synthesized both the S- and the R-forms
of b-Me PNA monomers and incorporated them individually into a
10-residue mixed-base PNA sequence. PNA containing the S-form
chiral units was well suited to form a right-handed hybrid duplex
with DNA, whereas incorporation of the R-form units was detri-
mental to hybridization with DNA. CD spectra suggested that b-
backbone modification of PNA induced a right-handed helix. The
results reported here would contribute to a better understanding
of PNA chemistry and provide a basis for the future design of
PNA with improved properties.
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structure (PDB code: 1NR8) in Ref. 18 was used as an initial structure.
Models were built by just replacing beta-hydrogen with methyl group through
automatically editing mode without optimizing calculation.
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21. See Supplementary data.
This work was supported in part by Grants-in-Aid for Scientific
Research (KAKENHI, No. 20590099 to T.S. and No. 21590022 to
A.K.) from Japan Society for the Promotion of Science. The authors
would like to thank Professor Akira Suyama and Dr. Koh-ichiro
Shoda of the University of Tokyo for UV-melting measurements.
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Supplementary data
23. PNA1 calcd for [M+2H]2+ 1427.5997, found 1427.5997; PNA2 calcd for
[M+2H]2+ 1448.6232, found 1448.6225; PNA3 calcd for [M+2H]2+ 1448.6232,
found 1448.6237.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.017.