Synthesis and characterization of naphthalimide-containing peptide nucleic acid

Article abstract of DOI:10.1016/S0040-4039(02)01164-4

Naphthalimide-containing PNA monomer unit 6 was synthesized for the preparation of PNAs containing a naphthalimide chromophore (NPNA). PNA oligomers were purified by reversed-phase HPLC and characterized by MALDI-TOF MS and UV spectra. Thermodynamic analyses revealed that the PNA oligomer containing a naphthalimide moiety at the amino terminal stabilizes PNA-DNA hybrids.

Full text of DOI:10.1016/S0040-4039(02)01164-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5525–5528
Synthesis and characterization of naphthalimide-containing
peptide nucleic acid
Hisafumi Ikeda,a Yushin Nakamuraa and Isao Saitob,*
aDepartment of Biological Science & Technology, Faculty of Industrial Science and Technology, Science University of Tokyo,
2641 Yamazaki, Noda, Chiba 278-8510, Japan
bDepartment of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, CREST, Japan Science
and Technology Corporation, Yoshida, Sakyo, Kyoto 606-8501, Japan
Received 27 March 2002; revised 10 June 2002; accepted 14 June 2002
Abstract—Naphthalimide-containing PNA monomer unit 6 was synthesized for the preparation of PNAs containing a naphthal-
imide chromophore (NPNA). PNA oligomers were purified by reversed-phase HPLC and characterized by MALDI-TOF MS and
UV spectra. Thermodynamic analyses revealed that the PNA oligomer containing a naphthalimide moiety at the amino terminal
stabilizes PNA–DNA hybrids. © 2002 Elsevier Science Ltd. All rights reserved.
Peptide nucleic acid (PNA) is a DNA analogue in
which the phosphate backbone is replaced by N-(2-
aminoethyl)glycine units.1 The electrostatically neutral
peptide backbone prevents inter-strand repulsion of the
complementary strand, allowing sequences to be recog-
nized with high affinity and selectivity with respect to
hybridization with DNA or RNA.2 The use of PNAs is
also advantageous because they are extremely stable to
cellular nucleases and proteases.3 The PNA oligomer
can be easily synthesized by solid-phase tBoc/Fmoc
peptide chemistry.4 Several groups have carried out
modifications of PNA backbones to improve the bind-
ing characteristics to DNA and RNA as well as to
improve its membrane permeability.5 Concurrently,
synthesis of several functionalized PNA oligomers con-
taining functionalized molecules in place of nucleobases
have been reported to improve the water solubility and
to study the electron transfer chemistry through DNA
duplex.6 Recently, we have demonstrated a strategic
synthesis of a photosensitizable PNA monomer that
enables the introduction of photoactive molecules into
PNA oligomers at predetermined sites.7
a
d
O
N
O
O
O
N
O
O
OR
O
O
O
Boc
N
N
H
OR
O
1: R = t-Bu
2: R = H
O
H
N
5: R = Et
6: R = H
b
c
Boc
N
H
OEt
e
4
3: R = Pfp
Scheme 1. Preparation of naphthalimide-containing PNA monomer. Reagents and conditions: (a) t-butyl glycinate hydrochloride,
triethylamine, xylene, reflux, 4 h, 87%; (b) HCOOH, rt, 2 h, 96%; (c) pentafluorophenol, DCC, DMF, rt, 4 h; (d) 4,
diisopropylethylamine, DMF, 10 h, 72% from 3; (e) 2N aq. NaOH, EtOH, 2 h, then Dowex 50Wx8 (Py+) treatment, 92%.
* Corresponding author.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01164-4

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H. Ikeda et al. / Tetrahedron Letters 43 (2002) 5525–5528
Naphthalimides are well-known as potential anti-tumor
agents as exemplified by mitonafide, amonafide, and
azonafide.8 Numerous naphthalimide analogues have
been synthesized, and qualitative structure–activity
relationships (QSARs) were studied to shed light on
their intercalative inhibition of topoisomerase II.9
Recently, related compounds, bis-naphthalimide LU
79553 and DMP-840, were reported to bisintercalate
into DNA at the major groove and inhibit topoiso-
merase II.10 Establishment of an effective method for
introducing intercalating reagents such as naphthal-
imide derivatives at specific oligomer sites is particu-
larly useful for studying PNA–DNA hybrid stability
aimed at the development of antisense oligonucleotides.
The protocol for the preparation of PNAs containing a
hybrid stabilizer at the desired sites has not been
described. Here we report the preparation of a naph-
thalimide-containing PNA monomer and the character-
ization of naphthalimide-containing PNA oligomers.
purified PNA oligomer was characterized by MALDI-
TOF MS (Table 1) and UV spectra (Fig. 1). UV
absorptions of NPNA1–2 at 348 nm indicated that
naphthalimides had been introduced into PNA
oligomers. The intensity at 348 nm of NPNA2 was
about twice that of NPNA1, indicating that NPNA2
has two naphthalimide moieties in a PNA oligomer.
However, the intensity of NPNA7 was less than three
times that of NPNA1, suggesting that any
hypochromicity caused by the three consecutively incor-
porated naphthalimide moieties might be indicative of
the restricted space within the interior region of
NPNA7.
Naphthalimide-containing PNA monomer 6 was syn-
thesized as shown in Scheme 1. A mixture of 1,8-naph-
thalic anhydride and t-butyl ester of glycine was
refluxed in xylene, evaporated, and recrystallized from
EtOH to give 1 (87%). Ester 1 was acid-hydrolyzed and
the residue was flash-chromatographed to give 2 (96%).
Treatment of 2 with pentafluorophenol and DCC fol-
lowed by condensation with ethyl N-(2-((t-
butoxy)carbonylamino)ethyl)glycinate11 (4) and diiso-
propylethylamine gave 5 (72% from 2). Ester 5, which
was hydrolyzed by 2N aq. NaOH, was directly flash-
chromatographed on DOWEX 50Wx8 (pyridinium
form) column, evaporated, and recrystallized with
EtOH to give 6 (92%). It was possible to monitor the
incorporation of the naphthalimide chromophore into
PNA oligomers by simply measuring UV spectra hav-
ing a UV absorption at ca. 340 nm which was not
overlapped with PNA nucleobases, as shown in Fig. 2.
Figure 1. HPLC profile of the crude NPNA2. Each crude
PNA oligomer was purified by reversed-phase HPLC on a
Wakosil II 5-ODS-AR column (30×150 mm) using acetoni-
trile/water/0.1% TFA solvents system. Bold N means naph-
thalimide PNA unit.
Several PNA oligomers were synthesized by solid-phase
tBoc chemistry as previously described.12 A Ninhydrin
test showed that each coupling of PNA monomers with
the MBHA resin using HBTU was satisfactory. HPLC
analysis showed that the crude PNA oligomers could be
effectively prepared together with only a small amount
of shorter PNA oligomers as by-products (Fig. 1). Each
Figure 2. UV spectra of PNA1, NPNA1, 2, and 7. Each
sample (5 mM, strand concentration) dissolved in water/
CH3CN (1:1) was measured in 10 mm quartz cuvette on a
UV/VIS spectrophotometer. Each molar extinction coefficient
(m, cm2/mmol) at 348 nm was inserted in parenthesis.
Table 1. MALDI-TOF MS and melting temperaturesa of naphthalimide-containing PNA NPNA1–7
MALDI-TOF MS
Found
Melting temperaturea
Calcd
Tm (°C)
DTm (°C)
H-p(G TTA GGG TTA G)-NH2
H-p(N TTA GGG TTA G)-NH2
H-p(N NTA GGG TTA G)-NH2
H-p(G TTA GGG TTA N)-NH2
H-p(G TTA GGG TTN N)-NH2
H-p(G TTA GNG TTA G)-NH2
H-p(G TTA NNG TTA G)-NH2
H-p(G TTA NNN TTA G)-NH2
PNA1
3088.89
3134.96
3206.03
3134.96
3197.02
3134.96
3181.02
3227.08
3088.51
3134.52
3206.21
3135.50
3196.88
3135.31
3180.98
3227.11
64.5
65.0
67.8
56.7
60.3
35.2
27.5
N.D.
NPNA1
NPNA2
NPNA3
NPNA4
NPNA5
NPNA6
NPNA7
0.5
3.3
−7.8
−4.2
−29.3
−37.0
N.D.
a Each sample was heated from 20.0 to 90.0°C in 1.0°C increments with 1 min equilibration. Concentration of PNA–DNA, 5%-d(C TAA CCC
TAA C)-3%, hybrid was 5 mM (each strand) in 50 mM cacodylate buffer (pH 7.0) and 100 mM Na+. Bold N means naphthalimide PNA unit.

H. Ikeda et al. / Tetrahedron Letters 43 (2002) 5525–5528
5527
tributes to the stabilization of the PNA–DNA hybrid.
These results are in agreement with the thermal stability
of the flavin-containing PNA oligomers previously
reported.7a
In order to investigate the basis for PNA–DNA hybrid
stabilization, we carried out molecular mechanics
(MM) calculations of the PNA–DNA hybrids using
AMBER* force field as previously described.13 Mini-
mization energy of the NPNA1–DNA hybrid (−3398.42
kcal/mol) and NPNA2–DNA (−3371.22 kcal/mol)
hybrid was smaller than that of the PNA1–DNA hybrid
(−3161.12 kcal/mol); nevertheless, a naphthalimide moi-
ety pushed out the cytosine as a base pair partner (Fig.
3A). The p–p interaction by the naphthalimide moiety
extended to near adenine with 5%-d(C TAA CCC TAA
C)-3% in addition to the neighboring thymine in the
PNA oligomer (Fig. 3B). In NPNA2, two adjacent
naphthalimides were strongly stacked with a slight
spread such as a folding fan, suggesting that the naph-
thalimides could effectively form the p–p interaction
with the neighbor base pairs and an unpaired adenine
base (data not shown). These results suggested that the
naphthalimide moiety played an important role in the
stabilization of the PNA–DNA hybrid by the extended
p–p interaction.
Figure 3. Molecular models of the NPNA1 and 5%-d(CG
AGA AGG GCG)-3% hybrid. (A) Horizontal view of [p(NT)/
d(AC)] region; (B) vertical view of [p(NT)/d(AC)] region.
These models were obtained through optimization of the
hybrid of NPNA1 and 5%-d(CG AGA AGG GCG)-3% by
AMBER* force field in water set by using MacroModel
version 6.0. After the energy minimization, other base pairs
were removed. Bold N, T, C, and A mean naphthalimide
PNA, thymine PNA, deoxycytidine, and deoxyadenosine,
respectively. van der Waals surface is shown in dotted green.
We also believe that the introduction of a naphthal-
imide moiety at the amino terminal of the PNA
oligomer promotes the stability of the PNA–DNA
hybrids as the terminal amino group is electrostatically
attracted to the nearest neighboring irregular phosphate
of the DNA oligomer.
In summary, we have synthesized naphthalimide-con-
taining PNA oligomers for the first time and demon-
strated the thermodynamic stability in hybridization
with the corresponding DNA oligomer. We believe that
naphthalimide-containing PNA oligomers would be
attractive as a potent anti-tumor agent and antisense
molecule.
The thermal stability of the hybrids of naphthalimide-
containing PNA oligomers (NPNA1–7) with 5%-
d(CTAA CCC TAA C)-3% was examined by measuring
melting temperatures (Tm, Table 1). NPNA1 containing
a naphthalimide moiety at the amino terminal of the
PNA oligomer was slightly more stable than PNA1.
NPNA2 containing two neighboring naphthalimide
moieties at the amino terminal was considerably more
stable than PNA1, even though there were fewer base
pairs in the NPNA2/5%-d(C TAA CCC TAA C)-3%
hybrid compared with PNA1. Although NPNA3–4 con-
taining naphthalimide moieties at the C-terminal was
less stable than PNA1, the stabilization effect of the
PNA–DNA hybrid by the two neighboring naphthal-
imide moieties was evident. In contrast, NPNA5–7 con-
taining naphthalimide moieties incorporated in the
interior of the PNA–DNA hybrids was significantly less
stable than PNA1. In particular, NPNA7 containing
three consecutive naphthalimide moieties could not
hybridize with 5%-d(C TAA CCC TAA C)-3%, indicating
that the introduction of naphthalimide moieties into the
interior of the PNA oligomer is not suitable for the
stability of the PNA–DNA hybrid as a consequence of
the steric hindrance of the naphthalimide skeleton.
These results suggested that the naphthalimide moiety
introduced at the amino terminal region rather than the
C-terminal or the middle of the PNA oligomer con-
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