Construction of peptide conjugates with peptide nucleic acids containing an anthracene probe and their interactions with DNA

Article abstract of DOI:10.1016/S0968-0896(00)00329-1

We designed and synthesized the peptide nucleic acid (PNA)-peptide conjugates having anthracene chromophores and investigated their interactions with calf thymus DNA, [d(AT)₁₀]₂, [d(GC)₁₀]₂, and [d(AT)₁₀dA₆]₂. Considering the synthesis compatibility and expecting that a novel DNA analogue, PNA, can improve DNA binding properties of α-helix peptides, we attempted to attach thymine PNA oligomers at the C-terminus of a 14 amino acid α-helix peptide that contained a pair of artificial intercalators, anthracene, as a probe, and to examine their interactions with DNA using anthracene UV, fluorescence and circular dichroism properties. The results observed in this study showed that the designed peptide folded in an α-helix structure in the presence of calf thymus DNA, [d(AT)₁₀]₂, and [d(AT)₁₀dA₆]₂ with the chromophores at the side-chain being fixed with a left-handed chiral-sense orientation. The α-helix and the anthracene signals were not observed for [d(GC)₁₀]₂. Incorporation of thymine PNA oligomers into the designed α-helix peptide increased the DNA binding ability to [d(AT)₁₀dA₆]₂ with increasing the length of the PNA without changing the conformations of the peptide backbone and the anthracene side-chains.

Full text of DOI:10.1016/S0968-0896(00)00329-1

Bioorganic & Medicinal Chemistry 9 (2001) 1115±1121
Construction of Peptide Conjugates with Peptide Nucleic Acids
Containing an Anthracene Probe and Their Interactions with DNA
a
a
a
a,b,
Ganesan Balasundaram, Tsuyoshi Takahashi, Akihiko Ueno and Hisakazu Mihara *
aDepartment of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Yokohama 226-8501, Japan
b
Form and Function, PRESTO, Japan Science and Technology Corporation, Tokyo Institute of Technology,
Nagatsuta, Yokohama 226-8501, Japan
Received 6 September 2000; accepted 23 November 2000
AbstractÐWe designed and synthesized the peptide nucleic acid (PNA)±peptide conjugates having anthracene chromophores and
investigated their interactions with calf thymus DNA, [d(AT)10] , [d(GC)10] , and [d(AT)10dA ] . Considering the synthesis com-
2 2 6 2
patibility and expecting that a novel DNA analogue, PNA, can improve DNA binding properties of a-helix peptides, we attempted
to attach thymine PNA oligomers at the C-terminus of a 14 amino acid a-helix peptide that contained a pair of arti®cial inter-
calators, anthracene, as a probe, and to examine their interactions with DNA using anthracene UV, ¯uorescence and circular
dichroism properties. The results observed in this study showed that the designed peptide folded in an a-helix structure in the pre-
sence of calf thymus DNA, [d(AT)10
chiral-sense orientation. The a-helix and the anthracene signals were not observed for [d(GC)10
oligomers into the designed a-helix peptide increased the DNA binding ability to [d(AT) dA ] with increasing the length of the
]
2
, and [d(AT)10dA
6
]
2
with the chromophores at the side-chain being ®xed with a left-handed
]
2
. Incorporation of thymine PNA
1
0
6 2
PNA without changing the conformations of the peptide backbone and the anthracene side-chains. # 2001 Elsevier Science Ltd.
All rights reserved.
Introduction
helices with complementary PNA, DNA, and RNA
with the Watson±Crick base pairings.
6À8
Ithas been
shown that PNA molecules have a potential as thera-
peutic agents, tools in diagnostics and probes in mole-
The design of synthetic molecules that bind sequence-
speci®cally to the unique site of DNA and mimic the
action of proteins may have major implications for reg-
ulation of cellular reactions mostly mediated by the
9À12
cular biology.
Although many DNA analogues have
been studied extensively, PNA exhibits better advantages
such as stability to degradation by enzymes, and hybri-
dization property to complementary sequences with
higher anity and speci®city. Furthermore, from the
synthetic point of view, PNA oligomer synthesis is
compatible to standard solid-phase peptide synthesis
and that makes the synthesis easier either by manual or
by machine. Since PNA molecules are achiral, conjuga-
tion with chiral and structured peptides will lead to a
new property of PNA.
1
speci®c interaction between proteins and DNA. Peptides
with an a-helical conformation play an important role
in many biological systems, as this is a very important
protein secondary structure. For example, motifs such
as helix-turn-helix, helix-loop-helix, basic leucine-zipper,
and zinc ®ngers use a-helix structures in the framework
of DNA major groove interaction as most of the contacts
between amino acids and DNA bases are made within
2
such recognition helices. On the other hand, peptide
nucleic acid (PNA), a well established DNA mimic
developed by Nielsen et al., in which the entire sugar-
phosphate backbone has been replaced with a peptide-like
backbone, has attracted much attention in nucleic acid
In the design of small DNA binding molecules, many
DNA binding agents interact noncovalently with DNA
through intercalative association in which a planar,
3À5
13
science since itwas developed.
PNAs form double
hetero-aromatic moiety slides between base pairs.
Intercalation binding of small molecules with DNA has
been investigated at the molecular level, while inter-
1
4
calators are important as drugs against several diseases.
In general, intercalators are planar, and planarity is
*
5
Corresponding author. Tel.: +81-45-924-5756; fax: +81-45-924-
833; e-mail: hmihara@biotitech.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00329-1

1116
G. Balasundaram et al. / Bioorg. Med. Chem. 9 (2001) 1115±1121
proposed to be one of the important features for e€ective
intercalation into the DNA helix. So far, a variety of
anthracene derivatives have shown signi®cant antitumor
activity in animal models and many of them have been
was chosen because it can participate with no synthetic
obstacle for this purpose. In this study we used anthracene
as a probe to detect the chiral orientation of chromo-
phores and to adopt the intercalation properties to the
side-chains of the a-helix peptide. We investigated
interaction of the anthracene peptide±thymine PNA con-
jugates with calf thymus DNA, [d(AT) ] , [d(GC) ] ,
1
5
used for clinical testing. It has been also demonstrated
that anthracene ring system should have particularly
1
6
good stacking interactions with DNA base pairs. The
strong absorption and ¯uorescence property of the
anthracene group provide a sensitive spectroscopic
handle to study its intercalation with DNA. Changes in
the intensities can be used to ®nd the nature and the
strength of the interactions between molecules and
1
0 2
10 2
and [d(AT) dA ] using anthracene UV, ¯uorescence
1
0
6 2
and circular dichroism (CD) properties.
Results and Discussion
Design and synthesis
1
7
DNA.
In this work, we expected that the linkage of peptide
and PNA mightprovide a novel mechanism ot gauge
the interaction between peptide and DNA. For this
purpose, to combine the excellent properties of PNA
and a-helix peptides in the framework of peptide±DNA
interactions, we synthesized a 14 amino acid a-helix
peptide that conjugated with a series of PNA thymine
oligomers at the C-terminus and that employed two
anthracenes as functional chromophores on two lysine
residues at 6th and 9th positions (Fig. 1). Thymine PNA
The 14 peptide was designed to take an amphiphilic
a-helix structure upon binding to DNA as shown in
Figure 1. The amino acids of this peptide sequence are
selected for their helical propensity. Four positively
charged lysine residues were employed for the interac-
tion with negatively charged DNA phosphate groups. A
pair of anthracene groups were introduced on two lysine
residues at the 6th and 9th positions to give the intercala-
tion function in the a-helix peptide and to examine how
1
8
Figure 1. Amino acid sequences of anthracene peptide±thymine PNA conjugates and chemical structures of anthracene moiety and thymine PNA
a). a-Helical wheel and schematic illustration of their interactions with DNA (b). DNAs used in this study (c).
(

G. Balasundaram et al. / Bioorg. Med. Chem. 9 (2001) 1115±1121
1117
the chromophores behave via DNA binding. Earlier, we
used similar a-helix peptide sequences with two anthra-
cene chromophores atdi€erentpos ii to ns and invesi t-
the left-handed arrangement as expected.25
Next, we studied the CD analysis with calf thymus DNA,
[d(AT) ] , [d(GC) ] . In addition to these DNAs we
1
9
gated their DNA binding properties.
The study
1
0 2
10 2
revealed that the presence of anthracene chromophores
on lysine residues at the 6th and 9th positions exhibited a
left-handed chiral-sense orientation and anthracene chro-
mophores at the 6th and 10th positions showed a right-
handed chiral-sense orientation upon DNA binding.
Moreover, the study showed that the peptide with the
left-handed chiral-sense of anthracenes displayed a higher
DNA binding ability for an AT sequence compared to the
right-handed chiral-sense peptide and the peptide with a
single chromophore. In order to evaluate the in¯uence of
introduction of PNA on DNA binding of peptides, we have
synthesized a series of peptides, AntT ±AntT , containing
used [d(AT) dA ] , expecting that the thymine PNA
10 6 2
partof ht e helix sequence may have any binding in¯u-
ence. All these measurements were carried out when the
peptides were completely bound to DNA. As shown in
the Figure 2b±d, at the amide region, an a-helix struc-
ture was strongly induced via binding to calf thymus
DNA, [d(AT) ] , and [d(AT) dA ] . The positive and
1
0 2
10
6 2
negative splitting was observed at the anthracene region
showing the left-handed chiral-sense orientation. These
chiral signals also indicate the a-helix formation of the
peptides as a result of interaction with DNA. Peptides
with [d(GC) ] did not induce any CD pattern at both
10 2
0
4
various lengths of thymine-PNA at the C-terminus of the
anthracene peptide. Two glycine residues were used
between the peptide and PNA as a spacer (Fig. 1).
the amide region and the anthracene region (Fig. 2c).
The peptide without the anthracene groups showed also
the a-helix induction by the binding with AT-rich
1
9
DNA. These results indicate that the a-helix and
anthracene signals are AT selective. However, though
the conjugates exhibited a similar CD pattern with a
particular DNA, they showed di€erent ratios in nega-
tive and positive peaks. [d(AT) ] and [d(AT) dA ]
6 2
The peptide±PNA conjugates were synthesized by the
solid-phase method using standard Fmoc strategy. In
our experiences, elongation of thymine PNA oligomers
to the amino terminus of this same a-helix sequence by
solid-phase synthesis encountered with coupling pro-
blems, probably due to the unwanted side reaction of
1
0 2
10
showed almostequal area of neg ai vt e and posi vt e
peaks, whereas with calf thymus DNA the positive peak
was found to be greater than the negative peak. These
changes in peak ratios are probably due to slightly dif-
ferent interaction modes with di€erent DNAs. Over-
all, the CD patterns at the amide region and the
anthracene region showed that there occurred no con-
formational changes by the presence of thymine PNA
oligomers, indicating that all anthracene peptide±PNA
conjugates (AntT ±AntT ) possess a similar binding
20
N-acyl transfer. However, this experiment showed that
thymine PNA oligomerisation at the carboxyl terminus was
achieved with single coupling. The synthesized products
were treated with tri¯uoroacetic acid (TFA) to remove
the peptide from resin. Anthracene groups were attached
on the lysine residues at the 6th and 9th positions with
9
-anthracene carboxylic acid using 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide (EDC)/1-hydroxybenzotri-
azole (HOBt). After con®rming the anthracene coupling
reactions, the 2-chlorobenzyloxycarbonyl (ClZ) protection
groups of other lysine residues were removed by tri-
methylsilyl tri¯uoromethanesulphonate (TMSOTf)/TFA
0
4
mode to each DNA. These results demonstrated that
the peptides fold in an a-helix structure and the chro-
mophore at the side chain being ®xed in a coincidental
manner as they are deployed, when they interact with
AT partof DNA.
2
1
treatment. The ®nal products were puri®ed by semi-
preparative reversed-phase HPLC (RP-HPLC) to give
high purity (>98% on analytical RP-HPLC). The con-
jugates were identi®ed by matrix assisted laser desorption
ionization time-of-¯ight mass spectrometry (MALDI-
TOFMS).
UV study of the peptide±PNA conjugates with DNA
Prior to investigating the anthracene peptide±DNA inter-
actions by ¯uorescence spectroscopy, we performed UV
measurements of the peptides with DNA. Figure 3
depicts the UV spectral changes that occur when
d[(AT) dA ] was added to a solution of AntT pep-
CD study of the peptide±PNA conjugates with DNA
1
0
6 2
4
To obtain the conformational information, we per-
formed CD spectroscopy. In general, intercalators
bound to DNA exhibit induced circular dichroism
spectra due to their asymmetric environment. From this
one can easily evaluate the relative orientation of the
tide. By the addition of [d(AT) ] , [d(GC) ] , calf-thy-
10 2 10 2
mus, and d[(AT) dA ] , the absorbance of anthracene
1
0
6 2
groups were decreased and shifted to a longer wave-
length range, indicating the interaction between the
aromatic chromophore and the DNA bases. Isosobestic
points were observed in all DNA cases, suggesting that
the free and bound ligands are spectroscopically distin-
guishable. Furthermore, the observed large hypochro-
mism shows a sign of close proximity of the anthracene
chromophore to the DNA bases due to a strong inter-
action between the electronic states of the intercalating
chromophore with respect to the DNA helix.2
this experiment, the CD study revealed that the peptides
AntT ±AntT ) were almost in a random structure in
2À24
In
(
0
4
aqueous bu€er (pH 7.4) with 40 mM NaCl and 2 mM
MgCl . On the contrary, in tri¯uoroethanol (TFE), an
2
a-helix forming solvent, the peptides showed an a-helix
CD pattern (Fig. 2a). The excitation coupling was
remarkably observed at the anthracene region (254 nm
wavelength) displaying the negative and positive split-
ting from the longer wavelength. This suggests that the
two anthracene groups were oriented in a chiral-sense to
2
6À29
chromophore and those of the DNA bases.
Vir-
tually, these UV patterns indicate that the anthracene
chromophore has a property of intercalating to the all
selected DNAs. Further insight about the binding ability
is discussed in the subsequent ¯uorescence study.

1118
G. Balasundaram et al. / Bioorg. Med. Chem. 9 (2001) 1115±1121
.
Figure 2. CD spectra of conjugates in TFE (solid line) and 20 mM Tris HCl (broken line) (a), with calf-thymus DNA (b), with [d(AT)10
]
2
, and
(solid line) are shown. Spectral measurements
.
2
C in 20 mM Tris HCl bu€er (pH 7.4) containing 40 mM NaCl and 2 mM MgCl . [Peptide]=5.0 mM, [DNA nucleoti-
[
were carried outat25
d(GC)10
]
2
(c), with [d(AT)10dA
6
]
2
(d). From (b) to (d) CD patterns of AntT
0
(broken line) and AntT
4
ꢀ
de]=300 mM.
Fluorescence study of the peptide±PNA conjugates with
DNA
constants were determined by making a curve-®tting ana-
lysis of ÁI/I as a function of DNA concentration (Fig.
0
1
9
5). The previous study showed that the same anthra-
Next we investigated the interactions of the anthracene
peptide±PNA conjugates with DNA quantitatively by
¯
and this study showed that a similar a-helix peptide
exhibited a binding preference with AT base pairs rather
than GC base pairs. In this study, in addition to
cene peptide displayed the 1:4 stoichiometry with base
pairs of [d(AT) ] , [d(GC) ] , and calf-thymus DNA
based on the site exclusion equation of McGhee and
1
0 2
10 2
uorescence measurements. Both the previous study19
3
0
Von Hippel. In this experiment, since [d(AT) dA ]
10 6 2
contains both the single strand and the double
strand, we used the nucleotide concentration instead
of base pair concentration for all DNAs uniformly.
Coincidently, the ¯uorescence results showed that for
the DNAs, the best curve-®tting was achieved with a
1:8 stoichiometry using eq. 1 in the experimental
section.
[
d(AT) ] , [d(GC) ] , and calf-thymus DNA, we used
10 2 10 2
[
d(AT) dA ] , expecting that the thymine PNA part of
1
0
6 2
the helix peptide may have any binding in¯uence. Figure
depicts the ¯uorescence titration for AntT peptide
with [d(AT) dA ] . In all DNA cases, except
4
4
1
0
6 2
[
d(GC) ] , the addition of DNA to the conjugates
10 2
increased the ¯uorescence intensity. The higher DNA
concentration part of the curve corresponds to the
saturation limit of ¯uorescence. However, unlike other
DNAs, the titration with [d(GC) ] showed decreases in
the ¯uorescence intensity due to the quenching by G. To
characterize the interaction quantitatively, the binding
Table 1 shows the binding constants calculated by the
¯uorescence titration for various DNAs. Interaction
with calf thymus DNA gave the binding constant value
5
À1
of 7.2Â10 M for AntT peptide. Although the spec-
1
0 2
0
1
7
tral measurements with calf thymus DNA explained the
binding properties of all peptides, there was not any

G. Balasundaram et al. / Bioorg. Med. Chem. 9 (2001) 1115±1121
1119
Figure 3. UV spectra of AntT
4
peptide in the presence of various
concentrations of [d(AT)10dA ] . From top to bottom the nucleotide
6 2
concentrations were 0, 35, 70, 105, 140, 175, 210 mM. (Pepti-
de)=5.0 mM, 25 C.
Figure 5. Curve-®tting analysis for the ¯uorescence intensities of con-
jugates with [d(AT)10dA ] at424 nm.
6 2
ꢀ
Interestingly, there were noticeable variations in binding
constants with [d(AT) dA ] by the increasing number
1
0
6 2
of thymine PNA. Figure 5 shows the curve-®tting ana-
lysis performed for conjugates with [d(AT) dA ] . As
shown in Figure 5 and Table 1, both the ÁI/I value and
1
0
6 2
0
binding constant were increased from AntT to AntT
0
4
5
À1
5
À1
(
11.0Â10 M to 16.5Â10 M ). These results suggest
that the thymine PNA can interact with the single
strand dA6 of [d(AT) dA ] and this interaction is
10
6 2
enhanced by increasing the number of thymine PNA
Fig. 6). Although, the addition of single thymine PNA
(
is not e€ective, further addition of thymine PNA
remarkably increased the binding ability of the con-
jugates to the DNA.
In order to verify the in¯uence of thymine PNA part of
AntT4 peptide with the single strand part of
[
formed in the presence of excess amount of Ac-(T4)PNA
d(AT) dA ] , the ¯uorescence inhibition test was per-
10 6 2
-
Gly-NH . The measurement was carried out by titrating
2
Figure 4. Fluorescence spectra of AntT peptide in the presence of
6 2
various concentrations of d[(AT)10dA ] . From bottom to top the
4
AntT peptide (2.0 mM) with [d(AT) dA ] in the pre-
4
10
6 2
sence of Ac-(T4)PNA-Gly-NH2 (100 mM) (data not
nucleotide concentrations were 0, 9, 18, 27, 36, 45, 54, 63, 72, 81,
ꢀ
5
À1
shown). A binding constant value of 8.1Â10 M was
observed, lower than the other anthracene peptide±PNA
conjugates with [d(AT) dA ] (Fig. 6) This suggests that
90 mM. (Peptide)=2.0 mM, lex=389 nm, 25 C.
1
0
6 2
.
the Ac-(T4)PNA-Gly-NH binds to the dA partof ht e
2
6
observed DNA binding in¯uence by the addition of
thymine PNA. Interaction of AntT0 with [d(AT) ]
[d(AT) dA ] and thereby decreases the overall binding
10 6 2
ability of AntT peptide with the DNA. This inhibition
4
10 2
À1
5
showed a binding constant value of 11.8Â10 M
.
However, the results of AntT through AntT displayed
experiment con®rms the in¯uence of thymine PNA part
of AntT4 peptide with [d(AT) dA ] . Additionally, Ac-
(T4)PNA-Gly-NH might a€ect the peptide binding to
1
4
10
6 2
similar binding constants at the maximum of
5
2
À1
1
observed with [d(GC) ] , indicating that the binding
3Â10 M
.
The lowest binding constants were
the double strand part of the DNA to some extent so
that the binding constant decreased to a lower level than
AntT . All these ¯uorescence results reveals that PNA
1
0 2
ability of this anthracene peptide to the AT sequence is
two times higher than that of the GC sequence. These
results showed that the presence of thymine PNA does
not discriminate the DNA binding ability in these cases
of double strand DNAs.
0
thymine oligomersation with the helix peptide has no
signi®cant in¯uence with calf thymus DNA, [d(AT) ] ,
and [d(GC) ] , whereas increase in the binding ability
1
0 2
1
0 2
was observed only with [d(AT) dA ] .
0
1
6 2

1120
G. Balasundaram et al. / Bioorg. Med. Chem. 9 (2001) 1115±1121
Table 1. Binding constants of anthracene peptide-PNA conjugates with DNA estimated from ¯uorescence studies
5
À1
)
K
b
(Â 10 M
AntT
0
AntT
1
AntT
2
AntT
3
AntT
4
Calf thymus
7.2 Æ 0.1
11.8 Æ 0.5
5.2 Æ 0.2
6.4 Æ 0.1
11.0 Æ 0.6
5.5 Æ 0.6
10.8 Æ 0.1
7.4 Æ 0.1
12.0 Æ 0.6
5.7 Æ 0.2
12.8 Æ 0.1
7.2 Æ 0.3
10.5 Æ 0.5
5.9 Æ 0.3
13.2 Æ 0.1
NDa
[
[
[
d(AT)10
d(GC)10
d(AT)10 dA
]
]
13.0 Æ 0.2
5.0 Æ 0.1
16.5 Æ 0.1
6
]
2
11.0 Æ 0.06
aNot determined due to the formation of aggregates.
without further puri®cation. Fmoc amino acid derivatives,
0 0
-(2 ,4 -dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy
4
resin (Rink amide resin),
(
3
2
1-hydroxybenzotriazole
HOBt), water soluble carbodiimide (EDC) were pur-
chased from Watanabe Chemical (Hiroshima, Japan).
Benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphonium
hexa¯uorophosphate (BOP) was purchased from
Novabiochem. 9-Anthracene carboxylic acid was pur-
chased from Aldrich. Boc protected thymine PNA
monomer was prepared according to the published
3
3
method. This was converted to the Fmoc derivative
using N-(9-¯uorenylmethoxycarbonyl succinimide)
(Fmoc-OSu). The synthesis of PNA±peptide conjugates
was carried out manually according to the method
3
4
available for standard solid-phase peptide synthesis.
Completion of the each acylation steps was monitored
3
5
by the Kaiser test. Recoupling was performed if the
couplings were incomplete. Capping of the unreacted
amino groups was achieved using acetic anhydride. The
following amino acid derivatives were used in this
experiment: Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Leu-
OH, Fmoc-Lys(ClZ)-OH, Fmoc-Lys(Boc)-OH, and
Fmoc-Gln(Trt)-OH. The lysines engaged in anthracene
introduction were incorporated as Fmoc-Lys(Boc)-OH
and other lysines were protected with ClZ groups. All
amino acids and thymine PNA couplings were per-
formed with BOP and HOBT. After addition of the last
amino acid (Ala) and removal of the Fmoc group, the
products were acetylated with acetic anhydride. The
protecting groups (except ClZ) and resin were removed by
TFA/m-cresol treatment. The coupling with 9-anthracene
carboxylic acid was achieved with EDC/HOBT in DMF
for 24 h. Finally all ClZ groups were removed by treating
Figure 6. Variation of binding constants derived from ¯uorescence
measurements of conjugates with [d(AT)10dA ] .
6 2
Conclusion
A new type of peptidyl molecule was designed and syn-
thesized which bound to DNA. In the a-helix peptide,
the anthracene chromophores were ®xed to arrange via
binding to DNA so as to interact with each other. These
interactions of anthracene groups enable the detection
of the conformational changes of the peptides by the
induced CD with DNAs. Incorporation of thymine
PNA oligomers at the carboxyl terminus of the helix
peptide showed an increased binding ability with
2
1
with TMSOTf/TFA/thioanisole mixture. TOFMS was
performed on a Shimadzu MALDI III mass spectrometer
by using sinnapinic acid as a matrix. Final puri®cation
was carried outby RP-HPLC on a YMC-Pack C18
A-323 column (10Â250 mm) to give the products with a
single peak on analytical HPLC [Wakosil 5C18
(4.6Â150 mm)] with a linear gradient of acetonitrile/0.1%
[
d(AT) dA ] by the increase of the thymine PNA
10 6 2
length without changing the conformational property of
the helix peptide at both the anthracene and amide
regions. This study demonstrates the feasibility of our
method in utilizing anthracene as a probe and func-
tionality for DNA binding and the possible use of PNA
in the framework of peptide±DNA interaction. Further-
more, the strategy involved in this study can be readily
adopted to use other natural peptides with PNA oligo-
+
TFA. MALDI-TOFMS found m/z (calcd [M+H] );
AntT , 2116.8 (2117.7); AntT , 2382.2 (2383.9); AntT ,
0
1
2
2649.5 (2650.1); AntT , 2916.7 (2916.3); AntT , 3180.8
3
4
3
1
mers to modify the DNA/RNA binding properties.
(3182.5).
Spectroscopic measurements
All spectroscopic measurements were carried out at
Experimental
ꢀ
.
General methods
25 C atpH 7.4 using 20 mM Tris HCl bu€er containing
0 mM NaCl and 2 mM MgCl . Calf thymus DNA was
4
2
All chemicals and solvents used for synthesis and ana-
lysis were of reagentgrade or HPLC grade and used
purchased from Pharmacia Biotech. Calf thymus DNA
(4 mg/mL) was dissolved by slowly strirring for 24 h in

G. Balasundaram et al. / Bioorg. Med. Chem. 9 (2001) 1115±1121
1121
.
0 mM Tris HCl bu€er (0.15 M NaCl; pH 7.4). The
2
7. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.;
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solution was sonicated for 30 min under Ar gas and ®l-
tered. Concentration of the DNA was determined spec-
troscopically using molar extinction coecient per base
pair with the extinction coecient of E260=6600 at
8
. Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis, D. G.
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9
2
60 nm. Synthetic DNA oligomers were purchased from
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Sawady Technology, and concentration of each of them
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coecients. UV spectra were recorded using Shimadzu
UV-3100 spectrophotometer using a quartz cell with
1
cm path-length. Fluorescence measurements were carried
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photometer using a 1 cm path-length quartz cell with
excited wavelength at 389 nm. The ¯uorescence intensity
was monitored at 424 nm. Binding constants of anthra-
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by the following equation by making a ®tting-curve to the
1
1
1
1
3. Lerman, L. S. J. Mol. Biol. 1961, 3, 18.
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ÁI/I as a function of nucleotide concentration with a
0
Williams, A. R. J. Med. Chem. 1990, 33, 1549.
1
:8 stoichiometry using Kaleidagraph (Synergy Software);
1
1
1
6. Miller, K. J.; Newlin, D. D. Biopolymers 1982, 21, 633.
7. Kumar, C. V.; Asuncion, E. H. J. Am. Chem. Soc. 1993,
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ꢀ
ÁImax=I †f‰P Š ‡ ‰8D Š ‡ 1=K À
18. Chou, P. Y.; Fasman, G. D. Biochemistry 1974, 13(2), 211.
9. Takahashi, T.; Ueno, A.; Mihara, H.; Tam, J. P.; Kaumaya,
P. T. P (Eds.) Peptides: Frontiers of Peptide Science (Proceedings
0
0
0
b
2
0:5
1
ꢀ
ꢀ‰P Š ‡ ‰8D Š ‡ 1=K † À 8‰P Š‰8D Š††
g
0
0
b
0
0
ÁI=I0 ˆ
ꢀ1†
2
‰P Š
0
of the 15th APS), Kluwer, Dordrecht, 1998, p.130.
2
0. Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen,
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Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg, R. H. J. Pep. Sci.
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ÁI=IÀI , where I and I are the ¯uorescence intensities
0
0
of the conjugates in the presence and the absence of
DNA, respectively. ÁImax=ImaxÀI , where I is the
2
1. Fujii, N.; Otaka, A.; Ikemura, O.; Akaji, K.; Funakoshi,
0
max
S.; Hayashi, Y.; Kuroda, Y.; Yajima, H. J. Chem. Soc., Chem.
Commun. 1987, 274.
22. Lyng, R.; Hard, T.; Norden, B. Biopolymers 1987, 26,
¯
is the total peptide concentration and [D ] is the total
uorescence intensity when peptide bound to DNA. [P ]
0
0
1
2
2
327.
3. Norden, B.; Tjerneld, F. Biopolymers 1982, 21, 1713.
4. Schipper, P. E.; Norden, B.; Tjerneld, F. Chem. Phys.
DNA nucleotide concentration. K is the binding con-
b
stant of the conjugates for DNA nucleotide. CD mea-
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polarimeter using a 0.1 cm quartz cell. All spectral data
were collected in the 200 nm to 280 nm wavelength
range as the average of 10 scans only when the anthra-
cene peptides completely bound to DNA.
Lett. 1980, 70, 17.
2
2
2
1
2
1
5. Harada, N.; Nakanishi, K. Acc. Chem. Res. 1972, 5, 257.
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