Analysis of coherent heteroclustering of different dyes by Use of threoninol nucleotides for comparison with the molecular exciton theory

Article abstract of DOI:10.1002/chem.200900962

To test the molecular exciton theory for heterodimeric chromophores, various heterodimers and clusters, in which two different dyes were stacked alternately, were prepared by hybridizing two oligodeoxyribonucleotides (ODNs), each of which tethered a diffe

Full text of DOI:10.1002/chem.200900962

DOI: 10.1002/chem.200900962
Analysis of Coherent Heteroclustering of Different Dyes by Use of
Threoninol Nucleotides for Comparison with the Molecular Exciton Theory
Taiga Fujii,[a] Hiromu Kashida,[a] and Hiroyuki Asanuma*[a, b]
Abstract: To test the molecular exciton
theory for heterodimeric chromo-
phores, various heterodimers and clus-
ters, in which two different dyes were
stacked alternately, were prepared by
hybridizing two oligodeoxyribonucleo-
tides (ODNs), each of which tethered a
different dye on d-threoninol at the
center of the strand. NMR analyses re-
vealed that two different dyes from
each strand were stacked antiparallel
to each other in the duplex, and were
located adjacent to the 5’-side of a nat-
ural nucleobase. The spectroscopic be-
havior of these heterodimers was sys-
tematically examined as a function of
the difference in the wavelength of the
dye absorption maxima (Dlmax). We
found that the absorption spectrum of
the heterodimer was significantly dif-
ferent from that of the simple sum of
each monomeric dye in the single
strand. When azobenzene and Methyl
Red, which have lmax at 336 and
480 nm, respectively, in the single
strand (Dlmax =144 nm), were assem-
bled on ODNs, the band derived from
azobenzene exhibited a small hyper-
chromism, whereas the band from
Methyl Red showed hypochromism
and both bands shifted to a longer
wavelength (bathochromism). These
hyper- and hypochromisms were fur-
ther enhanced in a heterodimer de-
rived from 4’-methylthioazobenzene
and Methyl Red, which had a much
smaller Dlmax (82 nm; lmax =398 and
480 nm in the single-strand, respective-
ly). With a combination of 4’-dimethyl-
ACHTUNGTRENNaUNG mino-2-nitroazobenzene and Methyl
Red, which had an even smaller Dlmax
(33 nm), a single sharp absorption band
that was apparently different from the
sum of the single-stranded spectra was
observed. These changes in the intensi-
ty of the absorption band could be ex-
plained by the molecular exciton
theory, which has been mainly applied
to the spectral behavior of H- and/or J-
aggregates composed of homo dyes.
However, the bathochromic band shifts
observed at shorter wavelengths did
not agree with the hypsochromism pre-
dicted by the theory. Thus, these data
experimentally verify the molecular ex-
citon theory of heterodimerization.
This coherent coupling among the het-
erodimers could also partly explain the
bathochromicity and hypochromicity
that were observed when the dyes were
intercalated into the duplex.
Keywords: DNA · exciton theory ·
heteroclusters · NMR spectroscopy ·
UV/Vis spectroscopy
Introduction
dye assemblies have the potential to contribute to the pro-
duction of nonlinear optical materials[2] and light-harvesting
systems,[3] the preparation of ordered dye assemblies and
their characterization is very important both from a scientif-
ic and a practical point of view. To date, various methodolo-
gies for the preparation of homo-assemblies composed of
identical dyes have been proposed, and their spectroscopic
behaviors, such as J- and H-bands, have been examined the-
oretically on the basis of molecular exciton theory.[1,4] How-
ever, due to the difficulty of their preparation, there have
only been a limited number of reports on the characteriza-
tion of hetero-assemblies, especially those of a predeter-
mined size and orientation.[5–7] Hence, theoretical investiga-
tion of the spectroscopic behavior and potential applications
of hetero-assemblies is less advanced than that of homo-
assemblies, at least in part because theoretical calculations
concerning their spectroscopic behavior cannot be experi-
Assembly of a dye induces characteristic spectroscopic be-
havior, such as band narrowing and band shifts, that cannot
be achieved with a monomeric dye.[1] Furthermore, since
[a] T. Fujii, Dr. H. Kashida, Prof. Dr. H. Asanuma
Department of Molecular Design and Engineering
Graduate School of Engineering, Nagoya University
Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)
Fax : (+81)52-789-2528
E-mail: asanuma@mol.nagoya-u.ac.jp
[b] Prof. Dr. H. Asanuma
Core Research for Evolution Science and Technology (CREST)
Japan Science and Technology Agency (JST)
Kawaguchi, Saitama 332-0012 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900962.
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FULL PAPER
mentally verified. As far as we know, there are only a few
examples that demonstrate a relationship between the ex-
perimental observation of the spectroscopic behavior of het-
eroaggregation (dimerization) and its theoretical prediction.
The first report of this type was by Kuhn et al. on the spec-
troscopic behavior of a heterodimer of cyanine dyes spread
on the water/air interface using the Langmuir–Blodgett
method.[6] Furthermore, a change in the absorption spectra
of a fluorescence resonance energy transfer (FRET) donor–
acceptor pair and/or an fluorescein isothiocyanate/dimethyl
aminophenylazo benzoic acid (FITC/Dabcyl; Methyl Red)
pair tethered at both the 3’- and 5’-termini of a molecular
beacon has been demonstrated.[7,8] However, since mutual
orientation of these heterodimers was not well-defined and
was difficult to control, they were not appropriate models to
verify the exciton theory experimentally. Furthermore, these
investigations lacked the viewpoint of the difference of lmax
,
which should significantly affect the coherency of the
dimers. Verification of the exciton theory will lead to the
design of new hetero-assemblies having special optical prop-
erties that cannot be realized by homo-assemblies.
Previously, we proposed a unique method for the prepara-
tion of a dye assembly by hybridization of two single-strand-
ed oligodeoxyribonucleotides (ODNs), each of which teth-
ered dyes to d-threoninols (threoninol nucleotides) at the
center of the strand.[9] The use of threoninol as a dye scaf-
fold allows easy programming of various dye assemblies that
are difficult to design based on the conventional self-associ-
ation of dyes. Alternating heteroclusters of a predetermined
size and orientation can be easily prepared by hybridization
of two complementary DNA–dye conjugates, each of which
is conjugated to a different dye. In the present study, we
prepared various heterodimers of Methyl Red with other
dyes using threoninol nucleotides. The dyes chosen for het-
erodimerization with Methyl Red were chosen with a focus
on the difference in lmax between Methyl Red and the
second dye (Dlmax), as shown in Schemes 1 and 2. Such a
Scheme 2. Sequence of the ODNs synthesized in this study.
lecular sizes of these dyes are very similar; 2) the p–p* tran-
sition (light absorption) of these dyes occurs in a similar
manner; 3) they have a single and fairly symmetrical absorp-
tion band; and 4) the absorption spectra reflect the exciton
coupling (coherency) of the dyes rather than fluorescence.
Five threoninol nucleotides with azobenzene (Z), 4’-methyl-
thioazobenzene (H), Methyl Red (M), 4’-dimethylamino-2-
nitroazobenzene (R), or Naphthyl Red (N) tethers were in-
corporated into ODNs (see Scheme 2 for the structures),
and various heterodimers were prepared to investigate their
spectroscopic behavior from both experimental and theoret-
ical viewpoints. These insights into heterodimerization will
lead to the design of more sensitive fluorophore-quencher
pairs of molecular beacons.
Scheme 1. Schematic illustration of the design of the heterodimer using
threoninol nucleotides as dye tethers. There are two possible dye loca-
tions: the dyes are located adjacent to a) the 5’-side (corresponding to M/
Z orientation in NMR-Ma/NMR-Zb) or b) the 3’-side of a natural nucle-
obase (corresponding to Z/M orientation).
Results
Structural determination of the heterodimers by NMR spec-
troscopy: As illustrated in Scheme 1, heterodimer formation
is based on the tentative base-pairing of threoninol nucleo-
tides at the center of paired ODNs. A dye residue is located
at the counterpart of the other dye residue in the comple-
mentary ODN sequence,[11] and these dye residues from the
two strands stack with each other, in an antiparallel orienta-
tion, by pseudo “base-pairing”. The ODNs synthesized for
this study are outlined in Scheme 2. We first conducted an
NMR analysis of a heteroduplex of ODNs conjugated with
Methyl Red and azobenzene (NMR-Ma/NMR-Zb, respec-
systematic investigation of the relationship between Dlmax
and coherency in a firmly stacked heterodimer of predeter-
mined orientation has not yet been reported. The stacked
structure of the heterodimer in the duplex was first deter-
mined by NMR analysis,[10] and the spectroscopic behavior
of the dyes was then investigated in detail in order to verify
qualitatively the predictions of the molecular exciton theory.
We used azo compounds as model dyes because: 1) the mo-
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H. Asanuma et al.
tively), in order to determine the location of the stacked M
and Z in the duplex. There are two possible locations of the
dyes: adjacent to the 5’-side (Scheme 1a, M/Z) or to the 3’-
side (Scheme 1b, Z/M) of the natural nucleobase.
Because the melting temperature of the NMR-Ma/NMR-
Zb duplex was determined to be 22.18C,[12] NMR measure-
ments were performed at 58C (278 K), a temperature at
which a stable duplex exists. In order to monitor imino pro-
tons, which are exchangeable with water molecules, NMR
was measured in H2O (H2O/D2O, 9:1) with
ACHTUNGTRENNUNG
a 3-9-19
WATERGATE pulse sequence for H2O suppression.[13]
When these data were combined with the NOESY, DQF-
COSY, and TOCSY spectra obtained in H2O, most of the
signals of the duplex could be assigned. The one-dimension-
al NMR spectrum measured in H2O at the region of imino
protons (11–14 ppm) is depicted in Figure 1. The NMR-Ma/
Figure 2. 2D NOESY spectrum (mixing time=150 ms) between the
imino-proton signal (11–14 ppm) and the aromatic-proton signal regions
(5.5–7.5 ppm) for the NMR-Ma/NMR-Zb duplex in H2O/D2O 9:1 at
278 K, pH 7.0 (20 mm phosphate buffer), in the presence of 200 mm
NaCl. Assignments of the Methyl Red and azobenzene protons are de-
noted on the 1D spectra (F1 axis) using the numbers designated in
Scheme 2. The NOE signals surrounded by broken circles demonstrate
intercalation of the Methyl Red and azobenzene.
Figure 1. 1D NMR spectrum of the NMR-Ma/NMR-Zb duplex at the
imino region in H2O/D2O 9:1 at 278 K (mixing time=150 ms), pH 7.0
(20 mm phosphate buffer), in the presence of 200 mm NaCl. The concen-
tration of NMR-Ma/NMR-Zb was 1.0 mm. Assignments of the imino-pro-
tons and the residue number are denoted at the top of the peak.
NMR-Zb duplex allowed six natural base-pairs (C1–G12, G2–
C11, A3–T10, G4–C9, T5–A8, and C6–G7), and one tentative
M–Z pair (Scheme 2). As expected, there were six individu-
al signals that could be assigned on the basis of the NOESY
and chemical shift of each signal, indicating that the non-
natural M and Z did not interrupt the base-pairing. The
imino proton signals of the terminal G7 and G12 were rather
broad, because of the rapid exchange with water, whereas
signals from the other residues remained sharp. Figure 2 de-
picts the NOEs between the imino proton signal (11–
14 ppm) and the aromatic proton signal (5.5–7.5 ppm) re-
gions. A distinct NOE signal was observed between the
imino proton of T10 and H8 (H12) of the Methyl Red pro-
tons, indicating that the Methyl Red moiety was located at
around T10. In addition, strong NOE signals were detected
between the imino proton of G4 and H2 (H6) and H8 (H12)
of the azobenzene protons, indicating that the azobenzene
moiety was located in the vicinity of G4. Thus, we could un-
ambiguously conclude that each dye was located adjacent to
the 5’-side of the natural nucleobase (M/Z, corresponding to
Scheme 1a).
Figure 3. 2D NOESY spectrum (mixing time=150 ms) between the re-
gions of 5.5–7.5 ppm and 1.0–2.6 ppm for the NMR-Ma/NMR-Zb duplex
in H2O/D2O 9:1 at 278 K (20 mm phosphate buffer), in the presence of
200 mm NaCl.
Evidence for antiparallel stacking of M and Z was given
by the NOESY spectrum between the regions of 5.5–
7.5 ppm and 1.0–2.6 ppm (Figure 3). Strong NOE signals
À
were observed between N CH3 of M and H2 (H6) of Z, as
combined NOE signals demonstrate that M and Z are
stacked antiparallel to each other and are located adjacent
to the 5’-side of the nucleobase, as depicted in Figure 4a,
À
well as between N CH3 of M and H3 (H5) of Z, indicating
that M and Z were stacked in an antiparallel manner. The
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Coherent Heteroclustering of Dyes
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Figure 4. a) Stacking manner and orientation of Z and M residues in the duplex determined from NOESY, and b) an energy-minimized structure of the
NMR-Ma/NMR-Zb duplex calculated with InsightII/Discover3 from the initial structure determined by NMR analyses. Broken lines in a) show the ob-
served NOE signals. The sticks in b) depict azobenzene and Methyl Red in the duplex.
which validates our design of the heterodimer. Computer
modeling of NMR-Ma/NMR-Zb, using the InsightII/discov-
er3 software, was entirely consistent with the NMR analyses
(Figure 4b).
Figure 5 shows the UV/Vis spectra of the single-stranded
dye conjugates synthesized in this study. These conjugates,
Z1a, H1a, M1b, R1a, and N1a, showed absorption maxima
at 336, 398, 480, 513, and 548 nm, respectively. The absorp-
tion maxima corresponded to a monomeric transition at
08C, which is controlled by the introduction of electron-
donating and electron-attracting functional groups to the
azobenzene scaffold.[14] We then prepared four different
kinds of heteroclusters by combining Methyl Red-conjugat-
ed ODNs (Mnb) with ODNs conjugated with the other
dyes, thus forming Zna/Mnb, Hna/Mnb, Rna/Mnb, and Nna/
Mnb heterodimers. A summary of the lmax of the single con-
jugates and the Dlmax between M1b and the other dye conju-
gates is shown in Table 1.
Spectroscopic behavior of the alternate heterodimers:
Having validated our heterodimer design and confirmed its
stacked structure, we next investigated spectral changes of
heterodimers composed of the various ODN–dye conjugates
shown in Scheme 2. This analysis focused on the difference
of lmax between the dyes (Dlmax) in the heterodimer.
Table 1. Absorption maxima (lmax) of single-stranded dye-conjugates cor-
responding to a monomeric transition, and the Dlmax between M1b and
the conjugates used in this study.
Sequences
lmax [nm] (nmax [cmÀ1])[a]
Dlmax [nm] (Dnmax [cmÀ1])[a]
M1b
Z1a
H1a
R1a
N1a
480 (2.08ꢁ104)
336 (2.98ꢁ104)
398 (2.51ꢁ104)
513 (1.95ꢁ104)
548 (1.82ꢁ104)[b]
–
144 (8.93ꢁ103)
82 (4.29ꢁ103)
33 (1.34ꢁ103)
68 (2.59ꢁ103)[b]
Figure 5. UV/Vis spectra of single-stranded Z1a, H1a, N1a, R1a, and
M1b at 08C that corresponded to a monomeric transition. Solution condi-
tions were as follows: [ODN]=5 mm, [NaCl]=100 mm, pH 7.0 (10 mm
phosphate buffer). For the measurement of N1a, 10 mm MES buffer at
pH 5.0 was used.
[a] Measurement conditions were pH 7.0 (10 mm phosphate buffer),
[ODN]=5 mm, [NaCl]=100 mm, at 08C. [b] Measurement conditions
were pH 5.0 (10 mm MES buffer), [ODN]=5 mm, [NaCl]=100 mm, at
08C.
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H. Asanuma et al.
Z/M combination (Dlmax =144 nm): Figure 6 shows the UV/
Vis spectra of Z1a/M1b, Z1a, M1b, and a spectrum calculat-
ed as the simple sum of the two single strands (sum-spec-
Figure 6. UV/Vis spectra of the Z1a/M1b duplex (solid line), single-
stranded Z1a (dashed-dotted line), M1b (dotted line), and a simple sum
of their spectra (sum-spectrum, Z1a+M1b, dashed line) at 08C. Solution
conditions were as follows: [ODN]=5 mm, [NaCl]=100 mm, pH 7.0
(10 mm phosphate buffer).
trum). Both single-stranded Z1a and M1b exhibited broad
bands (peaks) at 336 and 480 nm, respectively. The sum-
spectrum gave two bands because Dlmax of Z1a/M1b was as
high as 144 nm. The absorption spectrum of the Z1a/M1b
duplex also gave two bands at around 344 and 500 nm, al-
though the pattern was slightly but distinctly different from
the pattern for the sum of each spectrum. Dimerization of Z
and M induced a bathochromic shift in the lmax of both dyes.
In addition, the peak located at a shorter wavelength in
Z1a/M1b (derived from the single-stranded Z) showed hy-
perchromism. In contrast, hypochromism was observed for
the peak derived from the single-stranded M.
Figure 7. a) UV/Vis spectra of H1a/M1b duplex (solid line), single-strand-
ed H1a (dashed-dotted line), M1b (dotted line), and a simple sum of
their spectra (sum-spectrum, H1a+M1b, dashed line) at 08C, and b) the
effect of temperature on the absorption spectrum of H1a/M1b. Solution
conditions were as follows: [ODN]=5 mm, [NaCl]=100 mm, pH 7.0
(10 mm phosphate buffer).
further amplified with the H1a and M1b combination, which
had a smaller Dlmax. In order to rule out the possibility that
these spectral behaviors were due to an unexpected covalent
interaction of these dyes, thermal reversibility of the H1a/
M1b duplex was examined. The absorption spectrum of
H1a/M1b above 608C, a temperature at which the duplex
was completely dissociated, was similar to the sum-spectrum
of H1a and M1b (compare the dashed line in Figure 7a with
the dashed-dotted line in Figure 7b). Note that the melting
temperature (Tm) of H1a/M1b was 51.28C under the condi-
tions employed (see Supporting Information, Table 1). In
contrast, a reduction in temperature below Tm allowed a
large, reversible, spectroscopic change. Thus, the spectral
changes observed in the duplex were confirmed to be due to
a noncovalent interaction between H and M in the duplex.
Since further reduction in temperature from 40 to 08C
slightly changed their absorption spectra, chromophore mo-
tions would also affect the exciton interaction slightly.[15]
H/M combination (Dlmax =82 nm): An H/M combination of
the dyes gave a Dlmax of 82 nm, which was half that of the
Z1a and M1b combination. The absorption spectra of H1a/
M1b, H1a, M1b, and their sum-spectrum are depicted in Fig-
ure 7a. Interestingly, hybridization of these two conjugates
displayed an absorption spectrum that was apparently differ-
ent from that of the sum of their single strands. The UV/Vis
spectrum of the H1a/M1b duplex gave one main band at
422 nm and a shoulder band at around 480–560 nm, as de-
picted by the solid line in Figure 7a, whereas the sum-spec-
trum of H1a and M1b (dashed line in Figure 7a) showed
two broad bands with almost equal absorbance at 398 and
480 nm. Following hybridization, the absorption band at
398 nm, corresponding to H1a, showed both strong batho-
chromicity and hyperchromicity, whereas the band at
480 nm, corresponding to M1b, displayed bathochromicity
and hypochromicity (Figure 7a). Although this tendency
was similar to that observed with the Z1a and M1b combi-
nation, the spectral change induced by hybridization was
R/M and N/M combination (Dlmax =33 and 68 nm): The
combination of R and M had the smallest Dlmax (33 nm) of
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Figure 8. a) UV/Vis spectra of the R1a/M1b duplex (solid line), single-
stranded R1a (dashed-dotted line), M1b (dotted line), and a simple sum
of their spectra (sum-spectrum, R1a+M1b, dashed line) at 08C. Solution
conditions were as follows: [ODN]=5 mm, [NaCl]=100 mm, pH 7.0
(10 mm phosphate buffer). b) UV/Vis spectra of the N1a/M1b duplex
(solid line), single-stranded N1a (dashed-dotted line), M1b (dotted line),
and a simple sum of their spectra (sum-spectrum, N1a+M1b, dashed
line) at 08C. Solution conditions were as follows: [ODN]=5 mm,
[NaCl]=100 mm, pH 5.0 (10 mm MES buffer).
Figure 9. Effect of the number of dye pairs on the UV/Vis spectra of
a) Hna/Mnb, and b) Rna/Mnb combinations. Solution conditions were as
follows: 08C, [ODN]=5 mm, [NaCl]=100 mm, pH 7.0 (10 mm phosphate
buffer).
the shoulder band of the longer wavelength. Consequently,
H3a/M3b exhibited almost a single band with a broad
shoulder. In addition, prominent hypsochromism was in-
duced as the size of the aggregates increased. In the case of
Rna/Mna, the single sharp band that appeared at 483 nm in
R1a/M1b grew as the number of dyes incorporated per
strand increased (n=1–3) and was accompanied by a dis-
tinct hypsochromic shift, as depicted in Figure 9b. The spec-
troscopic behavior of these ODNs with higher numbers of
dyes incorporated was similar to that of homo H-aggregates,
which exhibit hypsochromic shifts and sharp bands.[16] N/M
pairs with a Dlmax of 68 nm also showed a similar spectro-
scopic behavior to R/M pairs with an increase in the cluster
size (see Figure 1 in Supporting Information).
all the combinations examined in this study. Hence, the
sum-spectrum of these two single strands gave not two, but
one broad single band, due to the proximity of the absorp-
tion maxima. When these two conjugates were hybridized,
however, a single sharp absorption band appeared at
483 nm, with a broad, weak shoulder at 550–650 nm, which
was entirely different from the pattern of the sum-spectrum,
as shown in Figure 8a. Similarly, N1a/M1b, which we previ-
ously demonstrated as an alternating heterocluster,[9] also
showed a single absorption band at 496 nm with a broad
weak shoulder (Figure 8b).
The effect of cluster size on circular dichroism (CD) spec-
tra is shown in Figure 10. Although heteroclusters were
stably formed below Tm for both Hna/Mnb and Rna/Mnb,
the induced CD (ICD) at the p–p* transition region was not
so strong. A small ICD is interpreted as an unwound struc-
ture of a stacked dye assembly on d-threoninol. In a Methyl
Red homocluster, this would result in a ladder-like structure
due to its flexibility, as previously demonstrated.[9b]
Effect of aggregate size on spectroscopic behavior of alter-
nating heteroclusters: We next examined the effect of alter-
nating heteroclustering of Hna/Mnb and Rna/Mnb, both of
which exhibited a large spectral change upon dimerization.
As shown in Figure 9a, an increase in dye number in Hna/
Mnb (n=1–3) increased the absorbance of the band of
shorter wavelength, whereas little increase was observed in
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H. Asanuma et al.
Figure 11. Illustration of the stacking manner of the dyes from each
strand. The number of atoms from the dye to the rigid five-membered
ring is five between the crotches of ribose and threoninol at the 3’-side,
whereas it is four at the 5’-side. The number labeled in the figure indi-
cates the atoms between the crotches of ribose and threoninol.
which facilitates the intercalation of the dye from the coun-
terstrand. Computer modeling also supported the 5’-side M/
Z orientation of the dye in the ODN dimer. In this model,
both dyes from each strand fitted well into the space at the
5’-side of each other and were stacked in an antiparallel
manner. Furthermore, stacked dyes of the unwound struc-
ture, shown in Figure 4b, are also consistent with the small
ICDs measured (Figure 10). As reported previously, dyes
conjugated to threoninols do not need winding in order to
form a firmly stacked structure, and the flexibility of this
structure allows the formation of a ladder-like structure.[9b]
It is also expected that a heterocluster of two different dyes,
such as Z3a/M3b, H3a/M3b, R3a/M3b, or N3a/M3b, would
be alternately aligned in the same manner as the 5’-side M/
Z orientation (Figure 2 in Supporting Information).
Figure 10. Effect of the number of dye pairs on the CD spectra of
a) Hna/Mnb, and b) Rna/Mnb combinations. Solution conditions were as
follows: 08C, [ODN]=5 mm, [NaCl]=100 mm, pH 7.0 (10 mm phosphate
buffer).
Discussion
Stacked structure of the heteroclusters: Our heterodimer
design is based on the pseudo “base-pairing” of threoninol
nucleotides incorporated at the center of an ODN. In con-
trast with natural A–T and/or G–C base-pairs, however,
paired threoninol nucleotides stack with each other through
hydrophobic stacking interactions. Therefore there are two
possible stacked structures in which dyes are located either
adjacent to the 5’-side (M/Z) or the 3’-side (Z/M) of the nat-
ural nucleobase (see Scheme 1). NMR analyses of an NMR-
Ma/NMR-Zb duplex clearly revealed that the dyes were lo-
cated adjacent to the 5’-side (M/Z), and other minor struc-
tures were rarely observed. This stacked structure can be ex-
plained by the space between the threoninol and natural nu-
cleotide, in which a dye at the counterstrand is inserted. As
shown in Figure 11, the number of atoms from the dye to a
rigid five-membered ring is five between the crotches of
ribose and threoninol at the 3’-side, whereas it is four at the
5’-side. Thus, the 3’-side is one carbon longer than the 5’-
side, due to the 5’-carbon of the natural ribose scaffold,
Comparison of the spectroscopic behavior of the heterodi-
mer with the molecular exciton model: As shown in Fig-
ures 6 to 8, all four heterodimers exhibited UV/Vis spectra
that were distinctly different from those calculated from the
sum-spectrum, and this difference depended on the Dlmax of
the combined dyes. Next is an explanation of these spectro-
scopic behaviors by the molecular exciton theory.[1,4a,5b,6,7]
Spectral behavior predicted from the molecular exciton
theory: Figure 12 shows a schematic illustration of the
energy diagram of two laterally stacked dyes of different
lmax. When monomer A of a higher transition energy is exci-
tonically coupled with a dye of lower transition energy, the
excited state splits into two energy levels.[5b,7,17] The higher
energy level corresponds to the in-phase transition in which
the transition dipole moments of the monomers A and B
have the same direction. In this case, the energy level be-
comes higher than that of monomer A due to the repulsive
interaction between the two transition dipoles. The absorp-
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Coherent Heteroclustering of Dyes
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However, the spectral shift corresponding to the in-phase
transition that we observed did not fit with the theory. Ac-
cording to the theory, an in-phase transition (higher transi-
tion energy) induces a hypsochromic shift with respect to
the transition of monomer A, whereas an out-of-phase tran-
sition (lower transition energy) induces a bathochromic shift
with respect to the transition of monomer B. Our experi-
mental results revealed that the out-of-phase transition dis-
played a bathochromic shift (Z1a/M1b and H1a/M1b, Fig-
ures 6 and 7), in accordance with the theory. However, the
in-phase transition also showed a bathochromic shift, which
is contrary to the theory. Other combinations that were ex-
amined, such as R1a/H1b and Z1a/H1b, also exhibited bath-
ochromicity of the in-phase transition (Figure 3 in Support-
ing Information). In order to rule out the possibility that
this bathochromicity was induced simply by intercalation
with the base-pairs of the ODN,[19] we analyzed the hybridi-
zation of H1a with a natural C strand, which does not con-
tain any threoninol nucleotides. Our previous NMR investi-
gations demonstrated that such a sequence design (H1a/C
duplex) facilitates intercalation of the dye tethered to threo-
ninol between the base pairs.[20] As shown in Figure 13,
Figure 12. Schematic energy diagram of the heterodimer depicted on the
basis of the exciton model. Each arrow, outlined by an ellipse or an
oblong, designates the transition dipole moment.
tion coefficient of the in-phase transition is expected to in-
crease due to the sum of the transitions A and B. In con-
trast, the energy level of the out-of-phase transition be-
comes lower than that of monomer B, due to the attractive
interaction. However, the absorption coefficient of this tran-
sition decreases because the transition moment is partially
cancelled due to its antiparallel orientation. The above exci-
tonic interaction (namely, coherency) should be enhanced
when the gap between the transition energies of monomers
A and B, Dlmax, decreases. In the case where the transition
moment of A is the same as that of B (i.e., A and B are the
same dye), an out-of-phase transition is forbidden due to
the complete cancellation of transition moment, whereas the
in-phase transition is maximized.[18]
Comparison with experimental results: The experimental re-
sults almost completely coincide with the above predictions
based on the molecular exciton theory, with the exception of
the results obtained for the spectral shift. In the case of Z1a/
M1b, the band of the shorter wavelength (higher transition
energy), derived from the azobenzene transition, displayed
hyperchromism, whereas that of the longer wavelength
(lower transition energy), derived from Methyl Red, dis-
played hypochromism. Both hyper- and hypochromism were
enhanced in the H1a and M1b combination, because the
Dlmax of this combination was smaller than that between
Z1a and M1b. These absorbance changes qualitatively coin-
cided with the prediction based on the molecular exciton
theory. In the case of R1a/M1b and N1a/M1b, which dis-
played a much smaller Dlmax, the out-of-phase transition
became much weaker and hence a single sharp band, corre-
sponding to an enhanced in-phase transition, appeared. The
broad, weak shoulder observed at the longer wavelength
may correspond to a weakened, out-of-phase transition.
Thus, the molecular exciton theory of heterodimer forma-
tion was qualitatively validated from the viewpoint of transi-
tion intensity.
Figure 13. UV/Vis spectra of the H1a/M1b duplex (solid line), the H1a/C
duplex (dashed line) and the single-stranded H1a (dotted line) at 08C.
Solution conditions were as follows: [ODN]=5 mm, [NaCl]=100 mm,
pH 7.0 (10 mm phosphate buffer).
single-stranded H1a, double-stranded H1a/C, and double-
stranded H1a/M1b had lmax at 398, 408, and 422 nm, respec-
tively. The bathochromicity induced by hybridization of H1a
with C was only 10 nm (compare the dotted and dashed
lines in Figure 13), indicating that the presence of the adja-
cent bases did not much affect the spectral shift. However,
H1a/M1b allowed a bathochromic shift as large as 24 nm
(compare the solid and dotted lines in Figure 13), which was
obviously larger than that induced by the H1a/C duplex.[21]
Hence, we conclude that the bathochromicity of the in-
phase transition cannot be explained by the conventional
molecular exciton theory.
Since the conventional molecular exciton theory is based
on the point-dipole approximation model, this may cause
problems in its application to a firmly stacked heterodimer,
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H. Asanuma et al.
because stacked dyes of 10 ꢂ size that are in close proximity
(3.5 ꢂ) cannot be approximated as a “point”.[1] A further
improved model (such as the extended dipole approxima-
tion[1] and/or molecular transition density[22]) will be needed
to explain the bathochromic shift of heterodimerization. At
present, we think that this discord may be due to the orbital
overlap between two dyes firmly stacked close to each other
in a duplex. Currently, quantum calculation (ab initio calcu-
lation) of the heterodimer is being applied to explain the
bathochromic shift.
tal results with theoretical predictions based on ab initio cal-
culations.
Our results partly explain the spectroscopic behavior of
intercalation. In general, dyes show batho- and hypochro-
micity when they are intercalated between the base pairs of
a duplex. According to Sarkar et al., this bathochromic shift
is due to the change in local polarity around the chromo-
phore.[19] However, we demonstrate here that exciton cou-
pling of a natural nucleobase (lmax ꢀ260 nm) with an inter-
calated dye (lmax should be longer than 260 nm) also partly
contributes to the weak but distinct bathochromic shift and
hypochromicity. Furthermore, the design of a fluorophore
and a quencher pair, tethered to d-threoninols, with a small-
er Dlmax should result in a highly sensitive molecular beacon
based on a firmly stacked fluorophore–quencher hetero-
Heteroclustering of the dyes: As shown in Figure 9, an in-
crease in the dye number resulted in intensification of the
in-phase transition together with a hypsochromic shift,
whereas the corresponding out-of-phase transition did not.
This spectroscopic behavior is very similar to that of homo
H-aggregates in which identical dyes are axially stacked. Al-
though the hypsochromic shift was smaller than that seen
for homo H-aggregates, these results clearly demonstrate
that strong coherent coupling occurred even among the het-
eroclusters. Even H3a/M3b, which had a Dlmax of 82 nm, ex-
hibited almost a single band due to the strong coherent cou-
pling within the heterocluster. We can predict from this
result that one- or two-dimensional clusters, composed of
two different dyes with different absorption maxima, should
exhibit essentially a single band when the dyes are assem-
bled in an orderly fashion, and should show strong coheren-
cy (exciton coupling).[4]
AHCTUNGTREGdNNUN imer with greater coherency. The molecular design of a
new heterodimer of a fluorophore–quencher pair as a com-
ponent of a molecular beacon is currently underway.
Experimental Section
Materials: All the conventional phosphoramidite monomers, CPG col-
umns, reagents for DNA synthesis, and Poly-Pak II cartridges were pur-
chased from Glen Research. Other reagents for the synthesis of the phos-
phoramidite monomer were purchased from Tokyo Kasei Co. and Sigma-
Aldrich.
Synthesis of DNA modified with H, M, N, R, or Z: All the modified
DNAs were synthesized by using an automated DNA synthesizer (ABI-
3400 DNA synthesizer, Applied Biosystems) with conventional and dye-
carrying phosphoramidite monomers. Azobenzene, Methyl Red, and
Naphthyl Red phosphoramidite monomers were synthesized according to
a previous report.[9b,23] The compounds 4’-methylthioazobenzene and 4’-
dimethylamino-2-nitroazobenzene, synthesized according to the litera-
ture,[24, 25] were converted to phosphoramidite monomers as described in
Scheme 1 of the Supporting Information. The coupling efficiency of the
monomers with modified residues was as high as that of the conventional
monomers, as judged from the intensity of the color of the released trityl
cation. After the recommended workup, the oligomers were purified by
reverse-phase HPLC and characterized by MALDI-TOFMS (Autoflex II,
Bruker Daltonics).
Conclusions
The NMR study revealed that different dyes incorporated at
the center of an ODN through tethering to d-threoninols
were stacked antiparallel to each other, and were located
adjacent to the 5’-side of a natural nucleobase.
Heterodimerization induced hyperchromism of the band
of the shorter wavelength (in-phase transition), but hypo-
chromism of the band of the longer wavelength, and these
absorbance changes were enhanced when the Dlmax of the
two dyes was decreased. Furthermore, both bands exhibited
a distinct bathochromic shift. These spectroscopic behaviors
were consistent with the shifts predicted from the molecular
exciton theory, except for the bathochromic shift of the in-
phase transition. This discordance might be due to limita-
tions of the point dipole approximation of the conventional
molecular exciton theory.
An increase in dye number dramatically intensified the
in-phase transition, which resulted in the spectrum of the
heterocluster being almost a single absorption band even
though the two dyes had different absorption maxima.
Thus, we have presented, for the first time, a qualitative
comparison of the actual spectroscopic behavior of hetero-
dimers with the spectroscopic behavior predicted for such
heterodimers by the molecular exciton theory, by systemati-
cally changing the Dlmax of the two dyes. We are now con-
ducting a quantitative comparison of the present experimen-
The MALDI-TOFMS data, observed (found) versus calculated (calcd.),
for the monomers were: Z1a: calcd for [Z1a+H]+: 4020; found: 4020;
Z2a: calcd for [Z2a+H]+: 4395; found: 4395; Z3a: calcd for [Z3a+H]+:
4770; found: 4770; H1a: calcd for [H1a+H]+: 4066; found: 4067; H2a:
calcd for [H2a+H]+: 4487; found: 4488; H3a: calcd for [H3a+H]+:
4908; found: 4909; R1a: calcd for [R1a+H]+: 4108; found: 4108; R2a:
calcd for [R2a+H]+: 4571; found: 4572; R3a: calcd for [R3a+H]+:
5034; found: 5035; N1a: calcd for [N1a+H]+: 4113; found: 4113; N2a:
calcd for [N2a+H]+: 4581; found: 4581; N3a: calcd for [N3a+H]+:
5049; found: 5050; M1b: calcd for [M1b+H]+: 4063; found: 4065; M2b:
calcd for [M2b+H]+: 4481; found: 4481; M3b: calcd for [M3b+H]+:
4899; found: 4899; NMR-Ma: calcd for [MMR-Ma+H]+: 2211; found:
2211; NMR-Zb: calcd for [MMR-Zb+H]+: 2167; found: 2168.
Spectroscopic measurements: The UV/Vis and CD spectra were mea-
sured on a JASCO model V-550 spectrophotometer and a JASCO model
J-820 spectropolarimeter, respectively, with a 10 mm quartz cell. Both
models were equipped with programmable temperature controllers. The
conditions of the sample solutions were as follows (unless otherwise
noted): [NaCl]=100 mm, pH 7.0 (10 mm phosphate buffer), [DNA]=
5 mm. For measurements at pH 5.0, 10 mm MES buffer was used. All sam-
ples of DNA–dye conjugates were heated at 808C for 5 min in the dark,
to thermally isomerize the cis form, which might be photoisomerized by
the ambient light, to trans form before spectroscopic measurement.[26]
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Coherent Heteroclustering of Dyes
FULL PAPER
Measurement of melting temperature: The melting curve of duplex DNA
was obtained by measurement of the change in absorbance at 260 nm
versus temperature (unless otherwise noted), using a spectrophotometer
as described above. The melting temperature (Tm) was determined from
the maximum of the first derivative of the melting curve. Both the heat-
ing and the cooling curves were measured, and the obtained Tm values
agreed to within 2.08C. The temperature ramp was 1.08CminÀ1. The con-
ditions of the sample solutions were the same as those described for the
above spectroscopic measurements.
d) A. Yamaguchi, N. Kometani, Y. Yonezawa, Thin Solid Films
2006, 513, 125–135; e) H. Bittermann, D. Siegemund, V. L. Malinov-
skii, R. Hꢃner, J. Am. Chem. Soc. 2008, 130, 15285–15287; f) I. V.
Astakhova, V. A. Kurshun, J. Wengel, Chem. Eur. J. 2008, 14,
11010–11026; g) Y. N. Teo, J. N. Wilson, E. T. Kool, J. Am. Chem.
Soc. 2009, 131, 3923–3933.
[6] V. Czikkely, G. Dreizler, H. D. Fçrsterling, H. Kuhn, J. Sondermann,
P. Tillmann, J. Z. Wiegand, Naturforsch, 1969, 24a, 1821–1830.
[7] a) S. Bernacchi, Y. Mꢄly, Nucleic Acids Res. 2001, 29, e62; b) S. Ber-
nacchi, E. Piꢄmont, N. Potier, A. V. Dorsselaer, Y. Mꢄly, Biophys. J.
2003, 84, 643–654.
[8] a) S. Tyagi, F. R. Kramer, Nat. Biotechnol. 1996, 14, 303–308;
b) M. K. Johansson, H. Fidder, D. Dick, R. M. Cook, J. Am. Chem.
Soc. 2002, 124, 6950–6956; c) E. Socher, L. Bethge, A. Knoll, N.
Jungnick, A. Herrmann, O. Seitz, Angew. Chem. 2008, 120, 9697–
9701; Angew. Chem. Int. Ed. 2008, 47, 9555–9559.
NMR measurements: NMR samples were prepared by dissolving three-
times-lyophilized DNA (modified and complementary DNA) in a H2O/
D2O 9:1 containing 20 mm sodium phosphate (pH 7.0), to give a duplex
concentration of 1.0 mm. NaCl was added to give a final sodium concen-
tration of 200 mm. NMR spectra were measured with a Varian INOVA
spectrometer (700 MHz) equipped for triple resonance at a probe tem-
perature of 278 K. Resonances were assigned by standard methods using
a combination of 1D, TOCSY (60 ms of mixing time), DQF-COSY, and
NOESY (150 ms of mixing time) experiments. All spectra in the H2O/
D2O 9:1 were recorded using the 3–9–19 WATERGATE pulse sequence
for water suppression.
[9] a) H. Kashida, H. Asanuma, M. Komiyama, Angew. Chem. 2004,
116, 6684–6687; Angew. Chem. Int. Ed. 2004, 43, 6522–6525; b) H.
Kashida, T. Fujii, H. Asanuma, Org. Biomol. Chem. 2008, 6, 2892–
2899.
Computer modeling: Molecular modeling by conformational energy min-
imization was performed with the InsightII/discover3 software (Molecu-
lar Simulation, Inc.) on a Silicon Graphics O2+ workstation with the op-
erating system IRIX64 Release 6.5, and the AMBER force field was
used for the calculations. The results of the NMR analyses served as a
starting point for the modeling. For the analysis, a dimer of Methyl Red
and azobenzene was prepared by positioning the two dye molecules in a
cofacial orientation and replacing two native base pairs.
[10] There are two possible structures for the heterodimer, as shown in
Scheme 1.
[11] a) C. Brotschi, C. J. Leumann, Angew. Chem. 2003, 115, 1694–1697;
Angew. Chem. Int. Ed. 2003, 42, 1655–1658; b) C. Brotschi, G.
Mathis, C. J. Leumann, Chem. Eur. J. 2005, 11, 1911–1923; c) Z.
Johar, A. Zahn, C. J. Leumann, B. Jaun, Chem. Eur. J. 2008, 14,
1080–1086; d) A. Zahn, C. J. Leumann, Chem. Eur. J. 2008, 14,
1087–1094.
[12] The melting temperature was measured in the presence of 100 mm
NaCl at pH 7.0 (10 mm phosphate buffer). The concentration of
NMR-Ma/NMR-Zb was 5 mm. The melting temperature of the
duplex under NMR measurement conditions should be much higher
because the concentrations of both NaCl and modified ODNs are
higher: a) R. Owczarzy, Y. You, B. G. Moreira, J. A. Manthey, L.
Huang, M. A. Behlke, J. A. Walder, Biochemistry 2004, 43, 3537–
3554; b) H. Long, A. Kudlay, G. C. Schatz, J. Phys. Chem. B 2006,
110, 2918–2926.
Acknowledgements
This work was supported by the Core Research for Evolution Science
and Technology (CREST) and Japan Science and Technology Agency
(JST). Partial support was provided by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science and
Technology, Japan and also the Mitsubishi Foundation (for H.A.). We
thank Dr. Kyoichi Sawabe (Department of Molecular Design and Engi-
neering, Nagoya University) for his helpful discussions on the molecular
exciton theory.
[13] M. Liu, X. Mao, C. Ye, J. K. Nicholson, J. C. Lindon, J. Magn.
Reson. 1998, 132, 125–129.
[14] a) E. Sawicki, J. Org. Chem. 1957, 22, 915–919; b) R. K. Burkhard,
R. D. Bauer, D. H. Klaassen, Biochemistry 1962, 1, 819–827; c) T.
Sueyoshi, N. Nishmura, S. Yamamono, S. Hasegawa, Chem. Lett.
1974, 1131–1134; d) P. C. Chen, Y. C. Chieh, J. C. Wu, J. Mol. Struct.
2005, 733-779, 183–189.
[15] Since the absorption spectra also slightly depended on the tempera-
ture being higher than the Tm at which the duplex was dissociated
into two single-strands, chromophore motion also affected the
ground-state spectra slightly.
[1] T. Kobayashi, J-aggregates, World Scientific, Singapore, 1996, pp. 1–
40.
[2] a) W. Lin, W. Lin, G. K. Wong, T. J. Marks, J. Am. Chem. Soc. 1996,
118, 8034–8042; b) O. R. Evans, W. Lin, Acc. Chem. Res. 2002, 35,
511–522; c) E. L. Botvinick, J. V. Shah, Methods Cell Biol. 2007, 82,
81–109.
[16] A. H. Herz, Photogr. Sci. Eng. 1974, 18, 323.
[17] N. Berova, K. Nakanishi, Circular Dichroism. Principles and Appli-
cations, (Eds: N. Berova, K. Nakanishi, R. W. Woody), Wiley-VCH,
New York, 2000, pp. 340–344.
[18] This is what we call the H-band, which displays distinct hypsochro-
micity.
[3] a) S. Bahatyrova, R. N. Freses, C. A. Siebert, J. D. Olsen, K.
Van Der Werf, R. Van Grondelle, R. A. Niederman, P. A. Bullough,
C. Otto, C. N. Hunter, Nature 2004, 430, 1058–1062; b) T. S. Bala-
ban, N. Berova, C. M. Drain, R. Hauschild, X. Huang, H. Kalt, S.
Lebedkin, J.-M. Lehn, F. Nifaitis, G. Pescitelli, V. I. Prokhorenko, G.
Riedel, G. Smeureanu, J. Zeller, Chem. Eur. J. 2007, 13, 8411–8427;
c) J. D. Olsen, J. D. Tucker, J. A. Timney, P. Qian, C. Vassilev, C. N.
Hunter, J. Biol. Chem. 2008, 283, 30772–30779.
[4] a) M. Kasha, Radiat. Res. 1963, 20, 55–71; b) J. L. Seifert, R. E.
Connor, S. A. Kushon, B. A. Armitage, J. Am. Chem. Soc. 1999, 121,
2987–2995; c) M. Wang, G. L. Silva, B. A. Armitage, J. Am. Chem.
Soc. 2000, 122, 9977–9986; d) N. R. Conley, A. K. Pomerantz, H.
Wang, R. J. Twieg, W. E. Moerner, J. Phys. Chem. B 2007, 111,
7929–7931; e) S. Ikeda, A. Okamoto, Chem. Asian J. 2008, 3, 958–
968.
[19] D. Sarkar, P. Das, S. Basak, N. Chattopadhyay, J. Phys. Chem. B
2008, 112, 9243–9249.
[20] X. G. Liang, H. Asanuma, H. Kashida, A. Takasu, T. Sakamoto, G.
Kawai, M. Komiyama, J. Am. Chem. Soc. 2003, 125, 16408–16415.
[21] Since exciton splitting depended on the Dlmax between these dyes
and DNA, the effect of natural base on the chromophore–chromo-
phore splitting might be small. Furthermore, since the dye has al-
ready been located next to the natural base in the single strand, the
adjacent natural base did not greatly affect the spectral change (ex-
citon coupling) of incorporated dyes by hybridization.
[22] K. Norland, A. Ames, T. Taylor, Photogr. Sci. Eng. 1970, 14, 295–
307.
[23] a) H. Asanuma, H. Kashida, X. G. Liang, M. Komiyama, Chem.
Commun. 2003, 1536–1537; b) H. Asanuma, K. Shirasuka, T. Takar-
[5] a) K. Murata, S. Kuroda, K. Saito, Thin Solid Films 1997, 295, 73–
76; b) B. Z. Packard, D. D. Toptygin, A. Koriya, L. Brand, J. Phys.
Chem. B 1998, 102, 752–758; c) R. A. Garoff, E. A. , Litzinger R. E.
Connor, I. Fishman, B. A. Armitage, Langmuir 2002, 18, 6330–6337;
Chem. Eur. J. 2009, 15, 10092 – 10102
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10101

H. Asanuma et al.
ada, H. Kashida, M. Komiyama, J. Am. Chem. Soc. 2003, 125, 2217–
2223.
[24] a) W. H. Nutting, R. A. Jewell, H. Rapoport, J. Org. Chem. 1970, 35,
505–508; b) M. A. Rahim, P. N. P. Rao, E. E. Knaus, J. Heterocycl.
Chem. 2002, 39, 1309–1314.
[25] K. A. Bello, Dyes Pigm. 1989, 11, 65–76.
[26] H. Nishioka, X. Liang, H. Kashida, H. Asanuma, Chem. Commun.
2007, 4354–4356.
Received: April 10, 2009
Published online: August 31, 2009
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Chem. Eur. J. 2009, 15, 10092 – 10102