Artificial DNAs based on alkynyl C-nucleosides as a superior scaffold for homo- and heteroexcimer emissions

Article abstract of DOI:10.1002/chem.200700559

DNA-like fluorescent oligomers composed of alkynyl β-D-ribofuranosides bearing pyrene, perylene, and anthracene as a fluorophore were synthesized by solid-phase DNA synthesis. The fluorescent oligomers possess the defined number and order of the fluoropho

Full text of DOI:10.1002/chem.200700559

DOI: 10.1002/chem.200700559
Artificial DNAs Based on Alkynyl C-Nucleosides as a Superior Scaffold for
Homo-and Heteroexcimer Emissions
Junya Chiba,[a] Sakiko Takeshima,[a] Kikyo Mishima,[a] Hajime Maeda,[b]
Yasuaki Nanai,[b] Kazuhiko Mizuno,[b] and Masahiko Inouye*[a]
In memory of Professor Yoshihiko Ito
Abstract: DNA-like fluorescent oligo-
mers composed of alkynyl b-d-ribofur-
anosides bearing pyrene, perylene, and
anthracene as a fluorophore were syn-
thesized by solid-phase DNA synthesis.
The fluorescent oligomers possess the
defined number and order of the fluo-
rophores. In these oligomers, the adja-
cent fluorophores efficiently interact
with each other by hydrophobic inter-
actions in their electronic ground states
in a face-to-face fashion. The predomi-
nant excimer emissions were observed
from not only the homooligomers
(pyrene–pyrene and perylene–perylene
systems) but also the heterooligomers
(pyrene–perylene, pyrene–anthracene,
and perylene–anthracene systems) in
aqueous media.
Keywords: artificial DNA · exci-
mers · fluorescence · nucleosides ·
photochemistry
Introduction
necting two or more fluorescent moieties.[2–4] The examples
include simple molecules possessing multiple fluorophores
with the aid of appropriate linkers,[2] fluorophore-attached
polymers as a pendant style,[3] and macrocycles such as cy-
clophanes containing fluorophores as a core.[4] Although
these approaches are valid for obtaining photophysical data
of the excimers, their syntheses are time-consuming and
lack the generality for introducing various fluorophores into
the skeletons at will.
The excimer formation has also been utilized for solving
critical problems of biological events. The conventional illus-
trations are concerned with protein–substrate and protein–
protein interactions,[5] hybridizations and conformational
changes of DNAs and RNAs,[6] and real-time dynamics of
cell membranes.[7] Recently, excimer-forming molecules
have been employed as a fluorescent tag for labeling biomo-
lecules.[6a,c,d,8] In the labeling experiments, these tags, strong-
ly lipophilic aromatic hydrocarbons, must be applied under
biological conditions, such as in aqueous buffer media. This
sometimes causes technical problems, especially in the case
of highly condensed tags with wider polyaromatic hydrocar-
bons.
Emissions from homo- and heteroexcimers of polycyclic aro-
matic hydrocarbons have long been attractive in terms of
their inherent and novel luminescent features. Inspiring re-
searchers in its own right, the photophysics of the emission
has been investigated in detail to elucidate the radiation and
nonradiation processes from the excimers.[1] Generally, exci-
mer emission is observed only in highly concentrated solu-
tions because the formation of an excimer requires the en-
counter of one electronically excited fluorophore with an-
other, usually that of the ground state, in an extremely short
time. To make fluorophores interact with each other in low
concentrations suitable for quantitative experiments, intra-
molecular strategies have been adopted by covalently con-
[a] Dr. J. Chiba, S. Takeshima, K. Mishima, Prof. Dr. M. Inouye
Graduate School of Pharmaceutical Sciences
University of Toyama
Sugitani 2630, Toyama 930–0194 (Japan)
Fax : (+81)76-434-5049
E-mail: inouye@pha.u-toyama.ac.jp
[b] Dr. H. Maeda, Y. Nanai, Prof. Dr. K. Mizuno
Department of Applied Chemistry
Kool and co-workers have reported their original strategy
for creating fluorescent tags by replacing DNA bases of oli-
gonucleotides with various fluorescence molecules.[9] In this
combinatorial manner for the DNA-like fluorescent oligo-
mers, a DNA synthesizer allows easy preparations of the
oligomers with the defined number and order of the fluoro-
Graduate School of Engineering
Osaka Prefecture University
Gakuen-cho 1–1, Sakai, Osaka 599–8531 (Japan)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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FULL PAPER
phores. Indeed, they synthesized a tetrameric fluorophore li-
brary composed of more than 14,000 members on beads,
and observed the multicolor emissions at once in aqueous
media. For synthetic convenience of the library, they used a
mixture of the a and b anomers of the fluorescent nucleo-
side analogues as a monomer, in which non-natural a anom-
ers are major components. Because of this structural diversi-
ty, however, the detailed photophysical properties remain to
be investigated from the viewpoint of the specific radiation
from the multiply interacting individual tetrameric fluoro-
phore.[9c]
fined number and order of the fluorophores but also the
same b configurations at all of their anomeric positions.
Water solubility of the oligomers is provided from the nega-
tive charges of the phosphodiester backbone. Even in dilut-
ed aqueous solutions, the adjacent hydrophobic fluoro-
phores in the oligomers would interact with each other ade-
quately to adopt suitable conformations in their electronic
ground states. This motion may be assisted by puckering
flexibilities of the deoxyribose and by an additional hydro-
phobicity of the acetylenic bond in the alkynyl C-nucleo-
sides.
To clarify each emissive characteristic from such fluores-
cent oligomers, chemical structures in the oligomers must be
well defined. Therefore, all of the anomeric configurations
in the oligomer should be the same, hopefully being b
anomers of natural nucleosides, as the conformation of the
oligomer can be easily speculated. We previously reported
the stereoselective synthesis of alkynyl C-2-deoxy-b-d-ribo-
furanosides as a versatile building block for many different
kinds of C-nucleotides.[10] By means of the synthetic useful-
ness of the acetylenic function, a variety of fluorophores
such as pyrene, perylene, and anthracene will be attached to
the deoxyribose through an acetylenic bond in high yields
with the b configuration. Here we report the synthesis and
detailed photophysical data of the fluorescent oligomers
composed of the alkynyl b-d-ribofuranosides (Scheme 1).
Synthesis and abbreviation of oligomers: DMTr-protected
fluorescent nucleoside analogues 4 were prepared by Sono-
gashira coupling of bromohydrocarbons with 3 derivatized
from the published 1 (Scheme 2).[10] The analogues 4 were
Scheme 2. a) 4,4’-Dimethoxytrityl chloride, 4-(N,N-dimethylamino)pyri-
dine, pyridine; b) nBu4NF, H2O, THF; c) 1-bromopyrene, [Pd
CuI, iPr2NH, THF; d) 3-bromoperylene, [Pd(PPh3)4], CuI, iPr2NH, THF;
e) 2-bromoanthracene, [Pd(PPh3)4], CuI, iPr2NH, THF; f) NC-
(CH2)2OPClN(iPr)2, iPr2NEt, CH2Cl2; g) p-toluenesulfonic acid monohy-
(PPh3)4],
AHCTREUNG
AHCTREUNG
A
ACHTREUNG
drate, CH2Cl2; h) phosphorus oxychloride, dioxane, pyridine.
Scheme 1. Chemical structure of DNA-like fluorescent oligomers based
on alkynyl C-nucleosides.
converted into the corresponding phosphoramidites 5 and
also into 5’-monophosphate derivatives 7 as reference water-
soluble monomers (see Supporting Information). The phos-
phoramidites 5 were then subjected to automated DNA syn-
thesis to give fluorescent oligomers on a solid support. After
having been released from the support, the oligomers were
purified by reverse-phase HPLC and characterized by
MALDI-TOF mass spectrometry (see Experimental Sec-
tion). In the sequence of the oligomers, the pseudonucleo-
side residues containing pyrene, perylene, and anthracene
are abbreviated as Py, Pe, and An, respectively. An abasic
nucleoside residue is abbreviated as ab, and this was intro-
duced into both the ends of the sequence to improve the
water solubility of the oligomers. The oligomer referred to
as ab-Py-Pe-ab, for instance, possesses the abasic, pyrene-at-
tached, perylene-attached, and abasic nucleoside residues
from 5’ (the left end) to 3’ (the right end) of the sequence,
This novel DNA-like skeleton provides a superior scaffold
for investigating homo- and heteroexcimer formation and its
radiation in aqueous media, and the results presented here
can be expected to lead to fruitful applications in biological
sciences.
Results and Discussion
Structure of fluorescent oligomers: Pyrene, perylene, and
anthracene were chosen as representative fluorophores.
Each of the fluorophore-attached b-d-ribofuranosides is oli-
gomerized by solid-phase DNA synthesis. Therefore, the
DNA-like fluorescent oligomers possess not only the de-
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8125

M. Inouye et al.
and the “hyphen” indicates a phosphodiester bond. Table 1
shows the oligomers examined in this study.
the absorption spectra of the pyrene-linked monomer 7(Py)
and homooligomers, ab-Py-Py-ab, ab-Py-Py-Py-ab, and ab-
Py-Py-Py-Py-ab. For an easy comparison of the spectroscop-
ic data, the concentration of each solution was normalized
against the chromophore concentration [Py]. The absorption
maximum of 7(Py) appeared at 360 nm, and its shape and
absorption coefficient (e) are very similar to those of the
simple monoalkynylpyrenes in our previous report.[12] How-
ever, as the number of the pyrene residues in the oligomers
increased, we observed significant hypochromism and small
red-shifts of the absorption bands with the broadening.
Peak-to-valley ratio (PA) was used to estimate the degree of
the p–p interaction between chromophores.[13] The value is
calculated by using the equation PA =Apeak/Avalley, in which
Apeak is for the absorption intensity of the band appearing at
the longest wavelength and Avalley for that of the valley be-
tween the longest wavelength band and the adjacent one.
The PA values of the oligomers are 1.13, 1.10, and 1.11 for
ab-Py-Py-ab, ab-Py-Py-Py-ab, and ab-Py-Py-Py-Py-ab, re-
spectively. The values are considerably smaller than PA =
2.07 of 7(Py), indicating strong face-to-face interactions for
the pyrene residues of the oligomers in their ground states.
The steady-state fluorescence spectra of the pyrene-linked
monomer and homooligomers were measured in degassed
water (Figure 1B). The monomer 7(Py) revealed a charac-
teristic monomeric fluorescence with emission maxima at
385 and 405 nm. The fluorescence quantum yield (Ff) of
7(Py) is 0.66, comparable to those of the simple alkynylpyr-
Table 1. Photophysical data for fluorophore-linked monomers and oligo-
mers.
Monomers
and
oligomers
Absorption[a]
Fluorescence
[b]
[a,c,d]
labs
e ”104
lem
[nm]
Ff[a,e]
ts[f]
[nm] [molÀ1 dm3 cmÀ1
]
[ns]
7(Py)
ab-Py-Py-ab
ab-Py-Py-Py-ab
360
365
364
5.41
3.62
4.99
5.80
2.70
3.23
0.47
0.80
2.48
1.83
1.95
2.80
385[c]
510[d]
521[d]
515[d]
468[c]
595[d]
398[c]
525[d]
522[d]
517[d]
500[d]
526[d]
0.66
35.1
29.3, 66.8
30.6, 75.1
28.0, 86.5
4.35
4.42, 14.2
6.06
6.81
ab-Py-Py-Py-Py-ab 365
7(Pe)
ab-Pe-Pe-ab
7(An)
ab-An-An-ab
ab-Py-Pe-ab
ab-Py-Pe-Py-ab
ab-Py-An-ab
ab-Pe-An-ab
454
462
387
391
464
469
365
461
0.66
0.37
7.88, 16.2
[g]
–
73.0, 104
18.1, 28.8
[a] [7(Py)]=[7(Pe)]=[7(An)]=[ab-Py-Pe-ab]=[ab-Py-Pe-Py-ab]=[ab-
Py-An-ab]=[ab-Pe-An-ab]=2.0”10À5 m, [ab-Py-Py-ab]=[ab-Pe-Pe-ab]=
[ab-An-An-ab]=1.0”10À5 m, [ab-Py-Py-Py-ab]=6.7”10À6 m, [ab-Py-Py-
Py-Py-ab]=5.0”10À6 m in H2O, degassed by bubbling of Ar. [b] labs is the
absorption band appearing at the longest wavelength. [c] lem is the fluo-
rescence band appearing at the shortest wavelength. [d] lem is the exci-
mer fluorescence band. [e] Fluorescence quantum yields of the mono-
mers in H2O, degassed by bubbling of Ar, measured at RT. Standards
used were 9,10-diphenylanthracene (for 7(Py) and 7(An)) and perylene
(for 7(Pe)), OD<0.02. Ff(9,10-diphenylanthracene)=0.95 in EtOH.[17]
Ff
(perylene)=0.92 in EtOH.[18] [f] Fluorescence lifetime in aerated H2O
G
(5.0”10À6 m). [g] Not determined.
Photophysical properties of
pyrene-linked homooligomers:
To elucidate the luminescent
property of each individual oli-
gomer, all photophysical meas-
urements must be carried out
below this concentration so that
the intermolecular association
of the oligomer is negligible.
Therefore, intermolecular asso-
ciation tendencies of the oligo-
mers were studied in advance
with ab-Py-Py-ab and ab-Pe-
Pe-ab. The absorption spectra
of these representative oligo-
mers were measured in water at
room temperature. The shapes
of the spectra are not sensitive
Figure 1. Absorption (A) and fluorescence (B) spectra of 7(Py) (black line), ab-Py-Py-ab (red line), ab-Py-Py-
Py-ab (green line), and ab-Py-Py-Py-Py-ab (violet line) in H2O at 258C (FI=fluorescence intensity). Concen-
trations: [7(Py)]=2.0”10À5 m, [ab-Py-Py-ab]=1.0”10À5 m, [ab-Py-Py-Py-ab]=6.7”10À6 m, and [ab-Py-Py-Py-
Py-ab]=5.0”10À6 m. Excitation wavelength is 350 nm. The inset expands the region of the excimer emission.
to their concentrations, and at ꢀ1.0”10À4 m, the absorbances
of these oligomers fit in proportion to the concentration
obeying Beerꢁs law. Thus, the contribution of the intermo-
lecular association of the oligomers can be ruled out below
that concentration. Taking this fact into account, the follow-
ing measurements were conducted.
First, a series of homooligomers with the pyrene residues
were chosen because pyrene is an well-known fluorophore
that readily exhibits excimer emission.[11] Figure 1A shows
enes.[12] The emission maxima depended on the concentra-
tion: at ꢁ1.0”10À4 m, a new emission (lmax =507 nm) that
was thought to be for an intermolecular excimer appeared
(Supporting Information Figure S1). On the other hand, the
oligomers (ab-Py-Py-ab, ab-Py-Py-Py-ab, and ab-Py-Py-Py-
Py-ab) exhibited broad and structureless emissions at
around 510 nm nearly without the monomer emissions. The
wavelengths of the emission maxima of the oligomers were
independent of their concentrations, so that the emissions
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Alkynyl C-Nucleosides
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must result from the intramolecular interactions between
the pyrene residues. The emissions should be radiated from
so-called “static excimers”, as in water the tethered pyrene
residues interact with each other in their ground states, as
mentioned above.[13] The excimer emissions of the oligomers
gradually decreased as the number of the pyrenes increased,
apparently being due to the self-quenching of the excimers.
Although in the trimer and tetramer, triply p, p, p stacking
or higher coordinations of the pyrene residues might be pos-
sible, an extra emission band from such higher coordinations
could not be found. Nevertheless, on the basis of these data,
our artificial DNA-like skeleton is demonstrated to be a su-
perior scaffold giving excimer emissions of tethered fluoro-
phores in water.
tailed to 800 nm. The emission should radiate from the in-
tramolecular perylene excimer by analogy with the pyrene-
type oligomers. Although perylene is known to show the ex-
cimer emission in its crystalline form,[15] little is known for
the emission in solutions, especially in diluted media.[9c,16] By
use of the present strategy, the excimer emission of ab-Pe-
Pe-ab could be detected at concentrations as low as approxi-
mately 1.0”10À7 m. These findings demonstrate the superior-
ity of our scaffold to realize excimer emissions of various
fluorophores.
Unfortunately, we observed only a weak excimer emission
for the anthracene dimer ab-An-An-ab in the fluorescence
spectrum (Figure 3). In ab-An-An-ab, the adjacent anthra-
cene residues overlapped each other insufficiently because
of a comparatively narrow plane of the anthracene ring (see
the following section on heterooligomers). This speculation
is suggested by the fact that the absorption spectrum of ab-
An-An-ab scarcely changed compared to that of the mono-
mer 7(An) with respect to the hypochromism, red-shifts,
and broadening of the bands within the wavelength region
of 300–400 nm.
Photophysical properties of perylene-and anthracene-linked
homooligomers: Next, perylene was introduced into the al-
kynyl C-nucleosides because excimer emissions of perylene
are rare in solutions. Figure 2A displays absorption spectra
of the perylene-linked monomer 7(Pe) and homodimer ab-
Pe-Pe-ab under the same conditions as described for the
pyrene-linked ones. The monomer 7(Pe) has an absorption
maximum at 454 nm and its ab-
sorption coefficient is e=2.70”
104 molÀ1 dm3 cmÀ1, so that it
exists as an intensely colored
orange–red solid. Substantial
hypochromism and small red-
shift of the longest wavelength
band were observed with the
broadening also in the case of
ab-Pe-Pe-ab. The PA values of
7(Pe) and ab-Pe-Pe-ab are 1.63
and 1.21, respectively, suggest-
ing that the perylene residues
in ab-Pe-Pe-ab interact with
each other in a manner similar
to those of the pyrene-linked
oligomers.
Figure 2. Absorption (A) and fluorescence (B) spectra of 7(Pe) (black line) and ab-Pe-Pe-ab (red line) in H2O
at 258C (FI=fluorescence intensity). Concentrations: [7(Pe)]=2.0”10À5 m, [ab-Pe-Pe-ab]=1.0”10À5 m. Excita-
tion wavelength is 430 nm.
During recording of the fluo-
rescence
measurements
of
7(Pe) and ab-Pe-Pe-ab, special
care was taken in the prepara-
tion of the sample solutions to
avoid them being exposed to
light. Thus, the spectra were re-
corded as soon as the sample
was available (Figure 2B).[14]
The monomer 7(Pe) emitted a
characteristic monomeric fluo-
rescence with emission maxima
at 468 and 495 nm, and its Ff
value is 0.66. On the other
hand, the dimer ab-Pe-Pe-ab
predominantly gave
and structureless emission band
a broad
Figure 3. Absorption (A) and fluorescence (B) spectra of 7(An) (black line) and ab-An-An-ab (red line) in
H2O at 258C (FI=fluorescence intensity). Concentrations: [7(An)]=2.0”10À5 m, [ab-An-An-ab]=1.0”10À5 m.
at around 600 nm, which even Excitation wavelength is 350 nm. The inset expands the region of the excimer emission.
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M. Inouye et al.
Photophysical properties of heterooligomers: Because the
homooligomers showed distinct excimer emissions, we then
examined heterooligomers consisting of different kinds of
polycyclic aromatic hydrocarbons. We chose ab-Py-Pe-ab,
ab-Py-An-ab, and ab-Pe-An-ab as sequences for this pur-
pose. The concentration of all heterooligomers is 2.0”
10À5 m, so that each of the chromophore residues Py, Pe, and
An exists at the same concentration. In the absorption spec-
trum of ab-Py-Pe-ab, substantial red-shifts were observed
within the wavelength region corresponding to the absorp-
tion bands for the perylene residue (400–500 nm). On the
other hand, the corresponding bands for the pyrene residue
(300–400 nm) shifted largely hypochromically (Figure 4A).
Such red-shifts and hypochromism were observed also in the
cases of ab-Py-An-ab and ab-Pe-An-ab, however, the exact
degree of hypochromism of these two spectra could hardly
be evaluated because of the overlap between the chromo-
phore residues (Figure S2). The chromophores in the hetero-
oligomers were found to intramolecularly interact with each
other, as in the cases of the homooligomers.
dues (Figure 4B, see also Figure S3). The emission maximum
is located between those of the pyrene excimer (510 nm,
Figure 1B) and the perylene excimer (595 nm, Figure 2B).
The emission spectra are almost the same when the oligo-
mer is excited at 350 and 430 nm, corresponding to the ab-
sorption regions of the pyrene and perylene chromophores,
respectively (Figure S3). Moreover, the excitation spectrum
monitored at 520 nm is structurally analogous to the absorp-
tion spectrum of ab-Py-Pe-ab (Figure S4A). On the basis of
these data, the emission proved to radiate from the hetero-
excimers consisting of the pyrene and perylene residues, and
not to be due to fluorescence resonance energy transfer
(FRET). Strikingly similar heteroexcimer emission was seen
in ab-Py-Pe-Py-ab, however, no characteristic emission from
triplex coordinations of the fluorophores was monitored in
this oligomer (Figure S5).
In contrast to ab-An-An-ab, the anthracene-linked heter-
ooligomers revealed fluorescent properties similar to those
for ab-Py-Pe-ab. Therefore, in the fluorescence spectra, ab-
Py-An-ab and ab-Pe-An-ab have broad peaks at around 500
and 525 nm, respectively (Figure 4C and D). These emis-
sions must result from the heteroexcimer of the pyrene or
perylene residue with anthracene, as evidenced by the simi-
larity between the excitation
The fluorescence spectrum of ab-Py-Pe-ab exhibited a
broad emission at around 522 nm, almost without both of
the monomer emissions from the pyrene and perylene resi-
and absorption spectra of these
oligomers (Figure S4B, C). In
these heterooligomers, the an-
thracene residue exists close to
the wider aromatic hydrocar-
bons of pyrene and perylene
rather than anthracene itself, as
in the case of ab-An-An-ab.
This situation is most likely to
be responsible for the effective
excimer formation in the an-
thracene-linked heterooligom-
ers. To the best of our knowl-
edge, these are first examples
for the predominant emission
from heteroexcimers of anthra-
cenes.
Fluorescence-lifetime measure-
ments: Fluorescence lifetimes
of the fluorophore-linked mon-
omers and oligomers were mea-
sured to reinforce the above
conclusions and are listed in
Table 1. Single exponential
decay was observed in the fluo-
rescence-lifetime measurement
of 7(Py), whereas fluorescence
of the three pyrene-linked olig-
omers decayed double-expo-
nentially. These two compo-
nents can be interpreted to cor-
respond to the monomer and
Figure 4. A and B: Absorption (A) and fluorescence (B) spectra of 7(Py) (lex =350 nm, black line), 7(Pe)
(lex =430 nm, violet line), and ab-Py-Pe-ab (lex =350 nm, red line) in H2O at 258C (FI=fluorescence intensi-
ty). C and D: Normalized fluorescence spectra of (C) 7(Py) (lex =350 nm, black line), 7(An) (lex =350 nm,
violet line) and ab-Py-An-ab (lex =350 nm, red line); (D) 7(Pe) (lex =430 nm, black line), 7(An) (lex =350 nm,
violet line), and ab-Pe-An-ab (lex =350 nm, red line) in H2O at 258C (NFI=normalized fluorescence intensi-
ty). Concentrations: [7(Py)]=[7(Pe)]=[7(An)]=[ab-Py-Pe-ab]=[ab-Py-An-ab]=[ab-Pe-An-ab]=2.0”10À5 m.
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Alkynyl C-Nucleosides
FULL PAPER
Spectroscopic measurements: Steady-state absorption and emission spec-
tra were recorded by using a JASCO V-560 UV/Vis spectrophotometer
and a JASCO FP-6500 spectrofluorometer, respectively. Fluorescence
lifetimes were measured by using a HORIBA NAES-550 nanosecond flu-
orometer equipped with a SSU-111A photomultiplier, SCU-121A optical
chamber, SGM-121A monochromator, and LPS-111 lamp power supply.
These measurements were carried out at 258C using a 1-cm pathlength
cell. Fluorescence quantum yields, Ff, of the monomers in H2O were de-
termined by using 9,10-diphenylanthracene[17] and perylene[18] as a stan-
dard with a known Ff in EtOH of 0.95 and 0.92, respectively. The fluo-
rescence quantum yields were calculated according to the following equa-
tion: Ff(spl) =Ff(std) ”[Astd/Aspl]”[Istd/Ispl]”[nstd/nspl]2. In this equation, Ff(spl)
and Ff(std) are the quantum yields of a sample and a standard, respective-
ly. Aspl, Ispl, and nspl are the optical density, the integrated emission inten-
sity at the excitation wavelength, and the value of the refractive index of
the sample, respectively. Astd, Istd, and nstd are those for the standard.
Fluorescence lifetimes were obtained by time correction, a single-photon
counting methodology, by means of a nanosecond fluorometer. Sample
solutions (5.0”10À6 m) were prepared in Milli-Q water under aerated con-
dition (not degassed). Excitation wavelengths were 275 nm (for 7(An)
and ab-An-An-ab), 350 nm (for 7(Py), ab-Py-Py-ab, ab-Py-Py-Py-ab, ab-
Py-Py-Py-Py-ab, ab-Py-Pe-ab, ab-Py-An-ab, ab-Pe-An-ab, and ab-Py-Pe-
Py-ab), and 430 nm (for 7(Pe) and ab-Pe-Pe-ab). Cut-off filters used
were UV35 (for 7(Py), ab-Py-Py-ab, ab-Py-Py-Py-ab, ab-Py-Py-Py-Py-
ab, 7(An), and ab-An-An-ab) and Y45 (for 7(Pe), ab-Pe-Pe-ab, ab-Py-
Pe-ab, ab-Py-An-ab, and ab-Pe-An-ab). All decay curves were calculated
by using single or double exponential on the basis of the equation I(t)=
excimer emissions for the shorter lifetime (minor compo-
nent) and the longer one (major component). Similar ten-
dency was observed in the perylene-linked monomer and
oligomer. However, in the case of ab-An-An-ab, the decay
corresponding to its excimer could hardly be detected be-
cause the excimer emission is very weak, as shown in Fig-
ure 3B. Upon applying the heterooligomers, we used a cut-
off filter (passing photons at >450 nm) to discard the pho-
tons emitted from the monomeric excited states in the oligo-
mers. Thus, both the components observed for the heteroo-
ligomers must be assigned to fluorescence lifetimes from the
heteroexcimers. This finding means that two or more confor-
mational isomers emitted individually in agreement with
their ground states deduced by the UV-visible spectra. Un-
fortunately, the emissive components in ab-Py-Pe-Py-ab
were too numerous to be analyzed.
Conclusion
Our artificial DNA skeleton based on alkynyl C-nucleosides
was found to be a superior scaffold for investigating homo-
and heteroexcimer formation in aqueous media. The fluo-
rescent oligomers made of the skeleton predominantly
showed excimer emissions not only from homoexcimers but
also from heteroexcimers. Because of the synthetic versatili-
ty of the alkynyl C-nucleosides, a large number of fluoro-
phores can be attached to the skeleton. Therefore, these flu-
orophore-linked oligomers may be used for labeling various
biomolecules, and such an approach is now underway.
ꢀAiexp
A
components, and Ai =fluorescence intensity of component i at t=0.
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Experimental Section
Synthesis of fluorescent oligomers: The fluorescent oligomers were syn-
thesized from 5 by using an Applied Biosystems 392 synthesizer using
standard b-cyanoethylphosphoramidite chemistry with the coupling reac-
tion time of 15 min. The solid support (Universal Support II), which
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(10”150 mm with an eluent of 5”10À3 m ammonium formate and the fol-
lowing CH3CN percentages of linear gradient (0–40 min) at a flow rate of
2.0 mLminÀ1: ab-Py-Py-ab (20–45%), ab-Py-Py-Py-ab (30–60%), ab-Py-
Py-Py-Py-ab (30–60%), ab-Pe-Pe-ab (30–50%), ab-An-An-ab (20–50%),
ab-Py-Pe-ab (20–50%), ab-Py-Pe-Py-ab (30–60%), ab-Py-An-ab (20–
40%), ab-Pe-An-ab (20–50%).
MALDI-TOF measurements: MALDI-TOF mass spectra were recorded
by using a Bruker-Daltonics-Autoflex mass spectrometer with 3-hydroxy-
picolinic acid as
a matrix. ab-Py-Py-ab: m/z: calcd for C56H54O18P3
[MH+]: 1107.25; found: 1107.27, ab-Py-Py-Py-ab: m/z: calcd for
C79H71O23P4 [MH+]: 1511.33; found: 1511.10, ab-Py-Py-Py-Py-ab: m/z:
calcd for C102H88O28P5 [MH+]: 1915.41; found: 1915.09, ab-Pe-Pe-ab: m/z:
calcd for C64H58O18P3 [MH+]: 1207.28; found: 1206.87, ab-An-An-ab:
m/z: calcd for C52H54O18P3 [MH+]: 1059.25; found: 1059.31, ab-Py-Pe-ab:
m/z: calcd for C60H56O18P3 [MH+]: 1157.27; found: 1156.87, ab-Py-Pe-Py-
ab: m/z: calcd for C83H73O23P4 [MH+]: 1561.35; found: 1561.02, ab-Py-
An-ab: m/z: calcd for C54H54O18P3 [MH+]: 1083.25; found: 1082.99, ab-
Pe-An-ab: m/z: calcd for C58H56O18P3 [MH+]: 1133.27; found: 1132.92.
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Received: April 10, 2007
Published online: July 3, 2007
8130
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Chem. Eur. J. 2007, 13, 8124 – 8130