Alkynylpyrenes as improved pyrene-based biomolecular probes with the advantages of high fluorescence quantum yields and long absorption/emission wavelengths

Article abstract of DOI:10.1002/chem.200500638

The photochemical properties of various alkynylpyrene derivatives have been investigated in detail with a view to developing a new class of pyrene-based biomolecular probes. The absorption maxima of the alkynylpyrenes were seen to be shifted to longer wav

Full text of DOI:10.1002/chem.200500638

DOI: 10.1002/chem.200500638
Alkynylpyrenes as Improved Pyrene-Based Biomolecular Probes with the
Advantages of High Fluorescence Quantum Yields and Long Absorption/
Emission Wavelengths
Hajime Maeda,[a] Tomohiro Maeda,[a] Kazuhiko Mizuno,*[a] Kazuhisa Fujimoto,*[b]
Hisao Shimizu,[b] and Masahiko Inouye[b, c]
Abstract: The photochemical proper-
ties of various alkynylpyrene deriva-
tives have been investigated in detail
with a view to developing a new class
of pyrene-based biomolecular probes.
The absorption maxima of the alkynyl-
pyrenes were seen to be shifted to
longer wavelengths compared with
those of the unsubstituted parent
pyrene. Fluorescence quantum yields
of the alkynylpyrenes dramatically in-
creased up to 0.99 in ethanol, and only
slight quenching of the fluorescence oc-
curred even under aerated conditions.
The alkynylpyrenes have been success-
fully introduced into representative
biomolecules such as peptides, proteins,
and DNAs. The detectabilities of the
labeled biomolecules were significantly
improved, with the unique photochemi-
cal characteristics of the pyrene nu-
cleus being maintained.
Keywords:
biosensors
alkynylpyrenes
· fluorescence
·
·
fluorophores · photochemistry
Introduction
rivatives have long been attractive by virtue of their inher-
ent chemical and photochemical characteristics.[2] Indeed,
the simple polyaromatic hydrocarbon skeleton does not re-
quire any protection when being incorporated into peptides
or oligonucleotides. Also inspiring are excimer formation in
these systems and their rather long fluorescence lifetimes,[2,3]
which have prompted researchers to explore various appli-
cations, such as the development of novel strategies for de-
tecting DNA and RNA,[4] observing protein–substrate and
protein–protein interactions,[5] demonstrating conformation-
al changes of proteins,[6] and monitoring the real-time dy-
namics of biological membranes.[7] Furthermore, unlike ben-
zopyrene, pyrene itself is nonmutagenic and has a high LD50
(50% lethal dose; 250 mgkgꢀ1, mice), which makes it a
more favorable candidate for possible use in applications in
vivo.[8] At the same time, however, these fascinating photo-
chemical properties of pyrene also result in serious draw-
backs, such as the substantial quenching of its fluorescence
by the presence of oxygen[9] and by electron-donating and
-accepting molecules that may exist in vivo,[10] and the low
fluorescence quantum yield in protic solvents.[4c,11] More-
over, the relatively short absorption wavelengths of pyrene
and its simple derivatives are unsatisfactory because several
biomolecules and biomolecular segments will also be excited
upon irradiation at the same wavelength. To overcome such
disadvantages of pyrene-based fluorophores, Berlin et al.
have very recently reported the utilization of (phenylethyn-
Fluorescent probes are useful in investigations of many as-
pects of biomolecules, such as their conformations and inter-
actions, in nonradioactive detection and visualization
modes.[1] This usefulness is largely due to the ready avail-
ability of fluorophores with a variety of fluorescence proper-
ties, their specificity and versatility in labeling, and the avail-
ability of highly sophisticated fluorescence detection instru-
ments. Among the available fluorophores, pyrene and its de-
[a] Dr. H. Maeda, T. Maeda, Prof. K. Mizuno
Department of Applied Chemistry
Graduate School of Engineering, Osaka Prefecture University
1-1 Gakuen-cho, Sakai, Osaka 599-8531 (Japan)
Fax : (+81)72-254-9289
E-mail: mizuno@chem.osakafu-u.ac.jp
[b] Dr. K. Fujimoto, H. Shimizu, Prof. M. Inouye
Faculty of Pharmaceutical Sciences
Toyama Medical and Pharmaceutical University
Sugitani 2630, Toyama 930-0194 (Japan)
Fax : (+81)76-434-5049
E-mail: fujimoto@ms.toyama-mpu.ac.jp
[c] Prof. M. Inouye
PRESTO, Japan Science and Technology Agency (JST)
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
yl)pyrenes as fluorescent dyes for DNA labeling.[12] Al-
though they pointed out that the (phenylethynyl)pyrenes ex-
hibit high fluorescence quantum yields, systematic and quan-
titative studies of their photochemical characteristics[13] as
well as comparisons with those of the parent pyrene remain
elusive, as is also the case for many other similar systems.
During the course of our studies on pyrene-based biomolec-
ular probes,[4a,14] we have also noted the influence of alkynyl
substituents on the photochemical properties of pyrene
nuclei. Thus, we report herein in detail the synthesis, photo-
chemical data, and biological applications of various alkyn-
ylpyrenes; the results presented here can be expected to
lead to fruitful applications in biological investigations.
Results and Discussion
Synthesis of the alkynylpyrenes: Most alkynylpyrenes
[15]
(pyrene CꢁC R), where R is pyridyl, bipyridyl,[15] terpyr-
idyl,[15] quinolyl,[16] thienyl,[17] ferrocenyl,[18] or some other
group,[19–21] have been synthesized by Sonogashira coupling
reactions.[22] We have prepared a series of alkynylpyrenes 2–
13 according to this method, as shown in Schemes 1 and 2.
ꢀ
ꢀ
Scheme 1. Synthesis of alkynylpyrenes 2–10.
Bromination of pyrene (1) with one to four equivalents of
bromine gave the mono-, di-, tri-, and tetrabromopyrenes,
respectively (1-bromopyrene (14), 1,6-dibromopyrene (15),
1,3,6-tribromopyrene (16), and 1,3,6,8-tetrabromopyrene
(17)).[23] Cross-coupling of these bromopyrenes with (tri-
methylsilyl)acetylene under the conditions of the Sonoga-
shira reaction afforded mono-, bis-, tris-, and tetrakis(trime-
thylsilylethynyl)pyrenes 2–5, respectively. Tetrakis(tert-
butylethynyl)pyrene 6 and (arylethynyl)pyrenes 7–9 were
synthesized in a similar manner by using the corresponding
Scheme 2. Synthesis of alkynylpyrenes 11–13.
acetylene derivatives. Unsymmetrical dialkynylpyrene 10,
designed as a water-soluble alkynylpyrene, was prepared in
a stepwise manner via monosubstituted 20 starting from 1,6-
diiodopyrene (18) and the hydrophilic acetylene 19.
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825

K. Fujimoto, K. Mizuno et al.
Alkynylpyrenes 11–13 for biomolecular probes were each
prepared from 1-bromopyrene (14). Sonogashira reactions
of 14 with 4-ethynylaniline and 3-(4-ethynylphenyl)propion-
ic acid (23) gave intermediates 21 and 24, respectively. Con-
densation of 21 with maleic anhydride afforded the male-
imide derivative 11, which is capable of reacting with the
Cys residues of peptides and proteins. The succinimidyl
ester 12 was synthesized by treating 24 with N,N’-disuccin-
imidyl carbonate. Intermediate 24 was also transformed to
the phosphoramidite 13 via the alcohol derivative 25. Both
12 and 13 were used for labeling DNAs.
UV-visible absorption spectra of the alkynylpyrenes: Table 1
lists the absorption maxima (labs) of the alkynylpyrenes 2–10
in ethanol, along with their absorption coefficients (e). The
absorption maxima of the (trimethylsilylethynyl)pyrenes 2–5
consecutively shift to longer wavelengths, and their absorp-
tion coefficients increase with increasing number of (tri-
methylsilyl)ethynyl groups (Figure 1A). As 5 has an absorp-
tion maximum at l=434 nm and its absorption coefficient is
1.32”105 molꢀ1 dm3 cmꢀ1, it exists as an intensely colored
orange-red solid. The absorption spectrum of 1,3,6,8-tetra-
kis(tert-butylethynyl)pyrene (6), the corresponding carbon
counterpart of 5, also appears in the visible region, but is
less bathochromically shifted compared with that of 5. The
effect of the silicon atoms in inducing this extra bathochro-
ꢀ
mic shift might be due to s–p interaction between the C Si
s and the acetylenic p bonds.[24] Although the absorptions of
(arylethynyl)pyrenes 7 and 8 also reveal bathochromic
shifts, their absorption coefficients are near to that of 1 (Fig-
ure 1B). On the other hand, the bandwidths of the spectra
at longer wavelengths are considerably broadened because
of increments due to different vibrational levels of the p-ex-
panded skeletons. The absorption maxima of 9 and 10 bear-
ing two alkynyl groups are further shifted, and their absorp-
Figure 1. UV-visible absorption spectra of A) 1–5 (1.0”10ꢀ5 m) and B) 1
and 7–10 (1.0”10ꢀ5 m) in aerated EtOH.
tion coefficients are larger than those for the monoalkynyl
derivatives 7 and 8.
Fluorescence spectra of the
alkynylpyrenes: Fluorescence
Table 1. Photochemical data of compounds 1–10.
spectra of the alkynylpyrenes
were measured in degassed eth-
anol (Figure 2, and Figure S1 in
the Supporting Information).
Pyrene
and
alkynylpyrene
Absorption[a]
Fluorescence (monomer)
Fluorescence
(excimer)
lem [nm]
[b]
[e]
[f]
labs
e (”104)
lem
Ff[c]
ts[d]
kf[e] (”106)
[sꢀ1
k
isc +knr
[nm] [molꢀ1 dm3 cmꢀ1
]
[nm]
[ns]
]
(”106) [sꢀ1
]
1
2
3
4
5
6
7
8
9
334
365
390
413
434
424
4.63
5.77
9.22
10.0
13.2438
11.7
391
4.03
6.14
6.83
373
386
399
421
0.32[g] 377
0.849
7.63
143
250
326
1.80
6.24
70.5
62.5
n.d.[h]
41.7
475
505
523
545
568
560
The fluorescence maxima (lem
)
0.55
0.67
72.1
4.68
3.20
3.04
3.36
appearing at the shortest wave-
lengths are listed in Table 1.
Monomer emissions of the al-
kynylpyrenes 2–10 are shifted
to longer wavelengths com-
pared with those of 1. As ex-
pected from the rigid structures
of the alkynylpyrenes, their
Stokes shifts are essentially
small. It is noteworthy that the
fluorescence quantum yields
(Ff) of 2–5 are substantial and
0.80
>0.99
0.86
428
0.78
397
423
407
256
3824.47
391
71.7021 8.9
0.55
0.90
2
509
281
545
47235.6
1.96
1.65
1.97
229
61.1
509
530
407
403
10
0.93
7
52
[a] 1.0”10ꢀ5 m in EtOH, labs is the absorption band appearing at the longest wavelength. [b] 1.6–2.5”10ꢀ7 m in
EtOH, degassed by bubbling of Ar, lem is the fluorescence band appearing at the shortest wavelength.
[c] Fluorescence quantum yield in EtOH, degassed by bubbling of Ar, measured at room temperature. Stand-
ards used were 9,10-diphenylanthracene (for 1–4 and 7–10) and coumarin 102(for 5 and 6), OD<0.02. Ff
(9,10-diphenylanthracene)=0.95 in EtOH.[25] Ff (coumarin 102)=0.74 in EtOH.[26] [d] Fluorescence lifetime,
solution degassed by freeze-pump-thaw cycles, 1.0”10ꢀ5 m in EtOH. [e] Calculated using the equations Ff =
kfts and ts =1/(kf+kisc+knr). [f] Saturated concentration in CH2Cl2. [g] Data from ref. [27]. [h] The correspond-
ing Ff value is too large to be used for the estimation.
increase
with
increasing
number of alkynyl groups
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Alkynylpyrene-Based Biomolecular Probes
FULL PAPER
gassed solutions were maintained. On the other hand, the
fluorescence of the parent pyrene was scarcely observed
under the same conditions (integral only about 6% of that
in the degassed solution; Figure S3 in the Supporting Infor-
mation). Fluorescence quantum yield can be described by
the equation Ff =kfts, or Ff =kf /(kf+kisc+knr +kq[O2]), in
which kf, kisc, knr, and kq represent the rate constants for
fluorescence radiation, intersystem crossing, nonradiative
decay, and fluorescence quenching by oxygen, respectively,
and ts is the lifetime (s=singlet). Table 1 summarizes the
values of kf and kisc +knr for 1–10, calculated on the basis of
the experimental Ff and ts values under the degassed condi-
tions, that is, assuming kq[O2]=0. As knr is known to be neg-
ligible in aromatic hydrocarbons,[28] kisc should be the major
component in kisc +knr.[29] Under aerated conditions, the rate
constant for quenching of the fluorescence by oxygen (kq) is
estimated to be nearly diffusion-controlled (up to 3.89”
1010 mꢀ1 sꢀ1 in hexane),[30] and [O2] is below 2.0”10ꢀ3 m in
usual solvents. These data imply a kq[O2] value of at most
8.0”107 sꢀ1. As can be seen in Table 1, the kf values of the
alkynylpyrenes, especially 4–6 and 8–10, are much larger
than the kq[O2] value. This situation is most likely responsi-
ble for the observed high Ff values of the alkynylpyrenes
even in the presence of oxygen. The fact that the alkynyl-
pyrenes continue to emit their fluorescence in the presence
of oxygen represents an additional useful feature in the
context of their possible deployment in biological applica-
tions.
Figure 2. Fluorescence spectra of 1–5 in EtOH degassed by bubbling of
Ar (RI=the relative intensity); lex =337, 346, 368, 388, and 408 nm for
1–5, respectively. Each concentration of 1–5 was adjusted to the condition
of OD=0.01 at the corresponding excitation wavelength.
(Table 1). Especially in the case of 5, the Ff value is >0.99,
meaning that the absorbed photons on 5 are exclusively
emitted as fluorescence. Conversely, the fluorescence life-
times of 2–5 decrease with increasing number of attached al-
kynyl groups. This shortening of the lifetimes is responsible
for the high Ff values (see below). Excimer emission is the
most characteristic photochemical feature of pyrene nuclei,
and is also seen for the alkynylpyrenes. Thus, at high con-
centrations in CH2Cl2, 2–10 show excimer emissions in the
wavelength region 500–570 nm (Figure 3, and Figure S2in
the Supporting Information). The emission maximum for
the excimer of 5 is close to 600 nm, and its spectral band
even tails to 700 nm. Therefore, 5 fluoresces with a bright-
yellow color under these conditions.
Application of the alkynylpyrenes as fluorescent probes: As
mentioned above, the investigated alkynylpyrenes exhibit
bathochromic shifts of their absorption spectra and high
fluorescence quantum yields even in the presence of oxygen.
These findings encouraged us to apply the alkynylpyrenes as
novel hydrophobic fluorescent probes for biomolecules. Be-
cause fluorescent probes are usually used in aqueous media,
the well-characterized photochemical properties of the alkyn-
ylpyrenes in organic solvents must be retained in aqueous
buffer solutions. To ascertain this in advance, the water-solu-
ble 10 was chosen, in which the long oxyethylene side chain
is expected to render the hydrophobic alkynylpyrene core
soluble in such media. Indeed, the solubility of 10 proved to
be high enough to permit exact evaluation of its photochem-
ical characteristics at up to 1.0”10ꢀ3 m in pure water. Both
the monomer (lmax =407 and 432nm) and the excimer
(lmax =527 nm) emissions of 10 were clearly observed, and
the excimer emission could be detected at concentrations as
low as about 1.0”10ꢀ6 m, conditions far more dilute than
could be used for 2, 7, 8, and 9 in CH2Cl2. Thus, the strong
tendency of 10 to self-associate in aqueous media offers an
advantage in that the alkynylpyrenes may be applied to the
detection of biomolecule–biomolecule interactions.
Figure 3. Fluorescence spectra of 1–5 at their saturated concentrations in
aerated CH2Cl2; lex =337 nm.
The influence of oxygen on the fluorescence emissions
was investigated. Integrations of the fluorescence spectra of
8–10 in aerated ethanol showed that 90%, 92%, and 89%
of the levels observed from the corresponding carefully de-
As probes for peptides and proteins: We developed a fluo-
rescent probe 11 composed of (phenylethynyl)pyrene and
maleimide units as fluorescent and labeling functionalities,
respectively. This structure was inspired by that of commer-
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K. Fujimoto, K. Mizuno et al.
cially available N-(1-pyrenyl)maleimide (26), in which the
maleimide group is attached directly to a pyrene core.[31]
The fluorescent probe 26 is used for labeling thiol side
chains on Cys residues of peptides and proteins. The inher-
ent fluorescence of the pyrene nucleus in 26 is quenched
before the labeling reaction, but fluorescence is then re-
stored by the formation of thiol adducts at the carbon–
carbon double bond of the maleimide. This type of off/on
switching property is extremely useful for monitoring the la-
beling reaction, and we expected that the fluorescent probe
11 would behave similarly. Indeed, the fluorescence of 11
itself was found to be so weak that it could hardly be recog-
nized visibly.
Keillor et al. have proposed that the off/on switching
property in maleimide-attached fluorophores may be due to
a reversal of the energy levels between the p orbitals on the
fluorophore moiety and the n orbital on the maleimide car-
bonyl group before and after the labeling reaction.[32] Intra-
molecular PET (photoinduced electron transfer), however,
is considered to be another possible mechanism for this
switching, whereby the excited electron on the fluorophore
falls to the LUMO of the maleimide group but not to that
of the corresponding thiol-added succinimide skeleton. To
clarify this aspect, the following experiments were carried
out. If the mechanism were to be based on PET, addition of
a maleimide molecule (2,5-pyrroledione) to a solution of 7
should cause fluorescence quenching through intermolecular
PET. In fact, the fluorescence intensity of 7 (2 mm) de-
creased to about 10% of the original level in the presence
of 0.1m maleimide in ethanol. On the other hand, no
quenching was observed under the same conditions in the
case of succinimide. From the Stern–Volmer plot of 7
against the maleimide concentration (see Figure S4 in the
Supporting Information), the kq value was estimated to be
about 1.5”1010 mꢀ1 sꢀ1, supporting the view that the quench-
ing event is induced by intermolecular PET because the
value is larger than the diffusion-controlled rate constant
(6.0”109 mꢀ1 sꢀ1) in ethanol. Thus, this finding suggests that
in our alkynylpyrene-based maleimide probe, the off/on
switching is most likely controlled by an intramolecular PET
mechanism.
Figure 4. Fluorescence spectra of 11 and 26 before and after the labeling
reaction with l-cysteine in 0.07m phosphate buffer solutions; [11]=1.2”
10ꢀ6 m, [26]=3.8”10ꢀ7 m, [11+l-cysteine]=9.3”10ꢀ7 m, [26+l-cysteine]=
5.7”10ꢀ7 m. lex =369 and 341 nm for 11 and 26, respectively. Each con-
centration was adjusted to the condition of OD=0.01 at the correspond-
ing excitation wavelength.
tion shows fluorescence spectra of the glutathione adducts,
and a similar off/on switching could again be seen.
Comparing the spectra of the adducts of 11 and 26, we
note the following points: the spectra of the adducts of 11
reveal bathochromic shifts (~17 nm) with broadening, and
the fluorescence intensities of the adducts of 11 are much
more enhanced. To obtain quantitative information, fluores-
cence quantum yields (Ff) were measured in a phosphate
buffer solution (pH 7.0) before and after the labeling reac-
tions of l-cysteine and glutathione (Table 2). Both 11 and 26
Table 2. Ff values for 11 and 26 before and after the labeling reactions.[a]
Adduct
Ff for 11
Ff for 26
before
l-cysteine
glutathione
ꢂ0
ꢂ0
0.57
0.48
0.03
0.02
[a] Quinine sulfate (Ff =0.55) in 0.5m H2SO4 used as a standard.
alone displayed negligible Ff values owing to the fluores-
cence quenching described above. Among the adducts, the
Ff values for those with 11 were much higher than for those
with 26. Fluorescence lifetimes of the adducts of 11 were de-
termined in order to investigate the reason for these high Ff
values. The lifetimes were found to be extremely short (ts =
2.05 ns for the cysteine adduct and 2.76 ns for the gluta-
thione adduct), and hence the adducts were able to radiate
their fluorescence before collision with oxygen as in the
cases of 3–10 (see above).[33]
Protein adducts of 11 and 26 were assessed by sodium do-
decyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE). The target protein was bovine serum albumin
(BSA), which has a molecular weight of around 67 kDa and
incorporates 35 Cys residues. After treating BSA (300 mg)
with 11 or 26, the mixture was analyzed by SDS-PAGE.
Figure 5 shows gel photographs visualized by Coomassie
blue staining (A) and by irradiation with a UV transillumi-
nator (B). In photograph A, bands corresponding to BSA
l-Cysteine, the simplest biomolecule possessing a thiol
group, was chosen as a test target molecule and was allowed
to react with 11 in a water/dimethyl sulfoxide mixed solvent.
To show that the probe fulfilled the criteria for practical use,
fluorescence spectra of the adducts were measured under
aerated conditions in water without exclusion of oxygen.
Strong fluorescence was observed for the cysteine adduct of
11 at a longer wavelength compared with that of 26
(Figure 4). Thus, the fascinating property of the off/on
switching was confirmed to also be operative in our alkynyl-
pyrene-based maleimides. As a next target molecule, gluta-
thione was selected. Glutathione is a biologically active pep-
tide consisting of Cys, Glu, and Gly and is found in most or-
ganisms. Glutathione was labeled with 11 and 26, and the
adducts were analyzed under the same conditions as de-
scribed for l-cysteine. Figure S5 in the Supporting Informa-
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Alkynylpyrene-Based Biomolecular Probes
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the basis of two emission bands can largely eliminate the un-
certainty in the detection that results from individual sample
factors such as pH, temperature, the probe concentration,
and localization of the probes, as well as from instrumental
factors such as the kind of spectrometer or the dependence
of excitation intensity on the light source. The fascinating
excimer-monomer switching probe 27, however, suffers from
the inherent drawbacks of pyrene fluorophores mentioned
in the introduction to this paper. Thus, we decided to intro-
duce alkynylpyrene skeletons instead of the parent pyrenes
into our excimer-monomer switching probes in order to im-
prove their efficiency in the context of in vivo applications.
A new DNA probe 28 possessing two (phenylethynyl)pyr-
ene moieties as fluorophores was prepared in a similar
manner as described for 27 (Figure 6).[4a] A single-stranded
oligonucleotide with a C3-alkylamino linker (3’- end), which
consists of the same sequence as that of 27, was modified at
the 3’- and 5’- ends with 12 and 13, respectively, to give 28.
The melting temperature (Tm) of 28 was found to be about
608C on the basis of its electronic absorption spectra,
almost the same as that for 27.
Figure 5. SDS-PAGE photographs of BSA and the adducts. Photographs
(A) and (B) were visualized by Coomassie blue staining and by irradia-
tion with light of wavelength 312nm, respectively; lane 1: molecular
weight markers, lane 2: BSA (3.0”10ꢀ6 g), lane 3: BSA labeled with 26
(1.3”10ꢀ8 g), lane 4: BSA labeled with 11 (1.8”10ꢀ8 g).
and the adducts could be confirmed in each of the lanes 2–
4. On the other hand, only the fluorescent band due to the
adduct of 11 (lane 4) is clearly visible in photograph B,
while the band due to the adduct of 26 (lane 3) is faint in
the same photograph. This SDS-PAGE experiment demon-
strates the favorable photochemical characteristics of the
alkynylpyrene skeletons, making 11 a promising practical
probe for labeling Cys residues.
To examine the ability of 28 to act as a switching probe,
fluorescence titration experiments were performed with the
fully complementary DNA 29 (Figure 7). We ensured that
As probes for DNAs: We recently reported an excimer-
monomer switching DNA probe 27 with two pyrene moiet-
ies as fluorophores at both the 3’- and 5’- ends of a single-
stranded oligonucleotide (Figure 6).[4a] This DNA probe can
Figure 7. Fluorescence titration spectra of 28 ([28], 200 nm; [MgCl2],
5 mm; [KCl], 50 mm; [Tris-HCl], 20 mm, at pH 8.0) in the presence of the
fully complementary target DNA 29 (0.1–1.0 equiv); lex =370 nm.
the probe 28 retained the characteristic excimer–monomer
switching property as in the case of 27. Thus, upon addition
of 29, the excimer emission (l=511 nm) decreased, while
the monomer emission (l=405 and 427 nm) increased, with
an isoemissive point at l=490 nm. A significant difference
between the spectral changes observed for 27 and 28 is the
extent of the increase in the monomer emission, which is
probably because of the high fluorescence quantum yield of
the alkynylpyrene skeleton on 28 in protic media. Therefore,
the probe 28 also exhibits a somewhat “signal-on”-like
change in the wavelength region in which the monomer
emits, resembling conventional FRET-type molecular bea-
cons. On the other hand, 27 can be regarded as a “signal-
off”-type probe: the extent of the decrease in the excimer
emission is larger than the increase in the monomer emis-
Figure 6. Sequences of a new DNA probe 28 modified with alkynylpyr-
enes, the previous DNA probe 27 modified with pyrenes, and the fully
complementary target DNA 29 for the loop region of 27 and 28.
be regarded as a so-called molecular beacon, consisting of a
stem-and-loop structure. In conventional molecular beacons,
the detection of target DNAs is based on the FRET (fluo-
rescence resonance energy transfer) mechanism. Compared
with conventional beacons, our DNA probe 27 has the ad-
vantages of synthetic simplicity, high signal-to-noise, and un-
ambiguous detectability. Furthermore, the characteristic
emission switching of 27 with an isoemissive point allows for
ratiometric measurements. Ratiometric measurements on
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K. Fujimoto, K. Mizuno et al.
solutions (1.0”10ꢀ5 m in EtOH) were carefully degassed by freeze-pump-
thaw cycles before the measurements. The excitation wavelength was 337
or 370 nm, and cut-off filters UV-32for 1 at 337 nm, UV-35 for 2, 3, and
7–10 at 337 nm, and L38 for 4–6 at 370 nm were used. All decay curves
were calculated by assuming a single exponential on the basis of the
equation If(t) =Aexp(ꢀt/t), in which If(t) =fluorescence intensity at time t
and A=fluorescence intensity at t=0.
sion.[4a] Thus, the probe 28 has the advantages of an inherent
switching ability that allows for ratiometric measurements
as well as of a simple “signal-on”-type property. Sensitivity
is one of the most important criteria for probes directed
toward biomolecular detection. The spectral changes of 28
could be followed at a concentration of 1.0”10ꢀ10 m, which
is about 100 times more dilute than with 27 in aqueous
buffer solutions.[34] The high sensitivity of 28 further illus-
trates the usefulness of the alkynylpyrene skeleton as a bio-
molecular probe.
Determination of the melting temperature (Tm) of 28: Electronic absorp-
tion spectra were recorded in the temperature range from 10 to 908C.
The solution used for the measurements contained 1 mm 28, 5 mm MgCl2,
50 mm KCl, and 20 mm Tris-HCl (pH 8.0).
Fluorescence titration: A Milli-Q solution of the complementary DNA
29 (10”0.1 equiv) was added to a 200 nm solution of 28 containing 5 mm
MgCl2, 50 mm KCl, and 20 mm Tris-HCl (pH 8.0) at 258C. Each mixture
was stirred at 258C until no further change in the fluorescence spectra
was observed (ca. 15 min). The excitation wavelength was 370 nm, and
fluorescence spectra were recorded from 350 to 700 nm following each
addition of 29.
Conclusion
Direct attachment of alkynyl groups to a pyrene core has
been shown to impart photochemical properties that render
these systems favorable as fluorescent probes for biomole-
cules. As the number of alkynyl substituents is increased,
the fluorescence intensities of the alkynylpyrene derivatives
are enhanced, and this is accompanied by bathochromic
shifts in their absorption spectra. Taking advantage of these
promising features, we have developed alkynylpyrene-based
probes for biological applications. A maleimide-modified
probe was designed for labeling Cys residues, and succin-
imidyl and phosphoramidite derivatives were devised for
constructing new DNA probes. These alkynylpyrene-based
biomolecular probes have been found to be more practical
than conventional pyrene-based ones as a result of excita-
tion at longer wavelengths and the high Ff values that are
maintained without rigorous exclusion of oxygen.
Acknowledgements
This work was partly supported by the Corporation of Innovative Tech-
nology and Advanced Research in Evolutional Area (CITY AREA)
from Wakayama Technology Promotion Foundation and by a Grant-in-
Aid for Young Scientists (B) (16750039) and Scientific Research (B)
(15350026) from the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan.
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Materials and general procedures: Reagents were purchased from com-
mercial sources and were used without further purification. The com-
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2
FfðsplÞ ¼ FfðstdÞ Â ½Astd=Asplꢃ Â ½Ispl=Istdꢃ Â ½nspl=nstd
ꢃ
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830
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Chem. Eur. J. 2006, 12, 824 – 831

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Received: June 6, 2005
Published online: November 3, 2005
Chem. Eur. J. 2006, 12, 824 – 831
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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