Characteristic fluorescence behavior of dialkynylpyrene derivatives in hydrophobic cavity of protein

Article abstract of DOI:10.1246/cl.2009.84

Water-soluble fluorescent molecules consisting of dialkynylpyrene skeleton with an oxyethylene unit were designed and synthesized for interacting with hydrophobic cavities of proteins. In the presence of serum albumin, the fluorescent spectra of dialkynyl

Full text of DOI:10.1246/cl.2009.84

84
Chemistry Letters Vol.38, No.1 (2009)
Characteristic Fluorescence Behavior of Dialkynylpyrene Derivatives
in Hydrophobic Cavity of Protein
Hideyuki Shinmori,1 Hirotoshi Furukawa,1 Kazuhisa Fujimoto,2 Hisao Shimizu,2 Masahiko Inouye,2 and Toshifumi Takeuchiꢀ1
1Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501
2Graduate School of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194
(Received October 16, 2008; CL-080996; E-mail: takeuchi@gold.kobe-u.ac.jp)
Water-soluble fluorescent molecules consisting of dial-
kynylpyrene skeleton with an oxyethylene unit were designed
and synthesized for interacting with hydrophobic cavities of pro-
teins. In the presence of serum albumin, the fluorescent spectra
of dialkynylpyrene derivatives were remarkably changed. These
behavior can be explained by monomer–excimer emission
switching and twisted intramolecular charge-transfer mecha-
nism.
absorption maxima and an increase of quantum yield compared
with the parent pyrene as mentioned above.7b The aniline-linked
dialkynylpyrene 2 was newly prepared for this study (Chart 1).
Since 2 possess an amino group as a donor at the para position
of the benzene ring in 1, a donor–acceptor ICT will appear in
2, in which the pyrene moiety works as an acceptor. Further-
more, ICT emission will be observed from 2 in the protein cavity
via a controlled TICT pathway. Bovine serum albumin (BSA) is
well-known to have several hydrophobic binding sites for guest
molecules, so that its interaction with various organic molecules
has been explored in detail.8–11 Therefore, BSA is mainly used
as a model protein indicating hydrophobic interaction with dial-
kynylpyrenes.
The two dialkynylpyrenes 1 and 2 designed as water-soluble
pyrene derivatives were synthesized by Sonogashira coupling re-
actions5c in a stepwise manner via a monosubstituted pyrene de-
rivative attached to an octa(ethylene glycol), followed by cou-
pling with ethynylbenzene and 4-ethynylaniline, respectively.
To investigate the binding ability of 1 to the hydrophobic
cavity of BSA, a fluorescence titration experiment was per-
formed in 10 mM HEPES buffer (pH 7.4) at 25 ꢁC. In the fluores-
cence spectrum of 1, both monomer (ꢀmax ¼ 410 and 433 nm)
and excimer (ꢀmax ¼ 528 nm) emissions were observed at a con-
centration of 4.7 mM. When BSA was added to 1, the fluores-
cence spectrum changed remarkably (Figure 1). As the BSA
concentration was increased, the emission intensity of the mono-
mer band increased at the cost of the excimer band at 528 nm.
The emission maximum in the monomer band was shifted from
410 to 415 nm. In circular dichroism (CD) measurements of 1
with BSA, induced CD bands were observed in the absorptive
region of the pyrene moiety (Figure S1).12 These bands were at-
tributed to binding of the pyrene within the chiral environment
of the BSA cavities. The UV spectra of 1 also changed upon ad-
dition of BSA passing through an isosbestic point (Figure S2),12
assuming that pyrene and BSA might form a 1:1 complex be-
cause of the presence of a large excess of BSA. Analysis of
the BSA-binding profile by a nonlinear least-squares method
Fluorescent molecules are essential for detecting biomole-
cules and analyzing their functionalities in biochemistry and
chemical biology.1 Among them, pyrene derivatives have been
extensively used as fluorescent molecular probes for various pro-
teins due to their inherent photophysical characteristics.2 In the
fluorescence spectra of pyrene derivatives, vibronic bands orig-
inating from monomer emission and excimer emission depend
on the concentration of the pyrene solution.3 Monomer to exci-
mer emission switching induced by the assembly of pyrene de-
rivatives can be used for ratiometric fluorescence analysis as a
highly sensitive assay.4 Also, pyrene derivatives attached to a
donor such as an amino group formed corresponding exciplexes
via intramolecular charge transfer (ICT).5 In particular, the
twisted intramolecular charge transfer (TICT) reflects structural
information between donors and acceptors.6 However, pyrene
has serious drawbacks; it has a relatively short absorption wave-
length and undergoes significant quenching of its fluorescence in
the presence of oxygen. Recently, it was reported that the intro-
duction of alkynyl groups into the pyrene skeleton induced a red
shift in the absorption maxima, as well as retaining strong fluo-
rescence intensity even in the presence of oxygen.7 Taking these
aspects into account, we expected that alkynylpyrenes bound to
hydrophobic cavities of protein might show intriguing fluores-
cence properties, which promise to be highly potential probes
for various proteins.
In this paper, we report the characteristic fluorescence of al-
kynylpyrenes in the presence of proteins. Recently, 1 has been
synthesized (Chart 1) and it was found that the introduction of
alkynyl groups into pyrene nuclei induced a red shift in the
OMe
O
8
1 : R = H
2 : R = NH2
380
430
480
530
580
630
Wavelength / nm
R
Figure 1. Fluorescence spectral changes of 1 (4.7 mM) upon the
addition of BSA (0–45 mM); ꢀex ¼ 341 nm.
Chart 1.
Copyright ꢀ 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.1 (2009)
85
40
35
30
25
20
15
10
5
(a)
BSA
lactalbumin
globulin
chymotrypsin
ribonuclease
lysozyme
0
0
10
20
30
40
50
380
430
480
530
580
630
Protein conc. / µM
Wavelength / nm
40
35
30
25
20
15
10
5
(b)
Figure 2. Fluorescence spectral changes of 2 (4.7 mM) upon the
addition of BSA (0–44 mM); ꢀex ¼ 363 nm.
BSA
lactalbumin
globulin
chymotrypsin
ribonuclease
lysozyme
gave an apparent association constant of 6:2 ꢂ 105 Mꢃ1. These
findings obtained from the CD and UV–vis analyses also confirm
that the excimer formation of the pyrene moiety is hindered in
the protein environment upon the addition of BSA. Thus, the
monomer–excimer emission for 1 can be controlled by complex-
ation with BSA.
0
20
Protein conc. / µM
40
0
10
30
50
Figure 3. Fluorescence response of l (a) at 415 nm and 2 (b) at
502 nm (4.7 mM) upon addition of a series of proteins.
With respect to the other derivative 2 including an aniline
moiety, the emission bands in aqueous buffer solution (pH 7.4)
appeared at 388 and 404 nm; 2 exhibited only monomer emis-
sion. The polar amino group on the benzene of 2 contributes
to the water solubility of 2 and perhaps leads the suppression
of its excimer formation in the buffer solution. Upon the addition
of BSA to 2, the broad emission band of 2 at around 502 nm sig-
nificantly increased with an isoemissive point at 417 nm, while
the monomer bands decreased (Figure 2). The broad band newly
appearing at 502 nm in the titration spectra differed by 26 nm
from that observed in the case of 1. This difference implies that
the band at 502 nm can not be attributed to excimer emission.
The increase in the emission at 502 nm can be rationalized by
TICT mechanism.5,6 In the TICT of 2, a twisted angle between
the aniline and pyrene planes should result from the regulated ro-
tation around the acetylene linkage by the encapsulation of 2 into
the BSA cavity. This supposition is consistent with the circular
dichroism spectra of 2 with BSA because an exciton coupling
was observed from the pyrene absorptive region in the CD spec-
tra of 2 unlike in those of 1 (Figure S1).12 The exciton coupling
of 2 could be attributed to the hetero combination of the pyrene
and aniline chromophores under the chiral environment of the
BSA cavity. Interestingly, a small difference in functional group,
i.e. the presence or absence of NH2, resulted in the pyrene mole-
cules having totally different photochemical characteristics in
terms of interaction with the protein.
To evaluate the selectivity of 1 and 2 for the binding to var-
ious proteins, fluorescence spectra were measured in the pres-
ence of BSA, lactalbumin, ꢁ-globulin, chymotrypsin, ribonu-
clease, and lysozyme. Figure 3 displays the fluorescence re-
sponse of 1 and 2 at 415 nm (monomer band for 1) and at
502 nm (TICT emission band for 2) in the presence of the pro-
teins. In both cases, the largest fluorescence changes were ob-
served upon the addition of BSA. On the other hand, the other
proteins hardly gave significant enhancements. This finding sug-
gests that the binding of 1 and 2 with the proteins may be driven
by hydrophobic interaction on the site having the high surface
hydrophobicity13 because the order of enhancement in fluores-
cence is independent of the pI values for the proteins.
to BSA, the excimer to monomer emission switching was ob-
served, and CD bands were newly induced from the pyrene moi-
ety of 1. Introduction of a polar amino group into the benzene
ring of 1 led 2 to the TICT emission and the exciton coupling
at the pyrene absorptive region. The photophysical properties
of the dialkynylpyrenes were found to be influenced by the steric
effect in the microenvironment arisen from the complexation
with BSA. The selective response of the dialkynylpyrenes to
BSA encourages us to develop a new class of fluorescent probes
directed toward various proteins because very simple functional
modification of the pyrene nuclei causes the fluorescent profiles
to be diverse.
References and Notes
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In summary, the two dialkynylpyrene derivatives used in
this study have shown specificity for BSA. In the binding of 1
Published on the web (Advance View) December 20, 2008; doi:10.1246/cl.2009.84