Phospholipid-induced aggregation and anthracene excimer formation

Article abstract of DOI:10.1021/ol8014418

(Chemical Equation) The zinc complex of anthryl bis(dipicolylamine) (1) aggregates upon binding with long-chain aliphatic phosphates and displays anthracene excimer fluorescence, which provides a new strategy toward detection of the biologically important lysophosphatidic acid in aqueous solution.

Full text of DOI:10.1021/ol8014418

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4401-4404
Phospholipid-Induced Aggregation and
Anthracene Excimer Formation
Kuan-Hung Chen, Jye-Shane Yang,* Chung-Yu Hwang, and Jim-Min Fang*
Department of Chemistry, National Taiwan UniVersity, Taipei, 106, Taiwan
jmfang@ntu.edu.tw; jsyang@ntu.edu.tw
Received June 26, 2008
ABSTRACT
The zinc complex of anthryl bis(dipicolylamine) (1) aggregates upon binding with long-chain aliphatic phosphates and displays anthracene
excimer fluorescence, which provides a new strategy toward detection of the biologically important lysophosphatidic acid in aqueous solution.
Lysophosphatidic acid (LysoPA) is a phospholipid that
influences many biochemical processes, including stimulation
of cell growth, promotion of platelet aggregation, and
induction of cancer cell invasion.1 LysoPA is found at a
significant level (approximately 10 µM) in ovarian cancer
cells2 and can be a useful marker for diagnosis of ovarian
cancer.3 In this context, a method of effective detection of
LysoPA is highly desirable. While fluorescent chemosensors
have the general advantages of high sensitivity, high through-
put, and real-time screening, examples of LysoPA chemosen-
sors are rare.4 In particular, one that can selectively
distinguish LysoPA from phosphate ions in aqueous solution
remains to be developed.
We report herein that the anthryl bis(dipicolylamine)-zinc
complex 1 previously reported by Hamachi and co-workers
displays an unusual and selective fluorescence response to
LysoPA in aqueous buffer solutions through a concentration-
dependent formation of aggregates and fluorescent anthracene
excimers. Complex 1 has been shown to display an off-on
fluorescence response to phosphorylated molecules5 and
phosphatidylserine-containing membranes,6 which can be
ascribed to the modulation of the photoinduced electron
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Kralj, L. J. Chromatogr. B 2007, 858, 287–291.
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I.; Kohira, T.; Tamaru, S.-I.; Hamachi, I. J. Am. Chem. Soc. 2005, 127,
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10.1021/ol8014418 CCC: $40.75
Published on Web 09/12/2008
2008 American Chemical Society

transfer (PET) from the amino group to the antrhacene
fluorophore. However, anthracene excimer emission of 1 has
been elusive in these studies. In view of the fact that
anthracene excimer vs monomer emission would allow a
more favorable ratiometric detection of LysoPA and that the
observation of anthracene excimer emission in solutions is
rare,7 it is desirable to understand the origin of LysoPA-
induced excimer formation for 1.
At the early stage when incremental amounts of LysoPA
are added to a solution of 1 in HEPES buffer, the fluores-
cence maximum shows red shifts by 30-50 nm along with
decreased fluorescence intensity (Figure 1a). However, when
cally investigated the fluorescence behavior of 1 in the
presence of phosphate ion (PO43-) and aliphatic phosphates
of different chain lengths, including hexyl phosphate
(C6H13OPO3H2), octyl phosphate (C8H17OPO3H2), decyl
phosphate
(C10H21OPO3H2),
dodecyl
phosphate
(C12H25OPO3H2), and tetradecyl phosphate (C14H29OPO3H2).
As shown in Figure 2, the fluorescence titration spectra
Figure 1. Fluorescence titration spectra of 1 (5 µM) with
incremental addition of LysoPA (as the sodium salt) (a) up to 4.0
equiv and (b) up to 100 equiv (spectra a-f are with 0, 10, 15, 20,
25, and 100 equiv of LysoPA, respectively) in HEPES buffer (10
mM, pH 7.22). The excitation wavelength was 380 nm. The inset
shows the ratio of the 432- and 480-nm fluorescence intensity (I432
/
I480) during titration.
Figure 2. Fluorescence titration of receptor 1 (5 µM) in HEPES
buffer (pH 7.22) by incremental addition of (a) (Bu4N)3PO4, (b)
C6H13OPO3H2, (c) C8H17OPO3H2, (d) C10H21OPO3H2, (e)
C12H25OPO3H2, and (f) C14H29OPO3H2, up to 5 equiv in each case.
The excitation wavelength was 380 nm.
the addition of LysoPA exceeds 10 equiv, the fluorescence
change is reversed, i.e., the fluorecence maximum shifts back
to 437 nm and the fluorescence intensity is recovered and
even enhanced (Figure 1b). To our knowledge, such a two-
stage and analyte concentration-dependent “reversible” fluo-
rescence sensing behavior is unprecedented.
highly depend on the length of the n-alkyl group. Whereas
PO43-, hexyl phosphate, and octyl phosphate enhance the
fluorescence without changing the wavelength of peak
maximum, decyl phospate simply induces fluorescence
quenching,8 and the long-chain dodecyl and tetradecyl
phosphates lead to a new and structureless emission band at
∼480 nm, similar to that observed in the case of LysoPA.
Accordingly, the formation of the 480-nm emission band is
To understand the distinct fluorescence sensing behavior
for 1 to LysoPA vs phosphoryl peptides,6 we have systemati-
(7) (a) Arman, S. A. V.; Czarnik, A. W. J. Am. Chem. Soc. 1990, 112,
5376–5377. (b) Shiraishi, Y.; Tokitoh, Y.; Nishimura, G.; Hirai, T. Org.
Lett. 2005, 7, 2611–2614. (c) Zhang, X.; Sasaki, K.; Kuroda, Y. J. Org.
Chem. 2006, 71, 4872–4877.
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Org. Lett., Vol. 10, No. 20, 2008

more likely associated with the behavior of its aliphatic
chains. It should also be noted that the phenomenon of
“fluorescence reversal” is also observed for both dodecyl and
tetradecyl phosphates, albeit in much higher concentrations
than that for LysoPA. The corresponding spectra are supplied
as Supporting Information.
It is known that aliphatic phosphates can undergo self-
assembly in aqueous media to form micelle by aggregation
of the hydrophobic hydrocarbon chains,9 and thus expose
their polar phosphate groups to the aqueous environment.
While such a micelle might not be formed when LysoPA is
at low concentration,10 the interactions of LysoPA with 1
could induce the formation of aggregates with a nonspecific
structure, which is responsible for the new emission band at
480 nm attributed to the excimer of the anthracene fluoro-
phore. Formation of micelle-type aggregation becomes
possible with increasing concentration of LysoPA,10 and the
chance of short contact between molecules of 1 is thus
reduced. This phenomena might account for the “reversed”
fluorescence changes (Figure 1b), and a schematic drawing
is shown in the vignette of the abstract. Indeed, the
concentration required for the second-stage fluorescence
reversal is in the order dodecyl phosphate (∼10 mM) >
tetradecyl phosphate (∼2.5 mM) > LysoPA (∼0.075 mM)
and comparable to but somewhat lower than their critical
micelle concentrations (cmc).10 It appears that the cmc value
of these phosphates is reduced in the presence of 1.
The aggregate formation for 1 in the presence of LysoPA,
dodecylphosphate, and tetradecylphosphoste is consistent
with the broadening and red-shift of their absorption and
excitation titration spectra. The typical spectra are provided
by the case of LysoPA as shown in Figure 3a. The elevation
of absorption in the long-wavelength region (>420 nm)
might reflect the formation of aggregates in the buffer
solution.11 In comparison, no such phenomena occur in the
corresponding spectra with hexylphosphate (Figure 3b).
Evidently, the antrhacene excimer results from the ground-
state preassociated species (i.e., a static excimer).
Figure 3. UV-vis titration of receptor 1 (10 µM) in HEPES buffer
(pH 7.22) by incremental addition of (a) LysoPA (as the sodium
salt, up to 5 equiv) and (b) C6H13OPO3H2 (up to 5 equiv). The
inset shows the normalized excitation spectra of receptor 1 (5 µM)
in HEPES buffer (pH 7.22) upon incremental addition of LysoPA
(up to 5 equiv). The emission wavelength was monitored at 480
nm.
Table 1. Fluorescence Lifetime and Composition in
Complexation of Receptor 1 with LysoPAa
τ1 (ns);
τ2 (ns);
τ3 (ns);
substrate
composition composition composition
The proposed fluroescence sensing mechanism can also
account for the observed changes in the fluorescence decay
times for 1 in the presence of LysoPA as compared with its
free form (Table 1).12 In the absence of LysoPA, there are
two distinct fluorescence lifetimes, 6.6 (88%) and 16.4 ns
(12%), for 1 in HEPES solutions. The major and shorter-
lived component could be attributed to a loose complex,
1
6.6; 88%
1.7; 16%
1.4; 14%
2.7; 36%
4.4; 0.4%
16.4; 12%
11.6; 51%
4.3; 23%
7.7; 39%
14.8; 99.6%
1+ 1.5equivofLysoPA
1+ 10equivofLysoPA
1+ 20equivofLysoPA
1+100equivofLysoPA
41.8; 33%
60.5; 63%
58.1; 25%
a Measurements were conducted in HEPES buffer (pH 7.22). The
excitation wavelength was 380 nm. The complexation on addition of
LysoPA was monitored at 480-nm fluorescence.
(8) The fluorescence quenching observed in Figure 2d could be attributed
to the formation of aggregates, in which the anthracene chromophores are
not sandwiched to form excimers but close enough to have significant
dipole-dipole interactions (i.e., H-type aggregates, see: Cornil, J.; Beljonne,
D.; Calbert, J.-P.; Bre´das, J.-L. AdV. Mater. 2001, 13, 1053-1067).
Aggregate formation for 1 in the presence of decylphosphate is supported
by the broadened absorption spectra (see the Supporting Information for
details).
where the PET process is incompletely inhibited. In the
presence of 1.5 equiv of LysoPA, the decay curve at 480
nm is deconvoluted to three lifetimes (Table 1). The long-
lived component (41.8 ns, 33%) can be ascribed to the
anthracene excimer due to the forbidden nature of optical
transition.13 The other two lifetimes are no longer than that
of the unbound 1 and thus they are more likely from the
anthracene monomer. Since energy transfer from the excited
anthracene monomer to anthracene dimer or unexpected
impurities is facile in aggregrates, the observation of short-
lived components (1.7 ns) is not unexpected. In a condition
(9) Singer, S. J.; Nicolson, G. L. Science 1972, 175, 720–731.
(10) The critical micelle concentrations (cmc) for LysoPA, dodecyl
phosphate, and tetradecyl phosphate are 0.35, 56, and 13 mM, respectively,
in water and 0.28, 42, and 9 mM, respectively, in the HEPES buffer (pH
7.22), which were determined by the previously reported fluorescence
method (see: Dominguez, A.; Ferna´ndez, A.; Gonza´lez, N.; Iglesias, E.;
Montenegro, L. J. Chem. Edu. 1997, 74, 1227-1231).
(11) Hao, E.; Sibrian-Vazquez, M.; Serem, W.; Garno, J. C.; Fronczek,
F. R.; Vicente, M. G. H. Chem. Eur. J. 2007, 13, 9035–9042.
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Org. Lett., Vol. 10, No. 20, 2008
4403

with 10 equiv of LysoPA, the population of excimer increases
(63%) and the monomer lifetimes are further shortened,
presumably due to the more facile energy transfer to the
dimers. However, the excimer population drops to only 25%
with 20 equiv of LysoPA, and it becomes negligible with
100 equiv of LysoPA. The observed lifetimes for the latter
case are similar to the that of the unbound 1, but the shorter-
lived component, which corresponds to a loose complex, is
less than 1%. In other words, the LysoPA-bound 1 strength-
ens the dipicolylamime-Zn(II) interactions and thus reduces
the PET process. This is consistent with the arguments of
Hamachi and co-workers for 1 with phosphorylated mol-
ecules,5 and accounts for the fluorescence behavior for 1 with
aromatic hydrocarbons,15 cations,16 and humidity.17 Depend-
ent on analytes, a functional polymer can be induced to form
varied aggregates to display analyte-dependent multistate
responses. Such induced aggregation of poly(thiophene) has
been used to identify structurally similar amines.18 The
polyanionic poly(p-phenylene ethynylene) has been utilized
to detect the natural polyamines through aggregation-
enhanced exciton migration.19 In addition to organic mol-
ecules, several functionalized metallic nanoparticles also act
as colorimetric chemosensors through the aggregation mech-
anism.20
In summary, we have uncovered that aggregation-induced
excimer formation is a novel fluorescence sensing mode for
the detection of biologically important LysoPA in aqueous
solution. The aggregation behavior relies on the hydrophobic
aliphatic chains in LysoPA, and the excimer emission results
from excitation of the ground-state preassociated anthracene
dimer or clusters. Our study may provide a protocol for the
development of aggregation-based fluorescence sensing.
3-
PO4 and short-chain alkyl phosphates (Figure 2a,c).
Although the conventional evaluation of binding constants
cannot be directly applied to the aggregate formation,
comparison of the intensity changes at 432-nm fluorescence
against the concentration of alkyl phosphates might be
informative. Nonlinear least-squares fitting of the curves (see
the Supporting Information) suggests the apparent “binding
constants” of 6.2 × 104, 4.0 × 104, 3.3 × 105, 9.0 × 105,
and 6.1 × 105 M-1 for C6H13OPO3H2, C10H21OPO3H2,
C12H25OPO3H2, C14H29OPO3H2, and LysoPA, respectively.
The phosphates bearing long chains (12 and 14 carbons)
appeared to exhibit about 10-fold higher affinity toward the
zinc complex. This enhanced binding might reflect certain
multivalent effects14 contributed by aggregation of the long-
chain phosphates.
Acknowledgment. We thank the National Science Council
for financial support.
Supporting Information Available: Synthetic procedures,
UV-vis titration, fluorescence titration, photokinetics studies,
and NMR spectra of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL8014418
The concept of aggregation-based fluorescence sensing has
been recently demonstrated in several systems. For example,
aggregation of dye molecules can be induced by large
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17, 2989–2991.
(13) The fluorescence lifetimes of anthracene excimers strongly depend
on the environment, solvent polarity, and the substituents. Lifetimes of
52-91 ns have recently been reported for the excimer of a 9,10-dimethylene-
substituted anthracene chromophore in a cyclodextrin-derived bianthracene
system in water (see: Zhang, X.; Sasaki, K.; Kuroda, Y. J. Org. Chem.
2006, 71, 4872-4877).
(18) Maynor, M. S.; Nelson, T. L.; O’Sullivan, C.; Lavigne, J. J. Org.
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16028.
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P. N.; Alkilany, A.; Kinard, B. E.; Hankins, P. Chem. Commun. 2008, 544–
557. (c) Zhao, W.; Chiuman, W.; Lam, J. C. F.; Brook, M. A.; Li, Y. Chem.
Commun. 2007, 3729–3731.
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Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998,
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