Two-photon sensor for metal ions derived from azacrown ether

Article abstract of DOI:10.1021/jo049124a

A two-photon sensor for the metal ions derived from azacrown ether as the receptor is reported. The sensor emits strong two-photon fluorescence when excited by 800 nm laser photons. Moreover, the binding constants measured by the one- and two-photon fluor

Full text of DOI:10.1021/jo049124a

upconversion lasing,9,10 two-photon fluorescence excita-
tion microscopy,11-15 three-dimensional optical data
storage,16-18 and photodynamic therapy.19
Tw o-P h oton Sen sor for Meta l Ion s Der ived
fr om Aza cr ow n Eth er
An important addition to such applications would be
the development of two-photon sensors for biological
applications. Currently, much research is being con-
ducted to develop one-photon fluorescence (OPF) probes
because they are useful tools for clarifying the functions
in biological systems.20 One of the major drawbacks of
such sensors is that the excitation wavelengths are in
the range of 350-560 nm, which may cause damage to
the substrates.21 The problem could be avoided if one
develops two-photon sensors, which allow visualization
of ions, small molecules, or enzyme activity in living cells
by employing two-photon-induced fluorescence (TPF)
microscopy that uses NIR photons as the excitation
source.11-15 More importantly, the two-photon process
would make it possible to determine the three-dimen-
sional distribution of the guest molecules in the living
cells in high resolution. Recently, there has been much
progress in the two-photon imaging of biological sub-
strates by using two-photon confocal laser scanning
microscopy (2PCLSM).22
A useful TPF sensor must possess a fluorophore with
large TPA cross section (δ) and a receptor for the guest
molecules or ions. The wavelength of the maximum two-
photon absorption (λ(m2a) x) for such fluorophore should be
close to 800 nm, because most of the TPF microscopy
experiments use an 800 nm laser beam. Recently, a
variety of bis(styryl)benzene derivatives has been shown
to exhibit large a two-photon cross section near 800 nm.2
As the first step toward developing a two-photon sensor
Hwan Myung Kim, Mi-Yun J eong, Hyun Cheol Ahn,
Seung-J oon J eon, and Bong Rae Cho*
Molecular Opto-Electronics Laboratory,
Department of Chemistry and Center for Electro- and
Photo-Responsive Molecules, Korea University,
1-Anamdong, Seoul, 136-701, Korea
chobr@korea.ac.kr
Received May 25, 2004
Abstr a ct: A two-photon sensor for the metal ions derived
from azacrown ether as the receptor is reported. The sensor
emits strong two-photon fluorescence when excited by 800
nm laser photons. Moreover, the binding constants measured
by the one- and two-photon fluorescence are similar. This
result may be useful for the design of efficient two-photon
fluorescence probes for biological substrates.
Organic materials exhibiting large two-photon absorp-
tion (TPA) behavior have drawn much interest because
they can be excited upon irradiation of low energy
photons with sufficiently high intensity.1-5 Many ap-
plications of such materials have become reality, some
of which include optical power limiting,6-8 two-photon
* To whom correspondence should be addressed. Phone: 82-2-3290-
3129. Fax: 82-2-3290-3121.
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10.1021/jo049124a CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/22/2004
J . Org. Chem. 2004, 69, 5749-5751
5749

SCHEME 1a
a
Reagent and conditions: (a) (i) NBS/CCl4, 80 °C, 2 h (61%); (ii) P(OEt)3/toluene, 120 °C, 5 h (98%); (b) (i) LDA (1 equiv)/THF, p-(N-
isoamyl-N-phenyl)aminobenzaldehyde, 6 h (62%); (ii) LDA (1 equiv)/THF, N-(p-formylphenyl)aza-15-crown-5, 45 °C, 6 h (72%).
F IGURE 1. Absorption and emission spectra of 1 in MeCN
(excitation at 425 nm) in the presence of (a) 0, (b) 1, (c) 3, (d)
12, or (e) 45 equiv of Ca(ClO4)2‚4H2O predissolved in MeCN
(0.01 M).
F IGURE 2. Two-photon excitation spectra of 1 in toluene or
MeCN and in the presence of 45 equiv of Ca2+
.
for metal ions, we have synthesized 1 with 1-cyano-2,5-
bis(styryl)benzene as the fluorophore and aza-15-crown-5
as the receptor. We now report that 1 is an effective TPF
sensor for the metal ions.
cross section of 1 in toluene is 320 GM at 740 nm. The
(2)
max
value of δ for 1 is slightly larger than 260 GM (λ
)
Synthesis of the sensor 1 is shown in Scheme 1.
Bromination of 2-cyano-1,4-dimethylbenzene followed by
phosphorylation produced A in high yield. Compound 1
was obtained in modest yield by successive condensation
of A with p-(N-isoamyl-N-phenyl)aminobenzaldehyde and
N-(p-formylphenyl)aza-15-crown-5.
Compound 1 is strongly fluorescent in MeCN (Φ )
0.47). Addition of Ca2+ to the solution of 1 in MeCN
induces a blue shift in the absorption spectra and a
gradual decrease in the absorption and fluorescence
intensity with a slight decrease in the fluorescent quan-
tum yield (Figure 1).
This may be due to the fact that complexation by a
metal ion reduces the donor character of the nitrogen
atom in the crown ether to induce the spectra changes.
The result of a J ob’s plot and the appearance of an
isosbestic point at 355 nm indicate the formation of 1:1
complex (Figure S1). The binding constant, expressed as
log K, estimated with the extent of fluorescence quench-
ing decreases in the order Ca2+ (4.26 ( 0.06) > Ba2+ (3.65
( 0.02) > Mg2+ (3.32 ( 0.03) > Na+ (3.24 ( 0.16) > K+
(2.40 ( 0.50) (Figures S2 and S4). Similar results were
previously reported.23
740 nm) reported for 1-cyano-2,5-bis(diethylaminostyryl)-
benzene.2c This may be due to the presence of the
diphenylamino group as the donor and/or the contribu-
tion of the excited-state excitation in the nanosecond
experiment. Moreover, δ also decreases by the change in
the solvent from toluene to MeCN. The origin of this
solvent effect is not clear at present. Nevertheless, the
intensity of the two-photon fluorescence was strong
enough for the measurement. When excess Ca2+ was
added, δ decreased even further, probably because the
electron-donating ability of the azacrown moiety is at-
tenuated upon complexation (Figure 2). Hence it is
possible to measure the metal ion concentration from the
decrease in the TPF upon addition of metal ions.
As expected, the TPF intensity decreased gradually
upon addition of metal ions (Figures S2 and S3). More-
over, the binding constants calculated with the TPF are
similar to those measured by OPF, i.e., Ca2+ (4.34 ( 0.08)
> Ba2+ (3.73 ( 0.04) > Mg2+ (3.57 ( 0.05) > Na+ (3.20 (
0.30) > K+ (2.10 ( 0.70). The larger standard deviation
for the two-photon process compared to that of the one-
photon process underlines the experimental difficulties
associated with measuring the weaker spectral intensi-
ties (Figures S2 and S3). On the other hand, the extent
of TPF quenching by the metal ions is slightly larger than
that of the one-photon process (Figure 3). Also, the two-
The two-photon cross section (δ) of 1 was determined
by using the two-photon-induced fluorescence measure-
ment technique.2a,5a Figure 2 shows that the two-photon
photon process shows slightly larger selectivity for Ca2+
.
Nevertheless, the trends observed in both experiments
are more or less the same. These results seem to indicate
(23) Fery-Forgues, S.; Le Bris, M.-T.; Guette´, J .-P.; Valeur, B. J .
Phys. Chem. 1988, 92, 6233.
5750 J . Org. Chem., Vol. 69, No. 17, 2004

CDCl3): δ 7.58 (s, 1H), 7.50 (s, 2H), 4.08 (m, 8H), 3.34 (d, 2H, J
) 9.0 Hz), 3.10 (d, 2H, J ) 9.0 Hz), 1.27 (m, 12H).
2-Cya n o-4-(d ieth ylp h osp h or yl)m eth yl-4′-[(N-isoa m yl-N-
p h en yla m in o)stilben e. LDA (1.5 M in c-Hex, 3.3 mL) was
slowly added to a solution containing A (2.0 g, 4.9 mmol) in THF
(50 mL) at -78 °C under Ar. To this solution was added p-(N-
isoamyl-N-phenyl)aminobenzaldehyde (1.2 g, 4.5 mmol) in THF
(20 mL), and the mixture was stirred for 6 h. The reaction was
quenched with water, and the product was extracted with ethyl
acetate. The organic layer was dried with MgSO4, and the
solvent was removed in vacuo. The product was purified by
column chromatography using ethyl acetate as the eluent.
Yield: 1.6 g (62%). 1H NMR (300 MHz, CDCl3): δ 7.70 (d, 1H,
J ) 9.0 Hz), 7.50 (m, 2H), 7.41 (d, 2H, J ) 9 Hz), 7.32 (t, 2H, J
) 9 Hz), 7.20 (d, 2H, J ) 9 Hz), 7.12 (m, 3H), 6.68 (d, 2H, J )
9 Hz), 4.06 (m, 4H), 3.74 (t, 2H, J ) 8 Hz), 3.10 (d, 2H, J ) 21
Hz), 1.57 (t, 3H, J ) 8 Hz),1.28 (t, 6H, J ) 6 Hz), 0.93 (d, 6H, J
) 6 Hz).
Com p ou n d 1. Synthesized by the same procedure as above.
The residue was purified by column chromatography using ethyl
acetate/hexane ) 2:1 as the eluent. Yield: 0.60 g (72%). Mp: 52
°C. IR: 2223 (CN). 1H NMR (300 MHz, CDCl3): δ 7.69 (d, 1H,
J ) 9 Hz), 7.65 (s, 1H), 7.59 (d, 1H, J ) 9 Hz), 7.41 (d, 2H, J )
9 Hz), 7.36 (d, 2H, J ) 9 Hz), 7.34 (d, 1H, J ) 18 Hz), 7.31 (s,
1H), 7.20 (d, 2H, J ) 9 Hz), 7.12 (d, 2H, J ) 9 Hz), 7.09 (d, 1H,
J ) 18 Hz), 7.02 (d, 1H, J ) 18 Hz), 6.84 (d, 2H, J ) 9 Hz), 6.77
(d, 1H J ) 18 Hz), 6.64 (d, 2H J ) 9 Hz), 3.76 (t, 2H, J ) 6 Hz),
3.64 (m, 20H), 1.60 (m, 3H), 0.93 (d, 6H, J ) 6 Hz). Anal. Calcd
for C44H51N3O4: C, 77.05; H, 7.49; N, 6.13. Found: C, 77.02; H,
7.46; N, 6.15.
F IGURE 3. A comparison of the percent quenching of one-
and two-photon excited fluorescence of 1 in MeCN in the
presence of 1 equiv of metal ions. The excitation wavelengths
for one- and two-photon processes are 425 and 800 nm,
respectively.
that similar mechanisms are operating in both one- and
two-photon sensors. Furthermore, this result suggests an
interesting possibility that efficient TPF probes for
biological substrates could be designed by using the same
strategy developed for the synthesis of one-photon sen-
sors by using an appropriate TPA chromophore.
In conclusion, we have synthesized a two-photon sensor
for the metal ions by using bis-styrylbenzene and aza-
crown ether as the fluorophore and receptor, respectively.
The sensor emits strong two-photon fluorescence when
excited by 800 nm laser photons. Moreover, the fluores-
cence is quenched upon complexation with metal ions,
and the binding constants measured by the one- and two-
photon fluorescence are similar. This result provides a
useful design strategy for the synthesis of the two-photon
sensors for biological applications.
Gen er a l P r oced u r e for Sp ectr oscop ic Stu d ies. The fluo-
rescence quantum yield of 1 was determined in MeCN by using
fluorescein (Φ ) 0.90) as the reference.24 It decreased from 0.47
to 0.45 upon addition of excess amount of Ca2+. The TPA cross
sections of 1 (5.0 µM) in toluene and MeCN both in the presence
and absence of Ca2+ were measured by the two-photon-induced
fluorescence method by using fluorescein (80 µM, pH ) 11) as
the reference.2a,5a One photon absorption and one- and two-
photon fluorescence titration experiments were performed by
adding various amounts of metal ions to a solution containing
5.0 µM of 1 in MeCN (Figures S2-S4). The binding constant,
log K, for each metal ion was calculated from the change in the
spectral intensities by using the Excel program as reported.25
Exp er im en ta l Section
Syn th esis. 2-Cya n o-1,4-bis(br om om eth yl)ben zen e. To a
solution containing 2-cyano-1,4-dimethylbenzene (10.0 g, 76.2
mmol) in CCl4 (300 mL) were added NBS (30.0 g, 168 mmol)
and benzoyl peroxide (0.40 g, 1.7 mmol). The mixture was
vigorously stirred under reflux for 2 h. After the usual workup,
the product was purified using flash column chromatography
on silica gel using hexane/ethyl acetate ) 10:1 as the eluent.
Ack n ow led gm en t. This work is supported by NRL-
MOST and CRM-KOSEF. H.M.K. and H.C.A. were
supported by BK21 program.
Su p p or tin g In for m a tion Ava ila ble: J ob’s plot, one-
photon absorption and emission, and two-photon fluorescence
spectra of 1 in the presence of Ba2+, Mg2+, Na+, and K+, and
plots of A0/A of one- and two-photon fluorescence vs the molar
equivalent of metal ions. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
Yield: 13 g (61%). H NMR (300 MHz, CDCl3): δ 7.69 (d, 1H, J
) 3.0 Hz), 7.60 (dd, 1H, J ) 3.0, 9.0 Hz), 7.53 (d, 1H, J ) 9.0
Hz), 4.62 (s, 2H), 4.46 (s, 2H).
2-Cyan o-1,4-bis[(dieth ylph osph or yl]m eth yl]ben zen e (A).
A solution of 2-cyano-1,4-R,R′-dibromomethylbenzene (3.70 g,
12.8 mmol) and P(OEt)3 (10.6 g, 64.0 mmol) in toluene were
heated to 120 °C for 5 h. Excess P(OEt)3 was removed in vacuo,
and the product was extracted with ethyl acetate. After drying
of the solution with MgSO4, the solvent was evaporated to obtain
slightly yellow oil. Yield: 5.0 g (98%). 1HNMR (300 MHz,
J O049124A
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J . Org. Chem, Vol. 69, No. 17, 2004 5751