m-Benziporphodimethene: A new porphyrin analogue fluorescence zinc(II) sensor

Article abstract of DOI:10.1039/b714412a

m-Benziporphodimethene is presented here as a long-wavelength Zn ²⁺ specific chemosensor; this sensor shows fluorescence switch-on upon Zn²⁺ binding with no apparent background fluorescence. The Royal Society of Chemistry.

Full text of DOI:10.1039/b714412a

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m-Benziporphodimethene: a new porphyrin analogue fluorescence zinc(II)
sensorw
Chen-Hsiung Hung,*a Gao-Fong Chang,a Anil Kumar,a Geng-Fong Lin,b
Li-Yang Luo,ac Wei-Min Chinga and Eric Wei-Guang Diauc
Received (in Cambridge, UK) 18th September 2007, Accepted 3rd December 2007
First published as an Advance Article on the web 21st December 2007
DOI: 10.1039/b714412a
m-Benziporphodimethene is presented here as a long-wavelength
Zn2+ specific chemosensor; this sensor shows fluorescence
switch-on upon Zn2+ binding with no apparent background
fluorescence.
11,16-Bis(phenyl)-6,6,21,21-tetramethyl-m-benzi-6,21-porphodi-
methene (1), a porphyrin analogue, was synthesized through
an acid catalyzed condensation reaction of a,a-dihydroxy-1,3-
diisopropylbenzene, pyrrole and benzaldehyde at room tem-
perature, as shown in Scheme 1.30 Compound 1, isolated in
27% yield as a red solid, was purified by silica gel column
chromatography.z Because of the discrete conjugated system,
the absorptions in UV-Vis spectrum for 1 is broad. As shown
in Fig. 1 (top, black line), a higher energy Soret-like absorp-
tion band is observed at 349 nm, while broad low energy bands
are apparent in the visible region at 514 and 542 nm. An
extinction coefficient of 3.59 ꢀ 104 Mꢁ1 cmꢁ1 for p to p*
transition at 349 nm is about one order smaller than that of the
Soret band for tetraphenylporphyrin.31 Compound 1 is non-
fluorescent in free base form. As the formation of a Znꢂ1
complex occurred, after the addition of Zn2+, there was an
instant change in solution color from pink to greenish blue,
which accompanied the shifting of the visible band from 542 to
639 nm. Complex Znꢂ1 formation was confirmed by observing
the loss of the inner amino protons by NMR, and by the
observation of a mass spectrum peak of m/z 594.1880, corre-
sponding to [M ꢁ Cl]+ upon the addition of Zn2+ into a
solution of 1.z The X-ray single-crystal structurey of the
complex unambiguously confirmed the coordination of zinc
ion with benziporphodimethene through three pyrrolic nitro-
gens and an axial chloride (see ESIw). The structure of the zinc
complex gave a co-planar tripyrrin unit in contrast to the
distorted benziporphodimenthene core in the free-base
form, and this might contribute to its unusual fluorescence
turn-on.30
Fluorescent metal ion sensors have become increasingly im-
portant tools for the quantitative, real time monitoring of
metal ions in biological samples.1–10 Metal coordination that
increases the fluorescence intensity of the chromophore is
called chelation-enhanced fluorescence (CHEF). Most CHEF
sensors are based on fluorophores, such as fluorescein,11,12
dansyl,13–16 and anthracene,2,4 which emit at wavelengths
shorter than 600 nm. Sensors that are effective above 600 nm,
where background fluorescence is minimal, minimize light
induced tissue damage, penetrate better and scatter less in
optically diffuse samples should be further developed.17–19
Zinc is, after iron, the second most abundant transition
metal in mammals,20 where it plays important roles in various
biological processes such as neurotransmission, signal trans-
duction and gene expression.21,22 A large body of knowledge
exists on the structural chemistry of zinc, but little is known
about zinc homeostasis.23 This lack of knowledge can be
attributed to the spectroscopically silent nature of d10 Zn2+
.
The well-established 8-aminoquinoline-based zinc chemosen-
sors show about a 300-fold increase in fluorescence intensity
after zinc chelation.23 Lippard and co-workers recently re-
ported low background Zn2+ induced fluorescence turn-on
molecules that were suitable for biological imaging.24 Pro-
tein25 and peptide based26 sensing approaches have also
emerged. Other sensors including ACF,27 Rhod–Zn,28 and
ZinPyr,29 have also been developed, but a majority of the
available zinc sensors suffer from high background fluores-
cence and weak fluorescence enhancement. Herein, we provide
a novel m-benziporphodimethene based molecule, which im-
parts no background emission, and, upon Zn2+ binding,
increases fluorescence intensity. This compound represents a
turn-on probe for Zn2+ sensing.
Compound 1 has a superb chemosensory response when
Zn2+ is added. The non-fluorescent solution of the free-base
form changes to a red emitting solution with lem at 672 nm
upon the addition of Zn2+ (Fig. 1 bottom). The Stokes shift of
33 nm is not large, but is comparable to other chromophores.
The fluorescence quantum yield (FF) of 0.34 at the S1 state in
degassed toluene at room temperature for Znꢂ1 is calculated
a Institute of Chemistry, Academia Sinica, Nankang, Taipei 105,
Taiwan. E-mail: chhung@chem.sinica.edu.tw;
Fax: +886-2-27831237
b Department of Chemistry, Tamkang University, Tamsui, Taipei 251,
Taiwan
c The Department of Applied Chemistry, National Chiao Tung
University, Hsinchu, 300, Taiwan
w Electronic supplementary information (ESI) available: Synthetic
procedures for 1 and Znꢂ1, Job plot, and ORTEP plot of Znꢂ1. See
DOI: 10.1039/b714412a
Scheme 1
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Fig. 2 Hill plot from the spectrophotometric titration in Fig. 1 (top).
The data fitting to a straight line with a slope of 1 gives an intercept
of 5.312.
demonstrate that only Zn2+, Hg2+ and Cd2+ turn on fluor-
escence, whereas other physiologically important cations such
as Na+, Mg2+ and Ca2+ do not. As is observed for most of
Zn2+ chemosensors,29 Cd2+ and Hg2+ bind to 1 and enhance
fluorescence, but to a lesser degree than zinc. The red-shift of
lmemaixssionfrom 672 nm for Zn2+ to 682 and 706 nm for Cd2+
and Hg2+, respectively, in acetonitrile solution suggests that
colorimetric assay can differentiate the three fluorescence turn-
Fig. 1 UV-Vis spectra (top) and fluorescence spectra (bottom) show
changes upon titration of 1 (2 ꢀ 10ꢁ5 M in acetonitrile) with increasing
on metal ions. The ratio of fluorescence intensity for Zn2+
,
concentrations of Zn2+
. Excitation wavelength for fluorescence
Cd2+ and Hg2+ is 17 : 6 : 1, which is large compared to other
Zn2+ chemosensors.36,37 Since the concentrations of Cd2+
and Hg2+ in healthy cells are low, these ions should not
spectra is 564 nm, the isosbestic point in UV-Vis.
from the ratio of ts/tr, where ts is the fluorescence lifetime
obtained from fluorescence decay recorded by means of time-
correlated single photon counting and the radiative lifetime, tr,
is calculated using the Strickler–Berg relation.32 Using the
same method, fluorescence quantum yields of 0.21 and 0.05
were obtained for Cdꢂ1 and Hgꢂ1, respectively. In comparison,
the fluorescence quantum yields for 5,10,15,20-tetraphenyl-
porphyrin free base and its zinc complex were reported to be
0.11 and 0.033, respectively.33 Importantly, chelation turn-on
resulted in fluorescence enhancement to a level that was higher
than what was previously observed for other zinc specific
chemosensors.10,27,29,34,35 Based on Job plot analysis, com-
pound 1 forms a 1 : 1 complex with Zn2+ (see ESIw)
interfere with the probing of Zn2+
.
Two competitive analyses were carried out to further under-
stand the selectivity of 1 for Zn2+ using either 1 in the presence
of a variety of metal ions, treated with Zn2+, or using a
solution of Znꢂ1 complex treated with other metal ions. As
indicated by red and green bars shown in Fig. 3, a significant
level of fluorescence enhancement was observed for zinc ion
A spectrophotometric titration of 1 with Zn2+ is included in
Fig. 1. The sharp change in absorption spectra for 1 after the
addition of Zn2+ demonstrates the perturbation of the elec-
tronic structure of compound 1. Two isosbestic points are
observed at lmax = 438 and 564 nm, suggesting a neat
complexation process. Incremental addition of Zn2+ also
resulted in fluorescence enhancement that saturated at the
1.9 equiv. range when 20 mM of 1 were used for titration
(Fig. 1 bottom). The increment of UV-Vis spectrum (Fig. 1
top) was used to obtain the stability constant in a Hill plot, as
shown in Fig. 2. This analysis provided a Hill plot coefficient
of 1 and a stability constant of 2.05 ꢀ 105. The large stability
constant suggests that 1 can be used to detect Zn2+ at low
concentrations.
Fig. 3 Top: Fluorescence intensities after treating 1 in acetonitrile
with a variety of metal chlorides, and relative binding abilities of 1 to
different metal ions compared to Zn2+ (excitation at 600 nm).
Conditions: Black bar, 3 ꢀ 10ꢁ6 M of 1 with 20 equiv. of metal ion.
Red bar, 3 ꢀ 10ꢁ6 M of 1 in 20 equiv. of metal ion with 20 equiv. of
Zn2+ then added. Green bar, 3 ꢀ 10ꢁ6 M of 1 in 20 equiv. of Zn2+
with 20 equiv. of metal ion then added. Metal ions were dissolved in
water–methanol (1 : 1) at 298 K. Bottom: The fluorescence image of a
solution of 1 plus mixed metal chlorides excited by a commercially
available UV lamp (l = 365 nm).
The selectivity of compound 1 for Zn2+ makes it a reliable
reagent for future applications in biological systems. Fig. 3
shows the fluorescence intensities of 1 treated with physiolo-
gically important metal ions. The black bars in the upper panel
and the photograph inserted in the lower panel of Fig. 3
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y CCDC 662990. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/b714412a
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when competing with most metal ions. The highly elevated
fluorescence intensity in the presence of Zn2+ where binding
was competitive for Cd2+ and Hg2+ shows that Zn2+ forms a
more stable complex and can readily replace these ions in the
Cdꢂ1 or Hgꢂ1 complexes. Among the tested metal ions, Cu2+
,
Cr3+ and Ni2+, which bind strongly to 1 and quench fluor-
escence because of their paramagnetic effect, interfere with
Zn2+ detection. However, these ions did not provide a false
positive signal that would mimic the presence of Zn2+, and
they are not typically present at high concentrations in biolo-
gical systems.38 To further evaluate the interference of metal
ions to zinc sensing, the solution of 1 was added a solution
containing all the metal ions tested in Fig. 3, except Cd2+ and
Hg2+. The results in Fig. 4 demonstrated a rapid fluorescence
response with the intensity reaching a plateau after 5 min.
In conclusion, we show that m-benziporphodimethene, a first-
generation porphyrin analogue Zn2+ chemosensor, is non-fluor-
escent in the free-base form and exhibits fluorescence turn-on
when bound to Zn2+. This sensor exhibits an unusual low energy
absorption maximum at 594 nm and an emission wavelength at
672 nm. It is interesting to see that 1, a simple porphyrin
analogue, can readily be used as a zinc-specific sensor with no
background fluorescence, and with long wavelengths in both
excitation and emission lmax. Its utility in photophysical applica-
tions and physiological imaging are foci of ongoing work.
We thank the National Science Council of Taiwan for
research funding, researchers at the Mass Spectrometry Center
of the Institute of Chemistry at Academia Sinica for the mass
spectra measurements and the National Center for High-
Performance Computing for use of the computation facilities.
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Notes and references
z Znꢂ1: 1H NMR (400 MHz, acetone-d6, 298 K): d 1.97 (s, 6H; meso-
CH3), 2.14 (s, 6H; meso-CH3), 6.05 (s, 2H; 13,14-H), 6.77 (d, 3J(H,H) =
4.70, 2H; 9,18-H), 6.99 (d, J(H,H) = 4.70, 2H; 8,19-H), 7.31–7.53 (m,
3
13H; 2,3,4-H, meso-phenyl), 8.69 (s, 1H; 22-H); UV-Vis (CH3CN) [lmax
/
37 X.-A. Zhang, K. S. Lovejoy, A. Jasanoff and S. J. Lippard, Proc.
Natl. Acad. Sci. U. S. A., 2007, 104, 10780–10785.
38 N. C. Lim and C. Brueckner, Chem. Commun., 2004, 12,
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nm (log e)]: 350 (4.58), 593 (4.29), 639 (4.56); Anal. (%). Found (calc for
C38H32N3ZnCl): C 71.38 (72.48); H 5.57 (5.13); N 5.85 (6.68); HR FAB-
+
MS (m/z): calc. [M ꢁ Cl]+ = 594.1888; obs. [M ꢁ Cl] = 594.1880.
ꢃc
This journal is The Royal Society of Chemistry 2008
980 | Chem. Commun., 2008, 978–980