Quantitative fluorescent detection of pyrophosphate with quinoline-ligated dinuclear zinc complexes

Article abstract of DOI:10.1021/ic401605m

Dinuclear zinc complex [Zn₂(TQHPN)(AcO)]²⁺ exhibits characteristic fluorescence response (λ_ex = 317 nm and λ_em = 455 nm) toward pyrophosphate (PPi) with maximum fluorescence upon 1:1 Zn₂(TQHPN)-PPi co

Full text of DOI:10.1021/ic401605m

Communication
pubs.acs.org/IC
Quantitative Fluorescent Detection of Pyrophosphate with
Quinoline-Ligated Dinuclear Zinc Complexes
Yuji Mikata,*,†,‡ Anna Ugai,‡ Risa Ohnishi,‡ and Hideo Konno§
†KYOUSEI Science Center for Life and Nature and ‡Department of Chemistry, Faculty of Science, Nara Women’s University, Nara,
Nara 630-8506, Japan
§National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
S
* Supporting Information
ABSTRACT: Dinuclear zinc complex [Zn2(TQHPN)-
(AcO)]2+ exhibits characteristic fluorescence response (λex
= 317 nm and λem = 455 nm) toward pyrophosphate (PPi)
with maximum fluorescence upon 1:1 Zn2(TQHPN)−PPi
complex formation. The crystallographic investigation
utilizing P1P2−Ph2PPi revealed that the fluorescent
response mechanism is due to intramolecular excimer
formation of two quinoline rings.
Figure 1. (a) Structures of the ligands. (b) ORTEP plot for 1·(ClO4)2·
2CH3CN in 50% probability. Atoms of counteranions, hydrogens, and
solvents were omitted for clarity.
he development of anion recognition systems has become
T
an important field of research. Phosphorus anions including
pyrophosphate (P2O74−, PPi), adenosine triphosphate (ATP),
and phosphorylated proteins and carbohydrates play many
important roles in biology; thus, an accurate sensing system for
such phosphorus anions could provide new insight to the
phosphorylation process.1,2 Fluorescent detection with chemical
probes in the biological system is one of the ideal methodologies
for anion sensing in solution because of its high sensitivity and
high spatial resolution, as well as rapid response times. To be
useful in a biological environment, fluorescence probes need high
target specificity and applicability to quantitative analysis. The
ability of the probe to quickly respond to concentration changes
of the analyte would be ideal for real-time quantitative analysis.3,4
PPi-specific fluorescent probes have been exploited in recent
decades,1,2,5−17 and many of these sensing molecules contain a
dinuclear metal core for substrate binding interactions. In this
Communication, pyrophosphate-induced fluorescence changes
of a dinuclear zinc complex supported by quinoline-based
ligands, N,N,N′,N′-tetrakis(2-quinolylmethyl)-2-hydroxy-1,3-
propanediamine (HTQHPN; Figure 1a),18 have been inves-
tigated. This metalloligand generates a fluorescent 1:1 complex
with PPi (ϕ = 0.029) via intramolecular excimer formation.
The HTQHPN ligand afforded a dinuclear zinc complex upon
mixing with 2 equiv of Zn(AcO)2 (Figures S1 and S2 in the
Supporting Information, SI). Figure 1b shows the crystal
structure of the acetate-bridged complex [Zn2(TQHPN)-
(OAc)](ClO4)2 [1·(ClO4)2]. Both zinc centers are pentacoordi-
nate, exhibiting slightly distorted trigonal-bipyramidal geo-
metries (τ = 0.81 for Zn1 and 0.88 for Zn2).19 The dizinc
complex 1 in an aqueous N,N-dimethylformamide (DMF)
solution (1:1 DMF/H2O) exhibited weak fluorescence upon
excitation at 317 nm, but with the addition of PPi to this solution,
fluorescence enhancement was observed (Figure 2). The
fluorescence intensity at 455 nm in the presence of 1 equiv of
Figure 2. (a) Fluorescence spectra for 34 μM 1 in DMF/H2O (1:1) in
the presence of increasing amounts of PPi ranging from 0 to 680 μM
concentration at 25 °C (λex = 317 nm). (b) Plot of the fluorescence
intensity change at 455 nm.
PPi was enhanced 8-fold in comparison to 1 in the absence of
PPi. The addition of further equivalents of PPi to the zinc dimer
complex in solution leads to a decrease in the fluorescence
intensity (Figure 2b). In the presence of 15 equiv of PPi,
fluorescence completely disappears and UV−vis spectra shown
in Figure S3 in the SI reveal that free HTQHPN was formed by
the removal of zinc by excess PPi.20,21 This was also confirmed by
electrospray ionization mass spectrometry measurement (Figure
S40 in the SI). Large excess of PPi increased the pH of the
solution, but this is not the reason for decomplexation of
Zn2(TQHPN)−PPi because a parallel experiment employing a
buffer solution with constant pH (pH = 7.5) gave similar results
(Figure S42 in the SI).
The fluorescence enhancement is specific to PPi because a
much smaller response was observed for ATP (50% of PPi) and
adenosine diphosphate (30% of PPi) in the presence of 1 equiv of
the anion (Figures 3 and S4 in the SI). Other anions including
monophosphates (AMP and PO43−) did not induce any
Received: June 25, 2013
© XXXX American Chemical Society
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dx.doi.org/10.1021/ic401605m | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Communication
Figure 3. Plot of the fluorescence intensity of a 34 μM Zn2(TQHPN)
complex (1) at 455 nm (λex = 317 nm) in DMF/H2O (1:1) at 25 °C in
the presence of 1 equiv of anions (filled bars), 1 equiv of anion + 1 equiv
of PPi (gray bars), and 1 equiv of PPi + 10 equiv of anion (open bars).
fluorescence enhancement. Importantly, this fluorescent PPi
sensing is valid even in the presence of other anions (1 equiv),
indicating the preferred binding of PPi over other anions
including other phosphate species. However, a further addition
of excess (10 equiv) phosphate species results in considerable
fluorescence quenching because of the removal of zinc from the
ternary complex (Figure S41 in the SI). The fluorescence of
Zn2(TQHPN)−PPi was not perturbed by 10 equiv of non-
phosphate anions.
In many cases, fluorescent PPi sensing was achieved by
intermolecular excimer formation between two chromo-
phores6,13,15 or electrostatic perturbation of metal
center(s)9−11 upon PPi binding. However, there are very few
examples in which the spectroscopic response mechanism was
discussed based on structural analysis by X-ray crystallogra-
phy.9,16
All crystallization trials for the Zn2(TQHPN)−PPi complex
resulted in free ligand HTQHPN formation. Using the diphenyl
ester of PPi, P1P2−diphenyl pyrophosphate (Ph2PPi),22 as a
crystallizable substitute of PPi was successful. Fluorescence
enhancement of the Zn2(TQHPN) complex in the long-
wavelength region (433 nm) was also induced by Ph2PPi, and
in parallel, the monophenyl phosphate (PhOPO32−) exhibited
similar weak fluorescence spectral changes in comparison to
PO43− (Figure S5 in the SI).
Figure 4. ORTEP plot for (a) 2·ClO4·2THF·1.5H2O and (b) 3·ClO4·
THF·6H2O in 50% probability. Atoms of counteranions, hydrogens, and
solvents were omitted for clarity.
intramolecular interactions similar to acetate-bridged complex 1.
Figure S6 in the SI highlights the difference in interquinoline
interaction of the Ph2PPi complex 2 in comparison to the acetate
or monophosphate complexes 1 and 3 in the crystal structures.
Analogous excimer formation induced by PPi binding was
observed in the N,N,N′,N′-tetrakis(8-quinolylmethyl)-2-hy-
droxy-1,3-propanediamine (HT8QHPN; Figure 1a) system 4
(Figures S7−S9 and S12a in the SI). In this case, the dizinc center
of 4 was hexa- and pentacoordinate with P1P2 ligation by Ph2PPi,
missing the H2O ligand bound to Zn2 found in complex 2. In
contrast, for the N,N,N′,N′-tetrakis(1-isoquinolylmethyl)-2-
hydroxy-1,3-propanediamine (1-isoHTQHPN; Figure 1a) com-
plex 5, neither fluorescence enhancement nor excimer formation
in the crystal structure was observed (Figures S10−S11 and S12b
in the SI), likely because PPi binding does not promote close
interisoquinoline interaction, as observed in the crystal structure.
P1P2 oxygen chelation to a zinc center of the metalloligand is
indispensable for the excimer formation observed in the
Zn2(TQHPN) system.
A titration experiment of a PPi solution with 1 to determine
the concentration of PPi was conducted (Figures 5 and S13 in the
SI). Maximum fluorescence was achieved at [1] = [PPi], and
then excess 1 quenched the fluorescence probably because of
formation of the [Zn2(TQHPN)]2−PPi species, in which the
quinoline excimer structure was collapsed by the mono-
phosphate-like binding of PPi to each dizinc unit. As seen in
Figure 5, a very sharp titration curve suitable for determination of
the concentration of a PPi solution was obtained because of self-
quenching of Zn2(TQHPN) at a late stage of the titration
(Figure S43 in the SI).
Having confirmed that phenyl esters can be used as an
alternative for inorganic phosphate species in this work, the
crystal structure analyses for complexes of Zn2(TQHPN)−
2−
Ph2PPi (2) and Zn2(TQHPN)−PhOPO3 (3) were inves-
tigated (Figure 4). For complex 2, both zinc centers adopt a
hexacoordinate geometry ligated by three nitrogen atom and an
μ-alkoxo oxygen atom from TQHPN, a bridging phosphate
oxygen atom, and an oxygen atom from water or pyrophosphate
(Figure 4a). As a result of Ph2PPi binding, two quinoline groups
opposite to the PPi side are forced into a close parallel
arrangement that is suitable for enhancing excimer emission at a
lower-energy region. This is the first crystal structure in which
PPi-induced excimer arrangement of the probe(s) is clearly
shown. On the other hand, both zinc centers in the mono-
phosphate complex 3 were of pentacoordinate geometry because
of the lack of an extra phosphate moiety in comparison with the
diphosphate PPi (Figure 4b). This distorted trigonal-bipyramidal
dizinc center (τ = 0.61 for Zn1 and 0.78 for Zn2) of 3 allows for
an extended structure for all quinolines with limited quinoline
B
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Inorganic Chemistry
Communication
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedure for synthesis and titration experiment,
Tables S1 and S2, Figures S1−S43, and crystallographic data in
CIF format. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mikata@cc.nara-wu.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Prof. Tim Storr and John Thompson of
Simon Fraser University for their helpful discussion about X-ray
crystallography. This work was supported by the Research for
Promoting Technological Seeds, JST, Adaptable and Seamless
Technology Transfer Program through Target-driven R&D, JST,
Grant-in Aid for Scientific Research from the MEXT, Japan, and
the Nara Women’s University Intramural Grant for Project
Research.
C
dx.doi.org/10.1021/ic401605m | Inorg. Chem. XXXX, XXX, XXX−XXX