Dizinc phosphohydrolase model built on a m-terphenyl scaffold and its use in indicator displacement assays for pyrophosphate under physiological conditions

Article abstract of DOI:10.1002/ejoc.200800882

A dinucleating ligand {1,3-bis[2-(di-2-picolylaminomethyl)-phenyl]benzene, L2} built on a m-terphenyl scaffold was prepared. The dissociation constants for the dizinc complex of L2 (Zn2L2) binding to phosphate, pyrophosphate and commercially available com

Full text of DOI:10.1002/ejoc.200800882

FULL PAPER
DOI: 10.1002/ejoc.200800882
Dizinc Phosphohydrolase Model Built on a m-Terphenyl Scaffold and Its Use
in Indicator Displacement Assays for Pyrophosphate Under Physiological
Conditions
Michael K. Coggins,[a] Austa M. Parker,[a] Anshuman Mangalum,[a]
Gabriela A. Galdamez,[a] and Rhett C. Smith*[a]
Keywords: Biomimetic synthesis / N ligands / Enzyme models / Metalloenzymes / Receptors / Phosphohydrolase / Bimetal-
lic complexes / Sensors / Pyrophosphate
A dinucleating ligand {1,3-bis[2-(di-2-picolylaminomethyl)-
phenyl]benzene, L2} built on a m-terphenyl scaffold was pre-
pared. The dissociation constants for the dizinc complex of
assays with selectivity for pyrophosphate over other anions
were achieved with Zn2L2 as the receptor component. These
assays show good response characteristics for quantification
L2 (Zn2L2) binding to phosphate, pyrophosphate and com- of pyrophosphate concentrations as low as 2.5ϫ10–6
M
, sug-
mercially available complexometric indicators were deter- gesting their utility for measuring pyrophosphate levels in
mined under physiological pH [10 m N-(2-hydroxyethyl)pip- synovial fluid.
M
erazine-NЈ-2-ethanesulfonic acid (HEPES) buffer at pH 7.4].
Colorimetric and fluorescence-based indicator displacement
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
phate depositions form between joints,[28] and because there
are already numerous phosphate (Pi) sensors relative to PPi
sensors.[29]
Introduction
The indicator displacement assay (IDA, Scheme 1)
strategy is a simple and increasingly popular approach to
colorimetric and fluorescent chemosensing.[1,2] Early IDAs
were developed to recognize a variety of anions,[3–16] includ-
ing inorganic phosphates,[15,17] and have even been adapted
to challenging problems such as nitric oxide (NO) genesis
by living cells[18–20] and nerve-agent detection.[21] The inter-
play between IDA receptor, indicator, target analyte, and
potentially interfering species in the sample must be care-
Scheme 1. General indicator displacement assay strategy in which
fully controlled in order to achieve the desired sensitivity the receptor is a bimetallic complex (I = indicator, A = analyte).
This representation assumes indicator emission enhancement upon
and selectivity. Numerous multipodal and compartmental
displacement.
complexes have served as IDA receptors.[22–26] The design
of such receptors can be inspired by enzyme active sites
known to bind target analytes. Dizinc phosphohydrolase
Our previous work on IDAs[30,31] included a study[30]
which built on extensive work by others who established m-
models, for example, are attractive candidates for detection
xylylene scaffolded dizinc complexes as preeminent recep-
of inorganic phosphates or phosphorylated biomolecules
tors for phosphate derivatives under physiologic condi-
such as proteins or phospholipids, which are important bio-
tions.[16,22,32,33] Zn2L1 (the product of Zn2+ addition to
aqueous L1, Scheme 3), for example, has a Zn–Zn distance
(3.0 Å)[34] somewhat less than that in phosphotriesterase
(3.5 Å), which binds with high specificity to Pi deriva-
tives,[35] whereas Zn2L1 and related complexes bind both Pi
and PPi.[36,37] We tested Zn2L1 as an IDA receptor with
various commercial indicators, but the similar binding af-
finity of Pi and PPi to Zn2L1 made their differentiation dif-
ficult.[30] The Zn–Zn distance should play a key role in ana-
lyte preference. The dependence of phosphoester hydrolysis
activity on Zn–Zn distance in phosphohydrolase models
has been studied employing, for example, xylyl, naphthyl,
logical contributors in metabolic and signaling pathways.[27]
Pyrophosphate (PPi) is of particular interest to us because
synovial fluid [PPi] is a diagnostic marker used to differen-
tiate osteoarthritic conditions wherein calcium pyrophos-
[a] Department of Chemistry and Center for Optical Materials Sci-
ence and Engineering Technologies (COMSET), Clemson Uni-
versity,
Clemson, SC 29634, USA
E-mail: rhett@clemson.edu
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2009, 343–348
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R. C. Smith et al.
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anthryl, and biphenyl spacers between Zn-chelating moie- ported for L1.[30] The binding strength and optical changes
ties.[38] Although these spacers have provided some func- of commercial indicators (Scheme 3) upon binding to
tional models, an emerging theme in bimetallic enzyme Zn2L2 were probed to reveal any differences from m-xylyl-
modeling has been to position metals within a steric pocket ene scaffolded analogues. Data from absorption spectro-
to exert control over metal–substrate interactions. This scopic titrations were used to calculate dissociation con-
same strategy could prove useful for developing sensors as stants (Kd, Table 1) by a modification of the Benesi–Hilde-
well, because steric coordination control may alter analyte brand method (Benesi–Hildebrand plots are provided in the
selectivity and affinity. Notable bulky ligands for enzyme Supporting Information).[47] Binding data for several indi-
modeling include m-terphenyl carboxylates, particularly for cators with Zn2L2, as well as for m-xylylene-based Zn2L1
diiron and dicopper enzyme models.[39–42] This established (which has a bridging phenolate) and Zn2L3 (lacking a
success of m-terphenyl scaffolds for supporting bimetallic bridging ligand) are provided in Table 1. It is worth noting
model complexes led us to explore translocation of the that the measured affinity of indicators for Zn2+ complexes
metal-ligating units from their typical position on the cen- is several orders of magnitude lower than that of Zn2+ for a
tral aryl ring onto the flanking rings, i.e. at the benzylic dipicolylamine receptor,[48–50] precluding the possibility that
sites of 1 (Scheme 2). This substitution pattern could pro- the indicators may be demetallating the receptor. Despite
vide useful dinucleating ligands for a variety of enzyme anticipated differences in average Zn–Zn distances, a similar
models for both catalytic and chemosensor applications. range of Kd values were found for all three complexes
Herein we discuss a dizinc complex of one such m-ter- (Table 1). The similarities are presumably due to the flexi-
phenyl-scaffolded ligand (L2, Scheme 3) and report its util- bility of the L2 and L3 scaffolds, which allows access to
ity as the receptor component of IDAs that are selective a range of Zn–Zn distances in order to optimize binding
for PPi at physiological pH. Binding and selectivity of L2 interactions with a particular indicator.
complexes are compared with those of two related dinucle-
ating ligands (L1 and L3) that have been used in indicator
displacement assays in biological contexts.
Scheme 2. Preparation of L2: i. 1) 1.1 equiv. nBuLi, –78 °C; 2)
3.5 equiv. 2-tolylmagnesium bromide, ∆; 3) H+. ii. N-bromosuc-
cinimide, benzoyl peroxide, CHCl3, ∆. iii. bis(2-picolyl)amine, tri-
ethylamine, THF.
Results and Discussion
There is only one example of a m-terphenyl scaffold sup-
porting a bimetallic complex in which ligands are only pres-
ent on the flanking aryl rings (rather than on the central
ring), a bimetallic palladium complex with a Pd–Pd dis-
tance of 3.6 Å.[43] Molecular modeling, however, suggested
that such a scaffold is capable of supporting M–M distances
relevant for bimetallic enzyme modeling (typically 3–
5 Å).[44] We therefore sought to incorporate ligands for
binding biologically relevant metals, particularly zinc, onto
the m-terphenyl scaffold. The bis(2-picolyl)amino unit was
selected for our initial study on the basis of its strong affin-
ity for Zn2+ and its widespread use in previous models.[22]
Scheme 3. Binucleating ligands L1–L3 and complexometric indi-
cators tested for displacement by inorganic phosphates. Only one
protonation state/resonance structure is provided for each.
Ligand L2 was readily prepared (Scheme 2) from known
Competitive binding analysis of absorption data from ti-
precursor 2[45] in one step through a procedure analogous tration of indicator-Zn2L2 complexes with Pi and PPi re-
to that used to prepare L1.[46] The Zn2L2 complex used vealed Kd for Pi (3.1ϫ10–5 ) and PPi (2.4ϫ10–6 ) on ap-
in absorption spectroscopic studies was prepared in situ in proximately the same order of magnitude as those reported
HEPES buffer (10 m, pH 7.4), following the procedure re- for Zn2L1 complexes (9.1ϫ10–6  and 1.5ϫ10–6  for Pi
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Dizinc Phosphohydrolase Model
Table 1. Absorption data and photos demonstrating the range of ports that these anions can displace indicators from bime-
tallic receptors.[11] None of the indicators were displaced
colorimetric responses observed in free, bound and pyrophosphate-
displaced states. λfree is the absorption maximum for the indicator
from Zn2L2 to any appreciable extent by glutamate or as-
alone and λZn2L2 is the absorption maximum for the indicator when
partate, indicating that Zn2L2 maintains selectivity for
bound to the Zn2L2 complex. The photos under each absorption
phosphates over carboxylates, a property previously noted
wavelength is of the color observed in the cuvettes by eye at
5ϫ10–5  indicator concentration. All data are for solutions in
1 m HEPES buffer at pH 7.4. Absorption spectra for these experi-
ments are provided in the Supporting Information.
for xylylene-bridged dizinc analogues.[32]
Although colorimetric assays are convenient for assessing
analyte presence visually, fluorescent sensors exhibiting
emission enhancement in response to analytes are consider-
ably more sensitive and thus preferred for biological studies.
Esculetin (ESC, Scheme 3), a catechol-derivatized fluores-
cent indicator previously used in IDAs,[19] was thus tested
for detection of PPi by fluorescence spectroscopy. Esculetin
is effectively bound by Zn2L2 (Kd = 3.85ϫ10–5 ), and the
ESC-Zn2L2 complex is only 25% as emissive as free escule-
tin. Near full restoration of emission to that of the free
indicator was achieved upon addition of 1.5 equiv. PPi (Fig-
ure 1), corresponding to a 4.1-fold emission enhancement.
The fluorescent turn-on IDA using ESC-Zn2L2 has a mod-
est sensitivity limit (based on 5% increase in integrated
emission) estimated at 2.5ϫ10–6 . This is, however, within
the level required for detection of biologically relevant [PPi]
found in synovial fluid (8.6ϫ10–6  to 15.9ϫ10–6 ),
which is elevated to higher concentrations in arthritis
patients.[28,52] Because clinical methods for measuring syno-
vial [PPi] rely most commonly on radiological methods and
may not readily differentiate phosphate from pyropho-
phosphate,[28,53] a sensitive fluorescence assay could eventu-
ally prove more economical and convenient for some practi-
cal applications. Once PPi selectivity was confirmed in the
presence of 5 equiv. Pi per PPi in solution, the viability of
[a] Y = yes, N = no. Indicator is considered to be displaced when
there is Ͼ50% return in absorbance at λbound to the absorbance at
that wavelength for the unbound form.
and PPi, respectively).[51] It is notable that although PPi is the Zn2L2 IDA for providing quantitative data on [PPi] was
bound equally well by both dizinc species, Zn2L2 binds Pi examined. Calibration curves constructed from absorption
three times less strongly than does Zn2L1. The reduced af- (Figure 2, a) and fluorescence (Figure 2, b) spectroscopic
finity of Zn2L2 for Pi presumably stems from the larger Zn– data indicate a linear response as a function of [PPi] in
Zn separation accessible using the m-terphenyl scaffold vs. HEPES buffer at physiological pH. The operational range
that provided by the m-xylylene spacer and anchoring of obtainable from absorption (on the order of 10–4 ) vs.
zinc centers by the µ-phenolate in L1.
fluorescence (on the order of 10–6 ) data emphasizes the
The enhanced preference for binding PPi over Pi sug- approximate two orders of magnitude greater sensitivity of
gested that IDAs with improved selectivity for PPi should the later method; however, both techniques appear well
be possible using L2. Four commercial complexometric in- suited for quantitative measurements.
dicators previously shown[30] to exhibit notable differences
in their absorption spectra between free and dizinc-bound
states were tested (Table 1). All of these indicators provide
IDAs selective for PPi over Pi and other simple anions (no
displacement of indicator is observed upon addition of ex-
–
2–
–
cess F–, Cl–, Br–, I–, AcO–, NO3 , CO3 and HSO4 ). The
zinc centers in Zn2L2 are not anchored by an intramolecu-
lar bridging ligand (cf. L1), so the ability of Zn2L2 to ex-
pand and accommodate a larger anion such as triphosphate
may be envisioned. Addition of one equiv. of triphosphate,
however, did not significantly displace any of the indicators.
Because biological or environmental assays are targeted end
uses of these IDAs, other potentially interfering bipodal
biological anions were also tested. Biomolecules such as
glutamate and aspartate are the most obvious candidates
for testing in this regard due to their similarity in size (i.e.,
Figure 1. Change in emission intensity of ESC-Zn2L2 (2.5ϫ10–5
in pH 7.4 HEPES) as up to 1.5 equiv. PPi were added (λex
380 nm). The inset demonstrates the visual change in emission (λex

=
distance between anionic atoms) to PPi and previous re- = 365 nm).
Eur. J. Org. Chem. 2009, 343–348
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R. C. Smith et al.
FULL PAPER
tem. Literature procedures were employed for the preparation of
2,2ЈЈ-dimethyl-m-terphenyl (1)[54] and 2,2ЈЈ-bis(bromomethyl)-
1
1,1Ј:3Ј,1ЈЈ-terphenyl (2).[45] All H and 13C NMR spectra were ob-
tained with a Bruker Avance 300 spectrometer with operational
1
parameters set at 300 MHz for H nuclei and 75 MHz for 13C nu-
clei. All spectra were collected at 20 °C and referenced to either
tetramethylsilane (δ = 0 ppm) as an internal standard or the resid-
ual solvent peak. A Corning CHEK-MITE pH 25 sensor interfaced
with a Symphony Ag/AgCl pH electrode was used for measuring
the pH of HEPES buffer, which was calibrated to the target pH
value of 7.4 by the addition of NaOH (aq.). All absorption and
fluorescence spectra were collected at 20 °C using a Cary 50 spec-
trophotometer for absorbance, and a Cary Eclipse fluorescence
spectrophotometer for photoluminescence. Samples were prepared
in poly(methylmethacrylate) cuvettes (1.0-cm path lengths) from
Starna Cells, Inc.
Preparation of 2,2ЈЈ-Bis[(2,2Ј-dipicolylamino)methyl]-1,1Ј:3Ј,1ЈЈ-ter-
phenyl (L2): A stirred solution of 2 (0.630 g, 1.51 mmol) was ini-
tially prepared in THF (10 mL, anhydrous) and cooled to 0 °C in
an ice water bath under N2. Separately, a solution of triethylamine
(0.460 g, 4.56 mmol) and bis(2-picolyl)amine (0.600 g, 3.01 mmol)
was prepared in anhydrous THF (5 mL). The solution containing
triethylamine and bis(2-picolyl)amine was added dropwise via sy-
ringe to the stirring 2,2ЈЈ-bis(bromomethyl)-1,1Ј:3Ј,1ЈЈ-terphenyl
solution, resulting in an orange mixture. The solution was removed
from the ice water bath and allowed to slowly warm to room tem-
perature where it was permitted to continue stirring under N2 and
ambient temperature for approximately 48 h, over which time the
solution turned from orange to brown. The solution was filtered to
remove precipitated triethylammonium bromide, and the filtrate
was concentrated in vacuo to yield a dark brown oil. About 50 mL
of dichloromethane was added and the organics washed with satd.
NaHCO3(aq.) (50 mLϫ3). The organic layer was collected, dried
with Na2SO4 and concentrated under reduced pressure to yield a
red oil. Approximately 10 mL of acetone and two drops of concen-
trated HCl were added to the crude product. The solution was al-
lowed to stand at room temperature for 12 h, over which time a
white microcrystalline solid formed. The solid was collected by fil-
tration, rinsed with acetone, and dried in vacuo to yield the target
compound (0.270 g, 51%) as a hygroscopic off-white powder; m.p.
Figure 2. Calibration curves for [PPi] measured via displacement of
pyrocatechol violet measured by ratiometric absorption spec-
troscopy (a) and displacement of esculetin measured by increase in
emission intensity of fluorescence spectra (b) in HEPES buffer (pH
= 7.4). In both graphs, diamonds with error bars are original data
points and the black line is a linear fit of the data. Equations of
linear fits and corresponding R2 values are also provided on each
graph. Error bars represent error associated with absorption (a)
and fluorescnece intensity readings (b).
Conclusions
To summarize, a binucleating ligand utilizing a m-ter-
phenyl scaffold has been prepared. The dizinc complex of
this ligand is a suitable receptor for PPi at physiologic pH,
affording improved selectivity over Pi in IDAs vs. related
systems. IDAs that employ ratiometric absorption changes
and fluorescence emission enhancement have both been de-
veloped and provide linear responses to pyrophosphate at
physiological pH. Although tests reported herein have been
ex vivo, the fluorescence assay provides sensitivity appropri-
ate for monitoring biologically relevant pyrophosphate con-
centrations. Efforts are underway to elaborate the m-ter-
phenyl scaffold for additional applications in chemosensing
and enzyme modeling.
1
122–126 °C. H NMR (300 MHz, CDCl3): δ = 8.47 (d, J = 7 Hz,
4 H), 7.81 (d, J = 7 Hz, 2 H), 7.56–7.08 (m, 22 H), 3.76 (s, 8 H),
3.72 (s, 4 H) ppm. 13C NMR (CDCl3): δ = 159.7, 148.9, 142.3,
141.2, 136.4, 136.3, 130.3, 129.6, 127.9, 127.8, 127.5, 126.8, 122.6,
121.8, 60.1, 56.0 ppm. C44H41N6O (L2·H2O) (669.85): calcd. C
79.85, H, 6.24. N, 12.70; found C 79.96, H 5.96, N 12.58.
Absorption Spectroscopic Titration of Indicators with Zn2L2: Indi-
cator stock solutions (50 mL, 5ϫ10–5 ) were individually pre-
pared in a buffered solution of HEPES (10 m, pH 7.4) and used
for all titration experiments. A solution of Zn2L2 (1.5 m, 10 mL)
was prepared in 1:4 acetonitrile/HEPES (10 m, pH 7.4) and uti-
lized for all titration experiments. For delineation of respective
spectral responses from each indicator due to the introduction of
Zn2L2, 3 mL of the stock indicator solution was added to a cuvette
and the resulting spectrum was recorded in the absence of Zn2L2.
Subsequently, the indicator solution was titrated in situ with 10 µL
aliquots (0.10 equiv.) of the Zn2L2 solution; individual spectra were
recorded following the addition of each Zn2L2 aliquot until a total
of at least10 aliquots (1 equiv.) had been added. Titrations were
performed in identical fashions for all indicators under consider-
ation. Resulting absorption titration spectra for each indicator are
provided in the Supporting Information.
Experimental Section
Reagents and General Methods: All reagents were obtained from
Aldrich Chemical Co., TCI America, Alfa Aesar, Fischer Scientific,
Mallinckrodt, Baker and Adamson, or MP Biomedicals, LLC and
used as received. Solvents were purified by passage through alu-
mina columns under N2 using an MBraun solvent purification sys-
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Dizinc Phosphohydrolase Model
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say titrations. Solutions of sodium hydrogen phosphate (1.5 m,
5 mL) and sodium pyrophosphate decahydrate (1.5 m, 5 mL)
were prepared in HEPES buffer (10 m, pH 7.4). Initially 3 mL
of an indicator solution and 100 µL of the Zn2L2 solution (1:1
stoichiometric equivalence) were added to a cuvette, followed by
recording of the respective spectrum. For determination of the abil-
ity of the selected anion (phosphate or pyrophosphate) to displace
the indicator from the indicator/Zn2L2 complex and concomitantly
form an anion/Zn2L2 complex, the indicator/Zn2L2 solution was
titrated in situ with 10 µL aliquots (0.100 equiv. relative to both the
indicator and Zn2L2) aliquots of the anion solution. Individual
spectra were recorded following the addition of each anion aliquot
until a total of 20 aliquots had been added to the indicator solution
(resulting in a 2:1:1 anion/indicator/Zn2L2 stoichiometry). Ti-
trations were iterated in the same manner for all indicator/analyte
combinations. Absorption spectra resulting from the titration of
each indicator-Zn2L2 complex with PPi are provided in the Sup-
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Response of Indicator-Zn2L2 to Other Anions: Indicator-Zn2L2
solutions were prepared as described above for absorption spectro-
scopic studies, and up to 10 equiv. of NaF, NaCl, NaBr, NaI, Na-
O2CCH3, NaP3H10, Na2SO4, NaNO3, Na2CO3 sodium glutamate,
or sodium aspartate was added to each cuvette, followed by collec-
tion of an absorption spectrum. None of these samples exhibited
significant changes in absorption spectra.
[24]
[25]
[26]
[27]
Fluorescence Titration of Indicator-Zn2L2 Complex with Phospho-
rous-Containing Analyte: Fluorescence experiments were executed
in a manner similar to that described above for absorption spectro-
scopic experiments, with the exception that initial indicator concen-
trations of 2.5ϫ10–5  were used.
[28]
[29]
[30]
[31]
[32]
[33]
Supporting Information (see also the footnote on the first page of
this article): 1H and 13C NMR spectra, absorption spectra, fluores-
cence spectra and Benesi–Hildebrand plots.
Acknowledgments
[34]
The authors thank Brad P. Morgan for insightful suggestions and
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