Fluorescent and electrochemical sensing of polyphosphate nucleotides by ferrocene functionalised with two ZnII(TACN)(pyrene) complexes

Article abstract of DOI:10.1002/chem.201000882

The [Fc-bis{Zn^II(TACN)-(Py)}] complex, comprising two Zn ^II-(TACN) ligands (Fc = ferrocene; Py = pyrene; TACN = 1,4,7-triazacyclononane) bearing fluorescent pyrene chromophores linked by an electrochemically active ferrocene molecule has been synthesised in high yield through a multistep procedure. In the absence of the polyphosphate guest molecules, very weak excimer emission was observed, indicating that the two pyrenebearing Zn^II(TACN) units are arranged in a trans-like configuration with respect to the ferrocene bridging unit. Binding of a variety of polyphosphate anionic guests (PPi and nucleotides diand triphosphate) promotes the interaction between pyrene units and results in an enhancement in excimer emission. Investigations of phosphate binding by³¹P NMR spectroscopy, fluorescence and electrochemical techniques confirmed a 1:1 stoichiometry for the binding of PPi and nucleotide polyphosphate anions to the bis(Zn^II-(TACN)) moiety of [Fc-bis{Zn^II-(TACN)(Py)}] and indicated that binding induces a trans to cis configuration rearrangement of the bis(Zn^II(TACN)) complexes that is responsible for the enhancement of the pyrene excimer emission. Pyrophosphate was concluded to have the strongest affinity to [Fc-bis{Zn^II(TACN)(Py)}] among the anions tested based on a six-fold fluorescence enhancement and 0.1 V negative shift in the potential of the ferrocene/ferrocenium couple. The binding constant for a variety of polyphosphate anions was determined from the change in the intensity of pyrene excimer emission with polyphosphate concentration, measured at 475 nm in CH ₃CN/Tris-HCl (1:9) buffer solution (10.0 mm, pH 7.4). These measurements confirmed that pyrophosphate binds more strongly (K_b = (4.45 ± 0.41)×10⁶M^-1) than the other nucleotide di-and triphosphates (K^b = 1-50× 10⁵ M^-1) tested.

Full text of DOI:10.1002/chem.201000882

DOI: 10.1002/chem.201000882
Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides by
Ferrocene Functionalised with Two ZnII
ACTHNUGRTNENUG(TACN)ACHTUGNTREN(NUGN pyrene) Complexes
Zhanghua Zeng,[a, b] Angel A. J. Torriero,[a] Alan M. Bond,*[a] and Leone Spiccia*[a, b]
[Fc bisACTHNGUTERNNUG ACHTUGNRTEN(NUNG TACN)(Py)}] among the
{ZnII
ꢀ
ꢀ
Abstract: The [Fc bis
(Py)}] complex, comprising two ZnII-
(TACN) ligands (Fc=ferrocene; Py=
pyrene; TACN=1,4,7-triazacyclono-
nane) bearing fluorescent pyrene chro-
U
G
sion. Investigations of phosphate bind-
ing by 31P NMR spectroscopy, fluores-
cence and electrochemical techniques
confirmed a 1:1 stoichiometry for the
binding of PPi and nucleotide poly-
G
anions tested based on a six-fold fluo-
rescence enhancement and 0.1 V nega-
tive shift in the potential of the ferro-
cene/ferrocenium couple. The binding
constant for a variety of polyphosphate
anions was determined from the
change in the intensity of pyrene exci-
mer emission with polyphosphate con-
centration, measured at 475 nm in
CH3CN/Tris-HCl (1:9) buffer solution
(10.0 mm, pH 7.4). These measure-
ments confirmed that pyrophosphate
binds more strongly (Kb =(4.45ꢁ
0.41)ꢀ106 mꢀ1) than the other nucleo-
tide di- and triphosphates (Kb =1–50ꢀ
105 mꢀ1) tested.
E
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
mophores linked by an electrochemi-
cally active ferrocene molecule has
phosphate anions to the bisACHTGNUTRENNUNG
ꢀ
(TACN)) moiety of [Fc bis
E
been synthe
N
(TACN)(Py)}] and indicated that bind-
a multistep procedure. In the absence
of the polyphosphate guest molecules,
very weak excimer emission was ob-
served, indicating that the two pyrene-
ing induces a trans to cis configuration
rearrangement of the bis (TACN))
A
ACHTUNGTRENNUNG
complexes that is responsible for the
enhancement of the pyrene excimer
emission. Pyrophosphate was conclud-
ed to have the strongest affinity to
bearing ZnII
ACHTUNGTRENNUNG(TACN) units are arranged
in a trans-like configuration with re-
spect to the ferrocene bridging unit.
Binding of a variety of polyphosphate
anionic guests (PPi and nucleotides di-
and triphosphate) promotes the inter-
action between pyrene units and results
in an enhancement in excimer emis-
Keywords: bioinorganic chemistry ·
electrochemistry · ferrocene deriva-
tives · fluorescence spectroscopy ·
polyphosphate anions
Introduction
portant chemical and biological processes,[3,4] which include
transport across membranes, DNA synthesis, cell signalling,
energy or electron-transfer processes, kinase-catalysed pro-
tein phosphorylation and important cofactors of enzymatic
reactions.[5] For example, adenosine triphosphate (ATP) and
guanosine triphosphate (GTP) are sources of chemical
energy.[6] In the case of ATP, the universal energy source
available for cellular functions in living systems, PPi is re-
The development of receptors and sensors for physiological
anions represents a challenging and topical area of re-
search.[1,2] Phosphate anions, such as the pyrophosphate
anion (PPi) and DNA/RNA nucleotides, are an important
class of anions that play a central role in physiologically im-
leased in DNA polymerisation to produce adenACHTNUTRGNEoUNG sine mono-
phosphate (AMP), and also takes part in protein synthesis
and DNA replication.[6] The development of receptor-based
sensors for biologically important phosphate anions is essen-
tial to the understanding of their biological activity.
Given the biological importance of polyphosphate anion
species, it is not surprising that a number of chemosensors
and receptors for PPi and DNA/RNA nucleotides have
been reported.[7] Several polyammomium-based receptors
for PPi sensing utilise hydrogen bonding and electrostatic
forces.[7b,c,8] Recently, conjugates bearing a fluorescent chro-
[a] Dr. Z. Zeng, Dr. A. A. J. Torriero, Prof. A. M. Bond, Prof. L. Spiccia
School of Chemistry, Monash University
Clayton, Victoria 3800 (Australia)
Fax : (+61)399054597
E-mail: alan.bond@monash.edu
leone.spiccia@monash.edu
[b] Dr. Z. Zeng, Prof. L. Spiccia
Australian Centre of Excellence for Electromaterials Science
Monash University, Clayton, Victoria 3800 (Australia)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000882.
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FULL PAPER
mophores and two di(2-picolyl)-amine (DPA) zinc(II) com-
plexes have been reported, for which strong coordination of
two pyrene-bearing ZnII
ACTHNGUTERN(UNG TACN) complexes linked by ferro-
cene, which can be applied in the two channels (or dual)
sensing of biologically important phosphate anions. Detec-
tion of PPi and nucleotide di- and triphosphates has been
achieved through electrochemistry (ferrocene) and fluores-
cence emission spectroscopy (pyrene) in aqueous or partial-
ly aqueous media.
PPi and DNA/RNA nucleotides to the bisACTHUNRGTENNGU CAHTUNTGERN(NUGN DPA)) com-
(ZnII
plex units induces fluorescence and UV spectral changes.[9]
Yoon and co-workers also developed cationic imidazolium
compounds as receptors for phosphate anions that exploit
hydrogen bonding and electrostatic interactions between the
imidazolium cations and phosphate anions in DNA/RNA
nucleotides.[10] Tarraga and Molina reported a triazole-based
receptor for PPi that incorporated ferrocene (Fc; electro-
chemical activity) and pyrene (Py; fluorescence activity).
Binding of PPi was accompanied by significant electrochem-
ical and fluorescence changes.[11] However, this sensor could
only be used in organic solvents owing to poor solubility in
aqueous media.
1,4,7-Triazacyclononane (TACN) is a cyclic organic com-
pound, which coordinates strongly to transition-metal ions
through three amine nitrogen donor atoms.[12] Binding of
one TACN macrocycle to a metal ion generally leaves one
or more coordination sites occupied by weakly binding lig-
Results and Discussion
Synthesis of [Fc bis
{ZnII
E
E
ꢀ
ꢀ
for receptor [Fc bis
ACHTUNGTRENNUNG
Scheme 1. Reaction of tacnorthoamide[15] with bromoethane
ꢀ
ꢀ
gave ethyl TACN orthoamidinium bromide, which was hy-
drolysed to give 2. Compound 3 was obtained by the reac-
tion of 2 with 1,1’-bis(dichloromethyl)ferrocene[16] followed
by hydrolysis in base to give 4. In parallel, 1-pyrenecarbox-
aldehyde was converted to 1-pyrenemethanol, which was re-
acted with 4-bromobutyric acid in the presence of dicyclo-
hexylcarbodiimide (DCC) and a catalytic amount dimethyl-
ACHTUNGTRENNUNG
AHCTUNGTREGaNNNU minopyridine (DMAP) to afford 1. Reaction of compounds
A
4 and 1 gave the free ligand 5. Finally, reaction of 5 with
E
zinc(II) perchlorate gave the zinc(II) complex, [Fc bis
{ZnII-
ꢀ
quences have been shown to displace such weakly binding
ligands.[13] Exploiting these properties and recognising that
the application of ferrocene in the electrochemical sensing
of anions and DNA is well established,[14] we have devel-
ꢀ
(TACN)(Py)}]. Details of the characterisation of these com-
pounds are provided in the Experimental Section.
Fluorescence study: The complexation of the free ligand, 5,
oped a new ZnII
E
G
N
U
with Zn2+ to form the [Fc bis
was investigated by measuring changes in the fluorescence
{ZnII
N
ꢀ
(Py)}] shown in Scheme 1, as a receptor or chemosensor for
biological phosphate anions. This receptor is comprised of
emission of the pyrene units present in 5 on addition of in-
{ZnII
N
ꢀ
Scheme 1. Synthetic route used to prepare receptor [Fc bis
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A. M. Bond, L. Spiccia et al.
creasing concentrations of Zn2+ (Figure S1 in the Support-
ing Information). Application of the continuous variation
method determined the 5 to Zn2+ binding stoichiometry to
be 1:2 (Figure S2 in the Supporting Information). Fitting of
the variation in pyrene excimer emission with concentration
of Zn2+ to Equation (1) yielded values of K11 =7.6ꢀ108 mꢀ1
and b21=3.2ꢀ1016 mꢀ2 for the apparent stability constants for
the formation of the 1:1 and 1:2 5:ZnII complexes, respec-
tively (Figure S3 in the Supporting Information). This estab-
ꢀ
conclude that the fluorescence bands at 475 nm and at
375 nm arise from excimer and monomer emission,[17] re-
spectively. Titration of the receptor with up to 1.0 equivalent
of PPi results in a six-fold increase in the intensity of exci-
mer emission together with a 40% decrease in the monomer
emission intensity. The continuous variation method was
ꢀ
used to determine that the [Fc bisACTHNUTRGENNUG ACHTUGNTRENN(NGU TACN)(Py)}] to PPi
{ZnII
binding stoichiometry was 1:1 (Figure S4 in the Supporting
Information). Fitting of the variation in pyrene excimer
emission with concentration of PPi (Figure 1) to Equa-
tion (2) gave a PPi binding constant (Kb) value of (4.45ꢁ
0.41)ꢀ106 mꢀ1 (Figure S5 in the Supporting Information).
lished that the receptor [Fc bisACHTNUTRGENNGU ACHTUNTGREN(NGUN TACN)(Py)}] is stable
{ZnII
under the conditions chosen for the study of the binding of
phosphate anions.
ðFꢀF0Þ=F0 ¼ FbKb½PPiꢂ=F0f1 þ Kb½PPiꢂg
ð2Þ
2
2
F ¼ fF0 þ cMbK11½Mꢂ þ Fbb21½Mꢂ g=f1 þ K11½Mꢂ þ b21½Mꢂ g
ð1Þ
In Equation (2), F0 and F are the fluorescence intensities in
the absence and presence of the guest, respectively, and Fb
is the maximum fluorescence intensity.
The affinity of the receptor for a variety of other biologi-
cal inorganic anions and DNA/RNA nucleotides was also in-
vestigated by measuring their effect on the excimer emission
intensity. Thus, fluorescence titrations were carried out for
thymine (T); adenine (A); cytosine (C); guanine (G); thy-
midine mono-, di- and triphosphate (TMP, TDP, TTP);
In Equation (1), F0 and F are the fluorescence intensities in
the absence and presence of Zn2+, respectively, [M] is the
concentration of Zn2+, Fb is the maximum fluorescence in-
tensity, K11 and K21 are the first and second binding con-
stant, respectively, and b21 =K11K21.
{ZnII-
ꢀ
The interaction between the receptor [Fc bis
(TACN)(Py)}] and phosphate anions was investigated by
G
measuring changes in the fluorescence emission of the
pyrene units in the absence and presence of various guests.
When PPi was added to a CH3CN/Tris-HCl (1:9) buffer so-
ꢀ
adenACTHNGUTERNoUNG sine mono-, di- and triphosphate (AMP, ADP, ATP);
cytidine mono-, di- and triphosphate (CMP, CDP, CTP); and
guanosine mono-, di- and triphosphate (GMP, GDP, GTP)
and inorganic salts (phosphate (Pi), CH3COOꢀ and Fꢀ). For
lution (10.0 mm, pH 7.4) containing 1.0 mm [Fc bisACTHNUTRGNEUNG
{ZnII-
ꢀ
(TACN)(Py)}] (Figure 1), a large fluorescence enhancement
comparison, the fluorescence emission of solutions of [Fc
bis
A
R
PPi, ATP, ADP, AMP, Pi, CH3COOꢀ and Fꢀ is shown in
Figure 2. The binding constants were determined by measur-
ing the change in fluorescence with guest concentration and
fitting this titration data to Equation (1). The data are sum-
marised in Table 1. Titration data for ATP and ADP, as ex-
amples of di-, and triphosphate nucleotides, are shown in
Figure S6 in the Supporting Information. In all cases, the
Figure 1. Fluorescence emission spectra of [Fc bisACTHNUTRGENNUG ACHUTGNTREN(NUGN TACN)(Py)}]
{ZnII
ꢀ
(1.0 mm, excitation at 350 nm) recorded upon addition of PPi (0, 0.1, 0.2,
0.4, 0.5, 0.8, 1.0, 1.5, 2.5 and 4.0 equiv) in CH3CN/Tris-HCl (1:9) buffer
solution (10.0 mm; pH 7.4; T=(20ꢁ1)8C).
was observed at 475 nm together with a gradual decrease in
the intensity of the emission at 375 nm. This result indicates
that binding of PPi forces the two pyrene units in the recep-
ꢀ
Figure 2. Fluorescence emission spectra of [Fc bis
{ZnII
(TACN)(Py)}]
ꢀ
tor [Fc bisACHTUNGTRENNUNG ACHTUNGTRENNUNG(TACN)(Py)}] into closer proximity such
{ZnII
that interaction between the aromatic rings is promoted and
the static pyrene excimer emission at 475 nm is strongly en-
hanced. As in previous studies applying this fluorophore, we
(1.0 mm, excitation at 350 nm) measured in a 1:9 mixture of CH3CN/Tris-
HCl buffer (10.0 mm; pH 7.4; T=(20ꢁ1)8C) for solutions containing
1.0 mm of PPi, ATP and ADP, and 100 mm of A, AMP, Pi, CH3COOꢀ and
Fꢀ.
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Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides
FULL PAPER
{ZnII-
ꢀ
Table 1. Summary of the binding constants between [Fc bis
ACHTUNGTRENNUNG(TACN)(Py)}] and various anions.
Anion
Kb [mꢀ1
]
Anion
Kb [mꢀ1
]
PPi
(4.45ꢁ0.41)ꢀ106
(9.31ꢁ0.84)ꢀ104
(2.32ꢁ0.19)ꢀ105
(2.13ꢁ0.29)ꢀ105
(5.05ꢁ0.46)ꢀ105
(9.63ꢁ0.93)ꢀ103
(1.55ꢁ0.14)ꢀ104
(1.41ꢁ0.12)ꢀ104
TDP
AMP
CMP
GMP
TMP
Pi
(2.03ꢁ0.18)ꢀ104
ATP
CTP
GTP
TTP
ADP
CDP
GDP
ND[a]
ND[a]
ND[a]
ND[a]
ND[a]
Fꢀ
ND[a]
CH3COOꢀ
ND[a]
[a] Not determined (ND) owing to the small changes in fluorescence
emission upon addition of the analyte.
binding stoichiometry was found to be 1:1 (Figure S4 in the
Supporting Information).
{ZnII
N
ꢀ
The receptor [Fc bis
affinity to triphosphate nucleotides (e.g., Kb =(9.31ꢁ0.84)ꢀ
104 mꢀ1 for ATP) than diphosphate nucleotides (Kb =(9.63ꢁ
0.93)ꢀ103 mꢀ1 for ADP). The fluorescence change was negli-
gible for the addition of monophosphate nucleotides, the nu-
cleotide bases, Pi, CH3COOꢀ and Fꢀ, even when present at
a much higher concentrations (100 equiv). The fluorescence
ꢀ
results indicate that [Fc bisACTHNUGTRENNUG CAHTUNGTRNE(NUGN TACN)(Py)}] can selective-
{ZnII
ly bind to polyphosphate anions. The largest binding con-
stant was obtained for PPi (Kb =(4.45ꢁ0.41)ꢀ106 mꢀ1),
which probably arises from the higher negative charge per
phosphorous centre of PPi when compared with di- and tri-
phosphate nucleotides, and is reflected in stronger binding
ꢀ
Scheme 2. Proposed mode of binding between [Fc bis
and polyphosphate anions (PPi and XTP (X=A, T, C or G)).
{ZnII
N
ꢀ
to the 4+ charged [Fc bis{ZnII
ACTHNUGRTNENUG HCATUNGTERNN(UGN TACN)(Py)}] receptor. Hong
and co-workers also concluded that bis
A
R
ments were carried out to quantify the selectivity level of
the receptor for PPi. As shown in Figure 3, the addition of
100 equivalents of inorganic anions and monophosphate nu-
cleotides and 1.0 equivalent of di- and triphosphate nucleo-
tides does not interfere with PPi binding to the receptor
ꢀ
plexes exhibit selective binding to PPi over ATP owing to
the more negatively charged phosphorous in PPi.[9a,b,d,g]
To account for the enhanced excimer emission in the pres-
ence of phosphate anions, it is proposed that the two posi-
tively charged bis
G
N
[Fc bisACHTNGUTRENNUG ACTHUNGTREN(NUNG TACN)(Py)}]. In contrast, addition of 10 equiv-
{ZnII
are arranged in a trans-like configuration with respect to the
ferrocene bridging unit, to minimise electrostatic repulsion
and energy, as was concluded in previous studies that em-
ployed a receptor with a pairs of ZnII
ACTHNUTRGNE(UNG cyclen) units to detect
TpT.[16b] Thus, in the absence of the polyphosphate the dom-
ꢀ
inant band in the fluorescence spectrum of [Fc bis
{ZnII-
(TACN)(Py)}] measured in CH3CN/Tris-HCl (1:9) buffer so-
lution is from monomer rather than excimer emission. On
introduction of PPi, triphosphate or diphosphate nucleo-
tides, the interaction between these polyphosphate anions
and bis
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(ZnII
the two ZnII
ACHTUNGTRENNUNG
from a trans conformation (which minimises electrostatic re-
pulsion), to a cis conformation. This rearrangement brings
the pyrene units into much closer proximity, leading to
strong excimer emission (static) and quenching of monomer
emission, on the basis of the modified metal–anion coordi-
nation interaction (Scheme 2).
ACHTUNGTRENNUNG
{ZnII-
ꢀ
Figure 3. Fluorescence change (F/F0) at 475 nm for [Fc bis
(TACN)(Py)}] (1.0 mm, excitation at 350 nm) on addition of 1.0 mm of PPi
AHCTUNGTRENNUNG
and mixtures containing 1.0 mm of PPi and 100.0 mm of either Pi, Fꢀ,
AcOꢀ or AMP, 1.0 mm of either ADP or ATP; and 10.0 mm of either ADP
or ATP in CH3CN/Tris-HCl (1:9) buffer solution (10.0 mm; pH 7.4; T=
(20ꢁ1)8C). F0 and F are the fluorescence intensities in the absence and
presence of the guest/guest mixture, respectively.
After it was established that [Fc bis
(ZnII
R
ꢀ
binds more strongly to PPi than to mono-, di-, or triphos-
phate nucleotides and other anions, competition experi-
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A. M. Bond, L. Spiccia et al.
alents of ATP and ADP caused a decrease in the fluores-
cence by 20 and 15%, respectively, indicating that these
terestingly, the change was negligible for the signal of the P
centre in AMP (Figure 4d). Thus, the 31P NMR chemical
{ZnII
T
ꢀ
poly
G
shift changes indicate the receptor [Fc bis
binding site on [Fc bis
{ZnII
(Py)}] shows the highest affinity to PPi among the phos-
phate anions tested, which is consistent with the fluores-
cence experiment. Analysis of the change in 31P NMR chem-
ical shift as a function of added receptor confirmed 1:1 bind-
ing stoichiometry (Figure 4), which was proposed from the
fluorescence emission studies.
31P NMR study: To gain further insight into the mechanism
ꢀ
of binding of the phosphate anions to [Fc bis
A
{ZnII-
(TACN)(Py)}], 31P NMR spectroscopy was used to investi-
gate the interaction between bisACTHNUTRGENNUG ACHTUGNTRENN(GUN TACN)) complexes
(ZnII
and phosphate anions in CD3CN/Tris-HCl (1:9) buffer solu-
tion (10.0 mm; pD=7.6; T=(20ꢁ1)8C) with the addition of
85% H3PO4 as a reference.[18] In these experiments, changes
in the 31P NMR spectrum of PPi, ATP, ADP and AMP were
ꢀ
In the case of ATP, the chemical shift change in the inner
Pa is very small (0.2 ppm) compared with Pg and Pb (1.4 and
1.1 ppm, respectively). This implies that the two ZnII-
AHCTUNGTREG(NNNU TACN) units mainly bind to the outer and middle phospho-
recorded on addition of the [Fc bis
{ZnII
E
rous, Pg and Pb, and lends support to our proposed binding
ꢀ
ceptor (from 0.1 to 4.0 equiv) as shown in Figure S7 in the
Supporting Information. The 31P NMR chemical shift
change for PPi became 2.5 ppm upon addition of 1.0 equiv-
ꢀ
mode between [Fc bis
in Scheme 2. Hong and co-workers proposed a similar bind-
{ZnII
(TACN)(Py)}] and ATP, shown
ing mode for the bisACTHNUTRGENNGU ACHTUGNTRENNUGN
(ZnII(DPA)) complexes and ATP.[9a,b,d,g]
alent of [Fc bis
G
N
The bigger change in the chemical shift for the outer phos-
phorous than for the middle (ATP) and inner (ADP), indi-
chemical shift changes for the Pg, Pb, and Pa centres were
found to be 1.4, 1.1 and 0.18 ppm, respectively. For the Pb,
and Pa centres in ADP, the respective changes were 0.9 and
0.37 ppm in the presence of same amount receptor and, in-
cates that bisACTHNUTRGENNGU ACHTUNGTRNEN(UGN TACN)) complexes bind more strongly to
(ZnII
the phosphate with the greater negative charge (2ꢀ when
fully deprotonated) than the inner phosphate (1ꢀ). Given
31
{ZnII
(TACN)(Py)}] (from 0.1 to
ꢀ
Figure 4. Variation in the P NMR chemical shift of PPi, ATP, ADP and AMP (1.0 mm) upon addition of [Fc bis
4.0 equiv) in a mixture of CD3CN and Tris-HCl D2O buffer (1:9) solution (10.0 mm; pD=7.6; T=(20ꢁ1)8C). a) PPi; b) ATP; c) ADP; d) AMP mea-
sured with H3PO4 (85% in D2O) as the reference.
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Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides
FULL PAPER
that PPi has a negative charge of 4ꢀ when fully deprotonat-
ed, it would be anticipated to bind more strongly to the
tion and leads to a substantial increase in the complexity of
the voltammetry. Examination of cyclic voltammograms ob-
tained as a function of scan rate over the range 0.02 to
1.00 Vsꢀ1 in both CH3CN/Tris-HCl (1:9) and CH3CN/
HEPES (1:9) buffer solutions (50.0 mm; pH 7.4) at (20ꢁ
1)8C, revealed a clear dependence on scan rate. Thus, a
single nearly chemically reversible diffusion-controlled
redox process, with a mid-point potential (Em) of 0.45 V
versus Ag/AgCl (in NaCl, 3m) is observed at low scan rates.
However, a new process emerges at potentials that are
almost 0.10 V more positive when the scan rate is increased
up to 1.0 Vsꢀ1. These observations imply the existence of a
square-type mechanism outlined in Scheme 3, which interre-
ꢀ
[Fc bis
{ZnII
G
ceptor to PPi, broadening of the 31P NMR resonance is ob-
served (see Figure S7 in the Supporting Information),
whereas on addition of ATP, the 31P NMR signal of the
outer phosphorous atom broadens. This is likely to be a con-
sequence of slow exchange between free and bound poly-
phosphate and/or slow structural rearrangement between
different conformations of the receptor:polyphosphate
adduct (slow dynamic equilibrium on the NMR timescale).
ꢀ
Electrochemistry of [Fc bis
redox-active molecule, Fc, is present as the linker, it is possi-
{ZnII
G
ble to probe the electrochemical behaviour of ferrocene/fer-
{ZnII
(ZnII
(TACN)(Py)}] and to ex-
(TACN)) binding agent.
ꢀ
rocenium couple in [Fc bis
plore the influence of the bis
N
ACHTUNGTRENNUNG
Typically, a Fc centre undergoes a chemically and electro-
chemically reversible one-electron oxidation process to give
a ferrocenium (Fc+) centre. As Figure 5 shows, the addition
ꢀ
of ZnII {ZnII
ACHTUNGTRENNUNG(TACN)(Py) to Fc to give [Fc bisACHTUNRTGENNUNG ACHTNGUTREN(NUNG TACN)(Py)}]
decreases the stability of the Fc+ centre formed on oxida-
Scheme 3.
ꢀ
ꢀ
lates four chemical forms ([Fc bis
{ZnII
E
I ACHTUNGTRENNUNG[Fc
+
bis
{ZnII
R
R
R
/ACHTUNG-
ꢀ
ꢀ
[Fc bis
N
ACHTUNGTRENNUNG
or other structural forms in two oxidation states). The scan
rate data also indicate that chemical reactions are linked to
+
ꢀ
the electrochemically generated [Fc bis
A
(TACN)(Py)}]I
+
ꢀ
and [Fc bis
Scheme 3.
{ZnII
(TACN)(Py)}]II species, also as shown in
At very slow scan rates (Figure 5a) equilibrium conditions
are almost maintained for the [Fc bis
{ZnII
{ZnII
ACHTUNGTRENNUNGCHTUNGTRENNUNG
ꢀ
C
N
{ZnII
G
ꢀ
ꢀ
+
+
ꢀ
(Py)}]I
/
A
N
(TACN)(Py)}]II systems, because only
a single process is observed. At high scan rates (Figure 5b),
in which two oxidation and two reduction processes are ob-
servable, approximate Em1 and Em2 values can be obtained,
whereas at low scan rates an average Em value is found. This
process, which is split in to two processes under short time-
scales, was also found under conditions of square wave vol-
tammetry (SWV, Figure S8 in the Supporting Information).
The proposal of a square scheme is based on an analogy
with previously studied systems that encompass redox-de-
pendent isomerisation, ligand exchange and protonation.[19]
ꢀ
The oxidation of [Fc bisACTHNUTRGENNGU HCATUNGTRNE(NUGN TACN)(Py)}] at a GC rotat-
{ZnII
Figure 5. Cyclic voltammograms obtained at a glassy carbon electrode
(1 mm diameter) for oxidation of 1.00 mm of [Fc bis
{ZnII
N
ꢀ
ing-disc electrode (RDE) at a scan rate of 0.02 Vsꢀ1 and a
rotation speed (f’) of 100 rpm produced sigmoidal-shaped
I/E curves. By using the Levich equation,[20] a diffusion coef-
in CH3CN/Tris-HCl (1:9) buffer solution (50.0 mm; pH 7.4; T=(20ꢁ
1)8C) at scan rates of a) 0.02, 0.05, 0.10 and 0.20 Vsꢀ1; and b) 0.1, 0.2,
0.3, 0.4, 0.5 and 0.7 Vsꢀ1. Potentials are versus Ag/AgCl (NaCl, 3m).
Chem. Eur. J. 2010, 16, 9154 – 9163
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9159

A. M. Bond, L. Spiccia et al.
ficient of 4.22ꢀ10ꢀ6 cm2 sꢀ1 was estimated for [Fc bis
{ZnII-
ꢀ
(TACN)(Py)}]. However, a plot of log[I/
R
tential did not yield the straight lines predicted for a single-
electron transfer step, again consistent with the presence of
a complex reaction of the kind given in Scheme 3.
A titration experiment with PPi was conducted by the
higher resolution SWV method (Figures 6 and S9 in the
Supporting Information). A significant shift in the oxidation
Figure 7. Peak potential shifts obtained by SWV for the oxidation of
{ZnII
G
ꢀ
0.5 mm of [Fc bis
lution (50.0 mm; pH 7.4; T=(20ꢁ1)8C) in the absence (Ep =0 mV) and
presence of 0.5 mm designated bases, nucleotides, Pi and PPi. SWV pa-
rameters: amplitude 25 mV, frequency=100 Hz, DE=2 mV.
above, PPi is negatively charged, and shows the strongest
ꢀ
binding properties to the positively charged receptor [Fc
bis
A
R
the adduct is formed, the charge on the receptor is lowered
and the configuration changes from trans to cis, which alters
the electron density on the Fc/Fc+ linker.[16b] As indicated
above, the NMR spectra show evidence of line broadening,
which is consistent with the existence of a dynamic equilibri-
um. As can be seen in Figure 6, this equilibrium is also re-
flected in the electrochemistry, in which concomitantly with
the potential shift, a second process is detected at about
0.07 V more negative, the peak current value of which in-
ꢀ
{ZnII-
ꢀ
Figure 6. Square-wave
voltammograms
of
0.5 mm
[Fc bis
A
lents of PPi in CH3CN/Tris-HCl (1:9) buffer solution (50.0 mm; pH 7.4;
T=(20ꢁ1)8C). Potentials are versus Ag/AgCl (NaCl, 3m). The working
electrode is a 1 mm diameter GC-disc electrode. SWV parameters: am-
plitude 25 mV, frequency=100 Hz, DE=2 mV.
ꢀ
peak potential (DEp) of [Fc bis
A
A
60 and 100 mV towards less positive values was found at fre-
quencies of 15 and 100 Hz, respectively, as the concentration
of PPi increases from zero to 1.0 equivalent. The depend-
ence of the DEp values on frequency is related to differences
in the time scale, and therefore to the level of interaction of
creases as the ratio between [Fc bisACTHNGURTENNUG ACTHUNGTRNEN(UNG TACN)(Py)}] and
{ZnII
PPi approaches 1:1. This complexity could be associated
with a different conformational form of the same adduct.
Nucleotide polyphosphates can also induce configuration
changes, but presumably not as readily because a smaller
ꢀ
{ZnII
E
A
H
potential shift for oxidation of [Fc bisACTHUGNRTENNUG CAHTUNGTRNE(NUNG TACN)(Py)}] is
{ZnII
ꢀ
ꢀ
the [Fc bis
A
observed relative to that induced by PPi.
discussed in related work.[16b] Importantly, changes in DEp
when additional PPi is added beyond the 1.0 equivalence
are negligible. This infers the binding stoichiometries be-
ꢀ
Conclusion
tween [Fc bisACHTUNGTRENNUNG ACHTUNGTRENNUNG(TACN)(Py)}] and the oxidised form with
{ZnII
PPi are both 1:1 (Figure S9 in the Supporting Information).
A receptor consisting of two pyrene-bearing ZnII
ACHTUNGTRENNUNG(TACN)
Hence, this result provides data for the oxidised form, not
complexes linked by ferrocene, [Fc bis
{ZnII
(TACN)(Py)}],
ꢀ
ꢀ
available with fluorescence results in which only [Fc bis-
has been developed, which combines an electrochemically
active group with an off–on fluorescent chromophore, and
shows selectivity for biological polyphosphate anions. Fluo-
rescence, electrochemistry and 31P NMR spectroscopy estab-
ꢀ
A
R
Consistent with fluorescence titration data, the change of
peak potential is smaller (<20 mV) when single bases and
lished that PPi binds most strongly to [Fc bisACTHNGUTERNNUG ACHTUNGTRENNUNGCHTUNGTRENNUNG(TACN)-
{ZnII
ꢀ
monophosphate nucleotides are added to [Fc bis
{ZnII-
ACHTUNGTREN(NUGN Py)}] amongst the polyphosphate anions examined, due to
a higher negative charge density when compared with the
(TACN)(Py)}]. However, titration with di- and triphosphate
nucleotides induces a ꢃ0.05 V shift to less positive values.
nucleotide di- or triphosphate anions. The binding of the
The maximum negative shift found for PPi indicates that
polyphosphate anions to the [Fc bis
{ZnII
(ZnII
T
ACHTUNGTRENN(UNG Py)}] re-
ꢀ
ꢀ
[Fc bis
{ZnII
A
ceptor is proposed to force the bis
N
U
ative to other phosphate anions (Figure 7). As mentioned
same side of the ferrocene unit, giving rise to significant
9160
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9154 – 9163

Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides
FULL PAPER
changes in both electrochemical behaviour of the Fc/Fc+
centre (ꢄ0.1 V), due to major disruption in the structure,
and off–on fluorescence enhancement of pyrene excimer
emission induced by p···p stacking of the two aromatic py-
and acetone, and then dried with lint-free tissue paper. The solutions
used for voltammetric measurement were purged with solvent-saturated
nitrogen gas before each experiment and were blanketed with nitrogen
gas during the experiment.
Synthesis
ACHTUNGTRENNUNGrenes.
1-Pyrenecarboxaldehyde: Pyrene (2.1 g, 10 mmol) was added to a mixture
of POCl3 and N-methylformanilide at room temperature. The solution
was heated to 1008C under an atmosphere of nitrogen for 6 h. A yellow
precipitate was obtained after the reaction mixture was poured into an
ice-water mixture. The precipitate was collected through vacuum filtra-
tion. The solid was recrystallised from methanol to give yellow needles of
1-pyrenecarboxaldehyde. Yield: 1.5 g (70%); 1H NMR (400 MHz,
CDCl3): d=7.92–8.34 (m, 9H), 10.2 ppm (s, 1H); ESIMS: m/z: 231.1
[M++H].
Experimental Section
Materials: Unless otherwise specified, reagents and chemicals were pur-
chased from commercial sources and were used without further purifica-
tion. In the synthetic work, the solvents were used as received or dried
over 4 ꢂ molecular sieves. Deionised water was distilled prior to use. Tet-
rabutylammonium hexafluorophosphate (Bu4NPF6, Fluka) was recrystal-
lised from ethanol prior to use as the electrolyte in electrochemical stud-
ies. Tris-HCl buffer solutions (pH 7.4; 10.0 and 50.0 mm) were prepared
by dissolving tris(hydroxymethyl)aminomethane (Tris base) in distilled
water (500 mL) followed by titration to pH 7.4 with HCl (1.0m) solution,
measured by using a pH meter (HANNA instrument pH 211) at (20ꢁ
1)8C, and adjustment of the total solution volume to 1000 mL in a volu-
metric flask. 31P NMR titration experiments were carried out in Tris-HCl
D2O buffer solution (10.0 mm, pD=7.6, (20ꢁ1)8C), in which the pD
value was determined to pH meter value plus 0.4. The HEPES buffer so-
lution (pH 7.4; 50.0 mm) was prepared by dissolution of HEPES in dis-
tilled water (500 mL) and titrated to pH 7.4, by using NaOH (0.1m) solu-
tion at (20ꢁ1)8C, and then diluted with distilled water to 1000 mL in a
volumetric flask.
Instrumentation and methods: 1H NMR and 13C NMR spectra were re-
corded in deuterated solvents by using an Avance DRX400 Bruker spec-
trometer at 308C. The chemical shifts are reported in ppm (parts per mil-
lion). Tetramethylsilane (TMS) or the residual solvent peaks have been
used as an internal reference. The abbreviations for the peak multiplici-
ties are as follows: s (singlet), d (doublet), dd (doublet of doublets),
t (triplet), q (quartet), m (multiplet) and br (broad). Infrared spectra
were recorded a Perkin–Elmer 1600 series FTIR spectrophotometer at
4 cmꢀ1 resolution on samples doped in KBr pellets. CHN analyses were
performed by the Campbell Microanalytical Services, University of
Otago, Dunedin (New Zealand). Low-resolution electrospray mass spec-
tra were recorded with a Micromass Platform II Quadrupole mass spec-
trometer fitted with an electrospray source. Samples were introduced by
a syringe pump at a rate of 1 Lminꢀ1 and the capillary voltage was at
200 V. Thin layer chromatography (TLC) was performed by using silica
gel 60 F-254 (Merck) plates and basic alumina followed by preparative
column chromatography on silica gel and alumina. Fluorescence titration
experiments were carried out in Tris-HCl buffer solution (10.0 mm,
pH 7.4, (20ꢁ1)8C). Fluorescence spectra were recorded with a Varian
Cary Eclipse fluorescence spectrophotometer, with excitation and emis-
sion slit widths of 2.5 mm.
1-Pyrenemethanol: 1-pyrenecarboxaldehyde (1.15 g, 5.0 mmol) was dis-
solved in THF (30 mL) and a solution of NaBH4 (210 mg, 5.5 mmol) dis-
solved in methanol (10 mL) was added dropwise into the 1-pyrenecar-
boxaldehyde solution at room temperature. After stirring overnight, a
few drops of acetic acid were added to the reaction mixture to quench
the excess NaBH4. After the organic solvent was removed on a rotary
evaporator, the solid was extracted into chloroform twice and washed
twice with an aqueous solution. The collected organic solution was dried
with sodium sulfate and was concentrated to give yellow solid 1-pyrene-
methanol. Yield: 1.0 g (90%); 1H NMR (400 MHz, CDCl3): d=5.07 (s,
2H), 7.75–8.14 ppm (m, 9H); ESIMS: m/z: 233.2 [M++H].
Compound 1: Dicyclohexylcarbodiimide (460 mg, 2.2 mmol) and a cata-
lytic amount of 4-dimethylaminopyridine were added to a solution pre-
pared by dissolving 1-pyrenemethanol (464 mg, 2.0 mmol) and 4-bromo-
butyric acid (335 mg, 2.0 mmol) in CH2Cl2 (20 mL), and the mixture was
stirred overnight at room temperature. The mixture was filtered and the
solvent was removed from the filtrate to yield a yellow oil, which was pu-
rified by silica-gel column chromatography by using ethyl acetate and
hexane (1:2) as the eluent to afford a yellow solid 1. Yield: 565 mg
1
(75%); H NMR (400 MHz, CDCl3): d=2.15–2.36 (m, 4H), 3.49 (t, 2H),
5.68ACHTNUGRTNEUNG
(s, 2H), 7.72–8.16 ppm (m, 9H); ESIMS: m/z: 383.2, 381.2 [M++H].
Compound 2: Tacnorthoamide[15] (700 mg, 5.0 mmol) was dissolved in
THF (3 mL) and a solution of bromoethane (600 mg, 5.5 mmol) in THF
(3 mL) was added to this solution over a 10 min period. The mixture was
then stirred overnight and the precipitate that resulted was collected by
filtration and washed several times with THF. The precipitate was dis-
solved in water (20 mL) and the aqueous solution was heated at reflux
for 4 h. After the solution was cooled to room temperature, NaOH solu-
tion (10m) was carefully added dropwise to the reaction solution to
adjust the pH to 12. The product was extracted with CHCl3 (3ꢀ30 mL).
Removal of the organic solvent on a rotary evaporator gave a colourless
oil. Yield: 625 mg (67%); 1H NMR (400 MHz, CDCl3): d=2.15–2.36 (m,
4H), 3.49 (t, 2H), 5.68ACTHNUTRGNEUGN(s, 2H), 7.72–8.16 ppm (m, 9H); ESIMS: m/z:
771.2 [M++H].
Compound 3: 1,1’-bis(dichloromethyl)ferrocene (270 mg, 1.0 mmol),
which was obtained by a literature method,[16] and 2 (405 mg, 2.2 mmol)
were dissolved in acetonitrile (40 mL), and potassium carbonate (550 mg,
4 mmol) was added. The mixture was stirred for 1 hour at room tempera-
ture and was then heated to 808C overnight. The mixture was filtered
and the solvent removed from the filtrate in vacuo to yield a solid residue
which was dissolved in chloroform and was extracted twice with distilled
water. After removing the organic solvent, the crude product was isolated
as a brown crude solid. Further purification was carried out by chroma-
tography on a silica-column with MeOH/CH2Cl2 in a 1:22 ratio as the
eluent. The product was collected as a pure yellow oil after the solvent
was removed on a rotary evaporator. Yield: 290 mg (50%); 1H NMR
(400 MHz, CDCl3): d=1.28 (t, J=7.8 Hz, 6H), 2.39–2.46 (m, 4H), 2.55–
2.64 (m, 8H), 2.80–2.88 (m, 4H), 3.00–3.06 (m, 8H), 3.30–3.38 (m, 4H),
3.45 (s, 4H), 4.05–4.12 (m, 8H), 8.02 ppm (d, 2H); 13C NMR (100 MHz,
CDCl3): d=12.5, 47.2, 48.2, 51.7, 52.1, 52,8 53.6, 58.3, 68.3, 70.4, 84.4,
164.6 ppm; ESIMS: m/z: 291.2 [M++2H]/2, 581.2 [M++H].
Electrochemical Studies: Electrochemical experiments were carried out
at (20ꢁ1)8C in a standard three-electrode cell arrangement with a BAS
100B electrochemical workstation (Bioanalytical Systems, West Lafay-
ette, IN, USA). A platinum wire auxiliary electrode, and an aqueous Ag/
AgCl (NaCl, 3m) reference electrode were used for studies in the mixed
solvent CH3CN/Tris-HCl (50.0 mm; 1:9) and CH3CN/HEPES (50.0 mm;
1:9) buffer solutions (pH 7.4). A glassy carbon (GC) working electrode
with a diameter of 1.0 mm and an area of 0.72 mm2 was used for the
cyclic voltammetric experiments. Rotating-disc experiments employed a
GC working electrode (3.0 mm diameter, 7.10 mm2 effective area) and
were performed with a BAS RDE-2 accessory. The area or radii of the
voltammetric working electrodes was determined from cyclic voltammo-
grams by using the peak current from the oxidation of a 1.0 mm Fc solu-
tion (diffusion coefficient of 2.3ꢀ10ꢀ5 cm2 sꢀ1
) in CH3CN (Bu4NPF6,
0.1m) and with the application of the Randles–Sevcik equation. The area
of the rotating-disc electrode was similarly determined by using the
Levich equation. Prior to each voltammetric experiment, the working
Compound 4: Compound 3 (290 mg, 0.5 mmol) was dissolved in metha-
nol (5 mL) and sodium hydroxide solution (20 mL, 5m) was added. The
mixture was heated to reflux overnight, cooled to room temperature and
electrode was polished with 0.3 mm alumiACTHUNGTRENNnUNG a (Buehler, Lake Bluff, IL) on
a clean polishing cloth (Buehler), sequentially rinsed with distilled water
Chem. Eur. J. 2010, 16, 9154 – 9163
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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9161

A. M. Bond, L. Spiccia et al.
extracted three times with CHCl3. This solution was washed three times
with distilled water. After removing the organic solvent, a yellow oil was
obtained, which was purified by chromatography on a silica column with
MeOH/diethylamine in a 10:3 ratio as the eluent. Removal of the eluent
gave 4 as yellow oil. Yield: 220 mg (84%); 1H NMR (400 MHz, CDCl3):
d=1.28 (t, J=8.2 Hz, 6H), 2.42–2.86 (m, 28H), 3.42 (s, 4H), 4.04–
4.10 ppm (m, 8H); 13C NMR (100 MHz, CDCl3): d=12.9, 46.4, 47.1, 51.1,
51.3, 52, 52.4, 56.1, 68.3, 70.4, 84.4 ppm; ESIMS: 264.2 [M++2H]/2, 525.3
[M++H].
[3] a) W. N. Limpcombe, N. Strater, Chem. Rev. 1996, 96, 2375; b) D. J.
McCarty, Arthritis Rheum. 1976, 19, 275; c) A. Caswell, D. F. Guil-
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38.
[4] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Wlater.
Molecular Biology of the Cell, 4th ed., Garland Science, New York,
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[5] a) G. Burnstock, Pharmacol. Rev. 2006, 58, 58; b) P. Bodin, G. Burn-
stock, Neurochem. Res. 2001, 26, 959; c) Y. Chen, R. Corriden, Y.
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[7] a) For recent reviews and papers on PPi receptors, see: S. K. Kim,
D. H. Lee, J. Hong, J. Yoon, Acc. Chem. Res. 2009, 42, 23, and refer-
ences therein; b) C. Bazzicalupi, A. Bencini, A. Bianchi, M. Cecchi,
B. Escuder, V. Fusi, E. Garcia-EspaÇa, C. Giorgi, S. V. Luis, G. Mac-
cagni, V. Marcelino, P. Paoletti, B. Valtancoli, J. Am. Chem. Soc.
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C. Giorgi, S. Santarelli, B. Valtancoli, J. Am. Chem. Soc. 2008, 130,
2440; d) A. A. Ikeda, M. Nakasu, S. Ogasawara, H. Nakanishi, M.
Nakamura, J. Kikuchi, Org. Lett. 2009, 11, 1163; e) S. Aoki, E.
Kimura, J. Am. Chem. Soc. 2000, 122, 4542; f) S. Aoki, E. Kimura,
Chem. Rev. 2004, 104, 769; g) S. Aoki, M. Zulkefeli, M. Shiro, M.
Kohsako, K. Takeda, E. Kimura, J. Am. Chem. Soc. 2005, 127, 9129.
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b) A. Bencini, S. Biagini, C. Giorgi, H. Handel, M. Le Baccon, P.
Mariani, P. Paoletti, P. Paoli, P. Rossi, R. Tripier, B. Valtancoli, Eur.
J. Org. Chem. 2009, 5610.
[9] a) D. H. Lee, J. H. Im, S. U. Son, Y. K. Chung, J. I. Hong, J. Am.
Chem. Soc. 2003, 125, 7752; b) D. H. Lee, S. Y. Kim, J. I. Hong,
Angew. Chem. 2004, 116, 4881; Angew. Chem. Int. Ed. 2004, 43,
4777; c) H. N. Lee, Z. Xu, S. K. Kim, K. M. K. Swamy, Y. Kim, S. J.
Kim, J. Yoon, J. Am. Chem. Soc. 2007, 129, 3828; d) J. H. Lee, J.
Park, M. S. Lah, J. Chin, J. I. Hong, Org. Lett. 2007, 9, 3729; e) Y. J.
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Y. Kim, S. J. Kim, Y. J. Yoon, J. Yoon, Org. Lett. 2007, 9, 243;
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Chem. Soc. 2008, 130, 12095.
Compound 5: Compound
4 (131 mg, 0.25 mmol) and 1 (230 mg,
2.2 mmol) were dissolved in a mixture of acetonitrile and THF (25 mL,
10:1), and potassium carbonate (275 mg, 2.0 mmol) was added. The mix-
ture was stirred for 1 hour at room temperature and was then heated to
508C overnight. The precipitate was filtered and washed three times with
acetone. The collected solvent was removed in vacuo to give a solid resi-
due that was dissolved in chloroform and extracted twice with distilled
water. After removing the organic solvent a crude yellow solid was ob-
tained, which was purified by chromatography on
a basic alumina
column by using MeOH/CH2Cl2 in a 1:15 ratio as the eluent. A pure
yellow oil was collected after the solvent was removed on a rotary evapo-
rator. Yield of 5: 81 mg (29%); 1H NMR (400 MHz, CDCl3): d=1.26 (t,
J=7.3 Hz, 6H), 1.85–1.97 (m, 4H), 2.15–2.26 (m, 4H), 2.46–2.81 (m,
32H), 3.44 (s, 4H), 4.02–4.11 (m, 8H), 5.61 (s, 4H), 7.70–8.08 ppm (m,
18H); 13C NMR (100 MHz, CDCl3): d=12.9, 24.6, 32.5, 45.2, 47.5, 50.3,
51.7, 51.9, 52.6, 56.5, 57.9, 68.2, 69.1, 71.6, 85.2, 123.9, 124.9, 125.5, 125.9,
126.3, 126.9, 127.5, 128.2, 127.6, 128.2, 128.4, 131.3, 131.5, 131.8, 156.1,
168.4 ppm; ESIMS: m/z: 563.3 [M++2H]/2, 1125.7 [M++H].
Caution: Although no problems were encountered in this work, metal
perchlorate complexes are potentially explosive. They should be pre-
pared in small quantities and handled with care.
N
{ZnII
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(TACN)(Py)}] receptor: Compound 5 (57 mg, 0.05 mmol)
ꢀ
(10 mL) and the solution was heated at reflux for 30 min. After cooling
to room temperature, diethyl ether was added to precipitate the complex,
which was filtered, washed three times with a 1:1 mixture of diethyl ether
and ethanol and dried in vacuo. Yield: 61 mg (72%); 1H NMR
(400 MHz, CD3CN): d=1.35 (t, J=7.5 Hz, 6H), 1.88–1.98 (m, 4H), 2.20–
2.28 (m, 4H), 2.56–3.03 (m, 32H), 3.52 (s, 4H), 4.12–4.20 (m, 8H), 5.63
(s, 4H), 7.68–8.24 ppm (m, 18H); 13C NMR (100 MHz, CD3CN): d=13.6,
24.8, 32.2, 46.8, 48.8, 51.8, 53.8, 54.1, 55.2, 57.8, 59.2, 68.4, 69.5, 71.9, 85.5,
123.2, 124.5, 124.8, 125.6, 126.6, 126.8, 127.1, 127.5, 127.8, 128.1, 128.6,
131.1, 131.4, 131.6, 155.3, 169.1 ppm; Selected FTIR bands (KBr): v˜ =
3235 (b), 3102 (b), 2763 (b), 1724 (s), 1640 (s), 1419 (s), 1434 (b), 1225
(b), 1095 (b), 1027 (s), 894 (b), 751 cmꢀ1 (s); ESIMS: m/z: 888.2 [M+]/2;
ꢀ
elemental analysis calcd (%) for C74H108Cl4FeN6O26Zn2 [Fc bisACHTNUGRTNEUNG
{ZnII-
ACHTUNGTRENNUNG
[10] a) J. Y. Kwon, N. J. Singh, H. N. Kim, S. K. Kim, K. S. Kim, J. Yoon,
J. Am. Chem. Soc. 2004, 126, 8892; b) Z. Xu, N. J. Singh, J. Lim, J.
Pan, H. N. Kim, S. Park, K. S. Kim, J, Yoon, J. Am. Chem. Soc.
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3466.
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Received: April 8, 2010
Published online: July 7, 2010
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