Design, synthesis and evaluation of ratiometric probes for hydrogencarbonate based on europium emission.

Article abstract of DOI:10.1039/b400734b

A series of cationic, zwitterionic and anionic macrocyclic europium complexes has been prepared incorporating a N or C- linked acridone chromophore that allows sensitisation of Eu emission following excitation at 390-410 nm. Each of these complexes select

Full text of DOI:10.1039/b400734b

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Design, synthesis and evaluation of ratiometric probes for
hydrogencarbonate based on europium emission
Yann Bretonniere,a Martin J. Cann,b David Parker*a and Rachel Slatera
a Department of Chemistry, University of Durham, South Road, Durham, UK DH1 3LE
b School of Biological and Biomedical Sciences, University of Durham, South Road, Durham,
UK
Received 15th January 2004, Accepted 26th March 2004
First published as an Advance Article on the web 29th April 2004
A series of cationic, zwitterionic and anionic macrocyclic europium complexes has been prepared incorporating a N
or C- linked acridone chromophore that allows sensitisation of Eu emission following excitation at 390–410nm. Each
of these complexes selectively binds bicarbonate at physiological pH and reversible binding is signalled by a change in
the form and relative intensity of the Eu emission spectrum. Affinity for bicarbonate is regulated by overall complex
charge and falls within the range required for intracellular or extracellular analyses. Monitoring the ratio of the
intensity of Eu emission at up to three wavelengths, e.g. 618/588 or 618/702 nm allows the solution concentration of
bicarbonate to be deduced in a background of competing anions such as lactate, citrate and phosphate. Preliminary
screens reveal the complexes to be non-toxic to NIH-3T3 cells and to be taken inside the cell, encouraging further
study.
approach to the development of responsive lanthanide
Introduction
probes has emerged,8 coupled with a better understanding of
the relationship between complex structure and both NMR and
emission spectral form.9 Considerable interest is targeted on
examining complexes in which the analyte binds to the metal
complex, with an affinity that is in line with the local ion
concentration.
The bicarbonate ion is an essential component of biological
systems and is vital to many cellular processes in mammals,
such as intracellular pH homeostasis, kidney function and
sperm maturation.1 However, very little is known about how
bicarbonate concentrations fluctuate within a cell in response to
cell stimuli or stress, and such analyses are currently limited to
Of particular interest, therefore, have been studies examining
the reversible displacement of one or both waters in diaqua
complexes, associated with anion ligation to the lanthanide
centre.10 This event is signalled by changes in the intensity and
form of Ln emission, allowing ratiometric analyses to be under-
taken. Such methods are most appropriate in analyses of Eu
emission as the absence of degeneracy of the emissive 5Do state
leads to relatively simple spectra. The relative intensity of the
magnetic-dipole allowed ∆J = 1 transitions (around 590 nm) is
insensitive to the associated change in coordination environ-
ment, whilst the intensity of the electric-dipole allowed ∆J = 2
(near to 616 nm) and ∆J = 4 (around 702 nm) manifolds
changes considerably, particularly if the ‘hard’ axial water mol-
ecule is displaced by a more polarisable charged donor. The
definition of reversible anion binding in competitive aqueous
media at cationic lanthanide centres has led to suggestions that
a ratiometric method can be developed, as the form of the Eu
emission spectrum changes, and this is most evident in binding
to the bicarbonate anion.11 The structure of several of these
‘ternary’ complexes has been defined by X-ray crystallography
(e.g. in chelated adducts with lactate, citrate, several amino
acids, acetate)12 and in solution by NMR, CD and emission
studies, particularly examining Eu and Yb analogues.13 Relative
binding affinities for a range of common bioactive anions have
been assessed in simple competitive analyses or in fixed inter-
ferences studies. They reveal that in the millimolar range, it is
radiochemical measurement of overall H14CO3 uptake and
Ϫ
inferences based upon changes in intracellular pH. Information
about how HCO3 is localised and varies within cell compart-
Ϫ
ments is needed in order to understand the diverse physiological
processes it may control, for example in cyclic-AMP regulation,
through pH-independent, reversible binding to the soluble
adenylyl cyclase enzyme.2 Moreover, the pathological con-
sequences of perturbing some of these physiological processes
may be very significant: mis-expression of carbonic anhydrase
(CA) is linked to the presence of a variety of tumour types and
CA-II deficiency syndrome in humans is characterised by renal
tubular acidosis, osteoporosis and mental retardation. There is,
therefore, a pressing need for a direct method to allow changes
Ϫ
in HCO3 concentration to be measured within a cell, and to
monitor the changes in different compartments in real time.
Fluorescent probes are used to probe intracellular ionic con-
centrations.3 For practical applications, they should be non-
toxic and cell-permeable, susceptible to efficient excitation in
the near-UV/visible region, thereby avoiding biomolecule co-
excitation, and undergo spectral changes that allow ratiometric
analyses to be performed, so that the concentration dependence
of intensity-based systems is obviated. A limiting feature of
several conventional fluorescent probes (e.g. for pH, pCa) is
that they are sometimes prone to interference from scattered
light or auto-fluorescence, as they lack large Stokes’ shifts in
emission. Such problems are circumvented with longer-lived
probes as time-gating allows the unwanted short-lived emission
to decay to zero and the probe emission can be monitored after
a delay of 5 to 500 µs.4,5 Luminescent Eu and Tb probes offer
much scope in this respect, as they possess emission lifetimes
in the 0.1 to 5 ms range. Most applications of luminescent
lanthanide complexes in biochemistry and biology have related
to the development of time-resolved immunoassays6 or the
introduction of lanthanide complexes as long-lived donors in
FRET analyses.7 However, over the past 5 years, a mechanistic
Ϫ
the more polarisable (higher energy HOMO) oxygen in HCO3
(bound as CO32Ϫ) and various doubly charged phosphorylated
anions (e.g. glucose-6-O-phosphate; 2,3-bisphosphoglycerate;
AMP but not cAMP) which bind most avidly10,14 These results
are in line with measurements of preferred donor affinity in
which the binding order follows the relative polarisability of
the donor.9 The recent reports of long wavelength sensitisation
(λexc in range 370–420 nm) of Eu emission using diarylketone
and acridone chromophores15,16 prompted us to engineer such a
sensitiser into a heptadentate ligand based on the 12-N-4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Scheme 1 Reagents and conditions: (i) CuBr, 2,3-butanediol; (ii) PPA/110C; (iii) MeI, K2CO3, acetone; (iv) NaBH4, MeOH; (v) MsCl, Et3N, CH2Cl2;
(vi) cyclen (2 eq., CHCl3).
(cyclen) ring, as complexes of such ligands were originally used
in the identification of reversible anion binding.17 Accordingly,
the cationic complexes [EuL]3ϩ[L = L1–L6] were considered, in
which the (S)-amino acid side chain confers a ∆-helicity18 on
the complex. Hydrolysis of the peripheral ester groups, then
gives neutral (e.g. Ala) or trianionic (e.g. Asp, Glu) complexes,
allowing ligand-based modulation of the affinity for the target
anion.
in acetone in the presence of potassium carbonate gave the
N-methylated methyl ester and stepwise borohydride reduction
and mesylation yielded the benzylic mesylate. Alkylation of
excess cyclen in chloroform afforded the C-linked macrocycle,
4. Reaction of 4 with N-chloroethanoyl-ethyl alanate gave the
triester L6. Cationic complexes of Eu(CF3SO3)3 were prepared
in dry MeCN, and isolated by precipitation onto anhydrous
ether. The series of zwitterionic or anionic complexes was iso-
lated following controlled base-catalysed hydrolysis of the ester
groups, of L1–L6, giving L7–L12, followed by complexation with
Eu(OAc)3 in water.
Ligand and complex synthesis
Reaction of acridone with ethylene carbonate in hot DMF in
the presence of a trace of KOH19 afforded the N-hydroxyethyl
compound 1 and this was converted into the mesylate 2 under
standard conditions (THF, Et3N, MsCl). Addition of 2 to
excess 1,4,7,10-tetraazacyclododecane (cyclen) in boiling
acetonitrile gave the mono-alkylated compound 3 in reasonable
yield. Reaction of 3 with three equivalents (MeCH, Cs2CO3)
of the 2-chloroethanoyl derivatives of ethyl glycinate, alanate,
β-alanate, diethyl aspartate and diethyl glutarate afforded the
esters L1–L5, which were purified by chromatography on neutral
alumina. The constitutional isomer of 3, was also prepared
from 2-bromoterephthalic acid, Scheme 1. Reaction of aniline
in the presence of cuprous bromide in hot butanediol afforded
2-aminophenylterephthalic acid which cyclised in the presence
of polyphosphoric acid to give acridone-3-carboxylic acid, in
81% yield over the two steps. Subsequent reaction with MeI
Emission and NMR spectral behaviour on anion binding
The europium emission spectra for each complex were recorded
in 0.1 M MOPS buffer (0.1 mM complex pH 7.4, 295 K) in the
absence and presence of excess added anions. The form of the
metal based emission spectrum changed significantly, consist-
ent with the partial or total displacement of the coordinated
water molecule and the binding of the anion to the Eu centre.
Representative spectra for [EuL7] – the zwitterionic complex
with three peripheral Ala side-arms – in the presence of
(S)-lactate, bicarbonate and hydrogenphosphate, (Fig. 1), were
very similar to those obtained previously with the N-methylated
complex [EuL13].10a As discussed at length in earlier articles,9,10a
the form of the emission spectrum of the carbonate adduct is
the most distinctive. It is characterised by a large ∆J = 2/∆J = 1
Fig. 1 Europium emission spectra for [EuL7] in the presence of a 10-fold excess of added (S)-lactate, hydrogencarbonate or hydrogenphosphate
(pH 7.4, 0.1 M MOPS, 295 K, λexc 410 nm, 0.1 mM complex).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
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band intensity ratio (e.g. 618/588 nm), a relatively small separ-
ation of the A2 A1 and E A1 transitions in the ∆J = 1
and presence of added anion. With [EuL4]3ϩ as the aqua species
for example, kH O = 2.39 msϪ1, kD O = 1.02 msϪ1, consistent
2
manifold around 590 nm, and a weak ∆J = 0 transition at
579 nm. Proton NMR spectra (pD7, 500 MHz, 295 K) for these
anion adducts were examined and were also very similar to
those previously characterised with [EuL13], consistent with the
formation of chelated (lactate/carbonate) or mono-aqua
(phosphate) adducts, the structures of which had been revealed
by crystallographic, chiroptical, emission lifetime and detailed
NMR analyses.12 Similarly the emission and 1H-NMR analyses
of spectra obtained in the presence of excess citrate, acetate,
fluoride were consistent with earlier interpretations. Radiative
rate constants defining the decay of the Eu emission at 592
or 618 nm were measured in H2O and D2O, in the absence
with a hydration state of one.20 Foll2owing addition of ≥ 10 eq.
of NaHCO3, the values obtained (kH O = 1.80 msϪ1, kD O
=
2
1.18 msϪ1) are consistent with complete displacement of2 the
bound water molecule and formation of a carbonate chelate.
The overall quantum yields for Eu emission (295 K, H2O) were
measured for the carbonate adducts, and were 4.4% ( 0.8) for
[EuL7] and [EuL12] and 6% for [EuL3]3ϩ
.
These modest overall quantum yields are primarily limited by
radiative deactivation, i.e. competitive fluorescence from the
intermediate acridone singlet excited state. N-Alkyl acridones
possess low-lying n–π* and π–π* singlet excited states, and the
relative energy of the latter is particularly sensitive to solvent
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
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polarity. Thus, for N-methyl acridone itself, in non-polar media
Es∼ 300 kJmolϪ1, ꢀfluor = 2%, ꢀT = 96% (77 K). In polar solvents
(e.g. EtOH, H2O), the π–π* transition is lowered in energy
(Es∼283 kJmolϪ1) and the rate of inter-system crossing becomes
much slower relative to the rate of fluorescence (ꢀfluor = 98%
ꢀT = 0.1%).21 Thus, in the Eu complexes described herein, it is
the efficiency of the rate of fluorescent emission – competing
with formation of the aryl triplet state – that limits the metal
based emission quantum yield.
The emission wavelength for acridone fluorescence was found
to be a sensitive function of solvent polarity in the europium
complexes. For example, with [EuL3]3ϩ, the position of the
emission wavelength maximum varied as a function of solvent
polarity. Indeed a good correlation was found by plotting λfmax
(acridone emission) versus Reichardt’s empirical solvent polar-
ity parameter, ET(30)22 (Fig. 2). Parallel variations in the form
and relative intensity of ‘Eu’ excitation spectra were also
observed, in accord with the lowering of the energy of the π–π*
excited state in the more polar media. The form and relative
intensity of the ∆J = n bands (n = 0, 1, 2, 3, 4) in the europium
emission spectra of [EuL3]ϩ were also sensitive to solvent polar-
ity, but not because of variations in the solvation of the acrid-
one ground/excited states. The changes in this case reflect the
variation in the europium coordination environment as the
axial donor (the solvent) is changed.9 This variation gives rise to
a marked diminution of the splitting of the ∆J = 1 (∼590 nm)
manifold accompanied by a change in the form and an increase
in the relative intensity of the ∆J = 2 transition (∼620 nm) in a
sequence that echoes the relative polarisability of the axial
donor, i.e. DMSO > ROH > MeOH > THF/CHCl3. For the
least polar solvents, it is likely that the triflate counterion
coordinates. Such a sequence has been characterised previously
for related nine coordinate macrocyclic tetra-amide complexes
in which the axial donor is permuted.9
Affinity for bicarbonate in a simulated extracellular medium and
ratiometric analysis of binding
Incremental addition of sodium bicarbonate to a solution of
[EuL7] in the presence of competing anions (295 K, pH 7.4,
0.1 M MOPS; 100 mM NaCl, 0.9 mM Na2HPO4, 2.3 mM Na
lactate, 0.13 mM potassium citrate) was monitored by following
changes in the form of the europium emission spectrum, Fig. 3.
An increase in the relative intensity of the ∆J = 2 manifold was
observed, (e.g. at 618 nm) accompanied by the formation of
isoemissive points at 588 nm (∆J = 1) and at 702 nm (∆J = 4).
Such changes, allow the changes in intensity ratio at 618/588 (or
618/702 nm) to be plotted as a function of added bicarbonate.
This revealed an increase in the 618/588 nm ratio from an
initial value of five to fifteen, following addition of 30 mM
bicarbonate. The cationic complexes, e.g. [EuL1]3ϩ, [EuL4]3ϩ and
[EuL5]3ϩ bound most strongly (Fig. 4) and the anionic systems,
Fig. 4 Variation of the emission intensity ratio (at 618/588 nm) for
[EuL1]3ϩ (᭹) [EuL8] (ꢀ), [EuL4]3ϩ (᭜) and [EuL11]3Ϫ (᭿) as a function
of added NaHCO3 (pH 7.4, 0.1 M MOPS in a simulated extracellular
background; 100 mM NaCl, 0.9 mM Na2HPO4, 2.3 mM Na lactate,
0.13 mM K citrate).
Fig. 2 Variation of the acridone fluorescence emission wavelength
maximum for [EuL3]3ϩ(CF3SO3)3 as a function of Reichardt’s empirical
solvent polarity parameter, ET(30).
Fig. 3 Variation of the metal based emission in [EuL7] following incremental addition of NaHCO3 (0.1 M MOPS, pH 7.4, 295 K, 0.1 mM complex).
The spectra in the insets reveal the formation of isoemissive points at 588 nm (∆J = 1) and 702 nm (∆J = 4).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
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Ϫ
Table 1 Apparent affinity constants for complexation of HCO3
that occurs on bicarbonate binding falls very clearly in this
(295 K, 0.1 M MOPS, pH 7.4; 100 mM NaCl, 2.3 mM Na lactate,
0.9 mM Na2HPO4, 0.13 mM K citrate)
range, and has been shown to trigger the release of cyclic-AMP
in mammalian cells.23 The europium complexes discussed
herein could serve as intracellular probes for bicarbonate, pro-
vided that they are non-toxic and that they are able to enter the
cell. Accordingly, some preliminary toxicity and cellular uptake
experiments have been carried out.
Europium
Complex
Log Ka
[EuL1]3ϩ
[EuL2]3ϩ
[EuL4]3ϩ
[EuL5]3ϩ
[EuL7]
(AlaOEt)
(β-AlaOEt)
(GluOEt)
(GlyOEt)
(Ala)
(β-Ala)
(Gly)
(C-linked Ala)
(Asp)
(Glu)
1.95
2.09
1.69
2.27
1.49
1.42
1.28
1.48
0.99
0.80
Cell toxicity was assessed in a crude screen, by incubating
NIH 3T3 mouse fibroblast cells with a standard growth
medium containing 1 mM complex ([EuL1]3ϩ, [EuL7] and
[EuL11]3Ϫ) for 3 h. Cells were washed with phosphate buffered
saline (PBS) solution and were incubated (1 h) with Trypan
Blue (0.1% v/v) in PBS. The number of live cells was then
counted using a haemocytometer. The charged complexes
[EuL1]3ϩ/[EuL11]3Ϫ showed no evidence for toxicity (same num-
ber of live cells [95 2%] as control with no added complex),
while the neutral complex showed only a slight toxic effect (87%
live cells). In order to examine cell uptake, cells were mounted
on a slide and were examined by fluorescence microscopy, excit-
ing at 400 nm. Each complex revealed a similar distribution
of staining, examining the Eu emission selectively by the use
of appropriate cut-off filters. Indeed localisation and staining
appeared to be independent of the period of incubation, the
nature of the complex and a similar overall profile was observed
when the cells were electroporated. The images obtained (Fig. 7)
revealed a punctuate distribution of the complex, resembling
an endosomal/lysosomal localisation. Further co-localisation
studies are underway using organic fluorescent dyes whose
cellular localisation profile is established. In addition, the
nature and efficiency of uptake will be examined together with
measurements of the ratio of the intensity of Eu emission from
the localised complex, with appropriate control experiments to
assess regional pH.
[EuL8]
[EuL9]
[EuL12]
[EuL10]3-
[EuL11]3-
e.g. [EuL10]3ϩ and [EuL9]3Ϫ bound with the least affinity, in
accord with electrostatic modulation of anion affinity following
encounter with the peripheral ligand substitutents. Inspection
of the form of the initial Eu emission spectrum, i.e. in the pres-
ence of the simulated extracellular medium, revealed that for
the anionic and neutral complexes, the spectrum corresponded
very closely to the lactate adduct, whereas for the cationic sys-
tems both lactate and phosphate adducts seemed to be evident.
The apparent affinity constant characterising reversible bind-
ing of bicarbonate in this competitive medium was measured by
fitting the variation of emission intensity (e.g. 618 nm) with
added total carbonate. A representative binding isotherm is
given (Fig. 5) for the association with [EuL2]3ϩ, in which the
curve shows the least squares derived fit to the experimental
data, based on a 1 : 1 stoichiometry, using methods previously
reported 10a. Data for a range of complexes are tabulated (Table
1) and reveal some simple trends. Affinity for bicarbonate
follows the trend: cationic > neutral > anionic, consistent with
Coulombic modulation of affinity for the anionic species, and
within the series of cationic complexes, substitution in the side
chain gave rise to complexes of lower apparent affinity (e.g. Gly
> β-Ala > Ala > Glu-substituted systems).
Conclusion
The europium complexes examined afford a new method for
assessing the solution concentration of HCO3Ϫ, by measuring
the intensity ratio of the 618/588 or 618/702 nm emission bands.
Controlled modulation of the affinity for the target anion is
achieved by varying the overall charge on the complex and this
is most readily effected by variation of the three amino-acid
derived side-chains. Such systems, therefore, allow measure-
ments of HCO3Ϫ to be made in principle, either in extracellular
media or in an intracellular medium. The absence of toxicity
in a preliminary screen and the ability of the complexes to
enter the cells augurs well for future work directed at devising
practicable ratiometic imaging protocols.
Experimental
Fig. 5 Change in the intensity of the Eu emission band at 618 nm in
[EuL2]3ϩ as a function of added NaHCO3 showing the fit (line) to the
experimental data for 1 : 1 complexation (pH 7.4, 0.1 mM MOPS,
295 K, 0.1 mM complex).
Reagents and solvents
Acetonitrile was dried over calcium hydride before use. Water
and H2O refer to high purity water with conductivity ≤0.04 µS
cmϪ1, obtained from the PURITE purification system.
1,4,7,10-Tetraazacyclododecane is commercially available
(Strem) and was used as received.
Affinity in cellular media: preliminary cell toxicity/update and
microscopy
A parallel titration experiment was carried out, using a cellular
lysate (pH 7.4, 0.1 M MOPS) as background medium, which
contains various proteins as well as significant concentrations
of lactate and various phosphoylated species, e.g. 2,3-diphos-
phoglycerate. Mouse skin fibroblasts (NIH 3T3) were sonicated
and to the resultant lysate was added an 0.1 mM solution of
[EuL7] or [EuL11]3Ϫ. The emission intensity ratio change (618/
588 nm) was plotted as a function of added bicarbonate con-
centration. For [EuL11]3Ϫ, a 69% change in intensity ratio was
noted between 5 and 15 mM (or 130% between 3 and 20 mM).
Such a variation coincides well with the putative range of intra-
cellular bicarbonate concentration, (Fig. 6). Indeed, poten-
tiation of the activity of the soluble enzyme, adenylyl cyclase,
Chromatography
Column chromatography was carried out using “gravity” silica
(Merck). Cation exchange chromatography was performed
using Dowex 50W 50 × 4–200 strong ion exchange resin, which
had been pre-treated with 3 M HCl.
Spectroscopy
1H NMR spectra were recorded at 65.6 MHz on a 1.53T mag-
net connected to a Varian VXR400 console, at 199.99 MHz on
Varian Mercury-200 and at 299.91 MHz on Varian Unity-300.
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Fig. 6 Variation of the Eu emission intensity ratio (618/588 nm) with added NaHCO3 in a cell lysate medium (λexc 410 nm, 0.1 M MOPS, pH 7.4,
0.1 mM complex).
Fig. 7 Confocal fluorescence microscopy images of: [EuL1]3ϩ, (left); [EuL7] and [EuL11]3Ϫ (right), showing the localisation of each complex inside
NIH 3T3 cells – the oval-shaped dark centre is the cell nucleus.
13C NMR were recorded on the Varian Mercury-200 at 50.2
MHz and a Varian Unity-300 at 75.4 MHz. Mass spectra were
recorded using a VG Platform II electrospray mass spectro-
meter with methanol or water as a carrier solvent. Accurate
masses were determined at the EPSRC National MS Service at
Swansea; isotope calculations for Gd and Eu complexes refer to
153Eu and 158Gd. Luminescence measurements for europium
and terbium complexes were recorded using an Instrument SA
Fluorolog 3–11 using DataMax for Windows v2.1. Second
order diffraction effects were obviated by using a 375 nm cut-
off filter. pH measurements were made using a Jenway 3320 pH
meter (fitted with a BDH Glass ϩ combination electrode –
microsample) calibrated with pH 4, 7 and 10 buffer solutions.
t, NCH ), 3.82 (2 H, q, CH OH; δ (DMSO-d6): 177.2 (C᎐O),
᎐
2 2 C
142.8 (Ar C), 134.7 (Ar C), 127.3 (Ar C), 122.3 (Ar C), 121.9
(Ar C), 117.1 (Ar C), 59.0 (CH2), 48.3 (CH2); m/z (ESϩ): 262.1
[M ϩ Na]ϩ, 501.2 [2M ϩ Na]ϩ, 740.3 [3M ϩ Na]ϩ. νmax (KBr):
3319 (OH), 1628, 1608 (C᎐O); λ
(MeOH): 254, 384, 402.
᎐
max
Found: C, 75.16; H, 5.46; N, 5.80%. C15H13NO2 requires C,
75.30; H, 5.48; N, 5.85%.
N-(2-(Methylsulfonatoethyl)-acridone, 2. To a suspension of
N-(2-hydroxyethyl)-acridone (1.50 g, 6.46 mmol) in dry THF
(25 mL) under argon was added methanesulfonyl chloride
(625 µL, 8.1 mmol, 1.25 eq.) and dry triethylamine (1.25 mL,
7.9 mmol, 1.5 eq.). The mixture was stirred at room temper-
ature until the reaction was adjudged complete by TLC
analysis, completed (2 to 3 h). The solvent was removed under
reduced pressure, the resulting solid dissolved in dichloro-
methane (20 mL), and the solution washed successively with 1%
aqueous hydrochloric acid (10 mL), 50% saturated aqueous
sodium bicarbonate solution (10 mL) and water (10 mL), dried
(MgSO4), filtered and evaporated to dryness to afford the title
compound as a yellow solid, 1.96 g, 95%, which was used with-
out further purification, mp 162–164 ЊC, Rf (SiO2, 30% EtOAc
in CH2Cl2): 0.50.
Titrations
Luminescence titrations were carried out in a background of
constant ionic strength and pH (0.1 M MOPS buffer, pH 7.4,
295 K) on solutions with absorbances of <0.2 at wavelengths
≥λex in order to avoid any errors due to the inner filter effect.
Incremental addition of sodium carbonate solution was carried
out using Gilson micropipettes. The luminescence spectra were
recorded at each point (30–40 points per titration). Excitation
wavelengths of 410 nm (sensitisation via acridone chromo-
phore) or 397/355 nm (direct excitation at Eu) were used to
obtain the luminescence spectra. Excitation and emission slits
were 2–5 nm and 1 nm bandpass respectively. Points were
recorded at 1 nm intervals with a 0.25 s integration time.
δH (CDCl3): 8.51 (2 H, dd, J, H1-8), 7.71 (2 H, dd, ArH), 7.51
(2 H, d, ArH), 7.27 (2 H, t, ArH), 4.75 (2 H, t, –CH2–OS), 4.61
(2 H, t, –CH2–N), 2.93 (3 H, s, –CH3); δC (CDCl3): 177.9 (C9);
141.7, 134.4, 128.2, 122.6, 122.0, 114.2, 64.5, 44.4, 37.8. m/z
(ESϩ): 318.1 [M ϩ H]ϩ, 340.1 [M ϩ Na]ϩ, 656.9 [2M ϩ Na]ϩ,
974.5 [3M ϩ Na]ϩ. νmax (KBr): 3900 (Ar CH), 1596 (C᎐O), 1179
᎐
Synthesis
(SO2); λmax (MeOH): 216, 254, 382, 396.
N-(2-Hydroxyethyl)-acridone, 1. To a suspension of acridone
(2.5 g, 12.8 mmol) and ethylene carbonate (2.25 g, 25.5 mmol,
2 eq.) in dry DMF (20 mL) was added a catalytic amount of
potassium hydroxide. The mixture was heated at 130 ЊC under
argon overnight. The solvent was removed under reduced
pressure and the crude product was precipitated from the resi-
dual oil by adding water. The solid was filtered, washed with
water and recrystallised from methanol to furnish a yellow
solid, (2.36 g, 70%); mp 212–214 ЊC (lit.19 212.2–213.8 ЊC).
δH (DMSO-d6): 8.35 (2 H, dd, H1–8), 7.90 (2 H, d, ArH), 7.82
(2 H, td, ArH), 7.33 (2 H, td, ArH), 5.07 (1 H, t, OH), 4.60 (2 H,
N-(2-Acridylethyl)-1,4,7,10-tetraazacyclododecane, 3. A solu-
tion of N-(2-(methylsulfonyl)ethoxy)-acridone (1.96 g, 6.18
mmol) and excess cyclen (2.13 g, 12.36 mmol, 2 eq.) in dry
acetonitrile (20 mL) was boiled under reflux for 36 hours. After
removal of the solvent, the residue was partitioned between
hydrochloric acid (2 M, 75 mL) and dichloromethane (75 mL).
The acidic layer was separated, basified to pH 13.5 with concen-
trated aqueous sodium hydroxide solution and extracted with
dichloromethane (3 × 50 mL); under these conditions the excess
cyclen remains in the aqueous layer. The combined organic
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
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extracts were dried (MgSO4), filtered and evaporated to dryness
to give the title compound as a hygroscopic yellow solid, 1.96 g,
80%, mp 110–112 ЊC (lit.2 not reported). m/z (ESϩ): 394.3
[MH]ϩ; 406.3 [M ϩ Na]ϩ. δH (CDCl3): 8.56 (2 H, dd, ArH), 7.76
(2 H, td, ArH), 7.63 (2 H, d, ArH), 7.29 (2 H, td, ArH), 4.60 (2
H, t, –CH2–NAr), 3.00 (2 H, t, –NCH2–CH2–NAr), 2.9–2.6 (16
H, m, ring CH2), 2.1 (3 H, br m, NH).; δC (CDCl3): 178.3 (C9),
141.9, 134.4, 128.2, 122.7, 121.7, 114.9, 52.4 (CH2), 52.2 (CH2),
47.3 (CH2), 45.8 (CH2), 45.4 (CH2), 44.2 (CH2); νmax (KBr):
the organic layer extracted with aqueous ammonia solution
(0.5 M, 100 mL). The aqueous extract was acidified with con-
centrated hydrochloric acid to give a yellow solid, which was
collected by filtration, washed with hot water and dried, (15 g
90%), mp (sub) >180 ЊC. δH (DMSO-d6): 12.26 (2H, br s,
COOH ), 9.61 (1H, br s, NH ), 7.98 (1H, d, J 8.2, H6), 7.74
(d, 1H, J 1.4, H3), 7.40 (1H, ddd, J 8.2, 7.2, 1.8, H3Ј), 7.25 to
7.30 (3H, m, H5 ϩ H2Ј), 7.13 (1H, ddd, J 7.4, 7.2, 1.2, H4Ј.
δC (DMSO-d6): 169.0 (C7), 167.5 (C8), 147.2 (C2 or C4), 140.1
(C1Ј), 135.3 (C2 or C4), 131.9 (C6), 129.2 (2 C, C3Ј), 123.2 (C4Ј),
121.7 (2 C, C2Ј), 117.2 (C5), 115.1 (C3), 114.6 (C1). m/z (ESϪ):
256.0 [M Ϫ H]Ϫ; 534.9 [2M Ϫ 2H ϩ Na]Ϫ. Found: C, 63.62; H,
4.39; N, 5.01%. C14H11NO4 0.5 H2O requires: C, 63.16; H, 4.54;
N, 5.26%.
3445 (NH), 2823 (CH), 1631, 1597 (C᎐O); λmax (H2O) 255, 384,
᎐
400.
A typical procedure for the synthesis of an N-linked acridone
ligand follows.
N-(2-[(S,S,S )-4,7,10-Tris-(1-(1,3-diethoxycarbonyl)propyl
carbamoyl methyl)-1,4,7,10-tetraazacyclododecan-1-yl]ethyl)-
acridone, L3. (S)-N-(2-Chloroethanoyl)ethyl glutamate (1.00 g,
3.57 mmol, 3.1 eq.) and caesium carbonate (1.16 g, 3.57 mmol,
3.1 eq.) were added to a solution of N-(2-acridylethyl)-1,4,7,
10-tetraazacyclododecane (0.45 mg, 1.15 mmol) in acetonitrile
(10 mL) and the mixture was boiled under reflux for 48 h. The
reaction mixture was cooled, filtered, and the solvent evapor-
ated under reduced pressure to give an oily residue that was
purified by chromatography on neutral alumina (100% CH2Cl2
to 0.5% EtOH–CH2Cl2) yielding a hygroscopic yellow solid, 740
mg, 57%, mp 98–100 ЊC; Rf (Al2O3, 5% MeOH–CH2Cl2): 0.6.
δH (CDCl3): 8.51 (2 H, dd, ArH), 7.78 (2 H, d, NH), 7.69 (2H, t,
ArH), 7.52 (2 H, d, ArH), 7.49 (1 H, d, NH), 7.24 (2 H, t, ArH),
4.50 (5 H, m, CHNHCO ϩ ArNCH2CH2N), 4.01 (12 H, m,
OCH2CH3), 3.12 (6 H, m, NCH2CO), 2.95 (2 H, t, ArNCH2-
CH2N), 2.74–2.88 (16 H, br m, ring CH2), 3.12 (6 H, m,
CH2COO), 2.14 and 1.94 (3 H and 3 H, m and m, CHCH2CH2),
1.18 (18 H, m, OCH2CH3); δC (CDCl3,): 177.8, 172.6, 172.5,
172.1, 171.8, 170.8, 141.7, 134.2, 127.9, 122.4, 121.4, 114.4,
61.5, 60.6, 58.2, 53.6, 53.1, 52.5, 51.5, 30.5, 27.1, 14.1, 14.0;
Acridone-3-carboxylic acid. A suspension of finely powdered
2-(phenylamino)terephthalic acid (8.25 g, 32 mmol) in
polyphosphoric acid (70 mL) was heated at 120 ЊC with vigor-
ous stirring for ca. 2 to 3 hours until everything dissolved. The
solution was then slowly poured into boiling water. The precipi-
tate, which formed was collected by filtration, washed with hot
water and dissolved in a mixture of methanol (100 mL) and
aqueous sodium hydroxide solution (1 M, 100 mL). An in-
soluble black solid was filtered off whilst the solution was still
hot. The filtrate was acidified with glacial acetic acid, concen-
trated, cooled and the solid collected by filtration, washed with
water and dried under vacuum to yield a yellow solid (6.90 g,
90%) that was used without further purification; mp >260 ЊC.
δH (DMSO-d6): 13.38 (1H, br s, COOH ), 11.97 (1H, s, NH ),
8.31 (1H, d, J 8.0, H1), 8.24 (1H, dd, J 8, 1.5, H8), 8.18 (1H, d,
J 1.5, H4), 7.77 (1H, ddd, J 8, 7.5, 1.5, H6), 7.72 (1H, dd, J 8.5,
1.5, H2), 7.55 (1H, d, J 8.0, H5), 7.29 (1H, ddd, J 8, 7.5, 1.5, H7).
δC (DMSO-d6): 177.2 (C9), 167.4 (COO), 141.8 (C5Ј), 141.2 (C4Ј),
135.5 (C3), 134.6 (C6), 127.3 (C1), 126.7 (C8), 123.3 ( C1Ј), 122.2
(C7), 121.4 (C8Ј), 121.3 (C2), 119.8 (C4), 118.2 (C5).
νmax (KBr): 3268, 2981, 2828 (CH), 1735, 1672 (C᎐O), 2598
᎐
(NH)cmϪ1; λmax (MeOH) 258, 390, 408; HRMS (m/z): [M ϩ
3-Methoxycarbonyl-N-methyl-acridone. A suspension of
H]ϩ. Found: 1123.5927: C56H83N8O16 requires 1123.5916.
acridone-3-carboxylic acid (4.0 g, 16.7 mmol), methyl iodide
(5.2 mL, 83.5 mmol, 5 eq.) and K2CO3 (11.5 g, mmol, 5 eq.) in
acetone (100 mL) was boiled under reflux for 6 days. Dichloro-
methane (400 mL) and water (400 mL) were added and the two
phases separated. The organic extract was washed with water,
dried (MgSO4) and filtered. Removal of the solvents under
reduced pressure afforded the title compound as a yellow
solid, (4.0 g, 90%); Rf (SiO2, 2% CH3OH in CH2Cl2): 0.30;
H6L4. The hexaester L3 (453 mg, 0.40 mmol) was treated with
aqueous sodium hydroxide (0.5 M, 20 mL), and the solution
stirred at room temperature for 4 days. After the pH was
adjusted to 6 with concentrated hydrochloric acid, the solution
was concentrated by one half and passed down a strong cation
exchange column (DOWEX 50 W, Hϩ form), eluting with 12%
aqueous ammonia solution. The solvent was removed under
reduced pressure, the oil dissolved in water and the solution
freeze-dried to give a hygroscopic yellow solid, (400 mg, 89%),
m/z (ESϪ): 953.2 [L6H5]Ϫ. δH (D2O): 8.12 (2 H, d, ArH), 7.77 (2
H, t, ArH), 7.54 (2 H, d, ArH), 7.25 (2 H, t, ArH), 4.58 (3 H, br
m, CH–CONH), 4.17 (2 H, t, ArNCH2CH2–), 4.11 (2 H, br s),
3.86 (6 H, m), 3.29 (16 H, m, CH2 ring), 2.29 (2 H, t,
ArNCH2CH2–), 1.82–2.33 (12 H, m, CH2CH2CHCO);
δC (D2O): 181.0, 180.6, 180.4, 179.2, 178.4, 178.0, 141.1, 135.5,
126.8, 122.6, 121.1, 115.2, 55.2, 54.6, 54.4, 50.5, 33.3, 33.1, 32.9,
28.0, 27.8. HRMS (m/z): [M ϩ H]ϩ 953.3888. Found; C44H58-
N8O16 requires 953.3893.
mp >260 ЊC. νmax (KBr): 1727(ester CO), 1597 (CO) cmϪ1
.
λmax (CH3OH): 404 (7790), 420 (8330). δH (CDCl3): 8.54 (1H, d,
J 8.2, H1); 8.49 (1H, dd, J 8.0, 1.8, H8); 8.22 (1H, d, J 1.2, H4);
7.82 (1H, dd, J 8.2, 1.2, H2); 7.70 (1H, ddd, J 8.6, 7.0, 1.5, H6);
7.50 (1H, d, J 8.6, H5); 7.26 (1H, ddd, J 8.0, 7.0, 1.0, H7); 3.94
(3H, s, OCH3); 3.91 (3H, s, NCH3) δC (CDCl3): 178.0 (C9); 168.8
(COO); 143.1 (C5Ј); 143.0 (C4Ј); 134.5 (2C, C3 ϩ C6); 128.4 (C6);
128.1 (C8); 125.2 ( C1Ј); 123.0 (C8Ј); 121.9 (C7); 121.4 (C2); 117.2
(C4); 115.1 (C5); 52.8 (OCH3); 34.1 (NCH3). HRMS (ESϩ),
found: 290.0792; C16H13NO3Naϩ requires: 290.0793.
3-(Hydroxymethyl)-N-methyl-acridone. To a suspension of
the ester (2.43 g, 9.10 mmol) in freshly distilled dry ethanol
(50 mL) under argon was added NaBH4 (690 mg, 18.2 mmol,
2 eq.). The mixture was boiled under reflux overnight, (until
no more methyl ester was indicated by TLC: Al2O3–EtOAc),
cooled and concentrated by one half. Water (20 mL) was added
carefully to destroy the excess borohydride, and the mixture was
concentrated again by one half. This operation was repeated
twice. The resulting brown solution was extracted with dichloro-
methane (3 × 50 mL). At this stage, some of the product pre-
cipitated and was collected by filtration (0.72 g, 32%). The
combined organic phases were dried (MgSO4), filtered and
evaporated to dryness. The resulting brown oil was triturated
with diethyl ether and the product which precipitated was
2-(Phenylamino)terephthalic acid. A mixture of 2-bromo-
terephthalic acid (15 g, 61.2 mmol), aniline (8.5 mL, 93 mmol,
1.5 eq.), copper() bromide (1.5 g.), 2,3-butanediol (70 mL) and
toluene (50 mL) was heated and stirred at 120 ЊC for around
30 minutes with toluene being allowed to evaporate. Triethyl-
amine (30 mL) was added, and the mixture heated at 130 ЊC
overnight. The cooled solution was diluted with aqueous
ammonia solution (0.5 M, 70 mL) and treated with activated
charcoal. After filtration through Celite (washing well with
water) the dark coloured solution was acidified to pH = 2 with
hydrochloric acid (2 M) and extracted with 3 portions of ethyl-
acetate (150 mL). An insoluble precipitate was filtered off and
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
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collected by filtration as a fine yellow powder (0.5 g, 22%), mp
196–198 ЊC. δH (DMSO-d6): 8.33 (1H, d, J 8.0, 1.5, H8); 8.29
(1H, d, J 8.0, H1); 7.80 to 7.86 (2H, m, H5 ϩ H6); 7.76 (1H, s,
H4); 7.33 (1H, ddd, J 8.0, 7.5, 1.0, H7); 7.29 (1H, d, J 8.0, H2);
5.51 (1H, t, J 5.5, OH ); 4.72 (2H, d, J 5.5, CH2O); 3.94 (3H, s,
NCH3); δC (DMSO-d6): 171.0 (C9); 150.0 (C3); 143.1 (2C, C4Јϩ
C5Ј); 134.6 (C6); 127.2 (2C, C1 ϩ C8); 122.4 ( C8Ј); 121.8 (C7);
121.2 (C1Ј); 120.3 (C2); 116.8 (C5); 113.5 (C4); 63.6 (CH2O); 34.4
(NCH3). HRMS (ESϩ): found: 262.0846; C15H13NO2Na
requires: 262.0844.
freeze-dried to yield a hygroscopic yellow solid, (110 mg, 46%).
m/z (ES) : 778 [MH]ϩ; Found : 778.3648 C38H50N8O10 requires:
778.3650.
Europium(III) complexes
A typical procedure for Eu() complex formation with neutral
ligands follows methods previously reported, with represent-
ative characterization data.
[Eu(L4)](CF3SO3)3. Europium trifluoromethanesulfonate
(51 mg, 0.09 mmol) and L3 (80 mg, 0.07 mmol) were dissolved
in acetonitrile (5 mL) and the solution was boiled under
reflux for 18 h. The solution was cooled, filtered and added
dropwise with stirring to cold diethyl ether (50 mL). A yellow
solid precipitated which was recovered by centrifugation and
re-precipitated by following the same procedure twice more to
yield a very hygroscopic yellow solid. This was dissolved in
water (and the minimum amount of methanol) and the solution
freeze-dried, to give a bright yellow solid (88 mg, 72%). m/z
(ESϩ): 646.4 [M ϩ (OH)]2ϩ; νmax (KBr): 1734, 1718, 1700, 1685,
N-(3-Acridylmethyl)-1,4,7,10-tetraazacyclododecane. To
a
suspension of the alcohol 4 (470 mg, 1.96 mmol) in dry THF
(15 mL) under argon was added dry triethylamine (820 µL, 5.8
mmol, 3 eq.) and methanesulfonyl chloride (305 µL, 3.9 mmol,
3 eq.). The mixture was stirred at room temperature for 2 hours.
The solvent was removed under reduced pressure, the resulting
solid dissolved in dichloromethane (20 mL), and the solution
washed with aqueous hydrochloric acid (0.1 M, 10 mL), 50%
saturated aqueous sodium bicarbonate (10 mL) and water
(10 mL), dried (MgSO4), filtered and evaporated to dryness to
afford the mesylate as a yellow solid (574 mg, 92%) that was
used without further purification. Rf (SiO2, 5% CH3OH in
CH2Cl2): 0.50.δH (CDCl3): 8.53 (1H, d, J 8.4, H1), 8.50 (1H, dd,
J 8.1, 1.8, H8), 7.70 (1H, ddd, J 8.7, 7.2, 1.8, H6), 7.54 (1H, br s,
H4), 7.50 (1H, d, J 8.7, H5), 7.20 to 7.30 (2H, m, H2 ϩ H7), 5.34
(2H, s, CH2O), 3.87 (3H, s, NCH3), 2.96 (3H, s, SCH3).
1654, 1637 (C᎐O); λmax (H2O) 258, 390, 408.
᎐
[Eu(L6)](CF3SO3)3.. m/z (ESϩ): 508.7, 509.7 [EuL Ϫ H]2ϩ
,
1214 (EuL ϩ 2CF3SO3]ϩ. Found: 1315.2992 C46H66N8O16S2-
F6Eu requires: 1315.2998 [EuLX2]ϩ.
Eu() complexes of anionic ligands were prepared using the
lanthanide acetate salt.
A solution of the mesylate (390 mg, 1.23 mmol) in dry
chloroform (15 mL) was added dropwise over 3 hours to a
solution of cyclen in excess (867 mg, 5.04 mmol, 2 eq.) in dry
chloroform (30 mL). The solution was stirred at room temper-
ature for a further 24 hours before adding hydrochloric acid
(0.1 M, 80 mL). The acidic layer was separated, filtered and the
pH raised to 13 by adding concentrated aqueous potassium
hydroxide solution. The mixture was re-extracted with chloro-
form (3 × 50 mL), the organic phase dried (K2CO3), filtered and
evaporated to dryness to afford a very hygroscopic pale yellow
solid, (360 mg, 74%). δH (CDCl3): 8.50 (1H, dd, J 8.0, 1.4, H8),
8.43 (1H, d, J 8.2, H1), 7.65 (1H, ddd, J 8.6, 7.0, 1.4, H6), 7.58
(1H, br s, H4), 7.44 (1H, d, J 8.6, H6), 7.13 to 7.26 (2H, m, H2 ϩ
H7), 3.86 (3H, s, NCH3), 2.73 (2H, s, NCH2-Ar), 2.51 to 2.82
(16H, br m, ring CH2); δC (CDCl3): 178.1 (C9), 142.0 (C3); 141.1
ϩ 141.0 (2C, C4Јϩ C5Ј), 134.6 (C6), 127.2 (2C, C1 ϩ C8), 122.4
(C8Ј), 121.8 (C7), 121.6 (C1Ј), 120.5 (C2), 116.4 (C5), 114.2 (C4),
59.1 (NCH2Ar), 52.4 (NCH2), 52.2 (NCH2), 47.3 (NCH2), 45.9
(NCH2), 34.0 (NMe); νmax (KBr): 3445 (NH), 2823 (CH), 1631,
[Eu(L11H3)]. A solution of europium acetate (20 mg, 0.1
mmol) in water (2 mL) was added to a solution of H6L11
(100 mg, 0.10 mmol) in water (3 mL). A solid precipitated. The
mixture was heated under reflux for 18 h, cooled down and
filtered. The clear filtrate was freeze-dried to yield a very hygro-
scopic yellow solid, (100 mg, 85%). δH (D2O, pD 5.5, partial
analysis): 28.0, 19.8, 17.1, 12.2 (each 1H, Hax), 9.8(1H, Heq.),
9.0–7.3 (8H, br mult, ArH); 2.1, 0.4, Ϫ0.5 (12H, br s ϩ s ϩ s,
CMe ϩ CHC), Ϫ1.0, Ϫ2.1, Ϫ4.1, Ϫ6.0, Ϫ7.9, Ϫ10.0, Ϫ10.8,
Ϫ11.8, Ϫ13.0, Ϫ14.1, Ϫ14.7, Ϫ15.8, Ϫ16.7 (br s, HaxЈ ϩ NCH
ϩ CHeqЈ, 19H); νmax (KBr): 1773, 1734, 1717, 1685, 1654, 1637
(C᎐O); λmax (H2O) 258, 390, 408.
᎐
[Eu(L12)]. m/z (ESϪ): 928.4, 930.4, 931.3 [EuL]Ϫ; m/z (ESϩ):
953.4 [NaEuL]ϩ. Found: 953.268 C38H49N8O10EuNa requires
(15 Ϫ Eu): 953.2682; δH (D2O; pD6, partial analysis of aqua
complex): 30.6, 21.4, 19,2, 13.8 (CHax); 10.4 (2H, br s), 8.2, 7.6
(11H, br s ϩ br s, ArH ϩ CHeq); 0.3 ϩ 0.0 (s ϩ s, 9H, CMe);
Ϫ1.0 (1H), Ϫ1.8 (3H, s), Ϫ5.4 (2H, br s), Ϫ6.3 (1H), Ϫ7.8 (1H),
Ϫ11.3, Ϫ11.8, Ϫ12.3, Ϫ13.3, Ϫ14.7, Ϫ15.3, Ϫ16.3 (each 1H,
CHax ϩ NCHCO).
1596 (C᎐O); m/z (ESϩ): 394.3 [M ϩ H]ϩ; 406.3 [M ϩ Na]ϩ.
᎐
L6. A mixture of the monoalkylated cyclen (200 mg, 0.63
mmol), 2-chloroethanoyl alanine ethyl ester (402 mg, 2.08
mmol, 3.3 eq.) and caesium carbonate (634 mg, 1.95 mmol,
3.1 eq.) in dry acetonitrile (10 mL) was boiled under reflux
for 2 days. A white solid was filtered off, washed well with
acetonitrile and the filtrate and combined washings evaporated
to dryness. The resulting orange oil was dissolved in dichloro-
methane and the product purified by column chromatography
(SiO2, 5% triethylamine in CH2Cl2), to yield a yellow solid,
332 mg, 61%. Rf (SiO2, 5% Et3N in CH2Cl2): 0.1; m/z (ESϩ): 865
[MH]ϩ, 887 [M ϩ Na] ϩ. Found: 887.4642. C44H64N8O10Na
requires 887.4643.
Cell Culture and imaging
NIH 3T3 mouse fibroblast cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM) containing 10% (v/v) foetal
calf serum, 1 × non-essential amino acids, 100 U mlϪ1 peni-
cillin, and 100 µg mlϪ1 streptomycin. Cells were passaged prior
to confluence using 500 U trypsin and 180 µg mlϪ1 ethylene-
diaminetetraacetic acid tetrasodium salt in DMEM.
For cell loading experiments, cells were grown to approx-
imately 70% confluence on a sterile coverslip, washed twice with
1× phosphate buffered saline (PBS) or 1× tris buffered saline,
and incubated with growth medium containing a 1 mM concen-
tration of the appropriate indicator. Transfection of the cells
was performed using a 1 mM concentration of the indicator
using standard protocols. Loaded cells were mounted on a slide
and imaged using a Zeiss Axiovert 135 inverted fluorescent
microscope (excitation wavelength, 400 nm).
L12. A suspension of the triester (260 mg, 0.3 mmol) in
0.05 M aqueous potassium hydroxide (40 mL) was stirred at
room temperature for 2 days. The pH of the resulting clear
solution was adjusted to 6 with dilute hydrochloric acid and the
volume reduced to one third. The solution was loaded onto a
cation exchange column (DOWEX 50 W 4, Hϩ form). The
column was eluted with water, and the product eluted with 12%
aqueous ammonia. The solvent was removed under reduced
pressure and the resulting brown oil dissolved in water and
Toxicity was assessed by incubating indicator loaded cells
with 0.1% (v/v) Trypan Blue in 1x PBS and counting live cells in
a haemocytometer.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 2 4 – 1 6 3 2
1631

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Chem., 2003, 15, 505; (c) S. Faulkner, B. P. Burton-Pye, T. Khan,
Acknowledgements
L. R. Martin, S. D. Wray and P. Skabara, Chem. Commun., 2002,
1668; (d ) C. Li and W. T. Wang, Chem. Commun., 2002, 2034;
(e) T. Gunnlaugsson, R. J. H. Davies, M. Nienwenhuyzen, C. S.
Stevenson, R. Viguier and S. Mulready, Chem. Commun., 2003,
2136.
We thank the County Durham sub-regional partnership, the
Royal Society and the EPSRC high resolution MS service for
support.
11 A preliminary communication has appeared: Y. Bretonniere,
M. J. Cann, D. Parker and R. Slater, Chem. Commun., 2002, 1930.
12 R. S. Dickins, S. Aime, A. S. Batsanov, A. Beeby, M. Botta,
J. I. Bruce, J. A. K. Howard, C. S. Love, D. Parker, R. D. Peacock
and H. Puschmann, J. Am. Chem. Soc., 2002, 124, 12697.
13 A. Batsanov, R. S. Dickins, D. Parker, H. Puschmann and
S. Salamano, Dalton Trans., 2004, 70–80.
14 P. Atkinson, Y. Bretonniere and D. Parker, Chem. Commun., 2004,
438.
15 A. Beeby, L. M. Bushby, D. Maffeo and J. A. G. Williams, J. Chem.
Soc., Perkin Trans. 2, 2000, 1281.
16 A. Dadabhoy, S. Faulkner and P. G. Sammes, J. Chem. Soc., Perkin
Trans. 2, 2000, 2359; A. Dadabhoy, S. Faulkner and P. G. Sammes,
J. Chem. Soc., Perkin Trans. 2, 2002, 348.
17 R. S. Dickins, T. Gunnlaugsson, D. Parker and R. D. Peacock,
Chem. Commun., 1998, 1643.
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