Synthesis and characterization of coumarin-based europium complexes and luminescence measurements in aqueous media

Article abstract of DOI:10.1039/b907579h

A series of new ligands suitable for the formation of luminescent lanthanide complexes in water is described. The chelates are designed for analyte labeling and play the role of fluorescent donor in homogeneous time-resolved fluorescence assays using LEDs

Full text of DOI:10.1039/b907579h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and characterization of coumarin-based europium complexes
and luminescence measurements in aqueous media†
Cle´mentine Fe´au,a Emmanuel Klein,a Carsten Dosche,b Paul Kerthc and Luc Lebeau*a
Received 16th April 2009, Accepted 14th September 2009
First published as an Advance Article on the web 13th October 2009
DOI: 10.1039/b907579h
A series of new ligands suitable for the formation of luminescent lanthanide complexes in water is
described. The chelates are designed for analyte labeling and play the role of fluorescent donor in
homogeneous time-resolved fluorescence assays using LEDs as a light source for excitation at 370 nm.
Ligands 3–7 are constructed from a coumarin nucleus, for lanthanide sensitization, and different
aminomethylenecarboxy moieties are introduced in positions 7 and 5, 6, or 8 of the sensitizer. A
reactive spacer arm under biocompatible conditions (maleimide, azide) is introduced at position 3 for
ultimate bioconjugation purposes. The synthesis and characterization of the ligands are described,
together with the preparation of their corresponding europium complexes. Photophysical properties of
the complexes are investigated in water by means of UV-vis and luminescence spectroscopy.
centre) whereas deactivation is a complex phenomenon. A main
route of non-radiative decay occurs by energy transfer from the
Introduction
excited state of Ln(III) to the O–H vibrational oscillator.7 Thus a
good shielding of the metal from the surrounding O–H oscillators
of the solvent is key for building an efficient luminescent probe
that can be finely tuned by ligand design. The lanthanide ions
usually possess high coordination requirements and, for Eu(III)
and Tb(III), a coordination number of nine is very common.
Although such high coordination is difficult to fulfil using a single
coordinating ligand, the use of functionalized macrocyclic ligands
has come close.8-13 In such complexes, the antenna is tethered to
the chelate so as it can efficiently sensitize Ln(III).
In the course of our efforts to develop portable point-of-
care testing devices (POCT),14,15 we have been interested in
designing lanthanide-based probes to be excited by light-emitting
diodes (LEDs) for steady-state and time-resolved fluorescence
spectroscopy. The luminescent lanthanide chelate is ultimately
expected to play the role of FRET donor in homogenous time-
resolved fluorescence (HTRF) assays.2,16-18 LEDs as an excita-
tion source offer excellent stability, signal-to-noise ratio, power
efficiency and economy, and thus are especially suitable for
portable instrumentation manufacturing. Although commercially
available LEDs cover the whole visible range, only a few can
be found with emission wavelengths below 360–370 nm and
specific fluorophores are required. At the end of the 1990s, Selvin
developed quinolinone-based lanthanide complexes that proved to
be suitable for bioanalytical applications but required excitation at
337 nm.19-22 More recently, azaxanthone chromophores have been
described as europium sensitizers to be excited at 360–405 nm.23-26
Coumarins may also be interesting chromophores with regards to
their photochemical and photophysical properties. With emission
maximum in the range 350–450 nm, they are ideally suited for the
sensitization of lanthanides.27–30 Though coumarins so far have
scarcely been used in the design of lanthanide chelates, some
reports describe their conjugation with crown ethers and cryptands
or aminopolycarboxylic chelates and their use as fluorescent
probes for applications in metal ion detection,31,32 time-resolved
The rational design of molecules that exhibit fluorescence prop-
erties has gained much interest over the past decade. Among
them, luminescent lanthanide complexes with remarkable physical
and chemical properties have been developed and found many
applications, especially in the field of bioanalytical chemistry.1,2
For instance, the time-resolved fluorescence resonance energy
transfer (TR-FRET) technique takes advantage of the attracting
properties of lanthanide chelates. Indeed, their long-lived excited
states along with characteristic narrow emission bands make
them ideal for fluorescent background discrimination and delayed
emission signal detection. Long decay times result from forbidden
transitions involving 4f orbitals and, as a consequence, the
molar absorption coefficient of these complexes are very low
and typically less than 1 M-1 cm-1.3,4 Most of the time, effective
excitation cannot be performed directly, but via a light-harvesting
antenna that acts as a sensitizer. During that process (termed
the “antenna effect”), UV/visible light energy is collected by
an allowed antenna-centered absorption. Antenna excitation is
followed by a non-radiative intramolecular energy transfer from
the excited states of the antenna to Ln(III), resulting in a radiative
metal-centered luminescence.5,6 The luminescent properties of
lanthanide ions, such as characteristic narrow-line-like emission
bands, depend on how efficiently their excited state(s) can be pop-
ulated and non-radiative deactivation paths minimized. Excitation
mainly depends on antenna efficiency (high extinction coefficient,
efficient S1→T1 intersystem crossing rates, proximity to the metal
aLaboratoire de Chimie Organique Applique´e, C.A.M.B., UMR 7199
CNRS–UdS, Faculte´ de Pharmacie, 74 route du Rhin–BP 60024, 67401,
Illkirch Cedex, France. E-mail: llebeau@unistra.fr; Fax: +33 (0)390244306
bInstitute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25,
14476, Potsdam-Golm, Germany
cPreventor mTBC GmbH, Gernsheimer Strasse 46, 64319, Pfungstadt,
Germany
† Electronic supplementary information (ESI) available: 13C-NMR spectra
of 3b, 3c, 4a, 5a, 6a, 7a, 7d, 8–16, 18–21, 25–28, 30, and 31; [Eu(7a)]
emission spectrum.
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fluoroimmunoassays,33–35 site-specific protein labeling,36 distance
measurements in biomolecules,37 DNA sensing,38 or enzyme
activity reporting.39 Most of these coumarin-based probes suffer
from poor stability and relatively low emission quantum yield in
aqueous media. This stresses the necessity to develop additional
work based on ligand design for improving their properties, and
for allowing their use in bioanalytical applications.
Results and discussion
Design and synthesis of the ligands
Previously developed ligand 2 has a T-shape or branched structure
where 3 functional entities (i.e. metal-chelating moiety, antenna,
and reactive spacer) are distributed radially (Fig. 1). Eu(III) com-
plexation by the pyridine-2,6-diyl-bis(methyleneniminodiacetic
acid) unit leads to a heptadentate pattern that has been pre-
viously described and used in DNA labeling applications.40–44
In order to improve the quantum yield of metal sensitization,
we designed compounds 3–7 differing from 2 by their “linear”
geometry (Fig. 2). The coumarin antenna is inserted between the
chelating part and the reactive spacer, playing to some extent
the role of a scaffold. Iminodiacetic acid groups are directly
connected to the coumarin aromatic ring through different linkers
that can also participate in metal chelating. Compounds 3, 4,
and 6 can supposedly lead to heptacoordinate Eu(III) chelates
whereas 5 and 7 can potentially accommodate 10 and 14 different
coordination bonds with lanthanide ions. On the opposite side of
these molecules, the spacer R is connected at position 3 on the
coumarin moiety. The reactive spacer displays either an azide or
a maleimide group. Both functional groups have been extensively
used for bioconjugation purposes and are fully compatible with
ligand reactivity.45–48 Not all the synthesized compounds have
a reactive spacer. In some cases and for practical purposes,
ethyl esters have been prepared in place of amide-linked reactive
spacers, the photophysical evaluation of the compounds and their
corresponding Eu(III) chelates being better with a “silent” spacer
(vide infra).
Recent work by Lakowicz et al. validated the use of
LEDs as light sources in bioanalytical studies.30 The authors
used compound [Eu(1)],
a coumarin-sensitized europium
probe resulting from the consecutive functionalization of di-
ethylenetriamine pentaacetic acid dianhydride with 7-amino-4-
(trifluoromethyl)coumarin, the metal sensitizer, and dodecyl-
amine, the membrane anchor required for their study (Fig. 1).
Thus, the efficiency of a microscope-based fluorimeter with
UV/visible LED excitation could be demonstrated. These results
prompted us to design compound [Eu(2)] which incorporates a
metal chelator, a coumarin core as asensitizer, and afunctionalized
spacer.34 Functionalization of the spacer with a chemically reactive
group (azide) was necessary to allow further conjugation under
biocompatible conditions with the adequate biomolecule (AZT
derivative) for final assay setup. Although compound [Eu(2)] can
be satisfactorily excited at 360–370 nm and proved to be fairly
stable in water and human plasma, the fluorescence quantum yield
was modest. This particular result is attributed to the likely longer
distance between the coumarin moiety and the metal centre in
[Eu(2)], compared to [Eu(1)].
Fig.
1
Structure of 7-amino-4-(trifluoromethyl)coumarin-based eu-
ropium sensitizers.
In order to develop HTRF donor probes with improved
properties, we planned the relocation of an adequately function-
alized coumarin core in the close proximity of the metal centre.
Herein we describe our results concerning the design, synthesis,
and evaluation of photophysical properties of a series of new
functionalized chelating coumarins and of their corresponding
europium complexes.
Fig. 2 Structure of ligands 3–7.
The synthesis of ligands 3b and 3c was achieved according
to Scheme 1. The coumarin scaffold was elaborated starting
from 3-aminophenol. Bis-N-alkylation of the starting material
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followed by reaction of the intermediate triamine compound
with excess t-butyl bromoacetate led to pentaester 12 in 51%
yield. Compound 12 was quantitatively converted into acid 13
upon treatment with lithium hydroxide. Introduction of a reactive
spacer was achieved through condensation of 13 with 2-[2-(2-
azidoethoxy)ethoxy]ethylamine,52 and amide 14 was obtained in
68% yield. Direct treatment of tetra-t-butyl esters 13 and 14 with
trifluoroacetic acid (TFA) afforded the two ligands 3b and 3c.
We intended to transform bis-chlorocoumarin 10 into compound
12 using a one-step procedure (Scheme 2). All our attempts to
displace the two chloride substituents by iminodiacetic acid di-t-
butyl ester (directly or via intermediate iodide substitution) failed,
and yielded dialkyl piperazine 16.
Scheme
2
Reagents and conditions: (a) NaI, acetone, quant.;
(b) HN(CH2CO2tBu)2, DMF, 62%.
Scheme 1 Reagents and conditions: (a) ethylene oxide, AcOH, 88%; (b)
POCl3, DMF, 84%; (c) diethyl malonate, piperidine, EtOH, 94%; (d) NaN3,
NaI, DMF, 98%; (e) 1. PPh3, THF; 2. aq. HCl; 3. BrCH2CO2tBu, Na2CO3,
DMF, 51% (3 steps); (f) LiOH, quant.; (g) TFA, quant.; (h) DCC, NHS,
H2N(CH2CH2O)2CH2CH2N3, 68%.
Ligands 4a and 5a were prepared starting from 3-ethoxy-
carbonyl-7-hydroxycoumarin 1753 as
a common precursor
(Scheme 3). The iminodiacetate pendant arm was introduced via
a Mannich reaction with formaldehyde and iminodiacetic acid
di-t-butyl ester (Table 1). The latter compound was prepared
according to a procedure described by Achilefu.54 Compound
18 was obtained in 76% yield under the experimental conditions
described by Regnier et al.55 Alkylation of the phenol oxygen
with N-(2-hydroxyethyl)iminodiacetic acid di-t-butyl ester56 under
the Mitsunobu reaction conditions yielded compound 19, which
was quantitatively converted into ligand 4a upon treatment with
TFA. Compound 5a was prepared in a similar manner except
that the initial Mannich reaction was conducted with 2 molar
equivalents of iminodiacetic acid di-t-butyl ester and excess
formaldehyde, in N-methylmorpholine (NMM). The introduction
of the second aminomethylene pendant arm at position 6 on the
with ethylene oxide in aqueous acetic acid49 led to 3-[bis(2-
hydroxyethyl)amino]phenol 8 in 88% yield and proved superior
to the reaction with 2-chloroethanol reported by Li and Xie.50
Simultaneous bis-chlorination and formylation of 8 using the
Vilsmeier–Haack reagent formed in situ from DMF and phos-
phorus oxychloride was achieved in 84% yield. The resulting
compound 9 was subjected to a classical Knoevenagel conden-
sation reaction51 to give coumarin 10 in a nearly quantitative
yield. Nucleophilic displacement of the two chloride substituents
with sodium azide was achieved quantitatively in DMF to afford
compound 11. Reduction of the latter under Staudinger conditions
Scheme 3 Reagents and conditions: (a) 1.1 eq HCHO, 1.0 eq HN(CH2CO2tBu)2, 76%; (b) PPh3, DIAD, HOCH2CH2N(CH2CO2tBu)2, 47%; (c) TFA,
quant.; (d) 2.5 eq HCHO, 2.0 eq HN(CH2CO2tBu)2, 13%; (e) PPh3, DIAD, HOCH2CH2N(CH2CO2tBu)2, 43%.
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Scheme 4 Tentative route to 23.
Table 1 Reaction conditions for the preparation of coumarins 18 and 20
from 17
by the two bulky substituents at the central nitrogen atom in
triamine 22.
Ligands 6a, 7a, and 7d were elaborated from phlorogluci-
nol (Scheme 5). Reaction of the starting triol with diethyl
(N,N-diethylaminomethylene)malonate 24 (prepared according to
Momose et al.58) provided 3-ethoxycarbonyl-5,7-dihydroxy-
coumarin 2559 in 81% yield. The presence of a second hydroxyl
group at position 5 on the coumarin nucleus makes compound
25 much more reactive than the corresponding monohydroxy-
coumarin 17, and the double Mannich reaction with formaldehyde
and iminodiacetic acid di-t-butyl ester proceeded quantitatively to
yield compound 26. The latter was further bis-alkylated with N-(2-
hydroxyethyl)iminodiacetic acid di-t-butyl ester under Mitsunobu
reaction conditions. Ethyl ester hydrolysis in 27, followed by
carboxylic acid activation and reaction with 8-(N-maleimidyl)-3,6-
dioxaoctan-1-ol 31 afforded compound 28. Trifluoroacetic acid
treatment of 26, 27, and 28 gave quantitatively ligands 6a, 7a, and
7d, respectively.
The reactive spacer 31 was prepared as described in Scheme 6,
starting from 1-amino-3,6-dioxaoctan-1-ol.60 Protected amino-
alcohol 2961 was transformed into N-alkyl maleimide 30 by
reacting with maleimide under Mitsunobu reaction conditions.62
Removal of the carbamate protecting group was achieved under
standard conditions.
Europium complexes with ligands 3–7 were prepared from stoi-
chiometric amounts of ligand and europium chloride hexahydrate,
in water (pH 7.0).63,64
Entry Amine (eq) HCHO (eq) Solvent T (◦C) 18 (%)a 20 (%)a
1
2
3
4
5
6
2
2
4
2
2
2
2
EtOH
EtOH
tBuOH 83
NMM
NMM
NMM
78
78
86
79
82
54
62
56
0
0
0
6
13
11
12
12
2
115
115
115
2.5
6b
a Isolated yield. b A larger excess in aqueous formaldehyde did not improve
the conversion into 20.
coumarin nucleus proved to be difficult and was achieved in a
modest yield following a procedure described by Hinshaw et al.57
(Table 1, entry 5). Alkylation of phenol oxygen in compound 20
and subsequent removal of all t-butyl protecting groups in 21 led
to ligand 5a.
Due to the relatively easy introduction of the iminodiacetic
acid di-t-butyl ester moiety at position 8 on 3-ethoxycarbonyl-
7-hydroxycoumarin 17 via the Mannich condensation reaction,
we were interested in using a similar strategy to prepare coumarin
23 (Scheme 4). However, despite our efforts, we failed to find
any evidence for the condensation reaction between coumarin 17
and iso-diethylenetriamine pentaacetic acid tetra-t-butyl ester 22.54
This result may be attributed to some steric hindrance introduced
Scheme 5 Reagents and conditions: (a) AcOH, 81%; (b) 2.0 eq HCHO, 2.0 eq HN(CH2CO2tBu)2, 97%; (c) TFA, quant.; (d) PPh3, DIAD,
HOCH2CH2N(CH2CO2tBu)2, 51%; (e) 1. LiOH; 2. DCC, NHS, DIPEA, N-maleimidyl-(CH2CH2O)2CH2CH2NH3+ Cl- (31), 53% (2 steps).
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Table 2 Spectroscopic properties of compounds 3–7 measured in H2O, pH 7.0. UF, tF, and kF are fluorescence quantum yield, fluorescence lifetime, and
fluorescence rate constant, respectively
Compound
labs (nm)
lex (nm)
lem (nm)
lphos (nm)
UF (%)
tF (ns)
kF (106 s-1)
3c
396
342
402/332/302
405/366
407/318
394
344
402
411
407
453
401
443
457
451
481/549
479
482
491
481
36 (45)a
16b
2.4
0.9
3.5
3.1
3.8
150
170
9
10
16
4a
5a
2.5 (3.1)c
1.8
6a
7a (7d)c
6.0
a Measured at pH 5.0. b Measured at lex 350 nm. c Spectroscopic data for 7a and 7d are identical.
7a all display a longest wavelength absorption band at 400 nm
that corresponds to the fluorescence emission, as evident from
the excitation spectra. However, in the absorption spectra of 5a,
6a, and 7a, additional absorption bands can be observed that
have no counterpart in the excitation spectra (Fig. 3). Therefore,
different species have to be present in aqueous solution that
likely result from different protonated forms. According to the
excitation spectra, the fluorescence is due to the deprotonated
species that predominates at pH 7. Compared to those of the
other coumarins, absorption and fluorescence of compound 4a
are shifted about 50 nm towards shorter wavelengths. As expected
from the two hydroxyl groups attached to the chromophore
system, the absorption spectrum of 6a is pH-sensitive in the
range investigated (Fig. 3). For all the coumarin compounds,
phosphorescence emission around 480 nm could be observed at
80 K.
Scheme 6 Reagents and conditions: (a) (Boc)2O, quant.; (b) PPh3, DIAD,
maleimide, 74%; (c) TFA, 81%.
Photophysical properties
The photophysical data for compounds 3c, 4a, 5a, 6a, and 7a
are summarized in Table 2. Since all compounds have the same
chromophore system, the fluorescence spectra are all very similar
to each other, displaying one broad band with a maximum around
450 nm. The only exception is 4a, which also differs from the
others in other spectroscopic features. Considering the absorption
spectra, no clear trend can be found. Coumarins 3c, 5a, 6a, and
Fluorescence lifetime tF and quantum yield UF are drastically
influenced by type and position of the complexation arms on
the coumarin core. The quantum yield of the 7-aminocoumarin
derivative 3c exceeds the values of the 7-alkoxycoumarins 4a, 5a,
Fig. 3 Absorption (solid), fluorescence (dotted, lex 400 nm) and excitation spectra (dashed, lem 450 nm) of compounds 3c, 5a, 6a, and 7a in water,
pH 7.0 (grey line: pH 5.0).
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Table 3 Spectroscopic properties of europium complexes obtained from ligands 3–7 measured in H2O, pH 7.0
Stoichiometrya
pH 5
t
em (ms)b
UF (%)d
Ligand
lexc (nm)
pH 7
H2O
D2O
qc
Ligand
Eu3+
3c
4a
5a
7a
327
365
334
326
0.8 : 1
1 : 1
—
0.6 : 1
1 : 1
0.43
0.40
0.58
0.42
1.91
2.08
2.47
2.09
1.9
2.1
1.4
2.0
4.7
4.3
1.0
0.5
0.4
2.8
0.9
1.3
0.6–0.9 : 1e
1 : 1
1 : 1
a [Eu]/[ligand], determined at ligand concentration of 10-4 M. b t values are the mean of at least 3 independent measurements ( 0.02 ms). c The number of
coordinated water molecules at 300 K is obtained from Horrocks’ empirical equation,66 reviewed by Parker et al.:67 q= A[(1/tH - 1/tD O) - 0.25] with
2 O
2
A = 1.2 for Eu3+. The estimated uncertainty in q is approximately 0.5 water molecules. d Quantum yields of the complexes, separated in ligand centered
and europium centered emission. The error in quantum yield values is estimated to be 20%. e Depending on ligand concentration (see the main text).
6a, and 7a by one order of magnitude. This observation is in
agreement with data in the literature, since it is well established
that an electron-donor substituent in the 7-position enhances the
fluorescence intensity of coumarins.65-67 Modification of the pH
value induces a noticeable variation of UF, as can be seen for
coumarins 3c and 5a. This finding is consistent with the coumarin
pH-sensitivity reported earlier by Goodwin and Kavanagh.68 The
results for tF are quite different. The fluorescence lifetime of 3c
(2.4 ns) is similar to the tF of 5a, 6a, and 7a (3.1–3.8 ns), whereas
the tF of 4a is much shorter (0.9 ns). However, if we consider
the fluorescence rate constant kF calculated from these data, the
results are more homogenous and confirm the trend that is already
obvious from the UF values. Interestingly, no pH effect can be
observed for coumarin 6a (for tF or UF), although protolysis
Fig. 4 Intensity of the 5D0–7F2 transition peak as a function of Eu3+
added to a solution of ligand 7a (10-6 M) in water, pH 5.0 (square) and
pH 7.0 (circle).
reactions are expected.
In the europium complexes, energy transfer from the ligand
triplet state to the metal occurs and results in emission of the
characteristic europium bands when the complexes are excited
in the ligand absorption band (Table 3). However, for all the
complexes, partial decomposition in water can be observed (data
not shown). This is evident from the excitation spectra which show
an absorption band at 410 nm (exactly at the same position as for
the free ligands) when the wavelength of the emission channel is
set at 450 nm, and show a blue-shifted absorption around 340 nm
if the emission channel is set at 620 nm.
The stoichiometry of the Eu(III) complexes formed with ligands
3–7 was determined by a mole ratio method (Table 3).69 Complex
luminescence was monitored as a function of Eu3+ added to an
aqueous solution of the ligand of interest. A sharp break in the
titration curve reveals that a stoichiometric quantity of Eu(III)
has been added. Fig. 4 shows a typical plot of the relative Eu(III)
luminescence intensity versus equivalents of Eu3+ added to the
ligand 7a. Experiments were conducted at three different ligand
concentrations (10-4, 10-5, and 10-6 M). The stoichiometry of
complex [Eu(3c)] appears to be 1 : 2, probably revealing that
poorly nucleophilic aniline nitrogen does not participate in the
chelation of Eu3+. Complex [Eu(5a)] shows a 1 : 1 stoichiometry in
the micromolar concentration range. Surprisingly measurements
at higher ligand concentration (10-5 and 10-4 M) reveal a 1 : 2
stoichiometry that is in agreement with the law of mass action.
A mixture of both complexes is obtained at intermediate ligand
concentration. Complexes [Eu(4a)] and [Eu(7a)] have a 1 : 1
stoichiometry. Stoichiometry of [Eu(7a)] does not vary with ligand
concentration, as is the case for [Eu(5a)], although the two ligands
only differ in an additional chelating arm at position 5 for 7a.
Given the predominance of metal fluorescence quenching by
O–H oscillators in aqueous media, the number of water molecules
q in the inner coordination sphere may be derived from the
difference in the rate of quenching in H2O and D2O.70 We thus
measured luminescence lifetime tem of the europium complexes
in water and deuterium oxide at pH 7. According to Horrocks’
empirical equation7 (reviewed by Parker et al.71) about 2 water
molecules remain bound to the metal in complexes [Eu(3c)],
[Eu(4a)], and [Eu(7a)] (Table 3). The water content of [Eu(5a)]
appears significantly lower (1.4 molecules). From these results we
can conclude that whatever the number of donor atoms in the
ligand (6 to 12), not all water molecules can be removed from
the inner coordination sphere of the lanthanide cation, either in
the 1 : 1 or in the 2 : 1 complexes. However for a 2 : 1 europium
complex with 5a (high ligand concentration, vide supra), one (0.7)
additional water molecule could be displaced. From that, it can be
assumed that the chelating arm at position 5 in 7a is not available
for intramolecular cation complexation and that formation of a 2 :
1 complex is hindered due to the steric needs of that substituent.
For demonstrating the potentiality of complex [Eu(7a)] as a
donor in TR-FRET experiments, we used the diffusion-enhanced
fluorescence energy transfer method.72 The donor fluorescence
lifetime was decreased dose-dependently by addition of the
acceptor dye Cy5 (Fig. 5). The Stern–Volmer constant KD was
calculated as 7 ¥ 107 M-1 s-1 according to the equation F0/F =
t0/t = 1 + KD[Q] (where F0 and t0, and F and t, are respectively
the fluorescence intensity and lifetime of the donor in the absence
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Silica gel for flash chromatography (silica gel 60, 0.040–0.063 mm,
230–400 mesh ASTM) and TLC plates (0.25 mm silica gel 60
precoated plates, F254) were from E. Merck. Melting points were
1
determined using a Stuart SMP3 apparatus. H NMR and 13C
NMR spectra were recorded on Bruker WP-200-Sy and Avance-
DPX-300 instruments, and chemical shifts d are reported in ppm
relative to their standard reference (1H: CHCl3 at 7.27 ppm, HDO
at 4.63 ppm, CD2HOD at 3.31 ppm; 13C: CDCl3 at 77.0 ppm,
CD3OD at 49.0 ppm). Mass spectra (MS) were recorded on a
ZQ Waters/Micromass spectrometer using electrospray ionization
(ESI) mode. Mass data are reported in mass units (m/z). High-
resolution mass spectra (HRMS) were recorded on a Bruker
Daltonics MicrOTOF-Q spectrometer in ESI mode. IR spectra
were recorded on a Perkin-Elmer 1600-FTIR spectrometer in the
ATR mode and absorption values n are in wavenumbers (cm-1).
Abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; b, broad.
Fig. 5 Stern–Volmer plot for Cy5 quenching of [Eu(7a)].
and in the presence of the acceptor; [Q] is the concentration of the
acceptor). Such a value does reflect a high efficiency of fluorescence
energy transfer from [Eu(7a)] to Cy5.3 In addition, the linearity of
the Stern–Volmer plot indicates that only one type of quenching
occurs in the aqueous environment.
Luminescence measurements
Absorption spectra were recorded on a Cary 500 UV–vis–
NIR spectrophotometer (Varian Inc., Palo Alto, CA, USA).
Absorption maxima lmax are given in nm and molar extinction
coefficients e are in M-1 cm-1. For spectroscopic measurements,
fluorophore stock solutions were prepared in water (10-3 M) and
diluted to 10-4–10-6 M. Solutions were adjusted to pH 5 and
pH 7 with HCl or NaOH, respectively. Spectroscopic data for
the europium complexes were obtained from ligand solutions in
10-3 M europium chloride. All spectra were recorded in 1 cm
quartz cells. Stationary fluorescence and excitation spectra were
obtained with a Fluoromax 3 fluorimeter (Jobin Yvon, Edison, NJ,
USA). All spectra were corrected using the correction functions
incorporated in the spectrometer software. Absolute fluorescence
quantum yields were measured with an integration sphere system
(Hamamatsu) at an excitation wavelength of 400 nm. Fluorescence
lifetime measurements were performed with a FLS920 fluorimeter
(Edinburgh Instruments Ltd, Livingston, UK). A frequency-
doubled titanium sapphire laser system (Tsunami 3960, Spectra
Physics, Mountain View, USA) set at 400 nm was used as the
excitation light source. The original repetition rate of 80.2 MHz
was reduced to 500 kHz with a pulse picker (Pulse Select, APE,
Berlin, Germany). Fluorescence emission was detected with a
multichannel plate (ELDY EM1-132/300, Europhoton, Berlin,
Germany), providing a time response of ~100 ps. Phosphorescence
lifetime data were obtained with a Fluoromax P fluorimeter (Jobin
Yvon, Edison, NJ, USA). For phosphorescence measurements
at 80 K, the samples were prepared in a 5 ¥ 5 mm home-built
monolithic quartz cell, deoxygenated by flushing the samples for
15 min with argon, and then cooled to 80 K with an Optistate
DN1704 cryostat (Oxford Instruments, Wiesbaden, Germany)
fitted with an external controller (ITC4, Oxford Instruments). The
stoichiometry of the Eu(III) complexes was determined by a mole
ratio method as previously reported.69 TR-FRET experiments
with ligand 7a (10-4 M) were realized in the presence of EuCl3
(5 ¥ 10-4 M), with Cy5 as an acceptor (concentration ranging
up to 22.5 ¥ 10-6 M). Excitation was achieved at 350 nm with
an OPO system (Nd YAG Laser, Spectra-Physics, Mountain
View, CA, USA; OPO GWU-Lasertechnik, Erftstadt, Germany)
working at a 20 Hz repetition rate, with an average pulse energy
Conclusions
A series of 7 coumarin-based ligands have been prepared. These
ligands have been designed so they can potentially accommodate
6 to 14 coordination bonds to lanthanide cations and were syn-
thesized in 4 to 8 steps involving either Knoevenagel or Mannich
condensation. The europium(III) complexes have been prepared
from each characterized ligand and the resulting photophysical
properties have been investigated in water. Throughout the ligand
series, dodecadentate ligand 7a emerges. The related ligand 5a
lacking a chelating arm at the 5-position was revealed to be
more efficient at isolating the metal centre from the surrounding
water molecules. However, in that case, the stoichiometry of the
complex is 2 : 1, which might indicate that the additional chelating
arm in 7a does not directly participate in metal chelation but
prevents formation of a 2 : 1 complex, probably due to high steric
hindrance introduced by the chelating moiety at the 5-position.
The potential of [Eu(7a)] as a fluorescent donor in time-resolved
FRET experiments has been assessed by a diffusion-enhanced flu-
orescence energy transfer method. Additional work is required for
evaluating possible displacement of ligand donor atoms or inner
coordination sphere water molecules by coordinating groups of
proteins or endogenous anions present in biological samples. This
is currently underway, and maleimide-functionalized europium
complex [Eu(7d)], which presents similarly attractive features as
[Eu(7a)], is currently being evaluated in a homogenous time-
resolved fluorescence assay setup. Bioanalytical results will be
reported in due course.
Experimental
Materials
Starting materials were purchased from Sigma-Aldrich and Alfa
Aesar and used without purification. All reactions were carried
out with reagent-grade solvents. When required, solvents were
dried according to standard procedures as described elsewhere.61
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of ca. 15 mJ. Detection was achieved at 600–700 nm using an
iCCD camera system (iStar DH720, Andor, Belfast, UK) attached
to an imaging monochromator (MS257, LOT-Oriel, Darmstadt,
Germany).
(m, 2H); 1.25 (t, J = 6.9 Hz, 3H). 13C-NMR (D2O, 75 MHz) d
169.3; 169.1; 168.8; 164.0; 163.4; 157.6; 156.5; 149.9; 138.5; 121.5;
117.4; 116.7; 112.7; 71.1; 63.5; 57.2; 55.4; 53.8; 48.0; 47.6; 47.5;
13.7. IR (film) n 3391; 2987; 2904; 1682; 1614; 1384. UV-vis lmax
402 (H2O, pH 6.5). MS (ESI) m/z 684 [M + H]+; 706 [M + Na]+.
ESI-HRMS for C28H33N3O17: calcd. 684.1883 [M + H]+, found
684.1877.
Synthesis and characterization
7-(N,N -Bis{2-[bis(carboxymethyl)amino]ethyl}amino)-3-car-
boxycoumarin (3b). Compound 13 (96 mg, 0.13 mmol) was
stirred in CH2Cl2/TFA 1 : 1 (3 mL) at room temperature for 7 h.
Water (5 mL) was added and the solution was brought to pH 7
with 1 N NaOH, washed with CH2Cl2 (3 ¥ 5 mL) and evaporated
to dryness. The residue was triturated with MeOH, filtered and
dried to yield 3b (68 mg, 100%) as a yellow hygroscopic powder.
1H-NMR (D2O, 200 MHz) d 8.22 (s, 1H); 7.55 (d, J = 8.3 Hz, 1H);
6.90 (d, J = 8.3 Hz, 1H); 6.78 (bs, 1H); 3.93 (bs, 4H); 3.80 (s, 8H);
3.48 (bs, 4H). 13C-NMR (D2O, 50 MHz) d 170.9; 163.2; 162.7;
156.5; 151.1; 147.3; 131.6; 118.0; 115.1; 111.6; 110.7; 99.3; 58.1;
52.2; 45.9. IR (film) n 3391 (b); 2987; 2900; 1682; 1606. UV-vis lmax
375 (H2O, pH 6.5). MS (ESI) m/z 524 [M + H]+; 546 [M + Na]+.
ESI-HRMS for C22H25N3O12: calcd. 546.1330 [M + Na]+, found
546.1313.
6,8-Bis[bis(carboxymethyl)aminomethyl]-5,7-dihydroxy-3-eth-
oxycarbonylcoumarin (6a). Compound 6a (38 mg, 72%) was
obtained as an orange hygroscopic solid (TFA salt) from 26,
1
following the same procedure as for 4a (reaction time 3 h). H-
NMR (D2O, 300 MHz) d 8.93 (s, 1H); 4.73 (s, 4H); 4.35 (q, J =
6.9 Hz, 1H); 3.90 (s, 8H); 1.39 (t, J = 6.9 Hz, 3H). 13C-NMR
(CD3OD, 75 MHz) d 169.8; 165.8; 165.1; 163.5 (q, J = 35.2 Hz);
160.2; 158.1; 157.1; 147.7; 118.8 (q, J = 291.0 Hz); 111.6; 109.8;
105.3; 104.6; 69.7; 67.0; 65.1; 63.4; 15.4. IR (film) n 3084; 2973;
1731; 1614; 1574. UV-vis lmax 408 (H2O, pH 6.5). MS (ESI) m/z
541 [M + H]+; 563 [M + Na]+. ESI-HRMS for C22H24N2O14: calcd.
541.1300 [M + H]+, found 541.1291.
5,7 - Bis{2 - [bis(carboxymethyl)amino]ethoxy} - 6,8 - bis[bis(car -
boxymethyl)aminomethyl]-3-ethoxycarbonylcoumarin (7a). Com-
pound 7a (60 mg, 89%) was obtained as a yellow hygroscopic
solid from 27, following the same procedure as for 4a (reaction
time 15 h). 1H-NMR (CD3OD, 300 MHz) d 9.00 (s, 1H); 4.91 (s,
2H); 4.83 (s, 2H); 4.61 (s, 2H); 4.41–4.38 (m, 4H); 4.07 (s, 4H);
3.94 (s, 4H); 3.83 (s, 4H); 3.78 (s, 4H); 3.38 (m, 4H); 1.39 (t, J =
7.2 Hz, 3H). 13C-NMR (CD3OD, 75 MHz) d 174.6; 174.1; 172.6;
171.5; 166.2; 165.2; 164.7 (q, J = 35,5 Hz); 162.0; 159.2; 157.8;
146.1; 118.7; 118.2; 118.1 (q, J = 289.6 Hz); 114.0; 112.4; 78.0;
76.0; 63.9; 57.4; 57.1; 56.8; 56.6; 15.4. IR (film) n 3441; 2980;
1731; 1596; 1393. UV-vis lmax 397 (H2O, pH 6.5). MS (ESI) m/z
860 [M + H]+; 882 [M + Na]+. ESI-HRMS for C34H42N4O22: calcd.
859.2363 [M + H]+, found 859.2353.
3-{N -2-[2-(2-Azidoethoxy)ethoxy]ethylaminocarbonyl}-7-(N,
N-bis{2-[bis(carboxymethyl)amino]ethyl}amino)coumarin
(3c).
Compound 3c (13 mg, 100%) was obtained as a yellow hygroscopic
powder from 14, following the same procedure as for 3b. 1H-NMR
(CD3OD, 300 MHz) d 8.61 (s, 1H); 7.56 (d, J = 8.1 Hz, 1H); 6.89
(d, J = 8.1 Hz, 1H); 6.74 (s, 1H); 4.01 (m, 10H); 3.89 (bs, 4H);
3.69 (bs, 8H); 3.44 (bs, 4H); 3.37 (bs, 2H). 13C-NMR (CDCl3,
75 MHz) d 169.3; 164.7; 163.1; 157.0; 152.5; 149.0; 132.5; 111.8;
111.6; 110.5; 98.8; 70.2; 70.1; 69.4; 65.8; 56.7; 52.5; 49.4; 46.0;
39.7. IR (film) n 3387 (b); 2972; 2101. UV-vis lmax 403 (H2O,
pH 6.5). MS (ESI) m/z 681 [M + H]+; 703 [M + Na]+. ESI-HRMS
for C28H37N7O13: calcd. 680.2522 [M + H]+, found 680.2518.
5,7 - Bis{2 - [bis(carboxymethyl)amino]ethoxy} - 6,8 - bis[bis(car -
boxymethyl)aminomethyl]-3-(2-[2- {2-(N -maleimidyl)ethoxy}-
7-{2-[Bis(carboxymethyl)amino]ethoxy}-8-[bis(carboxymethyl)-
aminomethyl]-3-ethoxycarbonylcoumarin (4a). Compound 19
(30 mg, 39 mmol) was stirred in TFA (0.3 mL) for 3 h at rt. The
resulting mixture was coevaporated 3 times with CHCl3 (5 mL)
and the residue was triturated in CH2Cl2. The precipitate was
filtered and dried under vacuum to yield 4a (21 mg, 70%) as
a yellow hygroscopic solid (TFA salt). Mp 189 ◦C. 1H-NMR
(CD3OD, 300 MHz) d 8.70 (s, 1H); 7.92 (d, J = 7.2 Hz, 1H); 7.19
(d, J = 7.2 Hz, 1H); 4.83 (bs, 2H); 4.50 (m, 2H); 4.36 (q, J =
7.2 Hz, 2H); 4.17 (bs, 4H); 3.99 (bs, 4H); 3,66 (m, 2H); 1.39 (t, J =
7.2 Hz, 3H).13C-NMR (CD3OD, 75 MHz) d 172.8; 170.5; 165.1;
164.9; 163.2 (q, J = 36.3 Hz); 158.5; 157.8; 151.4; 136.2; 118.7 (q,
J = 289.7 Hz); 116.5; 114.8; 116.6; 108.0; 67.4; 63.7; 57.4; 57.1;
56.1; 56.0; 15.4. IR (film) n 3473; 2987; 1738; 1605. UV-vis lmax
365 (H2O, pH 6.5). MS (ESI) m/z 539 [M + H]+; 561 [M + Na]+.
ESI-HRMS for C23H26N2O13: calcd. 539.1508 [M + H]+, found
539.1499.
ethoxy]ethylaminocarbonyl)coumarin
(7d). Compound
7d
(32 mg, 91%) was obtained as a yellow hygroscopic solid from
28, following the same procedure as for 4a (reaction time 60
1
h). H-NMR (CD3OD, 300 MHz) d 9.04 (s, 1H); 6.79 (s, 2H);
4,83 (bs, 2H); 4.70 (bs, 2H); 4.50 (bs, 2H); 4.44 (bs, 2H); 4.10
(s, 4H); 4.01 (s, 4H); 3.97 (s, 4H); 3.94 (s, 4H); 3.68–3.61 (m,
16H). 13C-NMR (CD3OD, 75 MHz) d 172.9; 172.5; 170.7; 165.1;
163.3; 161.9; 160.7; 157.9; 143.9; 135.4; 119.3; 118.1; 115.5; 112.0;
76.0; 74.8; 71.4; 71.3; 70.3; 68.9; 56.4; 56.3; 56.2; 55.8; 55.7; 55.6;
40.8; 38.2. IR (film) n 3359; 2973; 1728; 1392. UV-vis lmax 397
(H2O, pH 6.5). MS (ESI) m/z 1042 [M + H]+; 1064 [M + Na]+.
ESI-HRMS for C42H51N6O25: calcd. 1041.3055 [M + H]+, found
1041.3052.
3-Bis(2-hydroxyethyl)aminophenol (8). Ethylene oxide (13.5 g,
0.3 mol) was condensed using a cold trap in a mixture of 3-
aminophenol (3.3 g, 30.2 mmol) and aqueous AcOH 50% (50 mL)
6,8-Bis[bis(carboxymethyl)aminomethyl]-7-{2-[bis(carboxy-
◦
methyl)amino]ethoxy}-3-ethoxycarbonylcoumarin
(5a). Com-
at 0 C. The solution was allowed to warm to rt and was stirred
pound 5a (61 mg, 86%) was obtained as a yellow hygroscopic
solid (TFA salt) from 21, following the same procedure as for
for 24 h. The crude mixture was coevaporated twice with toluene,
dissolved in AcOEt and washed with water and brine. The organic
layer was dried over MgSO4, reduced under vacuum, and the
residue was purified by silica gel chromatography (AcOEt) to yield
1
4a (reaction time 15 h). H-NMR (D2O, 300 MHz) d 8.73 (s,
1H); 8.07 (s, 1H); 4.78 (s, 2H); 4.62 (s, 2H); 4.37 (m, 2H); 4.27
(q, J = 6.9 Hz, 2H); 4.22 (s, 4H); 4.09 (s, 4H); 4.03 (s, 4H); 3.91
1
8 (5.24 g, 88%) as a yellow oil. TLC Rf 0.35 (AcOEt). H-NMR
5266 | Org. Biomol. Chem., 2009, 7, 5259–5270
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(CD3OD, 300 MHz) d 6.96 (t, J = 8.1 Hz, 1H); 6.23 (dd, J = 8.1,
2.1 Hz, 1H); 6.19 (d, J = 1.8 Hz, 1H); 6.11 (dd, J = 8.1, 1.8 Hz,
1H); 3.70 (t, J = 6.0 Hz, 4H); 3.48 (t, J = 6.0 Hz, 4H). 13C-NMR
(CD3OD, 75 MHz) d 159.2; 150.7; 130.9; 105.2; 104.5; 100.3; 60.5;
55.1. MS (ESI) m/z 198 [M + H]+.
7 - (N,N - Bis{2 - [bis(tert - butoxycarbonylmethyl)amino]ethyl} -
amino)-3-ethoxycarbonylcoumarin (12). Compound 11 (0.46 g,
1.25 mmol) and triphenyl phosphine (0.66 g, 2.5 mmol) were
stirred at rt in anhydrous THF (5 mL) for 3 h. The resulting mixture
was added with 1 N HCl (7 mL) and refluxed for 15 h. Solvent was
removed under vacuum. The residue was coevaporated twice with
MeOH, dissolved in anhydrous DMF (8 mL), and Na2CO3 (1.09 g,
10.3 mmol) and tert-butyl bromoacetate (1.1 mL, 7.4 mmol)
were added. The suspension was stirred at 70–80 ◦C for 12 h.
Ethyl acetate (25 mL) was added and the organic layer was
washed with water, brine, and was dried over MgSO4. Solvent
was removed under vacuum and the residue was purified by silica
gel chromatography (AcOEt/n-hexane 30:70) to yield 11 (0.49 g,
51%) as a yellow oil. TLC Rf 0.6 (AcOEt/n-hexane 1 : 1). 1H-NMR
(CDCl3, 300 MHz) d 8.41 (s, 1H); 7.32 (d, J = 8.9 Hz, 1H); 6.73
(dd, J = 8.9, 1.9 Hz, 1H); 6.50 (d, J = 1.9 Hz, 1H); 4.37 (q, J =
7.1 Hz, 2H); 3.60 (t, J = 7.2 Hz, 4H); 3.47 (s, 8H); 2.91 (t, J =
7.2 Hz, 4H); 1.47 (bs, 36H); 1.39 (t, J = 7.1 Hz, 3H). 13C-NMR
(CDCl3, 50 MHz) d 170.3; 164.1; 158.2; 158.0; 153.2; 149.0; 130.9;
109.7; 109.2; 107.8; 96.7; 81.2; 61.0; 56.6; 51.1; 50.2; 28.0; 14.2.
IR (film) n 2977; 1732; 1616; 1587. MS (ESI) m/z 777 [M + H]+.
ESI-HRMS for C40H61N3O12: calcd. 776.4328 [M + H]+, found
776.4326.
4-Bis(2-chloroethyl)amino-2-hydroxybenzaldehyde (9). Freshly
distilled phosphorus oxychloride (1,80 mL, 19,0 mmol) was added
dropwise to anhydrous DMF (3 mL) at 0 ◦C. The mixture was
stirred for 20 min at rt before compound 8 (1.08 g, 5.5 mmol)
in DMF (3 mL) was added. The resulting mixture was heated
at 70–80 ◦C for 2 h and the orange solution was cooled down
to rt, dropped on iced water (50 mL), and stirred for another 2 h
period, maintaining pH at 6–7 by addition of aqueous NaOH. The
mixture was then extracted with ether and the organic layer was
dried over MgSO4, reduced under vacuum, and the residue was
purified by silica gel chromatography (Et2O/n-hexane 40 : 60) to
yield 9 (1.21 g, 84%) as a grey-purple powder. Mp 69 ◦C. TLC
Rf 0.35 (Et2O/n-hexane 50 : 50). 1H-NMR (CDCl3, 200 MHz) d
11.53 (s, 1H); 9.59 (s, 1H); 7.37 (d, J = 8.8 Hz, 1H); 6.31 (dd, J =
8.8, 2.4 Hz, 1H); 6.13 (d, J = 2.4 Hz, 1H); 3.82 (t, J = 6.0 Hz, 4H);
3.68 (t, J = 6.0 Hz, 4H). 13C-NMR (CDCl3, 50 MHz) d 192.9;
164.2; 153.1; 135.7; 112.7; 104.4; 97.9; 53.3; 39.9. IR (film) n 2930;
2848; 1627. MS (ESI) m/z 359 [M + H]+.
7 - (N,N - Bis{2 - [bis(tert - butoxycarbonylmethyl)amino]ethyl} -
amino)-3-carboxycoumarin (13). Compound 12 (0.15 g,
0.19 mmol) was stirred in EtOH (10 mL) with NaOH 1 N
(0.19 mL, 0.19 mmol) for 20 h at rt. Solvent was removed
under vacuum and the residue was filtered over a short silica gel
cartridge (AcOEt/MeOH 100 : 0 to 50 : 50) to yield carboxylic
acid 13 (0.14 g, 97%) as a yellow glassy solid. TLC Rf 0.1 (AcOEt).
1H-NMR (CDCl3/CD3OD 1 : 1, 200 MHz) d 8.55 (s, 1H); 7.42
(d, J = 8.8 Hz, 1H); 6.79 (dd, J = 8.8, 1.0 Hz, 1H); 6.57 (d, J =
1.0 Hz, 1H); 3.61 (t, J = 8.4 Hz, 4H); 3.44 (s, 8H); 2.89 (t, J =
8.4 Hz, 4H); 1.45 (bs, 36H). 13C-NMR (CDCl3/CD3OD 1 : 1,
75 MHz) d 171.5; 169.6; 164.4; 158.2; 153.9; 150.1; 131.8; 112.3;
111.2; 109.6; 97.4; 82.1; 82.1; 57.2; 51.8; 50.8; 28.4. IR (film) n
3330; 2979; 2935; 1727; 1614; 1582. MS (ESI) m/z 749 [M + H]+.
ESI-HRMS for C38H57N3O12: calcd. 770.3834 [M + Na]+, found
770.3818.
7-Bis(2-chloroethyl)amino-3-ethoxycarbonylcoumarin
(10).
Piperidine (50 mL, 0.50 mmol) was added to a mixture of
compound 9 (125 mg, 0.48 mmol) and diethyl malonate (72 mL,
0.48 mmol) in EtOH (1 mL). A yellow suspension immediately
formed that was stirred for 2 h at rt. The solvent was removed
under vacuum and the residue was purified by silica gel
chromatography (CH2Cl2/Et2O 100 : 0 to 50 : 50) to yield 10
(160 mg, 94%) as a yellow powder. Mp 157 ◦C. TLC Rf 0.4
(CH2Cl2/Et2O/n-hexane 1 : 1 : 1). 1H-NMR (CDCl3, 200 MHz)
d 8.45 (s, 1H); 7.45 (d, J = 8.8 Hz, 1H); 6.67 (dd, J = 8.8, 2.4 Hz,
1H); 6.54 (d, J = 2.4 Hz, 1H); 4.39 (q, J = 7.1 Hz, 2H); 3.86 (t,
J = 6.2 Hz, 4H); 3.70 (t, J = 6.2 Hz, 4H); 1.40 (t, J = 7.1 Hz,
3H). 13C-NMR (CDCl3, 50 MHz) d 163.7; 157.9; 157.4; 151.6;
148.9; 131.3; 111.5; 109.5; 109.0; 97.9; 61.4; 53.4; 39.9; 14.3. IR
(film) n 1729; 1693; 1614; 1584. UV-vis lmax 402 (H2O, pH 7.0).
MS (ESI) m/z 359 [M + H]+; 381 [M + Na]+. ESI-HRMS for
C16H17Cl2NO4: calcd. 358.0607 [M + H]+, found 358.0606.
3-(2-[2-{2-(Azido)ethoxy}ethoxy]ethylaminocarbonyl)-7-(N,N-
bis{2-[bis(tert-butoxycarbonylmethyl)amino]ethyl}amino)coumarin
(14). NHS (17 mg, 0.15 mmol), 2-[2-(2-azidoethoxy)-
ethoxy]ethylamine52 (28 mg, 0.16 mmol), and DCC (55 mg,
0.26 mmol) were successively added to carboxycoumarin 13
(81 mg, 0.11 mmol) in anhydrous Et2O (4 mL). The reaction
mixture was stirred at rt for 20 h, reduced under vacuum, and the
residue was purified by silica gel chromatography (Et2O) to yield
compound 14 (67 mg, 68%) as a yellow oil. TLC Rf 0.3 (Et2O).
1H-NMR (CDCl3, 200 MHz) d 8.98 (bs, 1H); 8.64 (s, 1H); 7.35
(d, J = 8.9 Hz, 1H); 6.73 (dd, J = 8.9, 1.6 Hz, 1H); 6.51 (d, J =
1.6 Hz, 1H); 3.65–3.50 (m, 14H); 3.42 (s, 8H); 3.36 (t, J = 5.4 Hz,
2H); 2.88 (t, J = 8.4 Hz, 4H); 1.43 (s, 36H). 13C-NMR (CDCl3,
75 MHz) d 170.3; 163.0; 162.3; 157.4; 152.8; 147.7; 130.9; 110.5;
110.0; 108.5; 96.6; 81.1; 70.5; 69.9; 69.7; 56.5; 51.0; 50.6; 50.2;
39.3; 28.0. IR (film) n 2976; 2106; 1735; 1616. MS (ESI) m/z
905 [M + H]+. ESI-HRMS for C44H69N7O13: calcd. 904.5026
[M + H]+, found 904.5003.
7-Bis(2-azidoethyl)amino-3-ethoxycarbonylcoumarin
(11).
Compound 10 (4.20 g, 11.7 mmol) was stirred in anhydrous
DMF (40 mL) with NaN3 (2.30 g, 35.4 mmol) and NaI (1.12 g,
7.5 mmol) at 90 ◦C for 6 h. The mixture was cooled to rt, added
with Et2O (50 mL), and washed with water (3 ¥ 50 mL). The
organic layer was dried over MgSO4 and reduced under vacuum
to yield analyti◦cally pure 11 (4.26 g, 98%) as an orange-yellow
powder. Mp 91 C. TLC Rf 0.4 (AcOEt/n-hexane 1 : 1). 1H-NMR
(CDCl3, 200 MHz) d 8.45 (s, 1H); 7.44 (d, J = 8.8 Hz, 1H); 6.69
(dd, J = 8.8, 2.6 Hz, 1H); 6.54 (d, J = 2.6 Hz, 1H); 4.38 (q,
J = 7.1 Hz, 2H); 3.72–3.55 (m, 8H); 1.39 (t, J = 7.1 Hz, 3H).
13C-NMR (CDCl3, 50 MHz) d 163.7; 157.9; 157.6; 151.9; 148.9;
131.2; 111.4; 109.7; 108.9; 97.9; 61.4; 50.7; 48.6; 14.3. IR (film)
n 2979; 2099; 1764; 1698; 1618; 1514; 1412; 1205. MS (ESI) m/z
372 [M + H]+; 394 [M + Na]+. ESI-HRMS for C16H17N7O4: calcd.
372.1415 [M + H]+, found 372.1410.
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7-Bis(2-iodoethyl)amino-3-ethoxycarbonylcoumarin
(15).
7.1 Hz, 3H). 13C-NMR (CDCl3, 75 MHz) d 171.5; 171.2; 164.1;
163.3; 157.1; 155.9; 149.6; 130.6; 116.1; 114.7; 112.4; 109.7; 81.7;
81.1; 68.9; 62.1; 57.5; 56.6; 53.9; 46.7; 28.7; 14.8. IR (film) n 2978;
2929; 1731; 1605. MS (ESI) m/z 764 [M + H]+; 786 [M + Na]+.
ESI-HRMS for C39H58N2O13: calcd. 763.4012 [M + Na]+, found
763.3992.
Coumarin 10 (0.11 g, 0.31 mmol) was stirred with NaI (97 mg,
0.64 mmol) in refluxing acetone for 16 h. The reaction mixture
was reduced under vacuum and the residue was purified by
chromatography (AcOEt/n-hexane 30 : 70 to 70 : 30) to yield 15
(0.16 g, 98%) as a yellow powder. Mp 160 ◦C. TLC Rf 0.6 (Et2O).
1H-NMR (CDCl3, 300 MHz) d 8.43 (s, 1H); 7.44 (d, J = 9.0 Hz,
1H); 6.60 (dd, J = 9.0, 2.1 Hz, 1H); 6.84 (d, J = 2.1 Hz, 1H); 4.37
(q, J = 7.2 Hz, 2H); 3.84 (t, J = 8.4 Hz, 2H); 3.25 (t, J = 8.4 Hz,
2H); 1.39 (t, J = 7.2 Hz, 3H). 13C-NMR (CDCl3, 50 MHz) d
163.6; 157.9; 157.4; 150.9; 148.8; 131.3; 111.4; 109.3; 109.1; 97.6;
61.4; 53.8; 14.3; –0.7. IR (film) n 3071; 2981; 2921; 1732; 1587 MS
(ESI) m/z 542 [M + H]+; 564 [M + Na]+.
6,8-Bis[N,N-di(tert-butoxycarbonylmethyl)aminomethyl]-3-eth-
oxycarbonyl-7-hydroxycoumarin (20). Aqueous formaldehyde
(0.48 mL, 5.8 mmol) was added to iminodiacetic acid di-tert-butyl
ester (1.15 g, 4.6 mmol) in MeOH/t-BuOH (10 mL). The resulting
solution was stirred at rt for 90 min. Then solvent was removed
under vacuum and the residue was coevaporated twice with
anhydrous MeOH (4 mL). Coumarin 17 (0.55 g, 2.3 mmol) was
then added, followed by N-methylmorpholine (1.5 mL, 13.3 mmol)
and the reaction mixture was stirred in a sealed vessel at 115 ◦C for
5 h and for 16 h at rt. Volatile was removed under reduced pressure
and the residue was dissolved in AcOEt, washed with water and
brine, dried over MgSO4, and purified by flash chromatography
(CH2Cl2/AcOEt 80 : 20 to 50 : 50) to yield compound 20 (0.23 mg,
13%) as a yellow solid. Mp 115 ◦C. TLC Rf 0.35 (CH2Cl2/AcOEt
80 : 20). 1H-NMR (CDCl3, 300 MHz) d 8.49 (s, 1H); 7.69 (s, 1H);
4.37 (q, J = 7.2 Hz, 2H); 4.20 (s, 2H); 3.97 (s, 2H); 3.47 (s, 4H);
3.34 (s, 4H); 1.47 (s, 36H); 1.38 (t, J = 7.2 Hz, 3H). 13C-NMR
(CDCl3, 75 MHz) d 170.2; 169.7; 162.6; 163.1; 156.5; 154.3; 149.4;
130.0; 123.6; 112.1; 110.1; 108.5; 81.7; 80.8; 61.0; 55.3; 55.0; 51.5;
47.0; 27.9; 27.8; 13.9. IR (film) n 2977; 2929; 1730; 1613; 1135.
MS (ESI) m/z 750 [M + H]+; 772 [M + Na]+. ESI-HRMS for
C38H56N2O13: calcd. 749.3855 [M + H]+, found 749.3841.
7-[(4-tert-Butoxycarbonylmethyl)piperazin-1-yl]-3-ethoxycar-
bonylcoumarin (16). Yellow powder. Mp 127 ◦C. TLC Rf 0.6
(AcOEt). 1H-NMR (CDCl3, 300 MHz) d 8.43 (s, 1H); 7.40 (d, J =
7.9 Hz, 1H); 6.80 (dd, J = 7.9, 2.1 Hz, 1H); 6.64 (d, J = 2.1 Hz,
1H); 4.37 (q, J = 6.9 Hz, 2H); 3.49 (m, 4H); 3.20 (s, 2H); 2.76
(m, 4H); 1.47 (s, 9H); 1.39 (t, J = 6.9 Hz, 3H). 13C-NMR (CDCl3,
50 MHz) d 169.2; 164.1; 158.1; 158.0; 155.2; 149.1; 130.8; 111.6;
111.5; 109.4; 99.7; 81.7; 61.5; 59.7; 52.3; 47.0; 28.3; 14.5. IR (film)
n 2966; 2923; 2850; 1732; 1590. MS (ESI) m/z 417 [M + H]+; 439
[M + Na]+. ESI-HRMS for C22H28N2O6: calcd. 417.2020 [M + H]+,
found 417.2018.
8-[N,N-Di(tert-butoxycarbonylmethyl)aminomethyl]-3-ethoxy-
carbonyl-7-hydroxycoumarin (18). Aqueous formaldehyde (37%,
0.19 mL, 2.50 mmol) was added to 3-ethoxycarbonyl-7-
hydroxycoumarin 17 (0.50 g, 2.10 mmol) and iminodiacetic acid
di-tert-butyl ester (0.53 g, 2.10 mmol) in tert-butanol (10 mL).
The mixture was refluxed for 15 h, solvent was removed under
vacuum, and the residue was purified by silica gel chromatography
(CH2Cl2) to yield compound 18 (0.79 g, 76%) as a yellow solid.
6,8-Bis[N,N-di(tert-butoxycarbonylmethyl)aminomethyl]-7-{2-
[bis(tert-butoxycarbonylmethyl)amino]ethoxy}-3-ethoxycarbonyl-
coumarin (21). DIAD (47 mL, 0.24 mmol) was added dropwise
at rt to a stirred solution of N-(2-hydroxyethyl)iminodiacetic acid
di-tert-butyl ester (0.55 mg, 0.19 mmol), coumarin 20 (118 mg,
0.16 mmol), and PPh3 (62 mg, 0.24 mmol) in anhydrous THF
(2 mL). The reaction mixture was stirred for 3 h, the solvent
was removed under vacuum and the residue was purified by
chromatography (toluene/CH2Cl2/AcOEt 3 : 5 : 0.5 to 3 : 5 :
2) to yield compound 21 (69 mg, 43%) as a yellow oil. TLC Rf 0.6
(toluene/CH2Cl2/AcOEt 3 : 5 : 2). 1H-NMR (CDCl3, 300 MHz)
d 8.49 (s, 1H); 7.92 (s, 1H); 4.38 (q, J = 7.2 Hz, 2H); 4.17 (t, J =
6.1 Hz, 2H); 4.15 (s, 2H); 3.99 (s, 2H); 3.55 (s, 4H); 3.53 (s, 4H);
3.42 (s, 4H); 3.19 (t, J = 6.1 Hz, 2H); 1.45 (s, 36H); 1.40 (s, 18H);
1.38 (t, J = 7.2 Hz, 3H). 13C-NMR (CDCl3, 75 MHz) d 170.6;
170.5; 170.4; 163.2; 162.7; 156.4; 154.4; 148.9; 130.6; 130.1; 120.6;
115.9; 114.3; 81.0; 80.8; 80.7; 74.8; 61.6; 56.4; 55.5; 55.3; 54.2; 51.7;
46.7; 28.2; 28.1; 14.2. IR (film) n 2977; 2931; 1728; 1139. MS (ESI)
m/z 1021 [M + H]+; 1043 [M + Na]+. ESI-HRMS for C52H81N3O17:
calcd. 1020.5639 [M + H]+, found 1020.5620.
◦
1
Mp 113 C. TLC Rf 0.6 (Et2O). H-NMR (CDCl3, 300 MHz) d
8.47 (s, 1H); 7.42 (d, J = 8.5 Hz, 1H); 6.87 (d, J = 8.5 Hz, 1H);
4.38 (q, J = 7.2 Hz, 2H); 4.20 (s, 2H); 3.41 (s, 4H); 1.49 (s, 18H);
1.39 (t, J = 7.2 Hz, 3H). 13C-NMR (CDCl3, 75 MHz) d 170.0;
165.0; 163.6; 156.9; 155.5; 149.9; 130.8; 115.3; 112.7; 110.8; 108.5;
82.6; 61.6; 55.6; 48.1; 28.3; 14.5. IR (film) n 3307 (b); 3227; 2979;
2930; 1753; 1599. MS (ESI) m/z 492 [M + H]+. ESI-HRMS for
C25H33NO9: calcd. 492.5382 [M + Na]+, found 492.5369.
7-{2-[Bis(tert-butoxycarbonylmethyl)amino]ethoxy}-8-[bis(tert-
butoxycarbonylmethyl)aminomethyl] - 3 - ethoxycarbonylcoumarin
(19). A mixture of DIAD (0.15 mL, 0.74 mmol) and PPh3
(0.17 g, 0.61 mmol) in anhydrous THF (4 mL) was added
by portion to coumarin 18 (0.20 g, 0.40 mmol) and N-
(2-hydroxyethyl)iminodiacetic acid di-tert-butyl ester (0.12 g,
0.40 mmol) in THF (4 mL) until starting material completely
disappeared. The reaction mixture was stirred for 2 h at rt before
the solvent was removed under reduced pressure. The residue
was triturated with Et2O and the precipitate was filtered off. The
filtrate was reduced under vacuum and the residue was purified
by chromatography (AcOEt/n-hexane 50 : 50) to yield 19 (0.14 g,
47%) as a yellow oil. TLC Rf 0.3 (AcOEt/n-hexane 50 : 50). 1H-
NMR (CDCl3, 300 MHz) d 8.41 (s, 1H); 7.44 (d, J = 8.7 Hz,
1H); 6.90 (d, J = 8.7 Hz, 1H); 4.35 (q, J = 7.1 Hz, 2H); 4.23
(t, J = 6.1 Hz, 2H); 4.15 (s, 2H); 3.56 (s, 4H); 3.51 (s, 4H); 3.19
(t, J = 6.1 Hz, 2H); 1.41 (s, 18H); 1.36 (s, 18H); 1.35 (t, J =
5,7-Dihydroxy-3-ethoxycarbonylcoumarin (25). Diethyl (N,N-
diethylaminomethylene)malonate (1.92 g, 7.90 mmol) was added
to anhydrous phloroglucinol (1.00 g, 7.90 mmol) in AcOH
(25 mL). The reaction mixture was refluxed for 8 h, then cooled
down to rt. The precipitate was filtered and washed with CHCl3 to
yield coumarin 25 (1.60 g, 81%) as an orange powder. Mp > 250 ◦C
(dec.). TLC Rf 0.4 (AcOEt). 1H-NMR (CD3OD, 300 MHz) d 8.85
(s, 1H); 6.24 (d, J = 1.8 Hz, 1H); 6.22 (d, J = 1.8 Hz, 1H); 4.33 (q,
J = 7.1 Hz, 2H); 1.37 (t, J = 7.1 Hz, 3H). 13C-NMR (DMSO-d6,
5268 | Org. Biomol. Chem., 2009, 7, 5259–5270
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50 MHz) d 166.7; 163.9; 159.4; 158.8; 157.6; 145.5; 109.2; 102.4;
99.4; 94.9; 61.5; 15.1. IR (film) n 3296 (b); 1726; 1592; 1356. UV-
vis lmax 362 (MeOH, pH 7.0). MS (ESI) m/z 251 [M + H]+; 273
[M + Na]+. ESI-HRMS for C12H10O6: calcd. 273.0370 [M + Na]+,
found 273.0351.
162.5; 161.6; 158.4; 155.1; 145.4; 134.8; 124.1; 117.6; 116.3; 111.6;
81.4; 81.2; 81.1; 76.3; 71.2; 70.7; 70.4; 68.6; 57.6; 56.9; 56.1; 55.1;
54.8; 49.8; 47.5; 40.2; 37.8; 28.8. IR (film) n 3323; 2923; 2851;
1714; 1625; 1145. MS (ESI) m/z 1491 [M + H]+; 1513 [M + Na]+.
ESI-HRMS for C74H116N6O25: calcd. 1489.8063 [M + H]+, found
1489.8039.
6,8-Bis[N,N -bis(tert-butoxycarbonylmethyl)aminomethyl]-5,7-
dihydroxy-3-ethoxycarbonylcoumarin (26). Aqueous formal-
dehyde (37%, 15 mL, 0,40 mmol) was added to a solution of
coumarin 25 (50 mg, 0.20 mmol) and iminodiacetic acid di-
tert-butyl ester (0.97 mg, 0.40 mmol) in tert-BuOH (1 mL).
The reaction mixture was refluxed for 5 h and reduced under
vacuum to yield compound 26 (0.19 g, 97%) as a yellow powder.
That compound was used without further purification. Analytical
sample was obtained by preparative TLC (CH2Cl2/AcOEt 6 : 4).
Mp 130 ◦C. TLC Rf 0.8 (CH2Cl2/Et2O 8 : 2). 1H-NMR (CDCl3,
300 MHz) d 8.98 (s, 1H); 4.35 (q, J = 7.2 Hz, 2H); 4.07 (s, 2H); 4.01
(s, 2H); 3.41 (s, 4H); 3.38 (s, 4H); 1.49 (s, 36H); 1.37 (t, J = 7.2 Hz,
3H). 13C-NMR (CDCl3, 75 MHz) d 170.6; 170.4; 164.1; 158.3;
157.9; 155.2; 146.1; 110.1; 105.1; 102.6; 99.9; 82.9; 82.8; 61.6; 55.9;
55.6; 48.8; 48.1; 28.6; 14.9. IR (film) n 2977; 2929; 1731; 1614;
1133. MS (ESI) m/z 766 [M + H]+. ESI-HRMS for C38H56N2O14:
calcd. 765.3804 [M + H]+, found 765.3788.
tert-Butyl 2-[2-(2-maleimidoethoxy)ethoxy]ethylcarbamate (30).
A freshly prepared mixture of DIAD (1.20 mL, 6.0 mmol) and
PPh3 (1.20 g, 4.5 mmol) in anhydrous THF (10 mL) was added
dropwise to a solution of protected aminoalcohol 29 (0.75 g,
3.0 mmol) and maleimide (0.73 g, 7.5 mmol) in THF (5 mL) at rt.
The reaction mixture was stirred for 2 h, reduced under vacuum,
and triturated with Et2O. Precipitated Ph3PO was removed by
filtration, the filtrate was reduced under vacuum, and the residue
was purified by chromatography (cyclo-hexane/AcOEt 9 : 1) to
yield 30 (0.73 g, 74%) as a colorless oil. TLC Rf 0.3 (cyclo-
1
hexane/AcOEt 7 : 3). H-NMR (CDCl3, 300 MHz) d 6.64 (s,
2H); 5.05 (m, 1H); 3.64 (t, J = 5.4 Hz, 2H); 3.55 (t, J = 5.4 Hz,
2H); 3.48 (m, 4H); 3.40 (t, J = 5.1 Hz, 2H); 3.18 (td, J = 5.1,
5.1 Hz, 2H); 1.35 (s, 9H). 13C-NMR (CDCl3, 75 MHz) d 171.1;
156.4; 134.6; 79.5; 70.6; 70.3; 68.2; 40.8; 37.5; 28.8. IR (film) n
3368; 2975; 2929; 2871; 1701; 1101. MS (ESI) m/z 329 [M + H]+;
351 [M + Na]+.
5,7-Bis{2-[bis(tert-butoxycarbonylmethyl)amino]ethoxy}-6,8-
bis[N,N -bis(tert-butoxycarbonylmethyl)aminomethyl]-3-ethoxy-
carbonylcoumarin (27). Compound 27 (42 mg, 51%) was ob-
tained as a yellow glassy solid, starting from 26 and following
the same procedure as for 19. TLC Rf 0.7 (n-hexane/AcOEt 1 : 1).
1H-NMR (CDCl3, 300 MHz) d 9.06 (s, 1H); 4.38 (q, J = 7.2 Hz,
1H); 4,29 (m, 2H); 4.19 (m, 2H); 4,12 (bs, 2H); 4.00 (bs, 2H); 3.59
(s, 4H); 3.56 (s, 4H); 3.54 (s, 4H); 3.44 (s, 4H); 3.22 (m, 4H); 1.44-
1.40 (m, 75H). 13C-NMR (CDCl3, 75 MHz) d 171.3; 171.2; 170.9;
165.3; 164.1; 158.2; 157.0; 155.8; 145.8; 123.7; 117.7; 116.0; 110.6;
81.6; 81.4; 81.3; 81.1; 76.5; 62.2; 57.6; 56.9; 56.1; 55.1; 55.0; 54.8;
48.2; 47.5; 28.8; 14.9. IR (film) n 2977; 2930; 1729; 1595; 1142.
MS (ESI) m/z 1308 [M + H]+; 1330 [M + Na]+. ESI-HRMS for
C66H106N4O22: calcd. 1307.7371 [M + H]+, found 1307.7349.
[2-(2-Maleimidoethoxy)ethoxy]ethylamine hydrochloride (31).
Compound 30 (0.14 g, 0.42 mmol) was stirred in TFA/CHCl3 (3 :
4, 4 mL) for 2 h at rt. Volatile was removed under reduced pressure
and the residue was coevaporated twice with CHCl3, dissolved in
Et2O, and washed with HCl 10%. The organic layer was dried
over MgSO4 and evaporated to dryness to yield 31 (0.11 g, 81%)
1
as a colorless glassy solid. H-NMR (CDCl3, 300 MHz) d 6.74
(s, 2H); 3.72–3.68 (m, 4H); 3.62–3.57 (m, 6H); 3.20–3.16 (m, 2H).
13C-NMR (CDCl3, 75 MHz) d 171.6; 134.9; 70.7; 70.3; 68.7; 67.2;
40.3; 37.8. MS (ESI) m/z 229 [M + H]+.
General procedure for the preparation of europium complexes
EuCl3·6H2O 0.1 M (1 eq) was added to a solution of the
appropriate ligand (20 mg, 1 eq) in water (1 mL) and the resulting
mixture was stirred for 20 h at rt. The solution was brought to pH 7
with NaOH 0.2 M and was lyophilized. The residue was triturated
with MeOH (2 ¥ 2 mL) and the precipitate was collected after
filtration or centrifugation.
5,7-Bis{2-[bis(tert-butoxycarbonylmethyl)amino]ethoxy}-6,8-
bis[N,N-bis(tert-butoxycarbonylmethyl)aminomethyl]-3-(2-[2-{2-
(N-maleimidyl)ethoxy}ethoxy]ethylaminocarbonyl)coumarin (28).
Coumarin 27 (0.37 g, 0.28 mmol) in tert-BuOH (5 mL) was
sonicated for 5 min with NaOH 1 N (0.56 mL, 0.56 mmol) and
stirred for 30 h at rt. Then HCl 1 N (0.56 mL, 0.56 mmol) was
added and the reaction mixture was evaporated to dryness. The
residue was suspended in anhydrous CH2Cl2 (2 mL), DCC (0.07 g,
0.34 mmol) in CH2Cl2 (2 mL) was added at 0 ◦C, followed by NHS
(0.04 g, 0.34 mmol), and the solution was stirred for 20 h at rt.
Compound 31 (0.12 g, 0.34 mmol) in anhydrous CH2Cl2 (2 mL)
and N,N-di-iso-propyl-ethylamine (58 mL, 0.34 mmol) were added
to the previously activated ester and the reaction mixture was
stirred at rt for 5 h, reduced under vacuum, and purified by silica
gel chromatography (cyclo-hexane/AcOEt 6 : 4) to yield coumarin
28 (0.22 g, 53%) as a yellow oil. TLC Rf 0.3 (cyclo-hexane/AcOEt
1 : 1). 1H-NMR (CDCl3, 200 MHz) d 9.29 (s, 1H); 8.95 (bs, 1H);
6.71 (s, 2H); 4.30–4.15 (m, 4H); 4.13 (s, 2H); 4.02 (s, 2H); 3.78–
3.66 (m, 10H); 3.60 (m, 10H); 3.56 (s, 4H); 3.54 (s, 4H); 3.45 (s,
4H); 3.26–3.21 (m, 4H); 1.45 (s, 18H); 1.43 (s, 18H); 1.39 (s, 36H).
13C-NMR (CDCl3, 75 MHz) d 171.4; 171.3; 171.2; 171.0; 165.0;
[Eu(3b)]. Yellow powder (91%). UV-vis lmax 329 (H2O, pH 6.5).
ESI-HRMS for C22H22EuN3O12: calcd. 674.0489 [M + H]+, found
674.0460.
[Eu(3c)]. Yellow powder (79%). UV-vis lmax 327 (H2O, pH 6.5).
ESI-HRMS for C28H34EuN7O13: calcd. 830.1500 [M + H]+, found
830.1468.
[Eu(4a)]. Yellow powder (93%). UV-vis lmax 341 (H2O, pH 6.5).
ESI-HRMS for C23H24EuN2O13: calcd. 689.0485 [M + H]+, found
689.0469.
[Eu(5a)]. Yellow powder (81%). IR (film) n 3391; 1592; 1393.
UV-vis lmax 334 (H2O, pH 6.5). ESI-HRMS for C28H30EuN3O17:
calcd. 834.0863 [M + H]+, found 834.0832.
[Eu(6a)]. Yellow
powder
(66%).
ESI-HRMS
for
C22H21EuN2O14: calcd. 691.0278 [M + H]+, found 691.0259.
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[Eu(7a)]. Yellow powder (86%). IR (film) n 3379; 1614; 1393.
UV-vis lmax 326 (H2O, pH 6.5). ESI-HRMS for C34H39EuN4O22:
calcd. 1009.1341 [M + H]+, found 1009.1319.
33 E. Brunet, M. T. Alonso, O. Juanes, O. Velasco and J. C. Rodriguez-
Ubis, Tetrahedron, 2001, 57, 3105–3116.
34 C. Fe´au, E. Klein, P. Kerth and L. Lebeau, Bioorg. Med. Chem. Lett.,
2007, 17, 1499–1503.
35 C. Fe´au, E. Klein, P. Kerth and L. Lebeau, Synth. Met., 2009, 159,
528–536.
[Eu(7d)]. Yellow powder (75%). UV-vis lmax 326 (H2O, pH 6.5).
ESI-HRMS for C42H49EuN6O25: calcd. 1191.2032 [M + H]+, found
1191.2011.
36 C. F. W. Becker, D. Clayton, G. Shapovalov, H. A. Lester and G. G.
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38 K. E. Borbas and J. I. Bruce, Org. Biomol. Chem., 2007, 5, 2274–2282.
39 M. Halim, M. S. Tremblay, S. Jockusch, N. J. Turro and D. Sames,
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