Emissive and cell-permeable 3-pyridyl- and 3-pyrazolyl-4-azaxanthone lanthanide complexes and their behaviour in cellulo

Article abstract of DOI:10.1002/hlca.200900122

A series of seven emissive europium(III) and terbium(III) complexes was prepared, incorporating a 3-pyridyl-4-azaxanthone or 3-pyrazolyl-4-azaxanthone sensitising moiety within a polydentate macrocyclic ligand. High overall emission quantum yields in aqueous media are attenuated in the presence of protein or certain oxy anions due to displacement of the N,N′-chelated sensitiser. Nevertheless, these complexes are taken into cells and tend to localise over the first few hours in mitochondria before being trafficked to endosomal compartments. Cell uptake studies, in the presence of competitive inhibitors or promoters of well-defined uptake pathways, reveal a common uptake mechanism involving macropinocytosis.

Full text of DOI:10.1002/hlca.200900122

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Emissive and Cell-Permeable 3-Pyridyl- and 3-Pyrazolyl-4-azaxanthone
Lanthanide Complexes and Their Behaviour in cellulo
by Craig P. Montgomerya), Elizabeth J. Newa), Lars O. Palssona), David Parker*a),
Andrei S. Batsanova), and Laurent Lamarqueb)
a) Department of Chemistry, Durham University, South Road, Durham, DH13LE, U.K.
b) CISbio International, BP 84175, F-30204 Bagnols-sur-Ceze
(fax: þ 44-191-3844737; e-mail: david.parker@dur.ac.uk)
Dedicated to Professor Jean-Claude Bꢀnzli on the occasion of his 65th birthday
A series of seven emissive europium(III) and terbium(III) complexes was prepared, incorporating a
3-pyridyl-4-azaxanthone or 3-pyrazolyl-4-azaxanthone sensitising moiety within a polydentate macro-
cyclic ligand. High overall emission quantum yields in aqueous media are attenuated in the presence of
protein or certain oxy anions due to displacement of the N,N’-chelated sensitiser. Nevertheless, these
complexes are taken into cells and tend to localise over the first few hours in mitochondria before being
trafficked to endosomal compartments. Cell uptake studies, in the presence of competitive inhibitors or
promoters of well-defined uptake pathways, reveal a common uptake mechanism involving macro-
pinocytosis.
Introduction. – H2O-soluble complexes of lanthanide(III) ions (Ln3þ) that are
amenable to sensitised emission have been studied intensively over the past few
decades for three main reasons, each related to a particular technological application.
First, they may serve as components in various time-resolved assays, most commonly
serving as a donor to a proximate acceptor lumophore, usually involving an
intermolecular energy-transfer process [1 – 4]. Second, they can function as responsive
probes, in which the energy lifetime or polarisation of the emitted light is monitored.
For example, ratiometric sensors have been devised, in which the relative intensity of a
pair of emission bands in a single complex (usually EuIII) reports on the change in the
composition of the local environment, on a millisecond timescale. These include
systems designed to report changes in pH [5] or the concentration of anions, such as
hydrogencarbonate [6] or citrate and lactate [7][8]. Alternative strategies have been
devised in which EuIII and TbIII complexes of a common ligand are used. In these cases,
it is the intensity ratio of red (Eu) to green (Tb) light that is measured. By careful
consideration of the nature of the sensitising moiety and the constitution of the ligand,
systems able to report on changes in pO2 or p[urate] have been developed [9][10].
Third, emissive probes suitable for application in living cells are being evaluated [11 –
15]. Probes for this purpose should possess the following characteristics: cell
permeability and minimal toxicity over the relevant range of concentrations for the
duration of the observation period; resistance to photofading and photobleaching;
kinetic stability with respect to chemical degradation, such as loss of the metal ion from
the ligand or rapid oxidative metabolism of the sensitiser or ligand; immunity, or at
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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least relative insensitivity, to quenching of the metal-based excited state and those
intermediate excited states associated with excitation of the integral sensitising moiety
[11][16 – 18].
A plethora of hepta-, octa-, and nonadentate ligands designed to form robust
complexes with lanthanide(III) ions has been reported [1 – 4][16][19 – 21], including
some elegant dinucleating systems [14]. The ligand should be designed to suppress
vibrational deactivation of the excited LnIII ion by proximate NH, OH, or CH
oscillators [22]. It also should include an aromatic or heterocyclic sensitising moiety,
possessing a small singlet/triplet energy gap, with a rapid rate of intersystem crossing
that facilitates efficient population of the triplet state. The 4-azaxanthone sensitisers 11)
have recently been introduced for this purpose [23] (4-azaxanthone1) ¼ 5H-[1]benzo-
pyrano[2,3-b]pyridin-5-one) and have proved to be effective sensitisers for Eu and Tb
emission in aqueous media [12][17][18]. In seeking to extend their scope, we resolved
to prepare bidentate analogues with an increased conjugation length, allowing
excitation at longer wavelengths [24]. Both 2,2’-bipyridines 2 and 2-pyrazolyl pyridines
3 are well-established chelating ligands in Ln coordination chemistry [20][23][25] and
possess desirable photophysical characteristics allowing their introduction into a
sensitising moiety. Accordingly, we set out to prepare these compounds and
incorporate them into well-defined, macrocyclic ligand frameworks, e.g., L1 – L6, to
define a new sub-set of nonadentate ligands. Here, we describe their synthesis and
characterisation and report the outcomes of various studies to assess their utility as
time-resolved assay components and as selective cellular probes. A preliminary account
of part of this work has been published [24].
Ligand and Complex Synthesis and Characterisation. – The synthetic intermediate
4 was identified as a key primary target for the preparation of chromophores 2 and 3
t
(Scheme 1). It included a Bu group at the 7-position to aid solubility, without unduly
perturbing the energy of the S1/T1 excited states. Nucleophilic attack of 4-(tert-
butyl)phenoxide on 2-chloronicotinic acid, followed by electrophilic cyclisation
mediated by polyphosphoric acid (PPA), afforded 4a in 77% overall yield. N-
Methylation with methyl triflate and subsequent anion exchange yielded the H2O-
soluble chloride salt. Oxidative hydrolysis, following a literature protocol [26] using
basic ferricyanide, gave the N-methylpyridinone in 74% yield. Conversion to the 2-
chloro-derivative 4b was undertaken with POCl3 in 49% yield after chromatographic
purification. A Stille coupling of 4b with 6-methyl-2-(tributylstannyl)pyridine
([Pd(PPh3)4]/PhMe; 61% yield) afforded the bipyridine derivative 2a, whilst reaction
of 4b with 3-methyl-1H-pyrazole (NaH/THF; 90% yield) yielded the pyrazolyl-
pyridine intermediate 3a. Selective benzylic bromination of both 2a and 3a (NBS/CCl4)
gave the bromomethyl derivatives 2b and 3b, respectively. These reactions also led to
significant quantities of the corresponding dibrominated products 2c or 3c, typically in
ca. 30% isolated yield, following separation by column chromatography. Each
dibrominated compound could subsequently be converted into the desired mono-
brominated compound by reaction with diethyl phosphite in the presence of Hꢀnigꢂs
1
)
Arbitrary atom numbering; for systematic names, see Exper. Part. Notice that compounds of type 1
have also been designated as 1-azaxanthones.

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Scheme 1. Synthesis of the Key Intermediates 4a and 4b
base in THF. The constitution of compounds 2a and 3b was confirmed by single-crystal
X-ray-analysis (Fig. 1)2).
The absorption and emission spectral properties of 2a and 3a were examined in
MeOH. The absorption spectrum of the pyridyl system 2a (lmax 348 nm, e 10100 mꢀ1
cmꢀ1) was slightly broader in form than that of the pyrazolyl analogue 3a (lmax 346 nm, e
15400 mꢀ1 cmꢀ1) and extended further to the red, absorbing 5 times more strongly at
365 nm, for example. Each gave rise at room temperature to a very weak and
unstructured fluorescence emission band in the range 390 – 430 nm. Phosphorescence
emission spectra (77 K, Et2O/isopentane/EtOH glass) revealed a lowest-energy T1 – S0
transition at 20960 and 23470 cmꢀ1 for 2a and 3a, respectively. Fine structure was
observed in each phosphorescence spectrum (Fig. 2), with three distinct bands
observed at higher energy than the zero – zero transition, separated by 1650 cmꢀ1.
Such a spectral form is characteristic of C¼O vibrational fine structure, consistent with
dominant np* character for the triplet state [27][28]. Therefore, given that the excited-
state energies of the emissive states of Tb (5D4) and Eu (5D0) are at 20400 and
17200 cmꢀ1, respectively, only 3a is suitable as a sensitiser for both ions, without the
prospect of thermally activated back energy transfer.
The lanthanide(III) complexes of ligands L1 and L2 were prepared by using similar
methods to those defined for the tetraazatriphenylene analogue L3 [12]. For example,
stepwise reaction of 2b with 1,4,7-tri(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7-
triacetate (MeCN, Cs2CO3; 90% yield), followed by treatment with CF3COOH
(CH2Cl2, 208; 50% yield) afforded L1 as its protonated trifluoroacetate salt. Complex-
ation of the ligand with Eu(OAc)3 (pH 5.5, 50% aq. MeOH, 15 h, 808) gave the neutral
complex, [Eu(L1)], which was purified by chromatography (neutral Al2O3). The other
series of complexes that was prepared was based on the neutral ligands L4 – L6. Each
chiral ligand was prepared in enantiomerically pure form. Similar series of ligands
2
)
CCDC-724804 and -724805 contain the supplementary crystallographic data for 2a and 3b. These
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif.

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Fig. 1. X-Ray crystal structure of 2a and 3b, showing atomic displacement ellipsoids at the 50%
probability level. Arbitrary atom numbering.
Fig. 2. Phosphorescence emission spectra for 2a (right) and 3a (77 K, Et2O/isopentane/EtOH frozen
glass)

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incorporating such chiral amide substituents have been studied quite intensively
[29][30] as they give rise to formation of preferred isomeric species in their LnIII
complexes, and these relatively hydrophobic complexes tend to resist excited-state
quenching processes well [17][23]. Reaction of 1,4,7-tri(tert-butyl) 1,4,7,10-tetraazacy-
clododecane-1,4,7-tricarboxylate with 2b, followed by (tert-butoxy)carbonyl(Boc)-
deprotection and N-alkylation with three equiv. of (S)-5a (MeCN, K2CO3) afforded L4a
(Scheme 2). Complexation of L4a (MeCN, 408) with an anhydrous lanthanide(III)
triflate salt, followed by anion-exchange chromatography, led to isolation of the H2O-
soluble cationic complex [Ln(L4a)]Cl3. Similar methods were used to prepare LnIII
complexes of ligands L5a and L6.
Scheme 2. Synthesis of the Cationic Europium Complex [Eu(L4a)]3þ
A different strategy was required for the synthesis of the complexes of L4b and L5b
bearing a p-(methoxycarbonyl)phenyl group at the amide substituent. This p-
(methoxycarbonyl) group is introduced to allow subsequent conjugation of the Ln
complex to different linkers or biomolecules. Reaction of the mono-Boc derivative 7
with 2b [31] (K2CO3, MeCN; 74% yield), followed by Boc deprotection afforded the
diamide 8 with a secondary amino group. N-Alkylation of the latter with (S)-5b under
basic conditions (MeCN, K2CO3; yield 64%) yielded the ligand L4b. The pyrazolyl
analogue L5b was prepared by a similar strategy, and the EuIII or TbIII complexes were
isolated, as described for [Ln(L4a)]X3. Various strategies are available for linking these
emissive probes to a particular protein or antibody [32], allowing selective FRET
experiments to be undertaken. An alternative approach has recently been devised by

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Covalys (ꢃSNAP-tagꢂTM technology), that simplifies the procedure and enables the
covalent labelling of proteins (e.g., for use as acceptors in a FRET system), with
compatible donor lumophores, labelled with an O-benzylguanine moiety. A mutant
DNA repair enzyme, human O6-alkylguanine-DNA alkyltransferase (LAGT) [33],
irreversibly and specifically reacts with O6-alkylguanine derivatives (e.g., BG – H, a
benzylguanine derivative) to form a thioether bond and release free guanine. Thus, the
BG conjugate of an emissive donor is transferred to a reactive cysteine residue on the
protein [34][35].
Hydrolysis of the methyl ester in [Eu(L4b)]3þ and [Tb(L5b)]3þ was undertaken in
basic aqueous MeOH (pH 10, MeOH/H2O 1:1, 2 h) (Scheme 3). After neutralisation,
the acid salt was converted into a succinimido ester (TSTU ¼ N,N,N’,N’-tetramethyl-O-
i
succinimidouronium tetrafluoroborate), Pr2NEt, DMF), monitoring conversion by
anal. HPLC. Direct reaction with excess O-[4-(aminomethyl)benzyl]guanine (¼ BG –
H) afforded the desired coupled amide which was purified by prep. reversed-phase
Scheme 3. Synthesis of the O-Benzylguanine Conjugate BGꢀ[Eu(L4)]3þ

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HPLC. These BG conjugates, BG – [Eu(L4)]3þ and BG – [Tb(L5)]3þ, possessed
virtually identical absorption and emission spectral properties to the precursor methyl
esters.
Photochemical Characterisation and Solution Stability. – Salient photophysical
data, defining the absorption and emission spectral characteristics of EuIII and TbIII
complexes are summarised in the Table. Measurements of radiative rate constants in
H2O and D2O allowed hydration states for each complex to be estimated. Only
[Eu(L2)] possesses a coordinated H2O molecule, i.e., [Eu(L2)(H2O)]. Its differing
coordination environment is also apparent in the relative form of the EuIII emission
spectrum, in comparison to those of [Eu(L1)] and [Eu(L4)]3þ (Fig. 3). With
[Eu(L2)(H2O)], the observed ligand fluorescence at 410 nm is quite strong (ffem
ꢁ
25%) and is barely shifted from that of the free ligand. This behaviour contrasts with
that observed with [Eu(L1)] and [Eu(L4a)]3þ. In each of these cases, the fluorescence
emission centred at ca. 400 nm is much weaker, and the form of the Eu spectral
fingerprint is very similar. This behaviour is consistent with the adoption of a common
coordination environment for the latter pair of complexes, very similar to that observed
for [Eu(L3)]. In this case, the 680 nm band is postulated to characterise axial
coordination of an N-atom donor in the 9-coordinate complex [12][36]. With
[Eu(L2)(H2O)], the pyrazolyl and pyridine N-atoms presumably do not chelate, and
L2 serves as a heptadentate ligand with one coordinated H2O molecule completing the
coordination sphere. The reduced quantum yield and the differing Eu emission spectral
form – resembling that of analogous (N-alkyl-DO3A) europium(III) complexes [37] –
and unshifted ligand fluorescence emission accords with this hypothesis (DO3A ¼
1,4,7,10-tetraazacyclododecane-1,4,7-triacetato).
Table. Photophysical Data for Selected Lanthanide(III) Complexes (295 K, pH 5.5)
kH Oa)b)
kD Oa)b)
f
[%]
em(H2O)c)
ET
Gd
Complex
lmax
e
2
2
[nm]
[mꢀ1 cmꢀ1
]
[msꢀ1
]
[msꢀ1
]
[cmꢀ1] (77 K)d)
[Eu(L1)]
356
345
348
347
347
356
356
348
349
348
10100
10100
10050
8300
1.00
1.43
0.44
0.95
0.64
1.00
0.98
0.50
0.45
0.44
0.74
0.57
0.37
0.63
0.58
0.74
0.73
0.42
0.36
0.40
14
1.2
15
18
40
24
25
46
61
54
20950
23450
23450
23800
23800
20950
20950
23470
23470
23450
[Eu(L2)(H2O)]
[Tb(L2)]
[Eu(L3)]
[Tb(L3)]
8300
[Eu(L4a)]3þ
[Eu(L4b)]3þ
[Tb(L5a)]3þ
[Tb(L5b)]3þ
[Tb(L6)]3þ
10050
10050
15050
15050
15050
a) Hydration numbers q are zero for each complex examined, except for [Eu(L2)(H2O)], for which q ¼
c
0.73 [22]. b) Errors in lifetimes are estimated to be ꢂ 10%, and ꢂ 15% for quantum yields. ) Note that
lifetime data and fem values may differ considerably as a function of pH in the presence of different
anions and/or protein in the aq. solution. For example, with [Eu(L4a)]Cl3, values were reduced to 3% in
the presence of 0.4 mm human-serum albumin and to 2% in the cellular growth medium. d) Triplet
energies were determined with Gd analogues in a frozen glass of Et2O/isopentane/EtOH or EtOH/
MeOH 2 : 1.

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Fig. 3. Total emission spectra for a) [Eu(L1)] (red) and [Eu(L2)(H2O)] (blue) compared to b)
[Eu(L4a)]3þ (295 K, aerated H2O, lexc 355 nm). L1 ¼ DO3A-pyridylazaxanthone, L2 ¼ DO3A-pyrazolyl-
azaxanthone.
The inability of the pyrazolyl-azaxanthone moiety to chelate to the EuIII centre in
[Eu(L2)] prompted an examination of the pH sensitivity of the LnIII complexes and
their ability to resist chelation by common oxy anions, such as HCOꢀ3 , HPO42ꢀ, lactate,
and citrate. Such anions are well known to form ternary adducts with LnIII complexes of
related heptadentate ligands, several of which have been characterised by X-ray
crystallography [38]. The emission spectra of [Eu(L1)] and [Eu(L4a)]3þ did not vary
over the pH range 3 – 9. With [Tb(L5a)]3þ, the intensity of emission at 542 nm decreased

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five-fold over the pH range 5 to 8, with an apparent pKa of 6.50 (ꢂ0.05) (Fig. 4). Such
behaviour may tentatively be assigned to partial dissociation of the chelating sensitising
moiety (Scheme 4). The measured q values at pH 5 and 8 were both zero. Similar
behaviour was exhibited by [Tb(L2)], and in each case, the spectral changes were
reversible.
Fig. 4. Variation of the ratio of LnIII emission intensity vs. residual ligand fluorescence with pH for
4a 3þ
[Tb(L5a)]3þ
( ) and [Eu(L )] ( ) (295 K, 0.1m NaCl)
&
^
Scheme 4. Dissociative Equilibrium Occurring with [Tb(L5)]3þ
Addition of a ten-fold excess of sodium lactate, phosphate, or citrate (pH 5.5) to
[Tb(L5a)]Cl3 resulted in a marked decrease in Tb emission intensity at 542 nm,
consistent with the observed pH instability. With [Eu(L4a)]Cl3, incremental addition of
aqueous solutions of lactate or citrate (pH 7.4), in up to a 10-fold excess, caused a
change in the form and a reduction in the intensity of the Eu emission (Fig. 5).
Addition of a 0.4 mm solution of human serum albumin (HSA) also changed the
spectral form, but in each of these cases less than a 10% reduction of the measured Eu
emission lifetime occurred. The changes to the form and relative intensity of the
hypersensitive DJ ¼ 2 (around 620 nm) and DJ ¼ 4 (680 – 710 nm) transitions were most

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distinctive, and the limiting spectra (e.g., strong DJ ¼ 2 transition) observed were
strongly reminiscent of ternary adducts observed in related triamide complexes
[10][36][38] based on an N4O3 (ligand) plus O2 (with one or more polarisable O-
atoms) donor array. The loss of the transition at 680 nm is also consistent with cleavage
of a Ln – N interaction. In addition, absorption spectra of solutions of [Eu(L4a)]Cl3, in
the presence of added citrate or HSA, exhibited a distinct shift (ꢁ 5 nm) to the blue of
the low-energy p – p* transitions at 356 and 325 nm, consistent with dissociation of the
chelating N-atoms of the sensitising moiety.
Fig. 5. Emission spectra for [Eu(L4a)]Cl3 (295 K, lexc 355 nm, 10 mm complex) (red) and in the presence
of 0.13m citrate (blue) and 0.4 mm human-serum albumin (HSA; green)
In summary, these competition experiments strongly indicate that for the LnIII
complexes examined here, the sensitising chromophore is likely to dissociate in cellulo,
lowering the overall emission quantum yield and modulating the emission spectral
form, notably for EuIII complexes. This tendency – not observed for complexes of
[Ln(L3)] and its congeners – will tend to restrict the range of applications of such
complexes as cellular probes but does not preclude their usage in cellulo, as they remain
emissive. This is particularly pertinent for the complexes of L4a and L1.
Behaviour as Cellular Probes. – In striving to develop LnIII complexes as cellular
probes, and not simply cellular stains or labels, it is important to understand their
mechanism of cell entry. In addition, the relationship between probe structure and its
cytotoxicity and compartmentalisation profile needs to be established [11][39]. Initial
work in Durham has examined over 70 emissive Eu and Tb complexes. This study has
revealed that uptake and compartmentalisation profiles are dependent upon the nature
and mode of linkage of the heterocyclic sensistising moiety, rather than upon complex
charge, lipophilicity, or relative protein affinity [11][12][18][40][41]. Three main
classes of behaviour have been observed so far. The majority of complexes are

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noncytotoxic and are taken up quite quickly and exhibit localisation to the perinuclear
endosomes/lysosomes [11]. A second set localises in protein-dense regions, such as
ribosomes or nucleoli and exhibit rather slow uptake/egress. Recently, it has been
suggested that the presence of these probes in the growth medium during incubation
may enhance membrane permeability [40]. A third series of complexes localises
quickly to mitochondria with subsequent shuttling to endosomal/lysosomal compart-
ments [41].
Cytotoxicity studies [39 – 41] were undertaken by using the ꢃMTTꢂ assay in mouse-
skin fibroblasts (NIH-3T3 cells), monitoring, over a 24 h period, the perturbation of
mitochondrial dehydrogenase enzymes in viable cells. Cytotoxicity values (IC50; with
the standard deviation in parenthesis) for [Eu(L4a)]Cl3, [Tb(L5a)]Cl3, and [Tb(L6)]Cl3
were 73(9), 42(9), and 101(13) mm, respectively. This is in the same range as other cell-
permeable complexes that exhibit similar cellular behaviour [18]. Cellular uptake and
localisation profiles of [Eu(L4a)]Cl3 and [Tb(L5a)]Cl3 were examined in Chinese-
hamster ovarian (CHO) cells and mouse-skin fibroblasts (NIH-3T3) by means of
fluorescence microscopy. Cultured cells were exposed to a growth medium with
complex concentrations of between 10 and 50 mm, and living cells were examined at
time intervals from 20 min to 24 h, post-incubation. Optical sections throughout the
observed cells were undertaken to ensure the probe was internalised, rather than
simply associated with the cell membrane. With [Eu(L4a)]Cl3, after a 20 min incubation
with a 50 mm solution, red Eu emission was observed clearly. Co-localisation with
Mitotracker GreenTM after 2 h confirmed this as a predominant mitochondrial
distribution, with merged images showing good correspondence (Fig. 6). Other
luminescence was observed, which was attributed to the lysosomes. For incubation
times of greater than 4 h, such merged images showed inconsistencies, and after more
than 16 h, the complex appeared to have migrated from the mitochondria to the
perinuclear endosomes/lysosomes. This hypothesis was indicated by a co-localisation
study with Lysotracker GreenTM (Fig. 7).
Fig. 6. Fluorescence microscopy co-localisation study with [Eu(L4a)]Cl3 (50 mm, 2 h): a) red Eu emission
(lobs > 600 nm), b) Mitotracker GreenTM emission (lobs 450 – 500 nm), and c) merged image showing
good correspondence (scale bar 20 mm)
Like [Eu(L4a)]Cl3, the pyrazolyl (S,S,S)-complex [Tb(L5a)]Cl3 exhibited time-
dependent intracellular trafficking between mitochondrial and endosomal compart-
ments. No difference in localisation behaviour was observed with the enantiomeric
complex (R,R,R)-[Tb(L5a)]Cl3. Furthermore, [Tb(L5a)]Cl3 and [Tb(L6)]Cl3 (the latter
with three amide substituents derived from l-phenylalanine replacing the (1-phenyl-

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Fig. 7. Fluorescence microscopy co-localisation study with [Eu(L4a)]Cl3 (50 mm, 24 h): a) Eu emission, b)
Lysotracker GreenTM, and c) merged image (scale bar 20 mm)
ethyl)amino moiety) also behaved more or less identically in their uptake and
localisation behaviour. Such behaviour lends further support to the hypothesis that the
nature of the pendant arms in homologous series of macrocyclic LnIII complexes does
not determine uptake/egress or intracellular localisation behaviour. Similar results
were obtained recently for a set of six complexes related to [Ln(L3)] [40]. The
observations reported here lend further support to the theory that it is the constitution
of the sensitising moiety that primarily determines uptake and trafficking. The
condensed aromatic moiety is the structural element that is most likely to be recognised
in protein association, which must be a key step in intramolecular trafficking involving
recycling vesicles.
Information on the speciation of EuIII complexes can be gleaned by analysis of the
form and relative intensity of the emission bands. With [Eu(L4a)]Cl3, following a 6-h
incubation in Chinese-hamster ovarian cells (50 mm, complex concentration in growth
medium), the cells were inspected by microscopy to confirm the mixed endosomal/
mitochondrial localisation profile. A set of cells (ca. 106) was then washed and
immediately suspended in an optical cell and the emission spectrum measured (Fig. 8).
The spectrum is consistent with a protein-bound ternary adduct, given its correspond-
ence to that measured in the presence of human-serum albumin in vitro (Fig. 5).
Measurement of Cell Uptake. – The cellular behaviour of [Eu(L4a)]Cl3 and
[Tb(L5a)]Cl3 was further examined by studying the effect of adding to the growth
medium various established inhibitors and promoters of defined cell uptake pathways
[40]. This was addressed by examining their effect the on observed image intensity
under standardised conditions by means of microscopy, as well as by measuring the
percentage of the complex in sorted and counted cell populations by means of flow
cytometry and ICP-MS to determine the Ln-ion concentration. Suppressed uptake
(30% (ꢂ10)) was observed at 48, consistent with an energy-dependent process such as
endocytosis [42]. The influence of the following was examined; wortmannin (blocking

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Fig. 8. Europium emission spectrum of [Eu(L4a)]Cl3 localised in Chinese-hamster ovarian cells (50 mm,
6 h incubation) revealing a spectral fingerprint consistent with formation of a ternary adduct with protein
(295 K, lexc 365 nm)
P1-3 kinase and hence macropinocytosis) [43], amiloride (blocking the Naþ/Hþ pump
and hence macropinocytosis) [44], sucrose (inhibiting clathrin-dependent endocytosis)
[45], chlorpromazine (interacting with clathrin), filipin (sequestering cholesterol,
thereby perturbing caveolae), chloroquine (increasing endosomal pH), phorbol esters
(activating protein kinase C and stimulating macropinocyotosis) [46], and long-chain
di-O-acylglycerols (stimulating macropinocytosis) [47]. Both wortmannin and amilor-
ide suppressed cell uptake of each complex (by up to 40%), whilst phorbol esters and
fatty-acid-derived di-O-acylglycerol enhanced uptake (by up to 100%). The other
treatments did not give rise to significant deviations from the control. Variations in cell-
image intensity in CHO, HeLa, and NIH-3T3 cells were consistent with this trend
(Fig. 9).
Taken together, these results reinforce the mechanistic conclusions derived very
recently [40] that pinpoint macropinocytosis as the common cell uptake pathway for
this class of complexes. Neither evidence for clathrin-mediated uptake, nor processes
involving caveolae was found.
Conclusions. – The EuIII and TbIII complexes of macrocyclic complexes incorporat-
ing a 3-pyridyl-4-azaxanthone or an analogous 3-pyrazolyl-4-azaxanthane sensitising
moiety function as effective emissive probes in aqueous media. Each complex displays
a tendency to undergo dissociation of the metal-coordinated sensitiser at elevated pH
(pH > 7) or in the presence of chelating anions or endogenous protein. Whilst this
tendency lowers the measured quantum yield in such media, it has not inhibited their
application as cellular probes. The complexes appear to enter the cell via macro-
pinocytosis. This is an attractive mechanism for cell labelling, as it is not receptor-
mediated, and the probe is not necessarily confined to endosomal or lysosomal
compartments [48]. Indeed, macropinosomes are generally regarded as rather leaky
compartments that tend to discharge their contents into the cell [48].

Helvetica Chimica Acta – Vol. 92 (2009)
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Fig. 9. Fluorescence microscopy images of Chinese-hamster ovarian cells in the presence of [Tb(L5a)]Cl3
(50 mm, 4 h), following a 30 min pretreatment with activators or inhibitors of endocytosis: a) control, b)
300 nm wortmannin, c) 500 ng/ml of 1,2-dipalmitoyl-derived rac-di-O-acylglycerol, d) 50 mm chlorpro-
mazine, and e) 1 mg/ml of filipin
The lower affinity of these chelating sensitisers for the Ln ion compared to those
based on a bipyridine subunit, may be related to the increased steric demand imposed
by the substituted azaxanthone moiety and the reduced basicity of the pyridine N-atom
donor atoms. This is particularly true of the azaxanthone ꢃpyridineꢂ N-atom that is
subject to the inductive effect of the adjacent endocyclic O-atom. Future probe design
should take heed of this behaviour.
We thank CISbio (C. P. M.), the Association of Commonwealth Universities for a scholarship
(E. J. N.), COST-Action D38, and the EC-network DIMI for support. It is a pleasure to acknowledge the
inspiration of the work of Prof. Jean-Claude Bꢀnzli, particularly for his indomitable enthusiasm and
passion for lanthanide chemistry, and his buoyant attitude to scholarship and education.
Experimental Part
1. General. Details of instrumentation, spectral acquisition and abbreviations, separation methods,
solvent purification, photochemical analyses, and flow cytometry have been reported earlier [39 – 41].
Cell culture methods, inhibition/complex uptake studies, and cytotoxicity measurements are detailed
below.

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Helvetica Chimica Acta – Vol. 92 (2009)
2. Cell Culture. Three cell lines were selected for cellular studies: CHO (Chinese-hamster ovary),
NIH 3T3 (mouse-skin fibroblast), and HeLa (human endothelial carcinoma cells). Cells were
maintained in exponential growth as monolayers in F-12 (Ham) medium, DMEM (Dulbeccoꢂs modified
eagle medium), and RPMI 1640 medium, resp. For each cell line, the medium was supplemented with
10% foetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells were incubated at 378, 20%
average humidity, and 5% (v/v) CO2/air.
3. Microscopy. Cells were seeded in 12-well plates on glass cover slips and allowed to grow to 40 –
60% confluence at 378 in 5% CO2. At this stage, the medium was replaced, and cells were treated
with drugs and complexes as described below. For NIH 3T3 and HeLa cells, the non-phenol-red-
containing DMEM was used. Following incubation, the cover slips were washed with phosphate-buffered
saline (PBS; pH 7.5), mounted on slides and sealed with nail varnish. Epifluorescence images were taken
on a Zeiss-Axiovert-200M epifluorescence microscope with a digital camera, and were processed with the
Zeiss Axiovision software. G365 filters (Zeiss) were employed for excitation of the complexes, with 546/
12 and 575 – 625 filters (Comar) used for emission of Tb and Eu, resp.
4. Uptake Studies. Cells were seeded in 6-well plates and allowed to grow to 80 – 100% confluence at
378 in 5% CO2/air. At this stage, the medium was replaced, and cells were treated with drugs and
complexes as described below. Following incubation, the medium was removed, and the cells were
washed three times with PBS. Lysis buffer (500 ml; 10 mm Tris, pH 7.5, 100 mm NaCl, 1 mm EDTA, 1%
Triton X-100, 0.1% protease inhibitor cocktail) was then added to each well, and the cells were incubated
at 58 for 15 min. Aliquots (3 ꢃ 25 ml) of the supernatant were taken from each well for the BCA
(bicinchoninic acid) assay, as described below, and a sample (400 ml) was submitted for ICP-MS analysis.
5. BCA Assay. Total protein content was determined in lysed cells by the bicinchoninic acid protein
assay (Pierce BCA protein assay kit). A BSA (bovine-serum albumin) standard curve was constructed in
the range 0.2 to 1 mg/ml. Standards (25 ml) and samples (25 ml) were transferred into triplicate wells of a
96-well plate, and the BCA reagent mix (200 ml) was added to each well. Absorbance at 540 nm was
measured after 10 h incubation at r.t. with a microplate reader. Protein concentration for each sample
was determined against the standard curve.
6. ICP-Mass Spectrometry. Inductively coupled plasma mass spectrometry (ICP-MS) determinations
of Eu or Tb concentrations were made by Dr. C. Ottley in the Department of Earth Sciences at Durham
University.
7. Drug Treatments. For the 58 treatment, cells were incubated in a refrigerator set at 58.
Concentrations of inhibiting or activating drugs used were 10 mm chloroquine, 50 mm chlorpromazine,
1 mg/ml of filipin, 2 mm monensin, 0.01% poly-l-lysine, 50 mm sucrose, 300 nm wortmannin, 3 mm
amiloride, 50 ng/ml of phorbol 12-myristate 13-acetate, and 500 ng/ml of rac-1,2-di-O-palmitoylglycerol.
Cells were subjected to appropriate treatments for 30 min prior to treatment with 50 mm complex for 4 h.
8. Cytotoxicity. IC50 Values were determined with the MTTassay, which makes use of the conversion
of MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) to a purple formazan
product by the mitochondrial dehydrogenase of viable cells. This insoluble formazan was quantified
spectrophotometrically upon dissolution in DMSO. About 1 · 104 NIH-3T3 cells in DMEM (100 ml) were
seeded into each well of flat-bottomed 96-well plates and allowed to attach overnight. Complex solns.
were added to triplicate wells to give final concentrations over a 2-log range. Following 24 h incubation,
MTT (1.0 mm) was added to each well, and the plates were incubated for a further 4 h. The culture
medium was removed, and DMSO (150 ml) was added. The plates were shaken for 20 s and the
absorbance measured immediately at 540 nm in a microplate reader. IC50 Values were determined as the
drug concentration required to reduce the absorbance to 50% of that in the untreated control wells, and
represent the mean for data from at least three independent experiments.
9. HPLC Analysis. Retention time tR in min. Anal. reversed-phase HPLC: at 298 K; Perkin-Elmer-
200 pump/autosampler/diode array detector with a fluorescence detector; Chromolith-RP18e column
(100 ꢃ 4.6 mm); flow rate 1 ml/min; Method A: running over 20 min, from H2O/MeCN 95 :5 (each with
0.1% HCOOH) to 100% MeCN at 15 min; Method B: running over 13 min, from H2O/MeCN 95 :5 to
H2O/MeCN 35 :65 at 8 min, and to 100% MeCN at 10 min; Method C: running over 13 min, from H2O/
MeCN 85 :15 to 100% MeCN at 10 min; Method D: running over 17 min, from H2O/MeCN 90 :10 to
H2O/MeCN 30 :70 at 11 min, and to 100% MeCN at 12 min.

Helvetica Chimica Acta – Vol. 92 (2009)
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Semi-prep. HPLC (Method E): Chromolith performance RP18e column (100 ꢃ 10 mm); flow rate
14 ml/min; running over 13 min, from H2O/MeCN 80 :20 to H2O/MeCN 45 :55 at 8 min, and to 100%
MeCN at 10 min.
10. Chromophore Synthesis. 2-[4-(tert-Butyl)phenoxy]nicotinic Acid (¼2-[4-(1,1-Dimethylethyl)-
phenoxy]pyridine-3-carboxylic Acid). To a stirred soln. of Na metal (1.02 g, 44.4 mmol) in anh. MeOH
(25 ml), 4-(tert-butyl)phenol (15.2 g, 101 mmol) was added to form a thick cream-coloured soln. The
solvent was evaporated to afford a cream-coloured residue. Then 2-chloronicotinic acid (3.31 g,
21.0 mmol) was added to the residue and the resulting mixture heated at 1908 with stirring for 20 h. The
mixture was allowed to cool to 1008 and poured into H2O (200 ml). The aq. soln. was extracted with Et2O
(2 ꢃ 150 ml) and then acidified to pH 5 by the addition of AcOH to afford a fine precipitate. The
precipitate was filtered, washed with H2O (100 ml), and dried under vacuum: colourless crystalline solid
1
t
(4.89 g, 86%). M.p. 180 – 1818. H-NMR (CDCl3, 500 MHz): 1.37 (s, Bu); 7.14 (d, J ¼ 8.5, HꢀC(2’,6’));
7.20 (dd, J ¼ 7.5, 5.0, HꢀC(5)); 7.49 (d, J ¼ 9.0, HꢀC(3’,5’)); 8.35 (dd, J ¼ 4.5, 2.0, HꢀC(6)); 8.55 (dd, J ¼
8.0, 2.0, HꢀC(4)). 13C-NMR (CDCl3, 125 MHz): 31.7 (Me3C); 34.8 (Me3C); 113.5 (C(3)); 119.7 (C(5));
121.4 (C(2’)); 127.1 (C(3’)); 143.8 (C(4)); 149.3 (C(4’)); 149.8 (C(1’)); 152.4 (C(6)); 161.5 (C(2)); 164.9
(C¼O). HR-ESI-MS (neg.): 270.1135 ([M ꢀ H]ꢀ, C16H16NO3ꢀ ; calc. 270.1130). Anal. calc. for C16H17NO3
(271.31): 70.83, H 6.32, N 5.16; found: C 70.54, H 6.20, N 4.91.
6-(tert-Butyl)-10H-9-oxa-1-azaanthracen-10-one (¼ 7-(1,1-Dimethylethyl)-5H-[1]benzopyrano[2,3-
b]pyridin-5-one; 4a). A stirred mixture of polyphosphoric acid (70 g) and 2-[4-(tert-butyl)phenoxy]ni-
cotinic acid (2.15 g, 7.93 mmol) was heated at 1208 for 16 h. The brown mixture was allowed to cool to r.t.,
before being poured onto iced water (400 ml) to afford a pale yellow soln. The pH of the aq. soln. was
adjusted to pH 7 by the addition of conc. aq. NaOH soln. The aq. soln. was extracted with Et2O (3 ꢃ
300 ml) and the combined extract dried (MgSO4) and concentrated: 4a (1.79 g, 89%). Yellow solid.
M.p. 97 – 988. 1H-NMR (CDCl3, 500 MHz)3): 1.42 (s, tBu); 7.45 (dd, J ¼ 7.5, 4.5, HꢀC(2)); 7.58 (d, J ¼ 8.5,
HꢀC(10)); 7.86 (dd, J ¼ 9.0, 3.0, HꢀC(9)); 8.30 (d, J ¼ 2.5, HꢀC(7)); 8.73 (d, J ¼ 7.5, HꢀC(1)); 8.76 (d,
J ¼ 7.5, HꢀC(3)). 13C-NMR (CDCl3, 125 MHz)3): 31.6 (Me3C); 35.1 (Me3C); 117.0 (C(4)); 118.4
(C(10)); 121.1 (C(2)); 121.1 (C(1)); 122.7 (C(7)); 133.9 (C(9)); 137.6 (C(3)); 148.2 (C(6)); 154.1 (C(8));
154.3 (C(12)); 160.6 (C(11)); 178.1 (C(5)). HR-ESI-MS (pos.): 254.1177 ([M þ H]þ, C16H16NO2þ ; calc.
254.1181). Anal. calc. for C16H15NO2 (253.30): 75.87, H 5.97, N 5.53; found: C 75.80, H 5.91, N 5.61.
6-(tert-Butyl)-1-methyl-10H-9-oxa-1-azoniaanthracen-10-one Trifluoromethanesulfonate (¼ 7-(1,1-
Dimethylethyl)-1-methyl-5-oxo-5H-[1]benzopyrano[2,3-b]pyridinium 1,1,1-Trifluoromethanesulfonate).
To a soln. of 4a (1.00 g, 3.95 mmol) in anh. toluene (20 ml) at 08, methyl trifluoromethanesulfonate (6 ml,
8.70 g, 53.0 mmol) was added over 10 min. The soln. was stirred at 08 for 1 h, to afford a fine precipitate
that was filtered and dried under vacuum: colourless solid (1.49 g, 91%). 1H-NMR (CD3OD, 400 MHz)3):
1.43 (s, tBu); 4.51 (s, MeN); 7.84 (d, J ¼ 9.0, HꢀC(10)); 7.99 (dd, J ¼ 8.0, 6.0, HꢀC(2)); 8.15 (dd, J ¼ 9.0,
2.0, HꢀC(9)); 8.33 (d, J ¼ 2.5, HꢀC(7)); 9.14 (dd, J ¼ 6.0, 2.0, HꢀC(1)); 9.30 (dd, J ¼ 8.0, 2.0, HꢀC(3)).
13C-NMR (CD3OD, 100 MHz)3): 30.3 (Me3C); 34.8 (Me3C); 41.7 (Me); 118.2 (C(10)); 120.4 (C(4));
120.8 (C(6)); 121.2 (C(2)); 122.6 (C(7)); 135.4 (C(9)); 145.9 (C(3)); 149.1 (C(1)); 151.1 (C(8)); 152.4
1
(C(11)); 156.3 (C(12)); 173.8 (C(5)). 19F-NMR (CD3OD, 188 MHz, H-decoupled 400 MHz): 80.5 (s,
CF3). HR-ESI-MS: 268.1332 (Mþ, C17H18NOþ2 ; calc. 268.1338).
6-(tert-Butyl)-1-methyl-10H-9-oxa-1-azoniaanthracen-10-one Chloride. Ion exchange of 6-(tert-
butyl)-1-methyl-10H-9-oxa-1-azoniaanthracen-10-one trifluoromethanesulfonate to the corresponding
chloride salt was undertaken to enhance H2O solubility. The trifluoromethanesulfonate (1.00 g,
2.40 mmol) was dissolved in H2O/MeOH 1:1 (20 ml) and added to an excess of a Dowex anion-
3
)
The atom numbering in the NMR spectra of 10H-9-oxa-1-azaanthracen-10-one (¼4-azaxanthone)
derivatives is arbitrary, see A.

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Helvetica Chimica Acta – Vol. 92 (2009)
exchange resin (0.5 g, 200 – 400 mesh, Clꢀ form). The mixture was stirred for 2 h, filtered, and the solvent
evaporated to yield the corresponding chloride salt. 1H-NMR (CD3OD, 500 MHz)3): 1.46 (s, tBu); 4.55 (s,
MeN); 7.88 (d, J ¼ 9.0, HꢀC(10)); 8.03 (t, J ¼ 6.5, HꢀC(2)); 8.18 (dd, J ¼ 9.0, 2.0, HꢀC(9)); 8.36 (d, J ¼
2.0, HꢀC(7)); 9.22 (d, J ¼ 6.5, HꢀC(1)); 9.33 (d, J ¼ 7.5, HꢀC(3)). 13C-NMR (CD3OD, 125 MHz)3): 30.4
(Me3C); 34.8 (Me3C); 41.8 (Me); 118.2 (C(10)); 120.5 (C(4)); 120.8 (C(6)); 121.2 (C(2)); 122.6 (C(7));
135.4 (C(9)); 145.9 (C(3)); 149.1 (C(1)); 151.1 (C(8)); 152.4 (C(11)); 156.3 (C(12)); 173.8 (C(5)).
6-(tert-Butyl)-1-methyl-1H-9-oxa-1-azaanthracene-2,10-dione (¼ 7-(1,1-Dimethylethyl)-1-methyl-
2H-[1]benzopyrano[2,3-b]pyridine-2,5(1H)-dione). A soln. of 6-(tert-butyl)-1-methyl-10H-9-oxa-1-azo-
niaanthracen-10-one chloride (0.361 g, 1.18 mmol) in H2O (10 ml) was added over 30 min to a stirred
soln. of K3[Fe(CN)6] (1.16 g, 3.54 mmol) in H2O (6 ml) at 08. This was followed by the dropwise addition
of an aq. soln. of NaOH (0.850 g, 21.2 mmol) in H2O (10 ml) to the stirred soln. over 30 min. The soln.
was allowed to warm to r.t. and stirred for 8 h. The soln. was acidified to pH 3 by the addition of conc.
H2SO4 soln. to afford a green precipitate. The precipitate was filtered and dissolved in CHCl3 (50 ml) and
the soln. washed with H2O (2 ꢃ 50 ml), dried (MgSO4), and concentrated: red solid (0.251 g, 74%). M.p.
243 – 2448. 1H-NMR (CDCl3, 500 MHz)3): 1.41 (s, tBu); 3.76 (s, MeN); 6.54 (d, J ¼ 9.5, HꢀC(2)); 7.47 (d,
J ¼ 8.5, HꢀC(10)); 7.79 (dd, J ¼ 9.0, 2.0, HꢀC(9)); 8.21 (d, J ¼ 9.5, HꢀC(3)); 8.29 (d, J ¼ 2.0, HꢀC(7)).
13C-NMR (CDCl3, 125 MHz)3): 28.5 (Me); 31.6 (Me3C); 35.2 (Me3C); 102.8 (C(4)); 116.0 (C(2)); 117.3
(C(10)); 121.6 (C(6)); 122.9 (C(7)); 132.3 (C(9)); 135.7 (C(3)); 149.7 (C(8)); 152.0 (C(11)); 156.5
(C(12)); 162.3 (C(1)); 174.2 (C(5)). HR-ESI-MS: 284.1281 ([M þ H]þ, C17H18NO3þ ; calc. 284.1287).
Anal. calc. for C17H17NO3 (283.32): C 72.07, H 6.05, N 4.94; found C 72.02, H 5.99, N 4.90.
6-(tert-Butyl)-2-chloro-10H-9-oxa-1-azaanthracen-10-one (¼2-Chloro-7-(1,1-dimethylethyl)-5H-
[1]benzopyrano[2,3-b]pyridin-5-one; 4b). A stirred soln. of 6-(tert-butyl)-1-methyl-1H-9-oxa-1-azaan-
thracene-2,10-dione (0.180 g, 0.636 mmol) in POCl3 (10 ml) and N,N-dimethylaniline (0.30 ml, 0.313 g,
2.59 mmol) was heated under reflux for 18 h. The mixture was allowed to cool to r.t. and slowly added to
iced water (300 ml). The aq. soln. was extracted with CH2Cl2 (2 ꢃ 200 ml), the combined org. phase
washed with aq. dil. K2CO3 soln. (100 ml), dried (K2CO3), and concentrated, and the residue dried over
silica gel (SiO2) and purified by column chromatography (CC) (SiO2, hexane ! 10% AcOEt/hexane by
1% AcOEt increments): 4b (0.090 g, 49%). Pink solid. Rf 0.33 (SiO2, hexane/AcOEt 9 :1). M.p. 132 –
1338. 1H-NMR (CDCl3, 500 MHz)3): 1.41 (s, tBu); 7.43 (d, J ¼ 8.0, HꢀC(2)); 7.54 (d, J ¼ 9.0,
HꢀC(10)); 7.86 (dd, J ¼ 9.0, 2.5, HꢀC(9)); 8.27 (d, J ¼ 2.5, HꢀC(7)); 8.65 (d, J ¼ 8.0, HꢀC(3)).
13C-NMR (CDCl3, 125 MHz)3): 31.5 (Me3C); 35.1 (Me3C); 115.6 (C(4)); 118.4 (C(10)); 121.1 (C(6));
121.9 (C(2)); 122.7 (C(7)); 134.1 (C(9)); 139.9 (C(3)); 148.8 (C(8)); 153.8 (C(11)); 155.6 (C(12)); 159.7
(C(1)); 177.2 (C(5)). HR-ESI-MS: 288.0785 ([M þ H]þ, C16H15ClNO2þ ; calc. 288.0791).
6-(tert-Butyl)-2-(6-methylpyridin-2-yl)-10H-9-oxa-1-azaanthracen-10-one (¼ 7-(1,1-Dimethylethyl)-
2-(6-methylpyridin-2-yl)-5H-[1]benzopyrano[2,3-b]pyridin-5-one; 2a). Under Ar, 4b (0.201 g,
0.696 mmol), 6-methyl-2-(tributylstannyl)pyridine (0.293 g, 0.766 mmol), and degassed toluene (5 ml)
were added to tetrakis(triphenylphosphine)palladium(0) (0.040 g, 0.034 mmol). The mixture was stirred
and heated under reflux under Ar for 16 h. After cooling to r.t. and filtering, solvent was evaporated to
afford a pale brown oil. The crude material was triturated with Et2O (10 ml) to yield a fine precipitate.
The solvent was decanted and the solid dried under vacuum: 2a (0.145 g, 61%). Colourless solid. M.p.
191 – 1928. 1H-NMR (CDCl3, 700 MHz)3): 1.37 (s, tBu); 2.60 (s, Me); 7.18 (d, J ¼ 8.0, HꢀC(5’)); 7.53 (d,
J ¼ 8.0, HꢀC(10)); 7.69 (t, J ¼ 8.0, HꢀC(4’)); 7.79 (dd, J ¼ 8.0, 2.0, HꢀC(9)); 8.26 (d, J ¼ 2.0, HꢀC(7));
8.29 (d, J ¼ 8.0, HꢀC(3’)); 8.54 (d, J ¼ 8.5, HꢀC(2)); 8.74 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR (CDCl3,
176 MHz)3): 24.8 (Me); 31.5 (Me3C); 35.0 (Me3C); 116.4 (C(4)); 118.3 (C(10)); 118.5 (C(2)); 119.7
(C(5’)); 121.3 (C(6)); 122.7 (C(7)); 124.9 (C(2’)); 133.6 (C(9)); 137.4 (C(4’)); 138.2 (C(3)); 148.0 (C(8));
153.6 (C(1’)); 154.2 (C(11)); 158.6 (C(6’)); 160.2 (C(1)); 160.6 (C(12)); 177.8 (C(5)). HR-ESI-MS:
345.1596 ([M þ H]þ, C22H21N2Oþ2 ; calc. 345.1603).
The structure of 2a was confirmed by single-crystal X-ray diffraction: C22H20N2O2, Mr 344.40,
monoclinic (P21/c); a ¼ 13.472(1) ꢄ, b ¼ 6.7227(7) ꢄ, c ¼ 19.449 (2) ꢄ, V¼ 1760.7(3) ꢄ3, b ¼ 91.702(6)8,
Z ¼ 4; m ¼ 0.08 mmꢀ1, Dcalc. ¼ 1.299 g cmꢀ3, T 120(2) K; 4671 independent reflections (Rint ¼ 0.028), R1 ¼
0.042, wR2 ¼ 0.118 (I > 2s(I)); CCDC 724804.
2-[6-(Bromomethyl)pyridin-2-yl]-6-(tert-butyl)-10H-9-oxa-1-azaanthracen-10-one (¼2-[6-(Bromo-
methyl)pyridin-2-yl)-7-(1,1-dimethylethyl)-5H-[1]benzopyrano[2,3-b]pyridin-5-one; 2b). A stirred mix-

Helvetica Chimica Acta – Vol. 92 (2009)
2205
ture of 2a (0.200 g, 0.581 mmol), N-bromosuccinimide (NBS; 0.129 g, 0.725 mmol), and benzoyl
peroxide (0.010 g, 0.041 mmol) in CCl4 (5 ml) was heated under reflux for 16 h (1H-NMR monitoring).
After 8 h, NBS (0.100 g, 0.562 mmol) and dibenzoyl peroxide (0.010 g, 0.041 mmol) were added, and the
mixture was heated under reflux for a further 16 h. The mixture was then allowed to cool to r.t. and
filtered, the filtrate concentrated, and the yellow residue purified by CC (SiO2; CH2Cl2): 2b (0.101 g,
41%). Colourless solid. Rf 0.35 (SiO2, CH2Cl2). M.p. 186 – 1878. 1H-NMR (CDCl3, 500 MHz)3): 1.42 (s,
tBu); 4.66 (s, CH2Br); 7.56 (J ¼ 7.5, HꢀC(5’)); 7.60 (d, J ¼ 9.0, HꢀC(10)); 7.85 (dd, J ¼ 8.5, 2.5, HꢀC(9));
7.90 (t, J ¼ 8.0, HꢀC(4’)); 8.32 (d, J ¼ 2.5, HꢀC(7)); 8.48 (d, J ¼ 8.0, HꢀC(3’)); 8.63 (d, J ¼ 8.0, HꢀC(2));
8.82 (d, J ¼ 8.0, HꢀC(3)). 13C-NMR (CDCl3, 126 MHz)3): 31.6 (Me3C); 34.0 (CH2Br); 35.1 (Me3C);
116.8 (C(4)); 118.3 (C(10)); 118.7 (C(2)); 121.3 (C(6)); 121.8 (C(5’)); 122.8 (C(7)); 125.1 (C(3’)); 133.8
(C(9)); 138.4 ((C(4’)); 138.6 (C(3)); 148.2 (C(8)); 154.0 (C(1’)); 154.2 (C(11)); 157.0 (C(6’)); 160.0
(C(1)); 160.2 (C(12)); 177.9 (C(5)). HR-ESI-MS: 423.0700 ([M þ H]þ, C22H2079BrN2O2þ ; calc. 423.0708).
6-(tert-Butyl)-2-[6-(dibromomethyl)pyridin-2-yl]-10H-9-oxa-1-azaanthracen-10-one (¼2-[6-Dibro-
momethyl)pyridin-2-yl]-7-(1,1-dimethylethyl)-5H-[1]benzopyrano[2,3-b]pyridin-5-one; 2c). As descri-
bed for 2b: 2c (0.061 g, 33%). Colourless solid. Rf 0.71 (SiO2, CH2Cl2). 1H-NMR (CDCl3, 500 MHz)3):
1.41 (s, tBu); 6.78 (s, CHBr2); 7.58 (d, J ¼ 8.5, HꢀC(10)); 7.84 (dd, J ¼ 9.0, 2.5, HꢀC(9)); 7.92 (d, J ¼ 8.0,
HꢀC(5)); 7.98 (t, J ¼ 8.0, HꢀC(4’)); 8.30 (d, J ¼ 2.5, HꢀC(7)); 8.50 (d, J ¼ 7.5, HꢀC(3’)); 8.59 (d, J ¼ 8.0,
HꢀC(2)); 8.81 (d, J ¼ 8.0, HꢀC(3)). 13C-NMR (CDCl3, 126 MHz)3): 31.6 (Me3C); 35.1 (Me3C); 41.6
(CHBr2); 116.9 (C(4)); 118.3 (C(10)); 118.7 (C(2)); 121.3 (C(6)); 122.8 (C(7)); 122.9 (C(3’)); 123.6
(C(5’)); 133.9 (C(9)); 138.7 (C(3)); 139.1 (C(4’)); 148.3 (C(8)); 152.7 (C(2’)); 154.2 (C(11)); 159.0
(C(6’)); 159.1 (C(1)); 160.2 (C(12)); 177.8 (C(5)). HR-ESI-MS: 500.9807 ([M þ H]þ, C22H1979Br2N2O2þ ;
calc. 500.9813).
6-(tert-Butyl)-2-(3-methyl-1H-pyrazol-1-yl)-10H-9-oxa-1-azaanthracen-10-one (¼ 7-(1,1-Dimethyl-
ethyl)-2-(3-methyl-1H-pyrazol-1-yl)-5H-[1]benzopyrano[2,3-b]pyridin-5-one; 3a). NaH (0.030 g,
1.25 mmol) was added to a stirred soln. of 3-methyl-1H-pyrazole (0.880 g, 1.07 mmol) in anh. THF
(5 ml) under Ar. A soln. of 4b (0.281 g, 0.97 mmol) in anh. THF (5 ml) was added, and the mixture was
heated at 658 for 16 h. The mixture was allowed to cool to r.t., and H2O (10 ml) was added to induce
precipitation. The precipitate was collected by centrifugation and the resultant solid triturated with Et2O
(10 ml). The solvent was decanted and the solid dried under vacuum: 3a (0.294 g, 90%). Colourless solid.
M.p. 128 – 1308. 1H-NMR (CDCl3, 400 MHz)3): 1.39 (s, tBu); 2.37 (s, Me); 6.31 (d, J ¼ 3.0, HꢀC(2’)); 7.51
(d, J ¼ 9.0, HꢀC(10)); 7.80 (dd, J ¼ 9.0, 3.0, HꢀC(9)); 7.99 (d, J ¼ 8.5, HꢀC(2)); 8.27 (d, J ¼ 3.0,
HꢀC(7)); 8.51 (d, J ¼ 3.0, HꢀC(1’)); 8.72 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR (CDCl3, 100 MHz)3): 14.2
(Me); 31.6 (Me3C); 35.1 (Me3C); 109.7 (C(2)); 110.1 (C(2’)); 113.9 (C(4)); 118.1 (C(10)); 121.4 (C(6));
122.7 (C(7)); 129.0 (C(1’)); 133.4 (C(9)); 140.1 (C(3)); 148.3 (C(8)); 153.7 (C(11)); 153.9 (C(12)); 154.0
(C(3’)); 160.0 (C(1)); 177.0 (C(5)). HR-ESI-MS: 334.1551 ([M þ H]þ, C20H20N3O2þ ; calc. 334.1556).
2-[3-(Bromomethyl)-1H-pyrazol-1-yl]-6-(tert-butyl)-10H-9-oxa-1-azaanthracen-10-one (¼2-[3-
(Bromomethyl)-1H-pyrazol-1-yl]-7-(1,1-dimethylethyl)-5H-[1]benzopyrano[2,3-b]pyridin-5-one; 3b).
Procedure A. A stirred mixture of 3a (0.212 g, 0.636 mmol), NBS (0.113 g, 0.635 mmol), and benzoyl
peroxide (0.010 g, 0.041 mmol) in CCl4 (15 ml) was heated under reflux for 16 h. The mixture was
allowed to cool to r.t. and filtered, the filtrate concentrated, and the pale yellow oil purified by CC (SiO2,
CH2Cl2): 3b (0.148 g, 56%). Colourless solid. Rf 0.28 (SiO2, CH2Cl2). M.p. 184 – 1858. 1H-NMR (CDCl3,
500 MHz)3): 1.42 (s, tBu); 4.56 (s, CH2Br); 6.62 (d, J ¼ 2.5, HꢀC(2’)); 7.56 (d, J ¼ 9.0, HꢀC(10)); 7.85 (dd,
J ¼ 9.0, 2.5, HꢀC(9)); 8.06 (d, J ¼ 8.0, HꢀC(2)); 8.31 (d, J ¼ 2.5, HꢀC(7)); 8.63 (d, J ¼ 2.5, HꢀC(1’));
8.80 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR (CDCl3, 125 MHz)3): 24.6 (CH2Br); 31.6 (Me3C); 35.1 (Me3C);
109.7 (C(2’)); 110.0 (C(2)); 114.6 (C(4)); 118.1 (C(10)); 121.4 (C(6)); 122.8 (C(7)); 129.8 (C(1’)); 133.6
(C(9)); 140.5 (C(3)); 148.6 (C(8)); 153.3 (C(11)); 153.7 (C(3’)); 153.9 (C(12)); 159.9 (C(1)); 177.0 (C(5)).
HR-ESI-MS: 434.0475 ([M þ Na]þ, C20H1879BrN3NaO2þ ; calc. 434.0480).
The structure was confirmed by a single crystal X-ray analysis. C20H18BrN3O2, Mr 412.28, monoclinic
(P21/c); a ¼ 17.044(3) ꢄ, b ¼ 13.303(2) ꢄ, c ¼ 8.079(1) ꢄ, b ¼ 97.124(15)8, V¼ 1817.6(3) ꢄ3, Z ¼ 4; m ¼
2.28 mmꢀ1, Dcalc. ¼ 1.507 g/cm3, T 120(2) K; 3201 independent reflections (Rint ¼ 0.104), R1 ¼ 0.073,
wR2 ¼ 0.125 (I > 2s(I)); CCDC-724805.
Procedure B. A soln. of 3c (0.150 g, 0.307 mmol), iPr2NEt (0.157 g, 0.211 ml, 1.22 mmol) and diethyl
phosphite (0.168 g, 0.157 ml, 1.22 mmol) in anh. THF (10 ml) was stirred at r.t. for 16 h. The solvent was

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Helvetica Chimica Acta – Vol. 92 (2009)
evaporated and the residue partitioned between CHCl3 (10 ml) and H2O (10 ml). The org. phase was
concentrated and the crude material purified by CC (SiO2, CH2Cl2): 3b (0.085 g, 67%). Colourless solid.
6-(tert-Butyl)-2-[3-(dibromomethyl)-1H-pyrazol-1-yl]-10H-9-oxa-1-azaanthracen-10-one (¼2-[3-
(Dibromomethyl)-1H-pyrazol-1-yl]-7-(1,1-dimethylethyl)-5H-[1]benzopyrano[2,3-b]pyridin-5-one; 3c).
As described for 3b: 3c (0.970 g, 31%). Colourless solid. Rf 0.33 (SiO2, CH2Cl2). 1H-NMR (CDCl3,
500 MHz)3): 1.42 (s, tBu); 6.79 (s, CHBr2); 6.89 (d, J ¼ 2.5, HꢀC(2’)); 7.56 (d, J ¼ 8.5, HꢀC(10)); 7.85 (dd,
J ¼ 8.5, 2.5, HꢀC(9)); 8.03 (d, J ¼ 8.5, HꢀC(2)); 8.31 (d, J ¼ 2.5, HꢀC(7)); 8.67 (d, J ¼ 2.5, HꢀC(3’));
8.81 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR (CDCl3, 125 MHz)3): 31.0 (CHBr2); 31.6 (Me3C); 35.1 (Me3C);
108.8 (C(2’)); 110.0 (C(2)); 114.9 (C(4)); 118.1 (C(10)); 121.3 (C(6)); 122.8 (C(7)); 130.1 (C(3’)); 133.8
(C(9)); 140.6 (C(3)); 148.6 (C(8)); 153.1 (C(12)); 153.9 (C(11)); 157.0 (C(1’)); 159.8 (C(1)); 177.0 (C(5)).
ESI-MS: 486.3 (100, [M þ H]þ).
11. EuIII Complex of Ligand L1. Ligand L1 Precursor 1,4,7-Tri(tert-butyl) 10-{{6-[6-(tert-Butyl)-10-
oxo-10H-9-oxa-1-azaanthracen-2-yl]pyridin-2-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate
(¼1,4,7-Tris(1,1-dimethylethyl) 10-{{6-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-
2-yl]pyridin-2-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate). A stirred mixture of 1,4,7-
tri(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (0.032 g, 0.062 mmol), 2b (0.024 g,
0.057 mmol), and Cs2CO3 (0.026 g, 0.080 mmol) in anh. MeCN (5 ml) was heated under reflux for
16 h. The mixture was allowed to cool to r.t. and syringe filtered, the filtrate concentrated, and the yellow
oil purified by CC (Al2O3, CH2Cl2 ! 2% MeOH/CH2Cl2, by 0.1% MeOH increments): triester precursor
of L1 (0.035 g, 74%). Pale yellow solid. Rf 0.22 (Al2O3, CH2Cl2/MeOH 19 :1). 1H-NMR (CDCl3,
700 MHz)3): 1.29 (s, tBu); 1.32 (br. s, Boc); 1.35 (br. s, Boc); 2.71 (br. s, 8 H, cyclen CH2); 3.24 (br. s, 8 H,
t
t
cyclen CH2); 3.50 (br. s, 2 CH2CO2 Bu); 3.55 (br. s, 1 CH2CO2 Bu); 3.69 (s, pyCH2); 7.25 (d, J ¼ 8.0,
HꢀC(5’)); 7.45 (d, J ¼ 8.5, HꢀC(10)); 7.69 (t, J ¼ 8.0, HꢀC(4’)); 7.71 (dd, J ¼ 9.0, 3.0, HꢀC(9)); 8.17 (d,
J ¼ 3.0, HꢀC(7)); 8.32 (d, J ¼ 8.0, HꢀC(3’)); 8.36 (d, J ¼ 7.0, HꢀC(2)); 8.64 (d, J ¼ 8.0, HꢀC(3)).
13C-NMR (CDCl3, 176 MHz)3): 28.3 (Me3C); 31.5 (2 Me3CO); 35.1 (1 Me3CO); 50.6 – 56.7 (cyclen CH2
and NCH2CO); 57.1 (pyCH2); 82.4 (4 Me3C); 116.4 (C(4)); 118.2 (C(10)); 118.3 (C(2)); 121.0 (C(3’));
121.2 (C(6)); 122.6 (C(7)); 126.2 (C(5’)); 133.6 (C(9)); 137.4 (C(4’)); 138.2 (C(3)); 148.0 (C(8)); 153.6
(C(1’)); 154.2 (C(11)); 158.6 (C(6’)); 160.2 (C(1)); 160.6 (C(12)); 177.8 (C(5)). ESI-MS (pos.): 857.5 (100,
[M þ H]þ). HR-ESI-MS: 857.5176 ([M þ H]þ, C48H69N6Oþ8 ; calc. 857.5177).
[Eu(L1)]. A soln. of the ligand L1 precursor (see above; 0.035 g, 0.0419 mmol) in CH2Cl2/CF3COOH
1:1 (2 ml) was stirred at r.t. for 16 h to afford an orange soln. The solvent was evaporated to yield a glassy
solid. The crude material was repeatedly dissolved 3 ꢃ in CH2Cl2 (5 ml) and the solvent evaporated
t
(removal of excess acid and BuOH). The desired ligand, L1 · 3 Hþ, as its trifluoroacetate salt, was
1
examined by H-NMR to ensure complete ester hydrolysis, and the material was used immediately for
complexation. The ligand was dissolved in MeOH/H2O 1:1 (4 ml), and Eu(OAc)3 · 6 H2O (0.017 g,
0.039 mmol) was added to the mixture. The pH of the soln. was raised to 5.5 by the addition of 1m aq.
KOH, then stirred and heated at 808 for 15 h. The mixture was allowed to cool to r.t. before raising the
pH to 10.0 with dil. aq. KOH soln. The mixture was stirred for 1 h to allow precipitation of excess Eu
metal as its hydroxide salt Eu(OH)3. The solid precipitate was removed by syringe filtration and the pH
of the colourless aq. filtrate lowered to pH 5.5. The solvent was removed by lyophilization to yield a
colourless solid. The material was purified by CC (neutral Al2O3, CH2Cl2/MeOH 8 :2): [Eu(L1)].
Colourless solid (0.024 g, 68%). Rf 0.41 (Al2O3, CH2Cl2/MeOH 7:3). lmax (H2O) 356 nm; t (H2O)
1.00 ms; t (D2O) 1.34 ms; fEu (H2O, pH 7.4, lexc 365 nm) 14%.
12. Complexes of Ligand L2. Ligand L2 Precursor 1,4,7-Tri(tert-butyl) 10-{{1-[6-(tert-Butyl)-10-oxo-
10H-9-oxa-1-azaanthracen-2-yl]-1H-pyrazol-3-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate
(¼1,4,7-Tris(1,1-dimethylethyl) 10-{{1-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-
2-yl]-1H-pyrazol-3-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate). As described for the li-
gand L1 precursor, with 1,4,7-tri(tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (0.075 g,
0.146 mmol), 3b (0.060 g, 0.146 mmol), Cs2CO3 (0.050 g, 0.153 mmol), and MeCN (5 ml). CC (SiO2,
CH2Cl2 ! 8% MeOH/CH2Cl2 , by 0.1% MeOH increments): triester precursor of L2 (0.090 g,
0.106 mmol, 73%). Pale yellow solid. Rf 0.36 (SiO2, CH2Cl2/MeOH 19 :1). 1H-NMR (CDCl3 ,
t
500 MHz)3): 1.40 (s, Bu); 1.51 (br. s, 3 Boc); 2.67 (br. s, 8 H, cyclen CH2); 3.27 (br. s, 8 H, cyclen
t
t
CH2); 3.44 (br. s, 2 CH2CO2 Bu); 3.51 (br. s, 1 CH2CO2 Bu); 3.55 (s, pzCH2); 6.57 (d, J ¼ 2.5, HꢀC(2’));

Helvetica Chimica Acta – Vol. 92 (2009)
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7.56 (d, J ¼ 8.0, HꢀC(10)); 7.86 (dd, J ¼ 9.0, 2.5, HꢀC(9)); 8.23 (d, J ¼ 8.0, HꢀC(2)); 8.27 (d, J ¼ 2.5,
HꢀC(7)); 8.57 (d, J ¼ 2.5, HꢀC(1’)); 8.62 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR (CDCl3, 125 MHz)3): 28.3
(Me3C); 31.5 (2 Me3CO); 35.1 (1 Me3CO); 50.6 – 56.1 (cyclen CH2, br.); 56.7 (2 cyclen CH2, pzCH2); 82.4
(4 Me3C); 110.8 (C(2)); 110.9 (C(2’)); 114.3 (C(4)); 118.2 (C(10)); 121.1(C(6)); 122.7 (C(7)); 129.6
(C(1’)); 133.9 (C(9)); 140.1 (C(3)); 148.7 (C(8)); 153.6 (C(11)); 153.9 (C(12)); 155.3 (C(3)); 159.8
(C(1)); 172.9 (3 C¼O of ester); 176.9 (C(5)). ESI-MS: 846.5 (100, [M þ H]þ).
[Tb(L2)]. As described for [Eu(L1)] (above), with the ligand L2 precursor (see above; 0.045 g,
0.053 mmol) in CH2Cl2/CF3COOH 2 :1 (3 ml) at r.t. in a sealed flask for 16 h: [Tb(L2)] (0.026 g, 61%).
Colourless solid. Rf 0.39 (Al2O3, CH2Cl2/MeOH 7:3). lmax (H2O) 348 nm; t (H2O) 2.24 ms; t (D2O)
2.65 ms; fTb (H2O, pH 6.0, lexc 355 nm) 15%.
[Eu(L2)H2O]. As described for [Tb(L2)]: [Eu(L2) (H2O)] (0.015 g, 52%). Colourless solid. lmax
(H2O) 346 nm; t (H2O) 0.70 ms; t (D2O) 1.76 ms.
13. EuIII Complex of Ligand L4a. Tri(tert-butyl) 10-{{6-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaan-
thracen-2-yl]pyridin-2-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (¼ Tris(1,1-dime-
thylethyl) 10-{{6-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-2-yl]pyridin-2-yl}meth-
yl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate). A stirred mixture of tri(tert-butyl) 1,4,7-tetraa-
zacyclododecane-1,4,7-tricarboxylate (0.117 g, 0.248 mmol), 2b (0.100 g, 0.236 mmol), and K2CO3
(0.049 g, 0.354 mmol) in anh. MeCN (4 ml) was heated under reflux and Ar for 16 h. The mixture was
allowed to cool to r.t. and syringe filtered, the filtrate concentrated, and the residual yellow oil purified by
CC (SiO2, CH2Cl2 ! 3% MeOH/CH2Cl2, by 0.1% MeOH increments): pale yellow solid (0.173 g, 90%).
1
t
Rf 0.70 (SiO2, CH2Cl2/MeOH 49 :1). H-NMR (CDCl3, 700 MHz)3): 1.29 (s, Bu); 1.32 (s, 1 Boc); 1.35
(br. s, 2 Boc); 2.71 (br. s, 4 H, cyclen CH2); 3.24 (br. s, 8 H, cyclen CH2); 3.50 (br. s, 4 H, cyclen CH2); 3.87
(s, pyCH2); 7.25 (d, J ¼ 8.0, HꢀC(5’)); 7.45 (d, J ¼ 8.5, HꢀC(10)); 7.69 (t, J ¼ 8.0, HꢀC(4’)); 7.71 (dd, J ¼
9.0, 3.0, HꢀC(9)); 8.17 (d, J ¼ 3.0, HꢀC(7)); 8.32 (d, J ¼ 8.0, HꢀC(3’)); 8.36 (d, J ¼ 7.0, HꢀC(2)); 8.64
(d, J ¼ 8.0, HꢀC(3)). 13C-NMR (CDCl3, 176 MHz)3): 28.6 (3 Me3CO); 31.5 (Me3C); 35.0 (Me3C); 47.3
(cyclen CH2); 47.7 (cyclen CH2); 48.0 (cyclen CH2); 48.4 (cyclen CH2); 50.1 (cyclen CH2); 51.1 (cyclen
CH2); 54.4 (cyclen CH2); 55.2 (cyclen CH2); 57.1 (pyCH2); 79.5 (3 Me3C); 116.4 (C(4)); 118.2 (C(10));
118.3 (C(2)); 121.0 (C(3’)); 121.2 (C(6)); 122.6 (C(7)); 126.2 (C(5’)); 133.6 (C(9)); 137.4 (C(4’)); 138.2
(C(3)); 148.0 (C(8)); 153.6 (C(1’)); 154.2 (C(11)); 155.9 (3 C¼O of Boc); 158.6 (C(6’)); 160.2 (C(1));
160.6 (C(12)); 177.8 (C(5)). HR-ESI-MS: 815.4710 ([M þ H]þ, C45H63N6O8þ ; calc. 815.4707).
6-(tert-Butyl)-2-[6-(1,4,7,10-tetraazacyclododec-1-ylmethyl)pyridin-2-yl]-10H-9-oxa-1-azaanthra-
cen-10-one (¼ 7-(1,1-Dimethylethyl)-2-[6-(1,4,7,10-tetraazocyclododec-1-ylmethyl)pyridin-2-yl]-5H-
[1]benzopyrano[2,3-b]pyridin-2-yl]-5-one). A soln. of tri(tert-butyl) 10-{{6-[6-(tert-butyl)-10-oxo-10H-
9-oxa-1-azaanthracen-2-yl]pyridin-2-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate
(0.173 g, 0.212 mmol) in CH2Cl2/CF3COOH 2 :1 (3 ml) was stirred at r.t. for 6 h (!orange soln.). The
solvent was evaporated to yield a glassy orange solid. The crude material was dissolved 3 ꢃ in CH2Cl2
t
(5 ml) and the solvent evaporated (removal of excess acid and BuOH). The residue was finally taken
into 1m aq. KOH (10 ml) and extracted with CH2Cl2 (3 ꢃ 5 ml). The combined org. extract was dried
1
t
(K2CO3) and concentrated: orange solid (0.065 g, 94%). H-NMR (CDCl3, 500 MHz)3): 1.36 (s, Bu);
2.54 (s, 4 H, cyclen CH2); 2.68 (s, 8 H, cyclen CH2); 2.77 (s, 4 H, cyclen CH2); 3.85 (s, pyCH2); 7.44 (d, J ¼
7.5, HꢀC(5’)); 7.54 (d, J ¼ 9.0, HꢀC(10)); 7.79 (dd, J ¼ 8.5, 2.5, HꢀC(4’)); 7.81 (t, J ¼ 7.5, HꢀC(9)); 8.26
(d, J ¼ 2.5, HꢀC(7)); 8.38 (d, J ¼ 7.5, HꢀC(3’)); 8.62 (d, J ¼ 8.0, HꢀC(2)); 8.73 (d, J ¼ 8.0, HꢀC(3)).
13C-NMR (CDCl3, 126 MHz)3): 31.5 (Me3C); 35.0 (Me3C); 45.3 (cyclen CH2); 46.6 (cyclen CH2); 47.4
(cyclen CH2); 51.8 (cyclen CH2); 60.8 (pyCH2); 116.4 (C(4)); 118.2 (C(10)); 118.6 (C(2)); 121.0 (C(5’));
121.3 (C(6)); 122.6 (C(7)); 124.6 (C(2’)); 133.7 (C(9)); 137.8 (C(4’)); 138.1 (C(3)); 148.1 (C(8)); 153.5
(C(1’)); 154.2 (C(11)); 159.7 (C(6’)); 160.2 (C(1)); 160.6 (C(12)); 178.0 (C(5)). ESI-MS (pos.): 289.4
(100, [M þ Zn]2þ). HR-ESI-MS: 515.3130 ([M þ H]þ; C30H39N6Oþ2 ; calc. 515.3134).
10-{{6-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-yl]pyridin-2-yl}methyl}-N1,N4,N7-tris-
[(1S)-1-phenylethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetamide (¼10-{{6-[7-(1,1-Dimethylethyl)-
5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-2-yl]pyridin-2-yl}methyl}-N1,N4,N7-tris[(1S)-1-phenylethyl]-
1,4,7,10-tetraazacyclododecane-1,4,7-triacetamide; L4a). A stirred mixture of 6-(tert-butyl)-2-[6-(1,4,7,10-
tetraazacyclododec-1-ylmethyl)pyridin-2-yl]-10H-9-oxa-1-azaanthracen-10-one (0.060 g, 0.116 mmol),
2-chloro-N-[(1S)-1-phenylethyl]acetamide ((S)-5a; 0.080 g, 0.408 mmol), and K2CO3 (0.064 g,

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Helvetica Chimica Acta – Vol. 92 (2009)
0.466 mmol) in anh. MeCN (5 ml) was heated under reflux for 16 h. The resultant mixture was allowed to
cool to r.t. and syringe filtered, the filtrate concentrated, and the residual yellow oil purified by CC
(neutral Al2O3, CH2Cl2 ! 2% MeOH/CH2Cl2, by 0.1% MeOH increments): L4a (0.074 g, 64%). Pale
yellow solid. Rf 0.21 (Al2O3, CH2Cl2/MeOH 19 :1). 1H-NMR (CDCl3, 700 MHz)3): 1.38 (s, tBu); 1.45 (br.
s, 3 Me); 2.60 (br. s, 8 H, cyclen CH2); 2.92 (br. s, 8 H, cyclen CH2); 3.06 (br. s, CH2CO); 3.54 (br. s,
CH2CO); 3.67 (br. s, CH2CO); 3.96 (br. s, pyCH2); 4.96 (q, 1 CH); 5.11 (q, 2 CH); 7.11 – 7.30 (m, 3 Ph);
7.45 (br. s, HꢀC(5’)); 7.58 (d, J ¼ 9.0, HꢀC(10)); 7.70 (br. s, HꢀC(4’)); 7.82 (dd, J ¼ 9.0, 2.0, HꢀC(9));
8.28 (d, J ¼ 3.0, HꢀC(7)); 8.43 (br. s, HꢀC(3’)); 8.62 (d, J ¼ 8.5, HꢀC(2)); 8.77 (d, J ¼ 8.5, HꢀC(3)).
HR-ESI-MS: 998.5661 ([M þ H]þ, C60H72N9O5þ ; calc. 998.5656).
[Eu(L4a)]Cl3. A soln. of L4a (0.040 g, 0.040 mmol) and Eu(OTf)3 · 6 H2O (0.034 g, 0.041 mmol) in
anh. MeCN (1 ml) was heated under reflux and Ar for 18 h. The resultant soln. was allowed to cool to r.t.
followed by evaporation of the solvent to afford a glassy orange solid. CH2Cl2 (10 ml) was added to the
solid and the mixture sonicated for 10 min. The solvent was then decanted and the solid material
dissolved in a minimum volume of MeCN (0.5 ml). The soln. was added slowly onto Et2O (25 ml) to
induce precipitation. The solid material was isolated by centrifugation, and the process of induced
precipitation repeated twice more to yield [Eu(L4a)](TfO)3. The off-white solid was rendered H2O-
soluble by the exchange of triflate anions for Cl anions with Dowex (1 ꢃ 8, 200 – 400 mesh, Clꢀ) resin. The
solid material was dissolved in H2O/MeOH 1:1 (16 ml) and prepared resin (0.8 g) was added to the soln.,
which was stirred at r.t. for 3 h. The resin was removed by filtration and the filtrate concentrated:
[Eu(L4a)]Cl3 (0.034 g, 68%). HPLC (Method A): tR 10.9. lmax (H2O) 356 nm; t (H2O) 1.00 ms; t (D2O)
1.34 ms; f (H2O, pH 7.4, lexc 365 nm) 24%.
14. Complexes of Ligand L5a. Tri(tert-butyl) 10-{{1-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthra-
cen-2-yl]-1H-pyrazol-3-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (¼ Tris(1,1-dime-
thylethyl) 10-{{1-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-2-yl]-1H-pyrazol-3-yl}-
methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate). A stirred mixture of 1,4,7-tri(tert-butyl)
1,4,7-tetraazacyclododecane-1,4,7-tricarboxylate (0.045 g, 0.109 mmol), 3b (0.057 g, 0.119 mmol), K2CO3
(0.019 g, 0.131 mmol), and a cat. amount of KI (2 mg) in anh. MeCN/CH2Cl2 1:1 (5 ml) was heated for
16 h. The mixture was allowed to cool and syringe filtered, the filtrate concentrated, and the residual oil
purified by CC (SiO2, CH2Cl2 ! 3% MeOH/CH2Cl2, by 0.1% MeOH increments): pale yellow solid
1
(0.075 g, 85%). Rf 0.41 (SiO2, CH2Cl2/MeOH 24 :1). H-NMR (CDCl3, 500 MHz)3): 1.44 (br. s, 3 Boc,
tBu); 2.78 (br. s, 4 H, cyclen CH2); 3.39 (br., 8 H, cyclen CH2); 3.59 (s, 4 H, cyclen CH2); 3.89 (s, pzCH2);
6.44 (d, J ¼ 3.0, HꢀC(2’)); 7.56 (d, J ¼ 8.5, HꢀC(10)); 7.86 (dd, J ¼ 9.0, 2.5, HꢀC(9)); 8.04 (d, J ¼ 8.5,
HꢀC(2)); 8.30 (d, J ¼ 2.5, HꢀC(7)); 8.60 (d, J ¼ 2.0, HꢀC(1’)); 8.78 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR
(CDCl3, 125 MHz)3): 28.7 (3 Me3CO); 31.5 (Me3C); 35.5 (Me3C); 47.5 (cyclen CH2); 47.9 (cyclen CH2);
48.4 (2 cyclen CH2); 50.1 (2 cyclen CH2); 53.6 (cyclen CH2); 54.0 (cyclen CH2); 55.3 (pzCH2); 79.5
(3 Me3CO); 109.9 (C(2)); 110.8 (C(2’)); 114.2 (C(4)); 118.1 (C(10)); 121.4 (C(6)); 122.8 (C(7)); 129.0
(C(1’)); 133.6 (C(9)); 140.3 (C(3)); 148.5 (C(8)); 153.6 (C(11)); 153.9 (C(12)); 155.6 (C(3)); 155.9
(3 C¼O of Boc); 159.9 (C(1)); 177.0 (C(5)). ESI-MS: 804.3 (100, [M þ H]þ).
6-(tert-Butyl)-2-[3-(1,4,7,10-tetraazacyclododec-1-ylmethyl)-1H-pyrazol-1-yl]-10H-9-oxa-1-azaan-
thracen-10-one (¼ 7-(1,1-Dimethylethyl)-2-[3-(1,4,7,10-tetraazacyclododec-1-ylmethyl)-1H-pyrazol-1-yl]-
5H-[1]benzopyrano[2,3-b]pyridin-5-one). A soln. of tri(tert-butyl) 10-{{1-[6-(tert-butyl)-10-oxo-10H-9-
oxa-1-azaanthracen-2-yl]-1H-pyrazol-3-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate
(0.110 g, 0.137 mmol) in CH2Cl2/CF3COOH 2 :1 (3 ml) was stirred at r.t. for 16 h, generating a red soln.
The solvent was evaporated to yield an orange glassy solid. The crude material was repeatedly (3ꢃ)
dissolved in CH2Cl2 (5 ml) and the solvent evaporated. The residue was taken into 1m aq. KOH (15 ml)
and extracted with CH2Cl2 (3 ꢃ 15 ml). The combined org. extract was dried (K2CO3) and concentrated:
pale orange solid that was used directly in the next step without further analysis (0.065 g, 94%). 1H-NMR
(CDCl3, 200 MHz)3): 1.39 (s, tBu); 2.70 (br. s, 4 H, cyclen CH2); 2.80 (br. s, 8 H, cyclen CH2); 2.85 (br. s,
4 H, cyclen CH2); 3.83 (s, CH2); 6.51 (d, J ¼ 3.0, HꢀC(2’)); 7.54 (d, J ¼ 8.5, HꢀC(10)); 7.82 (dd, J ¼ 9.0,
2.0, HꢀC(9)); 8.04 (d, J ¼ 8.5, HꢀC(2)); 8.28 (d, J ¼ 2.0, HꢀC(7)); 8.58 (d, J ¼ 2.0, HꢀC(1’)); 8.74 (d,
J ¼ 8.5, HꢀC(3)).
10-{{1-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-yl]-1H-pyrazol-3-yl}methyl}-N1,N4,N7-
tris[(1S)-1-phenylethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetamide (¼10-{{1-[7-(1,1-Dimethyleth-

Helvetica Chimica Acta – Vol. 92 (2009)
2209
yl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-2-yl]-1H-pyrazol-3-yl}methyl}-N1,N4,N7-tris[(1S)-1-phenyl-
ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetamide; L5a). A stirred mixture of 6-(tert-butyl)-2-[3-
(1,4,7,10-tetraazacyclododec-1-ylmethyl)-1H-pyrazol-1-yl]-10H-9-oxa-1-azaanthracen-10-one (0.039 g,
0.078 mmol), 2-chloro-N-[(1S)-1-phenylethyl]acetamide ((S)-5a) (0.050 g, 0.250 mmol), and Cs2CO3
(0.081 g, 0.251 mmol) in anh. MeCN (5 ml) was heated for 16 h. The mixture was allowed to cool and
filtered, the filtrate concentrated, and the orange glassy solid sonicated in Et2O (15 ml) to yield a fine,
pale yellow precipitate that was isolated via centrifugation. The material was sonicated in Et2O and
centrifuged twice more to yield L5a (0.062 g, 81%). Pale yellow solid. 1H-NMR (CDCl3, 500 MHz)3): 1.41
t
(s, Bu); 1.52 (br., 3 Me); 2.48 – 3.12 (br. m, 20 H, cyclen CH2, CH2CONH); 3.77 (s, CH2); 4.05 (s,
pzCH2); 5.01 (m, 3 CH); 6.38 (br. s, HꢀC(2’)); 7.03 – 7.19 (br. m, 3 Ph); 7.54 (d, J ¼ 8.0, HꢀC(10)); 7.84
(dd, J ¼ 8.0, 2.0, HꢀC(9)); 7.96 (d, J ¼ 8.0, HꢀC(2)); 8.30 (d, J ¼ 2.5, HꢀC(7)); 8.60 (d, J ¼ 2.0,
HꢀC(1’)); 8.81 (d, J ¼ 8.0, HꢀC(3)). ESI-MS: 998.4 (100, [M þ H]þ).
[Tb(L5a)]Cl3. A stirred mixture of L5a (0.044 g, 0.044 mmol) and Tb(OTf)3 · 6 H2O (0.030 g,
0.049 mmol) in anh. MeCN (1 ml) was heated under reflux for 16 h. The soln. was allowed to cool and the
solvent evaporated to afford a glassy orange solid. CH2Cl2 (5 ml) was added to the solid, and the mixture
was sonicated for 10 min. The solvent was decanted and the solid dissolved in a minimum volume of
MeCN (0.4 ml). The soln. was added slowly onto Et2O (15 ml) to induce precipitation. The solid material
was isolated by centrifugation, and the process of induced precipitation repeated twice more to yield
[Tb(L5a)](TfO)3. The off-white solid was made H2O-soluble by the exchange of triflate anions for Cl
anions with Dowex (1 ꢃ 8, 200 – 400 mesh, Clꢀ) resin: [Tb(L5)]Cl3 (0.040 g, 69%). Colourless solid.
HPLC (Method A): tR 11.0. lmax (H2O) 348 nm; t (H2O) 2.00 ms; t (D2O) 2.38 ms; fTb (H2O, pH 6.0, lexc
355 nm) 46%.
[Eu(L5a)]Cl3. An analogous procedure with L5a (0.042 g, 0.042 mmol) and Eu(OTf)3 · 6 H2O
(0.033 g, 0.046 mmol) in anh. MeCN (1 ml), following anion exchange, yielded [Eu(L5a)]Cl3 (0.027 g,
54%). Colourless solid. HPLC (Method A): tR 11.1. lmax (H2O) 348 nm.
15. Complexes of Ligand L6. N2,N2’,N2’’-{{10-{{1-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-
yl]-1H-pyrazol-3-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl}tris(1-oxoethane-2,1-diyl)}tris[l-
phenylalanine] Triethyl Ester (¼ N2,N2’,N2’’-{{10-{{1-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyra-
no[2,3-b]pyridin-2-yl]-1H-pyrazol-3-yl}methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triyl}tris(1-oxo-
ethane-2,1-diyl)}tris[l-phenylalanine] Triethyl Ester; L6). A stirred mixture of 6-(tert-butyl)-2-[3-
(1,4,7,10-tetraazacyclododec-1-ylmethyl)-1H-pyrazol-1-yl]-10H-9-oxa-1-azaanthracen-10-one (0.033 g,
0.065 mmol), N-(2-bromo-1-oxoethyl)-l-phenylalanine ethyl ester ((S)-6) (0.064 g, 0.206 mmol), and
Cs2CO3 (0.073 g, 0.231 mmol) in anh. MeCN (7 ml) was heated under reflux for 16 h. The mixture was
allowed to cool to r.t. and filtered, the filtrate concentrated, and the orange glassy solid sonicated in Et2O
(15 ml) to yield a fine pale yellow precipitate that was isolated by centrifugation. The material was
1
sonicated in Et2O and centrifuged twice more to yield L6 (0.028 g, 36%). Pale yellow solid. H-NMR
t
(CDCl3, 400 MHz)3): 1.13 (t, J ¼ 6.0, 3 MeCH2O); 1.35 (s, Bu); 2.41 – 2.60 (br. m, 12 H, cyclen CH2);
2.68 – 3.08 (br. m, 14 H, cyclen CH2, CH2CO; PhCH2); 4.02 (s, pzCH2); 4.06 (t, J ¼ 6.0, 3 MeCH2O); 4.76
(q, 3 CH); 6.38 (br. s, HꢀC(2’)); 7.03 – 7.19 (br. m, 3 Ph); 7.50 (d, J ¼ 8.0, HꢀC(10)); 7.77 (dd, J ¼ 8.0, 2.0,
HꢀC(9)); 7.94 (d, J ¼ 8.0, HꢀC(2)); 8.24 (d, J ¼ 2.5, HꢀC(7)); 8.52 (d, J ¼ 2.0, HꢀC(1’)); 8.92 (d, J ¼ 8.0,
HꢀC(3)). HR-ESI-MS: 1203.6241 ([M þ H]þ, C67H83N10O1þ1 ; calc. 1203.6243).
[Tb(L6)]Cl3. A stirred soln. of L6 (0.013 g, 0.011 mmol) and Tb(OTf)3 · 6 H2O (0.007 g, 0.012 mmol)
in anh. MeCN (1 ml) was heated under reflux for 10 h. The complex was prepared as described for
[Eu(L4a)]Cl3: [Tb(L6)]Cl3 (0.008 g, 56%). Colourless solid. HPLC (Method A): tR 11.2. lmax (H2O)
348 nm, t (H2O) 2.25 ms; fTb (H2O, pH 6.0, lexc 355 nm) 54%.
[Eu(L6)]Cl3. Similarly, [Eu(L6)]Cl3 was isolated as a colourless solid. HPLC (Method A): tR 11.2.
16. EuIII Complex of Ligand L4b. tert-Butyl 7-{{6-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-
2-yl]-pyridin-2-yl}methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}ethyl}-1,4,7,10-tetraazacyclodo-
decane-1-carboxylate (¼tert-Butyl 7-{{6-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyr-
idin-2-yl]pyridin-2-yl}methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}ethyl}-1,4,7,10-tetraazacyclo-
dodecane-1-carboxylate). A stirred mixture of tert-butyl 4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]ami-
no}ethyl}-1,4,7,10-tetraazacyclododecane-1-carboxylate (7; 0.325 g, 0.547 mmol), 2b (0.254 g,
0.601 mmol), and K2CO3 (0.113 g, 0.821 mmol) in anh. MeCN (10 ml) was heated under reflux for

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Helvetica Chimica Acta – Vol. 92 (2009)
16 h. The mixture was allowed to cool and filtered, the solvent evaporated, and the yellow oil purified by
CC (neutral Al2O3, CH2Cl2 ! 0.5% MeOH/CH2Cl2, by 0.1% MeOH increments): orange glassy solid
(0.379 g, 74%). Rf 0.74 (Al2O3, CH2Cl2/MeOH 49 :1). M.p. 87 – 898. 1H-NMR (CDCl3, 500 MHz)3): 1.38
(br. s, tBu, Boc); 1.46 (d, J ¼ 4.0, 2 Me); 2.61 (br. s, 8 H, cyclen CH2); 2.82 (br. s, 4 H, cyclen CH2); 3.07
(br. s, 4 H, cyclen CH2); 3.33 (br. s, 1 CH2CO); 3.40 (br. s, 1 CH2CO); 3.61 (br. s, pyCH2); 5.12 (br. s,
2 CH); 7.19 (d, J ¼ 8.0, HꢀC(5’)); 7.26 (br. s, 2 Ph); 7.46 (br. s, NH); 7.58 (d, J ¼ 8.5, HꢀC(10)); 7.64 (t,
J ¼ 8.0, HꢀC(4’)); 7.74 (br. s, NH); 7.82 (dd, J ¼ 9.0, 2.5, HꢀC(9)); 8.29 (d, J ¼ 2.5, HꢀC(7)); 8.41 (d, J ¼
8.0, HꢀC(3’)); 8.46 (d, J ¼ 8.0, HꢀC(2)); 8.78 (d, J ¼ 8.0, HꢀC(3)). 13C-NMR (CDCl3, 125 MHz)3): 21.3
(Me); 22.0 (Me); 28.7 (Me3CO); 31.5 (Me3C); 35.1 (Me3C); 48.0 (CH); 48.4 (CH); 52.5 (2 cyclen CH2);
53.2 (2 cyclen CH2); 53.8 (2 cyclen CH2); 54.6 (2 cyclen CH2); 59.4 (pyCH2); 59.8 (CH2CO); 60.6
(CH2CO); 80.2 (Me3CO); 116.6 (C(4)); 118.3 (C(10)); 121.1 (C(3’)); 121.3 (C(6)); 122.7 (C(7)); 124.8
(C(5’)); 126.5 (Co/m); 126.8 (Co/m); 127.3 (2 Co/m); 127.8 (2 Co/m); 128.7 (Co/m); 128.9 (Co/m); 133.8 (C(9));
137.6 (C(4’)); 138.5 (C(3)); 143.0 (Cipso); 143.8 (Cipso); 148.2 (C(8)); 153.8 (C(1’)); 154.2 (C(11)); 156.1
(C¼O of Boc); 158.0 (C(6’)); 160.1 (C(1)); 160.3 (C(12)); 170.1 (2 amide C¼O); 177.8 (C(5)). HR-ESI-
MS: 937.5331 ([M þ H]þ, C55H69N8O6þ ; calc. 937.5340).
4-{{6-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-yl]pyridin-2-yl}methyl}-N1,N7-bis[(1S)-1-
phenylethyl]-1,4,7,10-tetraazacyclododecane-1,7-diacetamide (¼4-{{6-[7-(1,1-Dimethylethyl)-5-oxo-5H-
[1]benzopyrano[2,3-b]pyridin-2-yl]pyridin-2-yl}methyl}-N1,N7-bis[(1S)-1-phenylethyl]-1,4,7,10-tetraaza-
cyclododecane-1,7-diacetamide; 8). A soln. of tert-butyl 7-{{6-[6-(tert-butyl)-10-oxo-10H-9-oxa-1-azaan-
thracen-2-yl]pyridin-2-yl}methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}ethyl}-1,4,7,10-tetraaza-
cyclododecane-1-carboxylate (0.320 g, 0.341 mmol) in CH2Cl2/CF3COOH 2 :1 (6 ml) was stirred at r.t.
for 12 h. The solvent was evaporated to yield a glassy solid. The crude material was repeatedly (3ꢃ)
dissolved in CH2Cl2 (5 ml) and the solvent evaporated. The residue was finally taken into 0.02m aq. KOH
(10 ml) and extracted with CH2Cl2 (3 ꢃ 15 ml). The combined org. layer was dried (K2CO3) and
1
concentrated: 8 (0.254 g, 89%). Pale yellow solid. M.p. 92 – 948. H-NMR (CDCl3, 500 MHz)3): 1.38 (s,
tBu); 1.47 (d, J ¼ 7.0, 2 Me); 2.58 (br. s, 8 H, cyclen CH2); 2.64 (br. s, 8 H, cyclen CH2); 3.09 (q, J ¼ 17.0,
2 CH2CO); 3.60 (d, J ¼ 15.0, 1 H, pyCH2); 3.68 (d, J ¼ 15.0, 1 H, pyCH2); 5.14 (q, J ¼ 7.5, 2 CH); 7.16 (q,
J ¼ 8.0, HꢀC(5’), 2 Hp); 7.24 (t, J ¼ 7.5, 4 Hm); 7.30 (d, J ¼ 7.5, 4 Ho); 7.56 (d, J ¼ 9.0, HꢀC(10)); 7.62 (t,
J ¼ 7.5, HꢀC(4’)); 7.82 (dd, J ¼ 8.5, 2.0, HꢀC(9)); 8.06 (d, J ¼ 8.5, NH); 8.28 (d, J ¼ 2.5, HꢀC(7)); 8.37
(d, J ¼ 7.5, HꢀC(3’)); 8.44 (d, J ¼ 8.5, HꢀC(2)); 8.77 (d, J ¼ 8.5, HꢀC(3)). 13C-NMR (CDCl3)3): 21.7
(2 Me); 31.5 (Me3C); 35.0 (Me3C); 47.4 (2 cyclen CH2); 48.6 (2 CH); 52.5 (2 cyclen CH2); 53.4 (2 cyclen
CH2); 53.7 (cyclen CH2); 54.8 (cyclen CH2); 58.7 (pyCH2); 60.2 (2 CH2CO); 116.6 (C(4)); 118.3 (C(2);
C(10)); 121.0 (C(3’)); 121.3 (C(6)); 122.7 (C(7)); 125.0 (C(5’)); 126.7 (4 Co); 127.5 (2 Cp); 128.8 (4 Cm);
133.8 (C(9)); 137.6 (C(4’)); 138.5 (C(3)); 143.5 (2 Cipso); 148.2 (C(8)); 153.6 (C(1’)); 154.2 (C(11)); 158.0
(C(6’)); 160.2 (C(1)); 160.3 (C(12)); 170.8 (2 amide C¼O); 177.8 (C(5)). HR-ESI-MS: 837.4817 ([M þ
H]þ, C50H61N8Oþ4 ; calc. 837.4816).
Methyl 4-{(1S)-1-{{2-{7-{{6-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-yl]pyridin-2-yl}-
methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}ethyl}-1,4,7,10-tetraazacyclododec-1-yl}acetyl}ami-
no}ethyl}benzoate (¼ Methyl 4-{(1S)-1-{{2-{7-{{6-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-
b]pyridin-2-yl]pyridin-2-yl}methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}ethyl}-1,4,7,10-tetra-
azacyclododec-1-yl}acetyl}amino}ethyl}benzoate; L4b). A mixture of 8 (0.131 g, 0.157 mmol), methyl 4-
{(1S)-1-[(2-chloroacetyl)amino]ethyl}benzoate ((S)-5b; 0.050 g, 0.196 mmol), and K2CO3 (0.043 g,
0.313 mmol) in anh. MeCN (7 ml) was heated under reflux for 18 h. After cooling and filtering, the
solvent was evaporated and the orange oil purified by CC (neutral Al2O3, CH2Cl2 ! 1.0% MeOH/
CH2Cl2, by 0.1% MeOH increments): L4b (0.105 g, 64%). Orange solid. Rf 0.27 (Al2O3, CH2Cl2/MeOH
49 :1). M.p. 87 – 898. 1H-NMR (CDCl3, 500 MHz)3): 1.40 (s, tBu); 1.44 (d, J ¼ 7.0, 2 Me); 2.71 (br. s, 16 H,
cyclen CH2); 3.25 (br. s, 3 CH2CO); 3.75 (s, pyCH2); 3.84 (s, COOMe); 5.05 (q, 3 CH); 7.16 (d, J ¼ 8.0,
2 H, HꢀC(5’)), Hp); 7.28 (m, 2 Ph); 7.33 (d, J ¼ 8.0, 2 H, Ho/m); 7.60 (d, J ¼ 8.5, HꢀC(10)); 7.73 (t, J ¼ 8.0,
HꢀC(4’)); 7.85 (dd, J ¼ 8.5, 2.5, HꢀC(9)); 7.89 (d, J ¼ 8.0, 2 H, Ho/m); 8.30 (d, J ¼ 2.5, HꢀC(7)); 8.43 (d,
J ¼ 8.0, HꢀC(2), HꢀC(3’)); 8.77 (d, J ¼ 8.0, HꢀC(3)). 13C-NMR (CDCl3, 125 MHz)3): 21.9 (3 Me); 31.5
(Me3C); 35.0 (Me3C); 48.9 (3 CH); 51.5 (2 cyclen CH2); 52.3 (COOMe); 52.5 (2 cyclen CH2); 53.0 (2
cyclen CH2); 53.7 (2 cyclen CH2); 58.5 (3 CH2CO); 60.2 (pyCH2); 116.6 (C(4)); 118.3 (C(2); C(10));
121.3 (C(3’)); 121.4 (C(6)); 122.7 (C(7)); 125.4 (2 C, Ph); 126.2 (2 C, Ph); 126.5 (4 C, Ph); 127.5 (C(5’));

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128.8 (4 C, Ph); 129.2 (Ph); 130.1 (2 C, Ph); 133.8 (C(9)); 137.8 (C(4’)); 138.6 (C(3)); 143.5 (2 Cipso); 147.7
(1 Cipso); 148.2 (C(8)); 153.6 (C(1’)); 154.2 (C(11)); 159.9 (C(6’)); 160.2 (C(1)); 160.3 (C(12)); 166.9
(COOMe); 170.8 (3 amide C¼O); 177.8 (C(5)). HR-ESI-MS: 1056.5703 ([M þ H]þ, C62H74N9O7þ ; calc.
1056.5711).
[Eu(L4b)]Cl3. The complex was prepared as described for [Eu(L4a)]Cl3: [Eu(L4b)]Cl3 (0.044 g,
64%). Pale yellow solid. HPLC (Method A): tR 10.9. lmax (H2O) 356 nm; t (H2O) 1.02 ms; t (D2O)
1.34 ms.
17. Complex of Ligand L5b. 4-{{1-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-yl]-1H-pyra-
zol-3-yl}methyl}-N1,N7-bis[(1S)-1-phenylethyl]-1,4,7,10-tetraazacyclododecane-1,7-diacetamide (¼4-{{1-
[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyrano[2,3-b]pyridin-2-yl]-1H-pyrazol-3-yl}methyl}-N1,N7-
bis[(1S)-1-phenylethyl]-1,4,7,10-tetraazacyclododecane-1,7-diacetamide). A stirred mixture of N1,N7-
bis[(1S)-1-phenylethyl]-1,4,7,10-tetraazacyclododecane-1,7-diacetamide [41] (0.104 g, 0.211 mmol), 3b
(0.087 g, 0.211 mmol), and NaHCO3 (0.020 g, 0.231 mmol) in anh. MeCN (5 ml) was heated at 608 for
18 h. The mixture was cooled and filtered, the solvent evaporated, and the yellow residue purified by CC
(neutral Al2O3, CH2Cl2 ! 0.5% MeOH/CH2Cl2, by 0.1% MeOH increments): pale yellow solid (0.124 g,
71%). M.p. 152 – 1548. 1H-NMR (CDCl3, 500 MHz)3): 1.40 (s, tBu); 1.47 (d, J ¼ 7.0, 2 Me); 2.58 (br. s, 4 H,
cyclen CH2); 2.79 (br. s, 4 H, cyclen CH2); 2.91 (br. s, 8 H, cyclen CH2); 3.36 (s, 2 CH2CO); 3.71 (s,
pzCH2); 5.07 (q, J ¼ 14.0, 7.5, 2 CH); 6.31 (d, J ¼ 2.5 HꢀC(2’)); 7.13 (t, J ¼ 7.5, 2 H, Ph); 7.21 (t, J ¼ 7.5,
4 H, Ph); 7.35 (d, J ¼ 7.5, 4 H, Ph); 7.54 (d, J ¼ 9.0, HꢀC(10)); 7.84 (dd, J ¼ 8.5, 2.5, HꢀC(9)); 7.91 (br. s,
2 NH); 7.98 (d, J ¼ 8.5, HꢀC(2)); 8.29 (d, J ¼ 2.5, HꢀC(7)); 8.55 (d, J ¼ 2.5, HꢀC(1’)); 8.76 (d, J ¼ 8.5,
HꢀC(3)). HR-ESI-MS: 826.4769 ([M þ H]þ, C48H60N9O4þ ; calc. 826.4768).
Methyl 4-{(1S)-1-{{2-{7-{{1-[6-(tert-Butyl)-10-oxo-10H-9-oxa-1-azaanthracen-2-yl]-1H-pyrazol-3-
yl}methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}methyl}-1,4,7,10-tetraazacyclododec-1-yl}acetyl}-
amino}ethyl}benzoate (¼ Methyl 4-{(1S)-1-{{2-{7-{{1-[7-(1,1-Dimethylethyl)-5-oxo-5H-[1]benzopyra-
no[2,3-b]pyridin-2-yl]-1H-pyrazol-3-yl}methyl}-4,10-bis{2-oxo-2-{[(1S)-1-phenylethyl]amino}ethyl}-
1,4,7,10-tetraazacyclododec-1-yl}acetyl}amino}ethyl}benzoate; L5b). A mixture of 4-{{1-[6-(tert-butyl)-10-
oxo-10H-9-oxa-1-azaanthracen-2-yl]-1H-pyrazol-3-yl}methyl}-N1,N7-bis[(1S)-1-phenylethyl]-1,4,7,10-
tetraazacyclododecane-1,7-diacetamide (0.060 g, 0.073 mmol), (S)-5b (0.021 g, 0.084 mmol), and Cs2CO3
(0.031 g, 0.095 mmol) in anh. MeCN (5 ml) was heated under reflux for 16 h. The mixture was cooled and
filtered, the solvent evaporated, and the yellow residue purified by CC (neutral Al2O3, CH2Cl2 ! 0.5%
MeOH/CH2Cl2, by 0.1% MeOH increments): L5b (0.060 g, 79%). Pale yellow solid. Rf 0.68 (Al2O3,
CH2Cl2/MeOH 49 :1). M.p. 187 – 1898. 1H-NMR (CDCl3, 400 MHz)3): 1.41 (br. s, tBu, 1 Me); 1.45 (d, J ¼
7.0, 2 Me); 2.57 (br., 16 H, cyclen CH2); 2.86 (br. s, 1 CH2CO); 2.99 (br. s, 2 CH2CO); 3.62 – 3.84 (br.,
pzCH2, CO2Me); 5.14 (q, J ¼ 7.0, 3 CH); 6.30 (d, J ¼ 2.5, HꢀC(2’)); 6.21 – 7.32 (br., 12 arom. H); 7.47 (d,
J ¼ 8.0, 2 arom. H); 7.55 (d, J ¼ 9.0, HꢀC(10)); 7.82 (dd, J ¼ 8.0, 2.5, HꢀC(9)); 7.98 (d, J ¼ 8.0, HꢀC(2));
8.31 (d, J ¼ 2.5, HꢀC(7)); 8.78 (d, J ¼ 8.0, HꢀC(3)). HR-ESI-MS: 1045.5706 ([M þ H]þ, C60H73N10O7þ ;
calc. 1045.5664).
[Tb(L5b)]Cl3. The complex was prepared as described for [Tb(L5a)]Cl3: [Tb(L5b)]Cl3 (0.037 g, 55%).
Pale yellow solid. HPLC (Method A): tR 10.9. lmax (H2O) 349 nm; t (H2O) 2.27 ms; fTb (H2O, pH 6.0, lexc
355 nm) 63%.
18. Conjugate Formation. Ester Hydrolysis. A soln. of [Tb(L5b)]Cl3 (1.3 mmol) in MeOH/0.02m aq.
KOH 1:1 (2 ml) was stirred at 208 and the progress of hydrolysis monitored by reversed-phase HPLC
(Chromolith performance RP18e column (100 ꢃ 4.6 mm), Method C): tR (ester) 6.07, tR (acid) 5.78. The
reaction ran to completion in ca. 2 h, and the mixture was neutralised with a dil. aq. HCl soln. The solvent
was evaporated and the crude residue used directly in the next step without further purification. ESI-MS:
594 ([M ꢀ H]2þ), 1187 ([M ꢀ 2 H]þ).
BG-[Tb(L5)]Cl3. To a stirred soln. of the above acid in anh. DMF (2 ml) was added TSTU and
iPr2NEt (50 ml of a 100 mm soln. in DMF in each case; 5 mmol). The progress of the reaction was
monitored by reversed-phase HPLC (Method C): tR (acid) 6.07, tR (NHS ¼ succinimide) 6.28. BGꢀH was
added to the stirred soln. of the active ester (1 mmol) derived from [Tb(L5b)]Cl3 in anh. DMF (400 ml).
The mixture was stirred and monitored by reversed-phase HPLC (Method C): tR (succinimido ester) 6.28,
tR (BGꢀ[Tb(L5b)]Cl3) 5.97. The reaction ran to completion in ca. 15 min. The product was purified by

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Helvetica Chimica Acta – Vol. 92 (2009)
reversed-phase semi-prep. HPLC (Method E; tR (conjugate) 5.23): BGꢀ[Tb(L5b)]Cl3 (380 nmol, 38%).
Colourless solid. ESI-MS: 721 ([M ꢀ H]2þ), 1440 ([M ꢀ 2 H]þ).
BG – [Eu(L4)]Cl3. This conjugate was prepared similarly to BG – [Tb(L5b)]Cl3. HPLC (Method E);
BG – Eu(L4)]Cl3 at tR 6.67. HR-ESI-MS: 697.7724 ([M ꢀ H]2þ, C68H78151EuN17O27þ ; calc. 697.7729.
REFERENCES
[1] J. Yuan, G. Wang, Trends Anal. Chem. 2006, 25, 490; J. P. Gasser, M. Hehl, T. A. Millward, Anal.
Biochem. 2009, 384, 49.
[2] H. Bazin, E. Trinquet, G. Mathis, Rev. Mol. Biotechnol. 2002, 82, 233; M. Gabourdes, V. Bourgine, G.
Mathis, H. Bazin, B. Alpha-Bazin, Anal. Biochem. 2004, 333, 105.
[3] I. Hemmila, V. M. Mukkala, Crit. Rev. Clin. Lab. Sci. 2001, 38, 441.
[4] P. R. Selvin, Annu. Rev. Biophys. Biomol. Struct. 2002, 82, 233.
[5] R. Pal, D. Parker, Org. Biomol. Chem. 2008, 6, 1020.
´
[6] Y. Bretonniere, M. J. Cann, D. Parker, R. J. Slater, Org. Biomol. Chem. 2004, 2, 1624.
[7] J. Yu, D. Parker, Chem. Commun. 2005, 3141.
[8] R. Pal, D. Parker, L. C. Costello, Org. Biomol. Chem. 2009, 7, 1525.
[9] D. Parker, J. A. G. Williams, Chem. Commun. 1998, 245; S. Blair, R. Kataky, D. Parker, New J. Chem.
2002, 26, 530.
[10] R. A. Poole, F. Kielar, D. Parker, S. L. Richardson, P. A. Stenson, Chem. Commun. 2006, 4084; D.
Parker, Coord. Chem. Rev. 2000, 205, 109.
[11] C. P. Montgomery, B. S. Murray, E. J. New, R. Pal, D. Parker, Acc. Chem. Res. 2009, 42, 925.
[12] G. Bobba, J.-C. Frias, M. J. Cann, D. Parker, C. J. Hutchison, Org. Biomol. Chem. 2003, 1, 905; R. A.
Poole, G. Bobba, M. J. Cann, J.-C. Frias, D. Parker, R. D. Peacock, Org. Biomol. Chem. 2005, 3, 1013;
J. Yu, D. Parker, R. Pal, D. Parker, J. Am. Chem. Soc. 2006, 128, 2294; R. A. Poole, C. P.
Montgomery, E. J. New, A. Congreve, D. Parker, Org. Biomol. Chem. 2007, 5, 2055.
[13] J. Wu, Z. Q. Ye, G. L. Wang, J. L. Yuan, Talanta 2007, 72, 1693; G.-L. Law, K. L. Wang, C. W. Y. Man,
W. T. Wang, S. W. Tsao, M. H. W. Lam, P. K. S. Lam, J. Am. Chem. Soc. 2008, 130, 3714; G. Marriott,
M. Heidecker, E. P. Diamandis, Y. Yanmarriott, Biophys. J. 1994, 67, 957.
[14] A.-S. Chauvin, C. D. B. Vandevyver, S. Comby, J.-C. G. Bꢁnzli, Chem. Commun. 2007, 1716; A. S.
Chauvin, S. Comby, B. Song, C. D. B. Vandevyer, J.-C. G. Bꢁnzli, Chem. – Eur. J. 2007, 13, 9515; A. S.
Chauvin, S. Comby, B. Song, C. D. B. Vandevyver, J.-C. G. Bꢁnzli, Chem. – Eur. J. 2008, 14, 1729; E.
Deiters, B. Song, A. S. Chauvin, C. D. B. Vandevyder, F. Gumy, J.-C. G. Bꢁnzli, Chem. – Eur. J. 2009,
15, 885.
[15] H. C. Manning, S. M. Smith, M. Sexton, S. Haviland, M. F. Bai, K. Cederquist, N. Stella, D. J.
Bornhop, Bioconjugate Chem. 2006, 17, 735.
[16] S. U. Pandya, D. Parker, J. Yu, Dalton Trans. 2006, 2757.
[17] F. Kielar, C. P. Montgomery, E. J. New, R. A. Poole, D. Parker, S. L. Richardson, P. A. Stenson, Org.
Biomol. Chem. 2007, 5, 2975.
[18] F. Kielar, G.-L. Law, E. J. New, D. Parker, Org. Biomol. Chem. 2008, 6, 2256,
[19] T. Gunnlaugsson, J. P. Leonard, Chem. Commun. 2005, 3114; R. Ziessel, N. Weibel, L. J.
´
Charbonniere, M. Guardigli, A. Roda, J. Am. Chem. Soc. 2004, 126, 4088.
[20] J.-C. G. Bꢁnzli, C. Piguet, Chem. Soc. Rev. 2005, 34, 1048; R. S. Dickins, C. Crossland, D. Parker, H.
Puschmann, J. A. K. Howard, Chem. Rev. 2002, 102, 1977.
[21] S. Petoud, E. Muller, E. G. Moore, J. D. Xu, J. Sokdnicki, J. P. Riehl, U. N. Le, S. M. Cohen, K. N.
Raymond, J. Am. Chem. Soc. 2007, 127, 77.
[22] A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker, L. Royle, A. S. de Sousa, J. A. G.
Williams, M. Woods, J. Chem. Soc., Perkin Trans. 2 1999, 493.
[23] P. Atkinson, K. S. Findlay, F. Kielar, R. Pal, D. Parker, R. A. Poole, H. Puschmann, S. L. Richardson,
P. A. Stenson, A. L. Thompson, Org. Biomol. Chem. 2006, 4, 1707.
[24] C. P. Montgomery, D. Parker, L. Lamarque, Chem. Commun. 2007, 3841.

Helvetica Chimica Acta – Vol. 92 (2009)
2213
[25] J. C. Rodriguez-Ubis, R. Sedano, G. Barroso, O. Juanes, E. Brunet, Helv. Chim. Acta 1997, 80, 86; E.
Brunet, O. Juanes, R. Sedano, J. C. Rodriguez-Ubis, Tetrahedron 2005, 61, 6757.
[26] J. Lewis, T. D. OꢂDonoghue, J. Chem. Soc., Dalton Trans. 1980, 736.
[27] J. C. Scaiano, D. Weldon, C. N. Pliva, L. J. Martinez, J. Phys. Chem. A 1998, 102, 6898.
[28] L. J. Martinez, J. C. Scaiano, J. Phys. Chem. A 1999, 103, 203.
[29] G. Bobba, J.-C. Frias, D. Parker, Chem. Commun. 2002, 890.
[30] C. P. Montgomery, E. J. New, D. Parker, R. D. Peacock, Chem. Commun. 2008, 4261.
[31] L.-O. Palsson, R. Pal, B. S. Murray, D. Parker, A. Beeby, Dalton Trans. 2007, 5726.
[32] M. Schwab, T. Gibbs, A. Brecht, Genet. Eng. News 2005, 25, 21.
[33] D. Maurel, L. Comps-Agar, C. Brock, M.-L. Rives, E. Bourrier, M. A. Ayoub, H. Bazin, N. Tinel, T.
Durroux, L. Prezeau, E. Trinquet, J.-P. Pin, Nat. Methods 2008, 5, 561.
[34] A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K. Johnsson, Nat. Biotechnol. 2003, 21,
86.
[35] A. Keppler, H. Pick, C. Arrivoli, H. Vogel, K. Johnsson, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9955.
[36] J. I. Bruce, D. Parker, D. J. Tozer, R. S. Dickins, Dalton Trans. 2003, 1264.
[37] S. Aime, E. Gianoli, E. Terreno, R. Pagliarin, G. B. Giovenzana, M. P. Lowe, M. Sisti, D. Parker, G.
Palmisano, M. Botta, J. Biol. Inorg. Chem. 2000, 5, 488.
[38] R. S. Dickins, A. S. Batsanov, M. Botta, J. I. Bruce, J. A. K. Howard, C. S. Love, D. Parker, R. D.
Peacock, H. Puschmann, J. Am. Chem. Soc. 2002, 124, 12697.
[39] E. J. New, D. Parker, Org. Biomol. Chem. 2009, 7, 851.
[40] E. J. New, D. Parker, R. D. Peacock, Dalton Trans. 2009, 672.
[41] B. S. Murray, E. J. New, R. Pal, D. Parker, Org. Biomol. Chem. 2008, 6, 2085.
[42] S. Kessner, A. Kronise, U. Rothe, G. Bendas, Biochim. Biophys. Acta 2002, 1514, 177.
[43] A. Arcaro, P. Wymann, Biochem. J. 1993, 295, 297.
[44] P. R. Hoffmann, A. M. de Cathelineau, C. A. Ogden, Y. Leverrier, D. L. Bratton, D. L. Dalecke,
A. J. Ridley, V. A. Fathok, P. M. Henson, J. Cell Biol. 2001, 155, 649.
[45] S. Veldhoen, S. D. Laufer, A. Trampe, T. Restle, Nucleic Acid Res. 2006, 34, 6561.
[46] L. J. Hewlett, A. R. Prescott, C. Watts, J. Cell Biol. 1994, 124, 689.
[47] H. U. Keller, J. Cell Physiol. 1990, 145, 465.
[48] T. Soldati, M. Schliwa, Nat. Rev. Mol. Cell Biol. 2006, 7, 897.
Received April 1, 2009