Polyazamacrocycles based on a tetraaminoacetate moiety and a (poly)pyridine intracyclic unit: Direct synthesis and application to the photosensitization of Eu(III) and Tb(III) ions in aqueous solutions

Article abstract of DOI:10.1016/j.tet.2010.09.052

A series of five new 15-, 18- or 21-membered polyazamacrocycles (L ₁-L₅) based on a pyridine, bipyridine or terpyridine unit and a triethylenetetraminetetraacetic acid (TTTA) skeleton is described. In ligands L₄ and L

Full text of DOI:10.1016/j.tet.2010.09.052

Tetrahedron 66 (2010) 8594e8604
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Polyazamacrocycles based on a tetraaminoacetate moiety and a (poly)pyridine
intracyclic unit: direct synthesis and application to the photosensitization of
Eu(III) and Tb(III) ions in aqueous solutions
,
,
,
,
,b,
Ghassan Bechara a b, Nadine Leygue a b, Chantal Galaup a b, Béatrice Mestre-Voegtlé a b, Claude Picard a
*
a CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, SPCMIB, UMR-5068, 118 Route de Narbonne, F-31062 Toulouse cedex 9, France
b Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules d0Intérêt Biologique, SPCMIB, 118 route de Narbonne, F-31062 Toulouse cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 June 2010
Received in revised form 10 September 2010
Accepted 15 September 2010
Available online 21 September 2010
A series of five new 15-, 18- or 21-membered polyazamacrocycles (L1eL5) based on a pyridine, bipyridine
or terpyridine unit and a triethylenetetraminetetraacetic acid (TTTA) skeleton is described. In ligands L4
and L5 the azaheterocycle contains an additional extracyclic functionality (ester group) suitable for co-
valently attachment to bioactive molecules. The synthetic procedure is based on the use of a linear tetra-
N-alkylated tetramine synthon incorporating masked acetate arms and an efficient metal template ion
effect, which controls the crucial macrocyclization step. In the case of L1eL3, the formation of lanthanide
complexes with europium(III) and terbium(III) was investigated and the fluorescence characteristics of
the complexes were established. In this series, the terbium(III) complex derived from the bipyridine
Keywords:
Polyazapolycarboxylic acid
Functionalized polyamine
Macrocyclic ligand
Polypyridine
ligand exhibits the highest lifetime and quantum yield values (
s
¼2.18 ms,
F
¼26%).
Ó 2010 Elsevier Ltd. All rights reserved.
Lanthanide
Luminescence
1. Introduction
a consequence lanthanide complexes derived from polyazamacro-
cycles fitted with acetic pendant groups have been fruitfully used
for the design of contrast enhancing agents for magnetic resonance
imaging (Gd3þ),1 radiopharmaceuticals carrying radionuclides such
as 86Y, 90Y, 153Sm, 177Lu for the diagnosis and therapy of tumours,2
long-lived fluorescent probes (Eu3þ, Tb3þ) for bioanalyses.3
N-Functionalized polyazamacrocycles represent an important
class of synthetic hosts, in particular when development of lan-
thanide complexes for biological and biomedical applications is
considered. These ‘predisposed ligands’ feature an intermediate
character between rigid receptors based on cryptands (‘lock and
key’ principle) and flexible receptors based on podands (‘induced
fit’ concept). They encapsulate Ln(III) ions in macrocyclic cavities,
while additional donor groups on the flexible arms can provide
further coordination to the metal. The large coordination number of
Ln(III) ions in solution (typically 8e10) may be thus fulfilled, and as
a consequence these Ln(III) complexes often exhibit high thermo-
dynamic and kinetic stabilities that are essential for bioanalytical
applications. In this respect, polyazamacrocycles with acetic acid
side chains pendant from amino groups are excellent complexing
agents for lanthanide ions. The aminoacetate group is an efficient
complexing unit leading to five-membered chelate rings and for-
mation of strong ionic bonds with the carboxylate unit. As
For the development of time-resolved fluorescent Ln(III) biop-
robes, the ligand must enhance the fluorescence properties of the
free metal ion by bearing a chromophoric unit, which balances the
inherently weak metal centred absorption band thus yielding
highly fluorescent Eu(III), Tb(III) species (antenna effect).4 A survey
of the literature highlights the use of macrocyclic fluorophores
based on DOTA type derivatives (DOTA¼1,4,7,10-tetraazacyclodo-
decane-1,4,7,10-tetraacetate) where
a chromophoric unit is
substituted to one acetic side arm.5 From our side, we focused our
attention towards the design of polyazamacrocyclic ligands in-
corporating both an intracyclic chromophoric unit and exocyclic
acetate groups. This choice is considered attractive for the follow-
ing reasons: (i) the heterocyclic moiety is expected to increase the
rigidity of the resulting complexes and thus their kinetic inertness
in biological media, (ii) chromophoric-to-lanthanide photosensiti-
zation step occurs between partners in a rigid conformation that
can improve the energy-transfer rates, (iii) functionalization of the
heterocyclic unit for bioconjugation purposes is expected to have
* Corresponding author. Tel.: þ33 5 61 55 62 96; fax: þ33 5 61 55 60 11; e-mail
address: picard@chimie.ups-tlse.fr (C. Picard).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.052

G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
8595
little effect on the chelation properties of the ligand. We have re-
2. Results and discussion
2.1. Synthetic strategies
cently reported the synthesis and photophysical properties of eu-
ropium and (or) terbium macrocyclic complexes derived from
a diethylenetriaminetriacetic acid core (DTTA) and an intracyclic
2,20-bipyridine, N,C-pyrazolylpyridine or 2,20:60,200-terpyridine
chromophore.6 These complexes exhibited good thermodynamic
stability (log Kcond ML>18 at pH 7.3) and kinetic inertness in serum
media. They showed also promising properties as fluorescent
probes or as dual lanthanide probes suitable for optical and mag-
netic resonance imaging. A pyridine analogue, the pyridine-con-
taining 12-membered tetraazatriacetate ligand (H3PCTA) has also
been described by several groups and has some attractive features
for use in biomedicine.7 Stability constants of about 20.3 log K units
were recently reported for the corresponding Ln(III) complexes.7b
The presence of two water molecules in the inner coordination
sphere of the metal in these complexes leads to a high water
relaxivity for Gd-PCTA, but a weak fluorescence emission for
Eu-,Tb-PCTA due to vibronic deactivation via the OeH oscillators of
coordinated water molecules.
In the course of our research on the design of lanthanide-
containing chelating systems with potential applications as fluo-
rescent tags, we report here on the synthesis and characterization
of five new macrocyclic polyamines comprising a triethylenete-
traminetetraacetic acid (TTTA) core and an intracyclic chromo-
phoric unit (pyridine, 2,20-bipyridine, 2,20:60,200-terpyridine). The
structures of these 15-, 18- and 21-membered macrocyclic ligands
are shown in Scheme 1.
As previously showed in the literature, two synthetic strategies
(Scheme 2) may be envisaged for the preparation of poly-
azamacrocycles comprising an intracyclic heterocyclic unit and
bearing appended acetic groups, depending on the stage at which
the pendant groups are branched. In both strategies, efficient
macrocyclization using two precursor molecules is a crucial step.
chromophore
CO2H
HO2C
N
N
N
N
HO2C
CO2H
(a)
(b)
chromophore
CO2R
H
H
N
N
N
N
N
N
H
H
CO2R
RO2C
N
N
RO2C
H
H
H
NH2
N
N
H
H2N
R
Scheme 2. General methodologies for the synthesis of polyazamacrocyclic ligands
incorporating both an intracyclic chromophoric unit and exocyclic acetate groups.
n
N
N
The first and traditional route (Scheme 2, route a) begins with the
formation of polyazamacrocyclic intermediates bearing secondary
amine sites. In a second step, carboxyalkylation of these amine sites
gives the desired ligand. In this strategy, representative examples of
important cyclization procedures include reaction of polyamines
and a dialdehyde fragment of the heterocycle,10 a pernosylated
amine with a dihydroxy derivative of the heterocycle11 or a perto-
sylated (pernosylated) amine with a bifunctional heterocyclic
electrophile (dibromide or ditosylate).12 The last reaction, known as
the RichmaneAtkins cyclization13 provides an efficient (cyclization
yields up to 70% without resorting high dilution) and time tested
means of preparing unsubstituted macrocyclic amines. On the other
hand, per-N-carboxyalkylation of the unsubstituted nitrogens is not
always straightforward, requiring optimization of reaction condi-
tions or contributing to a drop in the overall yield of the target
products.11b,12d,e,14
The second strategy (Scheme 2, route b) involves the use of linear
polyamines having pendant alkyl acetate groups as precursor mol-
ecules for the macrocyclization process. In previous reports, this
methodology exploits a metal template effect on the macro-
cyclization process with dibromo derivatives of heterocycles,
affording macrocyclic rings in 35e65% yield.6,15 An important ad-
vantage of this approach is that the acetate arms are incorporated
prior to cyclization, eliminating the need for a protection/depro-
tection sequence and subsequent N-carboxyalkylation reaction on
polyamine macrocycles. We decided to develop the second strategy,
which allows variations on the nature of chromophoric antenna
without the reengineering of the whole macrocyclic molecule.
HO2C
HO2C
L1 n = 0, R = H
N
N
CO2H
N
N
CO2H
L4 n = 0, R = COOMe
L5 n = 1, R = COOMe
L2 n = 1, R = H
L3 n = 2, R = H
Scheme 1. Structure of the polyaminocarboxylate macrocyclic ligands.
To the best of our knowledge, the use of a TTTA core in the field
of acyclic or macrocyclic chelating agents has not been reported
until now. Only four patents claimed its potential use, but no data
were reported.8 On the other hand, these polypyridine chromo-
phores are widely used as antenna groups for photosensitizing
lanthanide ions, enabling us to compare the optical properties of Ln
(III) complexes depending on the ligand structure. These ligands
are potentially nona-, deca- or undecadentate: i.e., eight co-
ordination sites from the TTTA host and 1e3 additional ones
coming from heterocyclic nitrogen atoms. Assuming a coordination
number of nine, commonly reported for Eu(III) and Tb(III) ions in
aqueous solutions, these ligands may preclude the coordination of
water molecules in the inner sphere of the metal and thereby
avoiding a partial quenching of the metal fluorescence. In addition,
two of them contain a pyridine ring functionalized with an ester
function suitable for conjugation to biological materials. To have
some insight into the fluorescence properties of the corresponding
Ln(III) complexes, we have also investigated the formation of Eu(III)
and Tb(III) complexes derived from the unfunctionalized ligands
and established their photophysical properties in aqueous solu-
tions. Preliminary results from this work have been published
previously.9
2.2. Starting material: tetramine bearing four acetate
pendant arms
In this work, the key building block for the synthesis of all
compounds shown in Scheme 1 is the tetramine 6 bearing four tert-

8596
G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
butyl acetate side chains and two terminal secondary amine groups
available for subsequent ring closure reactions with a dihalide
(Scheme 3). For the preparation of 6, prealkylated precursor mol-
ecules, N,N0-dialkylated ethylenediamine derivative 2 and N,N-
dialkylated bromide 4 were used. In this approach, tert-butyl ester
and benzyl groups were selected as protection for the acetic acid
chains and amine functions, respectively. The use of bulky tert-
butyl ester prevents intramolecular cyclization yielding six-mem-
bered lactams during the course of alkylation reactions. As a matter
of fact, it is well established that such side reaction occurs in the
case of polyamines bearing secondary amine sites and more re-
active esters, such as methyl, ethyl or benzyl esters.16 On the other
hand, benzyl protective groups of amine functions can be efficiently
removed in neutral conditions by hydrogenolysis, avoiding purifi-
cation of charged species.
bromo derivative 4 by treatment with NBS/PPh3 at rt. In this way,
compound 4 can be prepared in 82% overall yield.
The secondary amine groups of 2 were alkylated with 2 equiv of
bromide 4 to give tetraester 5 in high yield (92%) after purification
by column chromatography. Debenzylation of 5 was readily ach-
ieved in a quantitative yield by catalytic hydrogenation at rt under
hydrogen pressure, in methanol, using Pd/C (10%) as catalyst. The
total yield of this reaction sequence (Scheme 3) was 75% in com-
pound 6, starting from commercial materials. Preparation of com-
pound 6 following a more direct route involving a tetra alkylation of
N,N000-dibenzyltriethylenetetramine was less successful. In this case,
formation of polyalkylated by-products, which are difficult to
separate from the desired product by chromatographic purification
limited the yield (9% overall yield in
6
from commercial
triethylenetetramine).9
CO2H
NH
CO2H
H
N
HN
OH
N
N
H
H
d
a
b
CO2tBu
NH
CO2tBu
OH
c
e
Br
N
N
N
+
HN
N
2
3
4
1
CO2tBu
CO2tBu
CO2tBu
CO2tBu
f
CO2tBu
N
CO2tBu
N
H
g
H
N
N
N
N
N
N
CO2tBu
CO2tBu
tBuO2C
tBuO2C
tBuO2C
tBuO2C
6
5
Scheme 3. Synthesis of tetramine 6. Reagents and conditions: (a) CH3COOt-Bu, HClO4, rt, 12 d, 43%; (b) BrCH2CO2t-Bu (2 equiv), K2CO3, CH3CN, reflux, 24 h, 100%; (c) Pd/C (10%), H2
(5 bar), MeOH, rt, 12 h, 100%; (d) BrCH2COOt-Bu, i-Pr2NEt, DMF, rt, 24 h, 100%; (e) NBS/PPh3, CH2Cl2, rt, 4 h, 82%; (f) 4 (2 equiv), K2CO3, CH3CN, reflux, 24 h, 92%; (g) Pd/C (10%), H2
(5 bar), MeOH, rt, 12 h, 100%.
Although C.G. Pitt described a three-step method for the prep-
aration of di-tert-butyl ester 2, the procedure is cumbersome (use
of isobutene) and gives a poor yield (15%) of the final product,
starting from commercial ethylenediamine-N,N0-diacetic acid.17 By
using the same starting material, we have prepared 2 in a one-step
procedure by utilizing a transesterification process commonly used
in peptide chemistry.18 Preparation of 2 was realized by reacting
ethylenediamine-N,N0-diacetic acid in tert-butyl acetate as solvent
and in the presence of perchloric acid. The reaction occurred at rt
and the desired diester 2 was isolated by simple extraction pro-
cedure in 43% yield with satisfying purity allowing to use the crude
product. We have also employed an alternative method, starting
from commercial N,N0-dibenzylethylenediamine. A combination of
alkylation with tert-butyl bromoacetate followed by a deprotection
step of benzyl protecting group by catalytic hydrogenation pro-
duced the target compound 2 in 100% yield for the two steps. A
report that appeared after the completion of the work described
here outlines that this procedure can be adapted to a large-scale
preparation of 2 (multigram scale).19
The second precursor molecule 4 was prepared in two steps from
commercially available N-benzyl ethanol amine using modified
published procedure.20 Briefly, alkylation of the amine group of N-
benzyl ethanolamine was performed with tert-butyl bromoacetate
in the presence of N,N-diisopropylethyl amine (DIPEA) as a base. The
use of this hindered organic base prevents totally a lactonization
side reaction forming 4-benzyl-morpholin-2-one.9,21 The hydroxyl
group of the resulting compound 3 was then transformed into the
2.3. Synthesis of ligands L1eL5
With the tetramine precursor 6 in hand, the macrocyclization
step for getting the 15-, 18- and 21-membered macrocycles L1, L2
and L3, respectively, was then envisaged (Scheme 4). Because ease
n
N
N
N
n
tBuO2C
CO2tBu
a
N
N
N
N
Br
Br
N
CO2tBu
tBuO2C
7 n = 0
8 n = 1
9 n = 2
10 n = 0
b
11 n = 1
12 n = 2
n
n
N
N
N
N
N
-O2C
Na+.
c
-
CO 2
CO 2H
HO2C
Ln3+
N
N
N
N
N
N
N
-
-O2C
CO2
HO2C
CO 2H
L1.Eu, L1.T b n = 0
L2.Eu, L2.T b n = 1
L3.Eu, L3.T b n = 2
L1 n = 0
L2 n = 1
L3 n = 2
Scheme 4. Synthesis of ligands L1eL3 and their Eu(III) and Tb(III) complexes. Reagents
and conditions: (a) 6 (1 equiv), Na2CO3, CH3CN, reflux, 24 h, [reactants]¼2.7ꢀ10ꢁ3 M,
54% (10), 58% (11) and 39% (12); (b) HCOOH, 60 ꢂC, 24 h, 100% (L1) and 98% (L2) or
CF3COOH, CH2Cl2, rt, 24 h, 92% (L3); (c) EuCl3$6H2O or TbCl3$6H2O, H2O, rt, 24 h.

G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
8597
of ring closure may be dependent on the size of macrocycle, we
started the optimization of the macrocyclization process with the
18-membered macrocycle derived from 2,20-bipyridine.
complex mixture was observed for 12. This suggests that the sodium
template effect is less efficient for 12, probably due to the size of the
macrocycle to be formed (21-membered cycle). K2CO3 was also
tested in the preparation of 12, but the use of this alkaline carbonate
did not improve the selectivity of the macrocyclization reaction
towards the monomeric structure. Purification by column chro-
matography on alumina associated with EDTA treatment afforded
macrocycles 10 and 12 in 54 and 39% isolated yields, respectively.
Fifteen- and eighteen-membered macrocycles 15 and 16
(Scheme 5) tethered to a methyl ester group were also prepared by
using this synthetic approach. This aromatic ester group was se-
lected because its subsequent transformation to various function-
alities allows further grafting of biological material by classical
methods or click chemistry.24 In the cyclization step we used the
dibromide compounds 13 and 14. The preparation of 13 was carried
out following a standard synthetic scheme starting from commer-
cial 2,6-dimethylpyridine-N-oxide. This was achieved in five steps
according to literature procedures.25 As previously described,24 an
efficient access to 14 required the use of a palladium-catalyzed cross
coupling using a modified Negishi procedure and a Boeckelheide
rearrangement, starting from 2-chloro-6-methyl-isonicotinic acid.
Condensation of tetramine 6 with dibromide 13 or 14 was carried
out in CH3CN and by using Na2CO3 as previously described. The free
ligand 15 was isolated in 56% yield and the sodium complex 16$Na
in 40% yield. For the later, repeated treatments with EDTA did not
allow to get the free ligand 16 with a suitable purity degree.
Treatment of the tetramine 6 with 6,60-bis(bromomethyl)-2,20-
bipyridine 822 was carried out in refluxing acetonitrile and in the
presence of an excess of sodium carbonate and without using high-
dilution techniques (reactant concentration: 2.7ꢀ10ꢁ3 M). 1H, 13C
NMR and MS analyses of the crude reaction product evidenced the
presence of a major species, characterized as the sodium mono-
meric complex 11$Na. Attempted purification by column chroma-
tography led to the isolation of a mixture of 11$Na and free ligand
11 (45:55 ratio), as a result of alumina-mediated dissociation.
Treatment of this mixture of 11$Na and 11 with an excess of NaCl in
CH3CN or with a saturated EDTA aqueous solution furnished cleanly
as single species 11$Na or 11, respectively. It can be noticed that
these two species can be readily distinguished by their 1H NMR
spectra (Fig. 1). Especially, these spectra exhibited significant dif-
ferences for the heteroaromatic and tert-butyl hydrogens. The
heterocyclic moiety related resonances cover a chemical shift range
1.5 fold larger in 11$Na than in 11 and the signals of the tert-butyl
protons are shifted upfield by Dd>0.12 ppm upon complexation by
Naþ ion. It is interesting to note that the positions of H-3,30 hetero-
atomic hydrogens (7.86 and 7.94 ppm) indicate that the orientation
of the bipyridinyl moiety is approaching a syn conformation in
these two species, and thus support the monomeric cyclic struc-
ture.12b On the other hand, complexation by Naþ was accompanied
by a reduction in the frequency of the carbonyl infrared band
(5 cmꢁ1) and a bathochromic shift of the maximum in the bipyr-
idine ultraviolet spectrum (6 nm), suggesting that these two groups
are involved in complexation. Finally, when the mixture of 11 and
11$Na species was treated with saturated EDTA solution, the free
macrocycle 11 was readily obtained in 58% yield.
COOMe
COOMe
n
N
N
N
n
CO2tBu
tBuO2C
a
N
N
N
N
Br
Br
N
CO2tBu
15 n = 0
tBuO2C
13 n = 0
14 n = 1
b
16 n = 1
COOMe
n
N
N
HO2C
CO2H
N
N
L4 n = 0
L5 n = 1
N
N
HO2C
CO2H
Scheme 5. Synthesis of ligands L4 and L5. Reagents and conditions: (a) 6 (1 equiv),
Na2CO3, CH3CN, reflux, 24 h, [reactants]¼2.7ꢀ10ꢁ3 M, 56% (15) and 40% (16$Na); (b)
CF3COOH, CH2Cl2, rt, 24 h, 100% (L4) or HCOOH, 60 ꢂC, 24 h, 91% (L5).
Hydrolysis of tert-butyl ester groups of 10e12 under acidic
conditions with trifluoroacetic acid (at rt) or with formic acid (at
60 ꢂC) gave cleanly tetraacids L1eL3 in high yields. Selective
deprotection of tert-butyl ester groups of 15 and 16 (vs methyl ester
group) was also carried in the same experimental conditions.
It is important to note that tetraacid macrocycle L1 was pre-
viously prepared by using the RichmaneAtkins methodology for
the macrocyclization step and was isolated in 17% overall yield
starting from tetratosylated triethylenetetramine.12a Following our
direct method, L1 can be prepared in 54% overall yield, starting from
tetramine 6. This result highlighted the relevance of using route
b (Scheme 2) for the synthesis of such macrocycles.
Fig. 1. 1H NMR spectra of compounds 11 and 11$Na in CDCl3 at 300 MHz (*¼solvent
peak).
The 15- and 18-membered macrocycles 10 and 12 were obtained
by treating 6 with 2,6-bis(bromomethyl)pyridine 723 or 6,600-bis
(bromomethyl)-2,20:60,200-terpyridine 9.6c This macrocyclization
was carried out under batch-wise procedure and in a heterogeneous
reaction in refluxing CH3CN with Na2CO3, as described for 11. As in
the case of 11, the analyses of the crude reaction mixtures high-
lighted the formation of a single species, 10$Na, while a more
2.4. Photophysical properties of L1e3$Eu and L1e3$Tb
complexes
Next, we assessed the photophysical properties of complexes of
the ligands L1eL3 with Eu(III) and Tb(III), lanthanide ions of choice

8598
G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
(i) For both series of complexes, the excitation wavelength (Table 1)
is in agreement with the spectral signature of the pyridine, cis
2,20-bipyridine and cis-cis 2,20:60,200-terpyridine moieties. As
expected, these excitation wavelengths increase with an in-
creasing number of pyridine rings and are similar to those
reported for Ln(III) complexes derived from (poly)pyridine
antennae and bis(iminodiacetate) or DTTA chelating
moieties.6b,c,26e28 On the other hand, no transition correspond-
ing to the own Eu(III) and Tb(III) absorptions levels, especially
the 7F0/5L6 (393 nm) and 7F6/5L10 (369 nm) transitions are
observable in the excitation spectra, demonstrating efficiency of
energy transfer (antenna effect) in these complexes.
(ii) Lifetimes are in the millisecond range, in particular, the metal
luminescence lifetime of L2$Tb at rt is about 2.2 ms, a re-
markably high value in comparisonwith those found in aqueous
solutions for Tb(III) complexes of ligands containing bipyridine-
type chromophore.4,6b,22,29 On the other hand, upon solvent
deuteration, the lifetimes of some complexes are increased by
a factor two, indicative of some coupling between the metal ion
and OeH oscillators of the solvent, which favour radiationless
deactivation of the metal excited state. Using this well-estab-
lished isotope effect and the empirical relations of Parker
allowed an estimation of the apparent hydration state q of the
complexes.30 These analyses indicate that there is about one-
metal bound water molecule in L1$Ln and L3$Ln and no bound
water molecule in L2$Ln. The non-integer values observed can
be arise from the uncertainty of these empirical formulae or
from the presence of two species with different degrees of sol-
vation in exchange faster than the Ln(III) lifetime. Thus the in-
troduction of a TTTA core (vs DDTA) in the macrocyclic systems
derived from pyridine and bipyridine chromophores displaces
one water molecule from the first coordination sphere of the
lanthanide ion. In contrast, when the TTTA core is substituted for
the DTTA core in macrocyclic terpyridine system, a water mol-
ecule is allowed to bind the metal ion. In this case, the expansion
of the cavity size (21-membered vs 18-membered) is an
unfavourable factor for the shielding of Ln(III) ions, despite the
presence of two additional coordination sites.
in the design of luminescent probes. The complexes were prepared
by the addition of a stoichiometric amount of the lanthanide salt
(LnCl3$6H2O) to an aqueous solution of the corresponding ligand
(Scheme 4). These solutions were adjusted in Tris buffer (50 mM,
pH 7.4) at a final concentration of 1ꢀ10ꢁ4 M for absorption and
1ꢀ10ꢁ6 M for emission spectroscopies, respectively. These com-
plexes were characterized by UV, MS, HPLC and ligand-sensitized
Ln(III) fluorescence techniques. ESI mass spectrometry analyses
allowed a 1:1 ligand/metal stoichiometry to be established in
aqueous solutions.
Both Ln(III) complexes gave at rt classical europium- or terbium-
centred luminescence spectra, with the strongest transition at
620 nm (5D0/7F2 transition) or 545 nm (5D4/7F5 transition), re-
spectively, when photoexcited in the lowest energy absorption of
the heterocyclic chromophore. Representative emission spectra are
shown in Fig. 2 for L2$Eu and L2$Tb complexes.
(a)
800
600
400
200
(b)
(c)
0
λ(nm)
400
500
600
700
800
Fig. 2. Normalized (a) phosphorescence (.) spectrum of L2$Gd and (b), (c) fluores-
cence spectra of L2$Tb (- - -) and L2$Eu (d) complexes. The phosphorescence spectrum
was measured at 77 K in MeOH/EtOH (4:1 v/v) glassy matrix and the fluorescence
spectra at 298 K in Tris buffer (50 mM, pH 7.4).
(iii) The sensitization pathway, which seems to be general in Eu(III)
and Tb(III) complexes, proceeds through an intramolecular
energy transfer from the triplet state of the antenna chromo-
phore to the closest emitting level of the metal. Except for
L3$Tb, the Eu (5D0) and Tb (5D4) lifetimes remain approxi-
mately constant between 298 K and 77 K, indicating that
thermally activated processes are negligible for the in-
vestigated complexes. For L3$Tb, the temperature dependence
of the Tb (5D4) lifetime is larger (knr(T)¼400 sꢁ1),31 which
suggests a back-transfer process taking place between the li-
gand triplet-state and the 5D4 level, a deactivation pathway,
which is commonly observed for photosensitized terbium
complexes.4,26 This was supported by the triplet-state ener-
gies, measured as usual from the ligand phosphorescence
spectra of the corresponding Gd(III) complexes (Fig. 2, Table 1).
For L3$Tb, the energy gap between the triplet state of the li-
The similarity between the absorption and excitation spectra
proves an energy transfer from the excited states of these ligands to
the Ln(III) emission states. The temporal decay of the emission of
both complexes was rigorously monoexponential, suggesting the
presence of one discrete L$Ln species. The lifetimes data are
reported in Table 1, along with quantum yields, calculated hydra-
tion numbers (q) and triplet-state energies.
An inspection of these luminescence data reveals several points
of significance:
Table 1
Absorption and excitation maxima (lmax and lexc in nm), luminescence lifetimes
(s in ms), quantum yields (F, %), hydration states (q) and triplet-state energies (ET in
cmꢁ1) for L1e3$Eu and L1e3$Tb complexes.a
lmax lexc sH298 K sD298 K sH77 K sD77 K FH298 K F2D98 K qb
ET
c
gand (22,200 cmꢁ1
) and the resonance level of Tb(III)
(20,500 cmꢁ1) is 1700 cmꢁ1, which is slightly below the min-
imum value of 1850 cmꢁ1 proposed by Latva et al.26 to prevent
such a metaleligand reversible process.
L1$Eu 266 268 0.67
L1$Tb 266 268 1.54
L2$Eu 305 310 1.13
L2$Tb 305 310 2.18
L3$Eu 320 326 0.76
L3$Tb 320 326 0.89
1.80
3.00
1.81
2.35
1.73
1.25
0.92 1.97
1.94 3.00
1.21 1.93
2.34 2.87 26
0.87 1.86
1.85 2.78
0.6
8.5
5
1.6
13
6
27
10
14
0.8 27,550
1.3
0.1 22,550
ꢁ0.1
(iv) The emission quantum yields are higher for the Tb(III) com-
plexes than for the Eu(III) complexes. The higher susceptibility
of Eu(III) (vs Tb(III)) luminescence towards quenching by the
hydroxyl groups of the solvent cannot account for these re-
duced emission quantum yields. As a matter of fact, a similar
trend was observed in D2O. These results can be explained
either by a less efficient ligand-to-metal energy transfer or by
the presence of ligand-to-metal charge transfer (LMCT) excited
4.5
9.2
0.6 22,200
1.3
a
Data obtained in aerated Tris buffer (50 mM, pH 7.4), H2O (H) or D2O (D)
solutions.
b
Number of coordinated H2O molecules calculated using the equation q¼1.2((1/
s
H)ꢁ(1/
s
D)ꢁ0.25) for L1e3$Eu and q¼5((1/
s
H)ꢁ(1/
s
D)ꢁ0.06) for L1e3$Tb.27
c
From the structured phosphorescence profiles of L1e3$Gd complexes recorded at
77 K in a MeOH/EtOH (4:1 v/v) mixture. The energy level ET was derived in corre-
spondence to the highest energy band maximum.

G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
8599
MRI agents.38 Similar Kcond was found for L2$Tb and additional
experiments showed that this complex does not dissociate in hu-
man serum and is also an efficient emitter in 50 mM HEPES (pH 7.3)
and phosphate (pH 8) buffers.
Among this series of Ln(III) complexes, L2$Tb displays high lu-
minescence efficiency, long luminescence lifetime and reasonable
kinetic stability in aqueous and biological media. The properties of
this complex are compatible with the stringent requirements of
a lanthanide luminescent bioprobe.
states, the latter being in agreement with the fact that Eu(III)
can be easily reduced. The best ligand for sensitizing the Tb(III)
luminescence is L2 with an overall quantum yield of 26%. In
moving to L1 and L3, the quantum yield decreases to 8.5 and
9.2%, respectively. In these macrocyclic TTTA-based ligands,
the (poly)pyridine chromophore sensitizes the Eu(III) ion less
efficiently than in open-chain (bis iminodiacetic core) or
macrocyclic (DTTA core) analogues.6b,c,26,32,33 Particularly, al-
though the long lifetime observed, L2$Eu displays a relatively
weak emission (
F
¼5%) despite the fact that in this complex,
coordination around the metal ion is saturated by the ligand.
For example, the emission quantum yield found for the mon-
oaquo Eu(III) complex of bpyCTA[15], a macrocyclic ligand
based on 2,20-bipyridine and DTPA moieties, is two times
higher.6b On the other hand, the luminescence quantum yield
of L2$Tb is among the largest values reported in aqueous so-
lution for Tb(III) complexes containing one or more 2,20-
bipyridine chromophore.6b,22,26,29,34 Moreover, it is interesting
to note that this quantum yield is close to the one reported for
terbium-based commercial luminescent probe, DTPA-Cs124
3. Conclusion
In conclusion, we present here a direct and efficient approach
for the synthesis of a new class of polyazamacrocycles containing
four acetate pendant arms and an intracyclic heterocyclic unit. The
synthetic route involved the macrocyclization between a dibromo
fragment of the heterocycle and a tetramine incorporating four
masked acetate arms and two secondary amine groups. The last
compound, readily available, promises to be useful synthetic tool
not only for polyazamacrocycles with other heterocyclic units, but
also for a wider range of acyclic compounds, which may possess
interesting properties for chelating lanthanide and related ions. On
the other hand, the photophysical properties of Eu(III) and Tb(III)
complexes reported here will help in rationalizing the ligand design
of luminescent probes for bioanalytical applications.
(F
¼32%).35
As far as the stability of these complexes are concerned, no
change in their luminescence data (emission intensity, lifetime) in
aqueous solutions (Tris buffer, 50 mM, pH 7.4) was observed after
several days at rt, indicating that the complexes are resistant to
dissociation in this medium. We have also studied the resistance of
the Eu(III) complexes at pH 7.4 (Tris buffer) in the presence of
chelating agents such EDTA (log Kcond EDTA$Eu¼14.4 at pH 7.4). The
dissociation of the complexes was determined by luminescent ex-
periments, that is, by monitoring the disappearance of the 5D0/7F2
peak at 620 nm as a function of time (Fig. 3). L3$Eu was nearly
completely dissociated after two days in the presence of a 10-fold
excess of EDTA, suggesting that the EDTA$Eu complex has been
formed preferentially. L1$Eu and L2$Eu complexes were 28 and 22%
dissociated, respectively, after two days under the same conditions.
These behaviours indicate that these complexes based on a TTTA
macrocyclic system have a weaker kinetic stability in comparison to
their DTTA macrocyclic analogues.6b,c,7b From these experiments
and by using the Verhoeven analysis,36 log Kcond (pH 7.4) was
measured to be 16.6 for the formation of L2$Eu complex in water.37
This indicates a reasonable physiological stability, compared to the
lowest log Kcond value (14.9 at pH 7.4) found in commercially used
4. Experimental
4.1. General methods
2,6-Bis(bromomethyl)pyridine 7,23 6,60-bis(bromomethyl)-2,20-
bipyridine 8,22 6,600-bis(bromomethyl)-2,20:60,200-terpyridine 9,6c
4-carbomethoxy-2,6-bis(bromomethyl)pyridine 1325 and 4-carbo-
methoxy-6,60-bis(bromomethyl)-2,20-bipyridine 1424 were pre-
pared according to literature procedures. Reactions requiring an
inert atmosphere were run under Argon. Acetonitrile was freshly
distilled from P2O5, and CH2Cl2 was freshly distilled from CaH2.
Diisopropylamine was dried and distilled over KOH. Thin-layer
chromatography was performed on Merck silica or alumina plates
with a fluorescence indicator. Column chromatography was carried
out on silica gel (Merck, 60e200
(Macherey-Nagel, activity IV, 50e200
ꢀ
m
m, porosity 60 A) and on alumina
m
m).
Melting points were taken with a Büchi mel-temp apparatus.
Infrared spectra were recorded on a PerkineElmer FTIR 1725x
spectrophotometer. Samples were prepared as KBr pellets (solid
sample) or applied to NaCl plates (liquid sample). Selected char-
acteristic absorption frequencies are reported in cmꢁ1. 1H and 13C
NMR spectra were recorded on a Bruker Avance 300 MHz spec-
trometer; chemical shifts are given in parts per million according to
the solvent peak. Electrospray (ES) mass spectra were obtained on
a Q TRAP Applied Biosystems Spectrometer and high-resolution
mass spectra (HRMS) on an LCT Premier Waters spectrometer. DCI,
NH3 or CH4, mass spectra were obtained on a DSQ II Thermo Fisher
or a GCT Premier Waters spectrometer, respectively. Elemental
analyses were carried out by the ‘Service d’Analyse’, Laboratoire de
Chimie de Coordination (Toulouse). Absorption measurements
were done with a Hewlett Packard 8453 temperature-controlled
spectrophotometer.
400
300
200
L .Eu
2
L .Eu
1
100
0
The ligands (L1eL5) and lanthanide complexes (L1e3$Eu and
L .Eu
3
L1e3$Tb) were analyzed by RP-HPLC using a Waters Alliance 2695
system with a PDA 2996 detector and using a reversed-phase (RP)
C8 column (Phenomenex Luna C8(2), 5
The flow rate was 1 mL/min with UV monitoring at 260 nm for li-
gands and complexes derived from a pyridine unit or 315 nm for
ligands and complexes constructed on the basis of a bipyridine or
a terpyridine unit. Two analytical procedures were developed as
0
500
1000
1500
2000
2500
3000
ꢀ
m
m 100 A, 150ꢀ4.6 mm).
Time (min)
Fig. 3. Plot of emission intensity of the 5D0/7F2 transition (620 nm) of the complexes
L1$Eu, L2$Eu and L3$Eu in the presence of 10 mol equiv of EDTA added in Tris buffer
(50 mM, pH 7.4).

8600
G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
following. System A: solvents were 10 mM pH 4 ammonium for-
mate buffer (solvent A) and acetonitrile (solvent B); the compounds
were analyzed using the HPLC gradient system beginning with
a solvent composition of 100% A and following a linear gradient up
to 80% A:20% B from 0 to 18 min. System B: solvents were H2O
containing 0.1% TFA (solvent A) and acetonitrile (solvent B); anal-
yses were performed using the HPLC gradient system beginning
with a solvent composition of 95% A:5% B and following a linear
gradient up to 80% A: 20% B from 0 to 35 min.
(5.85 g, 12.48 mmol, yield 100%) as a white solid. Mp: 70e71 ꢂC. Rf
(silica gel, CH2Cl2/MeOH 99:01): 0.24. IR nmax: 1718 (C]O ester). 1H
NMR (300 MHz, CDCl3)
d
: 1.45 (s, 18H), 2.80 (s, 4H), 3.25 (s, 4H),
3.78 (s, 4H), 7.22e7.30 (m, 10H). 13C NMR (75 MHz, CDCl3)
d: 28.2
(CH3), 51.7 (CH2), 55.2 (CH2), 58.4 (CH2), 80.7 (Cq), 127.0 (CH), 128.2
(CH), 129.0 (CH), 139.3 (Cq), 171.0 (Cq). MS (ESIþ): m/z (%) 491.4 (7)
[MþNa]þ, 469.3 (100) [MþH]þ, 413.4 (16) [(MꢁC4H8)þH]þ, 357.3
(14) [(Mꢁ2ꢀC4H8)þH]þ. Anal. Calcd for C28H40N2O4: C 71.76, H
8.60, N 5.98. Found: C 71.50, H 8.30, N 5.89.
4.1.1. Luminescence measurements. Fluorescence and phosphores-
cence spectra were obtained with a LS-50B PerkineElmer and
a Cary Eclipse spectrofluorimeters equipped with a Xenon flash
lamp source and a Hamamatsu R928 photomultiplier tube. The
measurements were carried out at pH 7.4 in Tris buffer (50 mM)
and all samples were prepared with an absorbance between 0.01
and 0.05 at the excitation wavelength in order to prevent the inner-
filter effect. Phosphorescence spectra at 77 K of gadolinium com-
plexes were carried out in a MeOH/EtOH (4:1 v/v) mixture and
recorded with the LS-50B PerkineElmer spectrofluorimeter
equipped with the low-temperature accessory No. L2250136.
Spectra were corrected for both the excitation light source variation
4.2.2.2. Compound (2). A mixture of 1 (540 mg, 1.15 mmol) and
10% Pd/C (180 mg) in methanol (20 mL) was stirred overnight at rt
under H2 (5 bar). The reaction mixture was filtered over Celite and
the Celite pad was washed with methanol. The filtrate was con-
centrated in vacuo to give pure 2 (330 mg, 1.15 mmol, yield 100%) as
a yellow oil, which was identical with the product obtained by
route A.
4.3. tert-Butyl 2-(benzyl(2-hydroxyethyl)amino)acetate (3)
To
a stirred solution of N-benzyl ethanolamine (500 mg,
3.3 mmol) and N,N-diisopropylethylamine (426 mg, 3.3 mmol) in
anhydrous DMF (4 mL), at 0 ꢂC, was added dropwise tert-butyl
bromoacetate (644 mg, 3.3 mmol). The reaction mixture was
allowed to warm up to rt and stirring was continued for 24 h. The
solvent was removed under reduced pressure, the residue was
dissolved in CH2Cl2 (50 mL) and washed with water (4ꢀ20 mL).
The organic layer was dried (Na2SO4) and evaporated under re-
duced pressure to provide 3 (875 mg, 3.3 mmol, yield 100%) as
a yellow oil. Rf (silica gel, CH2Cl2/petroleum ether 50:50): 0.08. 1H
and the emission spectral response. Lifetimes
s
(uncertainty ꢃ5%)
are the average values from at least five separate measurements
covering two or more lifetimes made by monitoring the decay at
a wavelength corresponding to the maximum intensity of the
emission spectrum, following pulsed excitation. The luminescence
decay curves were fitted by an equation of the form I(t)¼I(0)exp(ꢁt/
s
) by using a curve-fitting program. High correlation coefficients
were observed in each cases (higher than 0.999). The luminescence
quantum yields (uncertaintyꢄ10%) were determined by the
method described by Haas and Stein,39 using as standards [Ru
NMR (300 MHz, CDCl3)
d
: 1.45 (s, 9H), 2.92 (t, 2H, J¼6), 3.27 (s,
2H), 3.63 (t, 2H, J¼6), 3.88 (s, 2H), 7.29e7.35 (m, 5H). 13C NMR
(bpy)3]2þ in aerated water (
quinine sulfate in 1 N sulfuric acid (
complexes.
F
¼0.028)40 for the Eu(III) complexes or
(75 MHz, CDCl3) d: 28.1 (CH3), 55.4 (CH2), 56.6 (CH2), 58.6 (CH2),
F
¼0.546)41 for the Tb(III)
59.0 (CH2), 81.4 (Cq), 127.4 (CH), 128.5 (CH), 129.0 (CH), 138.4 (Cq),
171.1 (Cq). MS (DCI/CH4): m/z (%) 266.2 (23) [MþH]þ, 210.1 (100)
[(MꢁC4H8)þH]þ.
4.2. Di-tert-butyl ethylenediamine-N,N0-diacetate (2)
4.4. tert-Butyl 2-(benzyl(2-bromoethyl)amino)acetate (4)
4.2.1. Route A. To a stirred solution of ethylenediamine-N,N0-diac-
etic acid (3 g, 17 mmol) in tert-butyl acetate (135 mL), at 0 ꢂC, was
added dropwise HClO4 (70%, 4.5 mL). The mixture was then
allowed to warm up to rt, and stirring was continued for 12 days.
The resulting milky solution was cooled to 0 ꢂC and washed with
HCl (0.5 N, aqueous) (5ꢀ60 mL). The combined aqueous phases
were neutralized with solid NaHCO3, extracted with ether
(6ꢀ100 mL). The combined organic phases were dried (Na2SO4),
and the solvent was removed under reduced pressure. The result-
ing yellow oil was dissolved in ether (100 mL), washed with water
(3ꢀ15 mL) and dried. Evaporation of the solvent afforded the title
To a stirred solution of 3 (3.2 g, 12 mmol) and N-bromosucci-
nimide (2.5 g, 14.0 mmol) in CH2Cl2 (50 mL), at 0 ꢂC, was added
portionwise triphenylphosphine (3.7 g, 14.1 mmol) over 1 h. The
resulting mixture was stirred at 0 ꢂC for 30 min, then it was
allowed to warm up to rt, and stirring was continued for 4 h. The
solvent was removed under reduced pressure and the crude
product was purified by chromatography over silica gel (petro-
leum ether/diethyl ether 95:5) to give bromide
4 (3.23 g,
9.84 mmol, yield 82%) as a yellow oil. Rf (silica gel, CH2Cl2/petro-
leum ether 50:50): 0.61. IR nmax: 1733 (C]O ester). 1H NMR
compound 2 (2.1 g, 7.28 mmol, yield 43%) as a yellow oil. IR nmax
1733 (C]O ester). 1H NMR (300 MHz, CDCl3)
: 1.48 (s, 18H), 2.71 (s,
4H), 3.30 (s, 4H). 13C NMR (75 MHz, CDCl3)
: 28.1 (CH3), 48.9 (CH2),
:
(300 MHz, CDCl3)
d
: 1.47 (s, 9H), 3.16 (t, 2H, J¼6), 3.30 (s, 2H), 3.38
d
(t, 2H, J¼6), 3.88 (s, 2H), 7.29e7.34 (m, 5H). 13C NMR (75 MHz,
d
CDCl3) d: 28.2 (CH3), 30.5 (CH2), 55.2 (CH2), 55.9 (CH2), 58.1 (CH2),
51.6 (CH2), 81.1 (Cq), 171.8 (Cq). MS (DCI/NH3): m/z (%) 289.5 (100)
81.2 (Cq), 127.3 (CH), 128.4 (CH), 128.8 (CH), 138.7 (Cq), 170.6 (Cq).
MS (DCI/CH4): m/z (%) 330.1 (20)/328.1 (25) [MþH]þ, 274.0 (80)/
272.0 (85) [(MꢁC4H8)þH]þ.
[MþH]þ.
4.2.2. Route B.
4.5. Tetra-tert-butyl N,N000-dibenzyltriethylenetetramine
N,N0,N00,N000-tetraacetate (5)
4.2.2.1. Di-tert-butyl
N,N0-dibenzylethylenediamine-N,N0-diac-
etate (1). To a solution of N,N0-dibenzylethylenediamine (3 g,
12.48 mmol) in anhydrous acetonitrile (300 mL) was added solid
K2CO3 (17 g, 123 mmol). The suspension was refluxed for 1 h, then
tert-butyl bromoacetate (4.87 g, 25 mmol) was added dropwise,
and the mixture was stirred at reflux for 24 h before filtration. The
solvent was removed under reduced pressure, then the solid resi-
due was treated with CH2Cl2 and the insoluble fraction was elimi-
nated by filtration. The filtrate was evaporated to dryness to give 1
To a stirred solution of diamine 2 (680 mg, 2.36 mmol) in an-
hydrous acetonitrile (85 mL) was added K2CO3 (3.3 g, 23.9 mmol).
The suspension was refluxed for 1 h, then bromide 4 (1.55 g,
4.72 mmol) was added in one portion and the mixture was stirred
at reflux for 24 h. After cooling to rt, the reaction mixture was fil-
tered, and the filtrate was concentrated in vacuo. The resulting oily
residue was purified by chromatography on silica gel (CH2Cl2/

G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
8601
MeOH 100:0 then 98:02) to give tetramine 5 (1.7 g, 2.17 mmol, yield
92%) as a yellow oil. Rf (silica gel, CH2Cl2/MeOH 98:02): 0.34, Rf
(alumina, CH2Cl2): 0.66. IR nmax: 1733 (C]O ester). 1H NMR
quantitatively the sodium complex 10$Na as a pale yellow solid. Mp
>250 ꢂC. IR nmax: 1728 (C]O ester). 1H NMR (300 MHz, CDCl3)
d
:
1.38 (s, 18H), 1.41 (s, 18H), 2.66 (m, 12H), 3.09 (s, 4H), 3.28 (s, 4H),
(300 MHz, CDCl3)
(m, 8H), 3.23 (s, 4H), 3.29 (s, 4H), 3.78 (s, 4H), 7.28e7.31 (m, 10H).
13C NMR (75 MHz, CDCl3)
: 28.2 (CH3), 52.1 (CH2), 52.6 (CH2), 55.2
d: 1.43 (s, 18H), 1.45 (s, 18H), 2.68 (m, 4H), 2.75
3.81 (m, 4H), 7.15 (d, 2H, J¼7.7), 7.65 (t, 1H, J¼7.7). 13C NMR
(75 MHz, CDCl3) d: 27.9 (CH3), 28.1 (CH3), 53.5 (CH2), 53.6 (CH2),
d
54.1 (CH2), 54.4 (CH2), 58.4 (CH2), 60.4 (CH2), 81.8 (Cq), 81.9 (Cq),
122.2 (CH), 138.1 (CH), 157.7 (Cq), 171.3 (Cq), 171.6 (Cq). MS (ESIþ):
m/z (%) 728.8 (100) [MþNa]þ.
(CH2), 56.1 (CH2), 58.4 (CH2), 80.6 (Cq), 80.7 (Cq), 126.9 (CH), 128.3
(CH), 128.9 (CH), 139.2 (Cq), 170.9 (Cq), 171.0 (Cq). Anal. Calcd for
C44H70N4O8: C 67.49, H 9.01, N 7.15. Found: C 67.10, H 9.15, N 7.10.
MS (DCI/NH3): m/z (%) 783.6 (100) [MþH]þ.
4.9. 8,11,14,17,23,24-Hexaazatricyclo[17.3.1.12,6]tetracosa-1
(23),2,4,6(24),19,21-hexaene-8,11,14,17-tetraacetic acid,
8,11,14,17-tetrakis(1,1-dimethylethyl) ester (11) and its sodium
complex 11$Na
4.6. Tetra-tert-butyl N,N000-triethylenetetramine N,N0,N00,N000-
tetraacetate (6)
A mixture of 5 (313 mg, 0.40 mmol) and 10% Pd/C (106 mg) in
methanol (14 mL) was stirred overnight at rt under H2 (5 bar). The
reaction mixture was filtered over Celite and the Celite pad was
washed with methanol. The filtrate was concentrated in vacuo to
give pure 6 (241 mg, 0.4 mmol, yield 100%) as a yellow oil. Rf
4.9.1. Compound 11. The reaction was carried out using tetramine 6
(150 mg, 0.25 mmol) and 6,60-bis(bromomethyl)-2,20-bipyridine 8
(85 mg, 0.25 mmol). The residue was purified by chromatography
on alumina (CH2Cl2/CH2Cl2/MeOH, 50:50) to give 113 mg of
a mixture of sodium complex 11$Na and free ligand 11 (45:55 ratio).
This mixture was dissolved in CHCl3 (50 mL) and sequentially
washed with saturated Na2EDTA aqueous solution (2ꢀ50 mL)
and water (2ꢀ50 mL). The organic layer was dried (Na2SO4) and
evaporated under reduced pressure to give 11 (114 mg, 0.145 mmol,
yield 58%) as a yellow oil. Rf (alumina, CH2Cl2/MeOH 98:02): 0.3. IR
(alumina, CH2Cl2/MeOH 98:02): 0.4. 1H NMR (300 MHz, CDCl3)
d:
1.44 (s, 18H), 1.46 (s, 18H), 2.81 (m, 4H), 3.01 (m, 8H), 3.40 (s, 4H),
3.59 (s, 4H). 13C NMR (75 MHz, DMSO-d6)
d
: 28.2 (CH3), 28.3 (CH3),
45.5 (CH2), 49.0 (CH2), 50.2 (CH2), 51.3 (CH2), 55.3 (CH2), 81.0 (Cq),
82.5 (Cq), 168.5 (Cq), 171.25 (Cq). MS (ESIþ): m/z (%) 603.5 (100)
[MþH]þ.
nmax: 1733 (C]O ester). UV/vis (CH3CN): lmax
(
3
, L molꢁ1 cmꢁ1)¼
237 (13,200), 289 (14,900) nm. 1H NMR (300 MHz, CDCl3)
d: 1.38 (s,
4.7. General synthetic procedure for the macrocyclization
reaction
18H),1.49 (s,18H), 2.34e2.43 (m, 4H), 2.70e2.74 (m, 8H), 3.00e3.30
(m, 4H), 3.42 (s, 4H), 4.00 (s, 4H), 7.40 (d, 2H, J¼7.5), 7.75 (t, 2H,
J¼7.6), 7.86 (d, 2H, J¼7.5). 13C NMR (75 MHz, CDCl3)
d: 28.1
To a stirred solution of tetramine 6 in anhydrous acetonitrile
(2.7ꢀ10ꢁ3 M) was added solid Na2CO3 (10 equiv). The suspension
was refluxed for 1 h under Argon, then dibromide derivative of the
heterocycle (1 equiv) was added in one portion and the mixture
was stirred at reflux for 24 h. The mixture was then cooled to rt,
filtered and concentrated in vacuo. The residue was treated with
CH2Cl2, filtered again and the solvent was removed under reduced
pressure to give a residue that was further purified as indicated
below for individual compounds.
(CH3), 28.2 (CH3), 51.4 (CH2), 52.5 (CH2), 52.7 (CH2), 56.2 (CH2), 58.1
(CH2), 60.5 (CH2), 80.6 (Cq), 81.0 (Cq), 120.7 (CH), 124.0 (CH), 137.0
(CH), 156.6 (Cq), 158.9 (Cq), 170.8 (Cq). MS (ESIþ): m/z (%) 805.6 (67)
[MþNa]þ, 783.7 (19) [MþH]þ, 299.4 (100) [(Mꢁ4ꢀC4H8)þHþK]2þ
.
4.9.2. Compound 11$Na. To a stirred solution of free ligand 11 in
acetonitrile was added solid NaCl (5 equiv). The suspension was
refluxed for 1 h, then cooled to rt, filtered, and the filtrate was
concentrated in vacuo. The residue was treated with CHCl3, filtered
again and the solvent was removed under reduced pressure to give
quantitatively the sodium complex 11$Na as a pale yellow solid. Mp
>250 ꢂC. Rf (alumina, CH2Cl2/MeOH 98:02): 0.20. IR nmax: 1728 (C]
4.8. 3,6,9,12,18-Pentaazabicyclo[12.3.1]octadeca-1(18),14,16-
triene-3,6,9,12-tetraacetic acid, 3,6,9,12-tetrakis(1,1-
dimethylethyl) ester (10) and its sodium complex 10$Na
O ester). UV/vis (CH3CN): lmax
(3
, L molꢁ1 cmꢁ1)¼242 (7800), 295
(9100) nm. 1H NMR (300 MHz, CDCl3)
d: 1.24 (s, 18H), 1.37 (s, 18H),
4.8.1. Compound 10. The reaction was carried out using tetramine
2.60e2.80 (m, 8H), 2.83e2.96 (m, 4H), 3.15e3.19 (m, 8H), 4.01 (s,
6
(150 mg, 0.25 mmol) and 2,6-bis(bromomethyl)pyridine 7
4H), 7.28 (d, 2H, J¼7.5), 7.87 (t, 2H, J¼7.5), 7.94 (d, 2H, J¼7.5). 13C
(66 mg, 0.25 mmol). The residue was purified by chromatography
on alumina (CH2Cl2/CH2Cl2/MeOH, 50:50) to give 100 mg of
a mixture of sodium complex 10$Na and free ligand 10 (25:75 ra-
tio). This mixture was dissolved in CHCl3 (50 mL) and sequentially
washed with saturated Na2EDTA aqueous solution (2ꢀ50 mL) and
water (2ꢀ50 mL). The organic layer was dried (Na2SO4) and evap-
orated under reduced pressure to give 10 (95 mg, 0.135 mmol, yield
NMR (75 MHz, CDCl3) d: 28.0 (CH3), 28.2 (CH3), 52.0 (CH2), 52.6
(CH2), 56.0 (CH2), 56.7 (CH2), 81.8 (Cq), 82.2 (Cq), 120.3 (CH), 123.7
(CH), 138.5 (CH), 154.7 (Cq), 157.8 (Cq), 170.8 (Cq), 171.4 (Cq). MS
(ESIþ): m/z (%) 805.7 (100) [MþNa]þ, 783.8 (17) [MþH]þ, 411.6 (92)
[(MþH)þK]2þ
,
383.6 (20) [(MꢁC4H8)þHþK]2þ
,
355.5 (25)
[(Mꢁ2ꢀC4H8)þHþK]2þ, 327.4 (21) [(Mꢁ3ꢀC4H8)þHþK]2þ, 299.5
(54) [(Mꢁ4ꢀC4H8)þHþK]2þ
.
54%) as a yellow oil. Rf (alumina, CH2Cl2/MeOH 99:01): 0.4. IR nmax
:
1733 (C]O ester). 1H NMR (300 MHz, CDCl3)
d
: 1.41 (s, 18H), 1.47 (s,
4.10. 13,16,19,22,28,29,30-Heptaazatetracyclo[22.3.1.12,6.17,11]
triacosa-1(28),2,4,6(29),7,9,11(30),24,26-nonaene-13,16,19,22-
tetraaceticacid,13,16,19,22-tetrakis(1,1-dimethylethyl)ester(12)
18H), 2.47 (s, 4H), 2.50e2.80 (m, 8H), 3.15 (s, 4H), 3.38 (s, 4H), 3.87
(s, 4H), 7.33 (d, 2H, J¼7.7), 7.60 (t, 1H, J¼7.7). 13C NMR (75 MHz,
CDCl3) d: 28.1 (CH3), 28.2 (CH3), 50.8 (CH2), 51.8 (CH2), 51.9 (CH2),
57.5 (CH2), 57.7 (CH2), 60.4 (CH2), 80.7 (Cq), 80.9 (Cq), 123.0 (CH),
The reaction was carried out using tetramine 6 (199 mg,
136.6 (CH), 158.4 (Cq), 170.7 (Cq), 170.8 (Cq). MS (ESIþ): m/z (%)
0.33 mmol) and 6,600-bis(bromomethyl)-2,20:6’,200-terpyridine
9
728.8 (28) [MþNa]þ, 260.5 (100) [(Mꢁ4ꢀC4H8)þHþK]2þ
.
(138 mg, 0.33 mmol). The residue was dissolved in CH2Cl2 (100 mL)
and sequentially washed with saturated Na2EDTA aqueous solution
(3ꢀ100 mL) and water (1ꢀ100 mL). The organic layer was dried
(Na2SO4) and evaporated under reduced pressure. The residue was
purified by chromatography on alumina (CH2Cl2/CH2Cl2/MeOH,
50:50) to give 12 (110 mg, 0.128 mmol, yield 39%) as a yellow oil. Rf
(alumina, CH2Cl2/MeOH 95:05): 0.3. IR nmax: 1733 (C]O ester). 1H
4.8.2. Compound 10$Na. To a stirred solution of free ligand 10 in
acetonitrile was added solid NaCl (5 equiv). The suspension was
refluxed for 1 h, then cooled to rt, filtered and the filtrate was
concentrated in vacuo. The residue was treated with CHCl3, filtered
again and the solvent was removed under reduced pressure to give

8602
G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
NMR (300 MHz, CDCl3)
d
: 1.36 (s, 9H), 1.48 (s, 27H), 2.47 (br s, 4H),
and the residue was re-dissolved three times in MeOH, then in
water and then rotary evaporated. The residue was then dissolved
in the minimum volume of MeOH and Et2O was added dropwise,
resulting in the formation of a precipitate, which was isolated after
centrifugation and dried under vacuum.
2.67e2.76 (m, 4H), 2.80e2.85 (m, 4H), 3.10 (s, 2H), 3.47 (br s, 6H),
4.09 (br s, 4H), 7.48e7.54 (m, 2H), 7.77e7.90 (m, 5H), 8.50 (br s, 2H).
13C NMR (75 MHz, CDCl3)
d: 28.0 (CH3), 28.2 (CH3), 51.6 (CH2), 52.3
(CH2), 52.7 (CH2), 55.9 (CH2), 56.9 (CH2), 59.7 (CH2), 80.5 (Cq), 80.9
(Cq), 120.8 (CH), 121.7 (CH), 123.1 (CH), 137.1 (CH), 137.4 (CH), 156.3
(Cq), 157.2 (Cq), 159.8 (Cq), 170.9 (Cq). MS (ESIþ): m/z (%) 898.4 (20)
[MþK]þ, 882.5 (100) [MþNa]þ, 860.5 (80) [MþH]þ. HRMS (ESIþ)
calcd for C47H69N7O8þHþ¼860.5286, found 860.5261 (100%).
4.13.2. Procedure B. The protected ligand precursor was added to
a
1:1 mixture of trifluoroacetic acid/CH2Cl2 (concentration
25ꢀ10ꢁ3 M) and the mixture was stirred at rt for 24 h. The mixture
was then treated as described in procedure A.
4.11. 3,6,9,12,18-Pentaazabicyclo[12.3.1]octadeca-1(18),14,16-
triene-3,6,9,12-tetraacetic acid,16-(methoxycarbonyl)-,
3,6,9,12-tetrakis(1,1-dimethylethyl) ester (15)
4.14. 3,6,9,12,18-Pentaazabicyclo[12.3.1]octadeca-1(18),14,16-
triene-3,6,9,12-tetraacetic acid (L1)
The reaction was carried out using tetramine 6 (150 mg,
0.25 mmol) and 4-carbomethoxy-2,6-bis(bromomethyl)pyridine
13 (80 mg, 0.25 mmol). The residue was dissolved in CH2Cl2
(100 mL) and sequentially washed with saturated Na2EDTA aque-
ous solution (3ꢀ100 mL) and water (1ꢀ100 mL). The organic layer
was dried (Na2SO4) and evaporated under reduced pressure. The
residue was purified by chromatography on alumina
(CH2Cl2/CH2Cl2/MeOH, 50:50) to give 15 (107 mg, 0.140 mmol,
yield 56%) as a yellow oil. Rf (alumina, CH2Cl2): 0.2. IR nmax: 1733
The reaction was carried out applying the procedure A on 10
(35 mg, 0.05 mmol). L1 was isolated as a pale yellow powder
(24 mg, 0.05 mmol, yield 100%). Mp >250 ꢂC. HPLC (System A):
tR¼6.83 min. UV/vis (Tris buffer, 50 mM, pH 7.4): lmax
(3,
L molꢁ1 cmꢁ1)¼264 (4300) nm. 1H NMR (300 MHz, D2O)
d: 3.42 (m,
4H), 3.55e3.66 (m, 8H), 3.71 (s, 4H), 3.87 (s, 4H), 4.67 (s, 4H), 7.59
(d, 2H, J¼7.8), 8.05 (t, 1H, J¼7.8). 13C NMR (75 MHz, D2O)
d: 51.0
(CH2), 51.6 (CH2), 53.0 (CH2), 54.8 (CH2), 56.7 (CH2), 58.1 (CH2),
124.7 (CH), 140.6 (CH), 150.4 (Cq), 170.3 (Cq), 172.3 (Cq). MS (ESIþ):
m/z (%) 558.3 (100) [MꢁHþ2 K]þ, 542.3 (59) [MꢁHþNaþK]þ. HRMS
(ESIþ) calcd for C21H31N5O8þHþ¼482.2251, found 482.2244 (100%).
(C]O ester). 1H NMR (300 MHz, CDCl3)
2.50e2.90 (m, 12H), 3.20e3.34 (m, 4H), 3.40 (s, 4H), 3.94 (s, 3H),
3.99 (s, 4H), 7.86 (s, 2H). 13C NMR (75 MHz, CDCl3)
: 28.2 (CH3),
d: 1.43 (s, 18H),1.50 (s, 18H),
d
50.9 (CH2), 51.9 (CH2), 52.0 (CH2), 52.5 (CH2), 56.1 (CH2), 57.4 (CH2),
57.5 (CH2), 60.1 (CH2), 80.9 (Cq), 81.1 (Cq), 122.1 (CH), 138.2 (Cq),
159.9 (Cq), 165.9 (Cq), 170.6 (Cq). MS (ESIþ): m/z (%) 786.8 (43)
[MþNa]þ, 764.9 (100) [MþH]þ.
4.15. 8,11,14,17,23,24-Hexaazatricyclo[17.3.1.12,6]tetracosa-1
(23),2,4,6(24),19,21-hexaene-8,11,14,17-tetraacetic acid (L2)
The reaction was carried out applying the procedure A on 11
(40 mg, 0.05 mmol). L2 was isolated as a pale yellow powder
(27.5 mg, 0.049 mmol, yield 98%). Mp >250 ꢂC. HPLC (System A):
4.12. 8,11,14,17,23,24-Hexaazatricyclo[17.3.1.12,6]tetracosa-1
(23),2,4,6(24),19,21-hexaene-8,11,14,17-tetraacetic acid, 4-
(methoxycarbonyl)-, 8,11,14,17-tetrakis(1,1-dimethylethyl)
ester, sodium complex (16$Na)
tR¼6.03 min. UV/vis (Tris buffer, 50 mM, pH 7.4): lmax
(3,
L molꢁ1 cmꢁ1)¼240 (7820), 304 (8950), 315sh (3340) nm. 1H NMR
(300 MHz, D2O) d: 3.20e3.40 (m, 8H), 3.49e3.60 (m, 4H), 3.63 (s,
4H), 3.67 (s, 4H), 4.63 (s, 4H), 7.88 (d, 2H, J¼7.4), 7.94 (t, 2H, J¼7.4),
The reaction was carried out using tetramine 6 (150 mg,
0.25 mmol) and 4-carbomethoxy-6,60-bis(bromomethyl)-2,20-
bipyridine 14 (100 mg, 0.25 mmol). The residue was purified by
chromatography on alumina (CH2Cl2/CH2Cl2/MeOH, 50:50) to
give 100 mg of a mixture of sodium complex 16$Na and free ligand
16 (70/30). This mixture was dissolved in CH3CN (9 mL), solid NaCl
(73 mg, 1.25 mmol) was added and the suspension was refluxed
for 1 h. The suspension was then cooled to rt, filtered, and the
filtrate was concentrated in vacuo. The residue was treated with
CHCl3, filtered again and the solvent was removed under reduced
pressure to give 16$Na (97 mg, 0.1 mmol, yield 40%) as a pale
yellow solid. Mp >250 ꢂC. Rf (alumina, CH2Cl2/MeOH 98:02): 0.25.
8.49 (d, 2H, J¼7.8). 13C NMR (75 MHz, D2O)
d: 51.0 (CH2), 51.2 (CH2),
51.9 (CH2), 54.5 (CH2), 55.6 (CH2), 57.1 (CH2), 123.7 (CH), 128.0 (CH),
147.9 (Cq), 151.4 (Cq), 165.7 (Cq), 172.6 (Cq). MS (ESIþ): m/z (%) 619.3
(61) [MꢁHþNaþK]þ, 597.5 (100) [MþK]þ. HRMS (ESIþ) calcd for
C26H34N6O8þKþ¼597.2075, found 597.2070 (50%).
4.16. 13,16,19,22,28,29,30-Heptaazatetracyclo[22.3.1.12,6.17,11]
triacosa-1(28),2,4,6(29),7,9,11(30),24,26-nonaene-13,16,19,22-
tetraacetic acid (L3)
The reaction was carried out applying the procedure B on 12
(65 mg, 0.075 mmol). L3 was isolated as a pale yellow powder
(44 mg, 0.069 mmol, yield 92%). Mp >250 ꢂC. HPLC (System A):
IR nmax: 1729 (C]O ester). 1H NMR (300 MHz, CDCl3)
d: 1.22 (s,
9H), 1.23 (s, 9H), 1.36 (s, 9H), 1.37 (s, 9H), 2.60e2.80 (m, 8H),
2.90e3.10 (m, 4H), 3.10e3.30 (m, 8H), 4.01 (s, 3H), 4.04 (s, 2H),
4.12 (s, 2H), 7.35 (d, 1H, J¼7.8), 7.80 (s, 1H), 7.92 (t, 1H, J¼7.8), 7.96
tR¼11.68 min. UV/vis (Tris buffer, 50 mM, pH 7.4): lmax
(3,
L molꢁ1 cmꢁ1)¼230 (14,900), 287 (10,700), 302 (9850) nm. 1H NMR
(300 MHz, DMSO-d6) d: 2.60e3.30 (m, 12H), 3.30e3.70 (m, 8H),
(d, 1H, J¼7.5), 8.41 (s, 1H). 13C NMR (75 MHz, CDCl3)
d: 27.90 (CH3),
3.90e4.20 (m, 4H), 7.53 (m, 2H), 7.80e8.26 (m, 5H), 8.45 (m, 2H).
27.95 (CH3), 52.1 (CH2), 52.9 (CH3), 55.9 (CH2), 56.0 (CH2), 56.9
(CH2), 81.8 (Cq), 81.9 (Cq), 82.3 (Cq), 119.4 (CH), 120.4 (CH), 122.7
(CH), 124.3 (CH), 138.6 (CH), 139.7 (Cq), 153.9 (Cq), 156.0 (Cq),
158.2 (Cq), 159.4 (Cq), 165.1 (Cq), 170.4 (Cq), 170.7 (Cq), 171.8 (Cq).
MS (ESIþ): m/z (%) 863.5 (100) [MþNa]þ, 841.6 (7) [MþH]þ, 328.1
13C NMR (75 MHz, DMSO-d6)
d: 50.2 (CH2), 50.5 (CH2), 51.5 (CH2),
55.0 (CH2), 55.7 (CH2), 59.2 (CH2),122.3 (CH),123.5 (CH),124.5 (CH),
138.7 (CH), 139.1 (CH), 156.5 (Cq), 157.3 (Cq), 158.7 (Cq), 170.8 (Cq),
172.6 (Cq). MS (ESIþ): m/z (%) 696.4 (73) [MꢁHþNaþK]þ, 674.5
(100) [MþK]þ, 658.5 (36) [MþNa]þ. HRMS (ESIþ) calcd for
C31H37N7O8þHþ¼636.2782, found 636.2754 (75%).
(65) [(Mꢁ4ꢀC4H8)þHþK]2þ
.
4.13. General synthetic procedure for hydrolysis of tert-butyl
ester functions
4.17. 8,11,14,17,23,24-Hexaazatricyclo[17.3.1.12,6]tetracosa-1
(23),2,4,6(24),19,21-hexaene-8,11,14,17-tetraacetic acid, 4-
(methoxycarbonyl) (L4)
4.13.1. Procedure A. The protected ligand precursor was added to
formic acid 99% (concentration 25ꢀ10ꢁ3 M) and the mixture was
stirred at 60 ꢂC for 24 h. The solution was concentrated in vacuo
The reaction was carried out applying the procedure B on 15
(50 mg, 0.065 mmol). L4 was isolated as a pale yellow powder

G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
8603
(35 mg, 0.065 mmol, yield 100%). Mp >250 ꢂC. HPLC (System A):
L molꢁ1 cmꢁ1)¼284 (9200), 292 (9500), 320 (7900) nm. Lumines-
cence (Tris buffer, lexc¼326 nm): lem (relative intensity, corrected
spectrum), 580 (2), 593 (31), 616 (100), 652 (5), 694 (76) nm.
tR¼6.68 min. 1H NMR (300 MHz, D2O)
d: 3.34 (s, 4H), 3.49 (m, 4H),
3.60 (m, 4H), 3.70 (s, 4H), 3.90 (s, 4H), 3.92 (s, 3H), 4.62 (s, 4H),
7.95 (s, 2H). 13C NMR (75 MHz, D2O)
d: 50.7 (CH2), 51.6 (CH2), 52.5
(CH2), 53.5 (CH3), 53.7 (CH2), 55.4 (CH2), 58.0 (CH2), 123.7 (CH),
140.6 (Cq), 152.4 (Cq), 166.0 (Cq), 169.8 (Cq), 171.5 (Cq). HRMS
(ESIþ) calcd for C23H33N5O10þHþ¼540.2306, found 540.2304
(100%).
4.19.6. Complex L3$Tb. MS (ESIꢁ): m/z (%) 790.3 (100) [(L3ꢁ3H)
TbꢁH]ꢁ. HPLC (System A): tR¼8.38 min. UV/vis (Tris buffer): lmax
(3,
L molꢁ1 cmꢁ1)¼284 (8000), 292 (8000), 320 (6800) nm. Lumines-
cence (Tris buffer, lexc¼326 nm): lem (relative intensity, corrected
spectrum), 488 (40), 544 (100), 585 (32), 621 (17) nm.
4.18. 8,11,14,17,23,24-Hexaazatricyclo[17.3.1.12,6]tetracosa-1
(23),2,4,6(24),19,21-hexaene-8,11,14,17-tetraacetic acid, 4-
(methoxycarbonyl) (L5)
References and notes
1. For examples, see: (a) Hermann, P.; Kotek, J.; Kubícek, V.; Luke, I. Dalton Trans.
2008, 3027e3047; (b) Port, M.; Raynal, I.; Vander Elst, L.; Muller, R. N.; Dioury,
F.; Ferroud, C.; Guy, A. Contrast Med. Mol. Imag. 2006, 1, 121e127; (c) Chan, K.
W.-Y.; Barra, S.; Botta, M.; Wong, W.-T. J. Inorg. Biochem. 2004, 98, 677e682; (d)
Aime, S.; Cavallotti, C.; Gianolio, E.; Giovenzana, G. B.; Palmisano, G.; Sisti, M.
Org. Lett. 2004, 6, 1201e1204.
2. For examples, see: (a) Wangler, C.; Schirrmacher, R.; Bartenstein, P.; Wangler, B.
Bioorg. Med. Chem. Lett. 2009, 19, 1926e1929; (b) Nayak, T. K.; Brechbiel, M. W.
Bioconjugate Chem. 2009, 20, 825e841; (c) Chong, H.-S.; Song, H. A.; Ma, X.;
Milenic, D. E.; Brady, E. D.; Lim, S.; Lee, H.; Baidoo, K.; Cheng, D.; Brechbiel, M.
W. Bioconjugate Chem. 2008, 19, 1439e1447; (d) De León-Rodríguez, L. M.;
Kovacs, Z. Bioconjugate Chem. 2008, 19, 391e402.
3. For examples, see: (a) Bunzli, J.-C. G. Chem. Rev. 2010, 110, 2729e2755; (b)
Montgomery, C. P.; Murray, B. S.; New, E. J.; Pal, R.; Parker, D. Acc. Chem. Res.
2009, 42, 925e937; (c) Gunnlaugsson, T.; Leonard, J. P. Chem. Commun. 2005,
3114e3131; (d) Sasamoto, K.; Horiguchi, D.; Nobuhara, M.; Mochizuki, H. Eur.
Pat. Appl. EP 493,745, 1992; Chem. Abstr. 1993, 118, 3409f.
4. Sabbatini, N.; Guardigli, M.; Lehn, J.-M. Coord. Chem. Rev. 1993, 123, 201e228.
5. For examples, see: (a) Atsumi, H.; Yoshimoto, K.; Saito, S.; Ohkuma, M.; Maeda,
M.; Nagasaki, Y. Tetrahedron Lett. 2009, 50, 2177e2180; (b) Hanaoka, K.; Kikuchi,
K.; Kobayashi, S.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13502e13509; (c)
Pandya, S.; Yu, J.; Parker, D. Dalton Trans. 2006, 2757e2766; (d) Faulkner, S.;
Carrié, M.-C.; Pope, S. J. A.; Squire, J.; Beeby, A.; Sammes, P. G. Dalton Trans.
2004, 1405e1409; (e) Quici, S.; Marzanni, G.; Cavazzini, M.; Anelli, P. L.; Botta,
M.; Gianolio, E.; Accorsi, G.; Armaroli, N.; Barigelletti, F. Inorg. Chem. 2002, 41,
2777e2784; (f) Beeby, A.; Bushby, L. M.; Maffeo, D.; Williams, J. A. G. Dalton
Trans. 2002, 48e54.
6. (a) Nasso, I.; Bedel, S.; Galaup, C.; Picard, C. Eur. J. Inorg. Chem. 2008,
2064e2074; (b) Nasso, I.; Galaup, C.; Havas, F.; Tisnès, P.; Picard, C.; Laurent,
S.; Vander Elst, L.; Muller, R. N. Inorg. Chem. 2005, 44, 8293e8305; (c) Galaup,
C.; Couchet, J.-M.; Bedel, S.; Tisnès, P.; Picard, C. J. Org. Chem. 2005, 70,
2274e2284.
7. (a) Tircso, G.; Tircsone-Benyo, E.; Hyun Suh, E.; Jurek, P.; Kiefer, G. E.; Sherry, A.
D.; Kovacs, Z. Bioconjugate Chem. 2009, 20, 565e575; (b) Tircso, G.; Kovacs, Z.;
Sherry, A. D. Inorg. Chem. 2006, 45, 9269e9280 and references cited therein.
8. (a) Maruyama, T.; Miho, T. Jpn Patent 63,128,017, 1988; Chem. Abstr. 1988, 109,
191057; (b) Gibby, W.A. U.S. Patent 4,822,594, 1989; Chem. Abstr. 1989, 111,
228130; (c) Nakabayashi, M.; Okabe, K.; Mishima, T.; Mano, H.; Haraya, K. Eur.
Patent 638,353, 1995; Chem. Abstr. 1995, 122, 191535; (d) Mori, T.; Ogawa, M.;
Amano, Y. Eur. Patent 708,335, 1996; Chem. Abstr. 1996, 125, 29567.
9. Bechara, G.; Leygue, N.; Galaup, C.; Mestre, B.; Picard, C. Tetrahedron Lett. 2009,
50, 6522e6525.
The reaction was carried out applying the procedure A on 16$Na
(33 mg, 0.035 mmol). L2 was isolated as a pale yellow powder
(19.5 mg, 0.032 mmol, yield 91%). Mp >250 ꢂC. HPLC (System B):
tR¼9.54 min. UV/vis (Tris buffer, 50 mM, pH 7.4): lmax
(3,
L molꢁ1 cmꢁ1)¼243 (9500), 315 (8050) nm. 1H NMR (300 MHz,
€
€
D2O) d: 3.00e3.24 (m, 4H), 3.26e3.43 (m, 4H), 3.44e3.58 (m, 4H),
3.63 (s, 2H), 3.70 (s, 2H), 3.75e3.93 (m, 4H), 4.04 (s, 3H), 4.52 (s,
2H), 4.87 (s, 2H), 7.90 (d, 1H, J¼5.1), 8.20 (br s, 1H), 8.51 (br s, 2H),
8.77 (br s, 1H). 13C NMR (75 MHz, D2O)
d: 50.2 (CH2), 50.9 (CH2),
52.0 (CH2), 52.5 (CH2), 53.6 (CH3), 53.8 (CH2), 54.6 (CH2), 55.1 (CH2),
55.5 (CH2), 55.9 (CH2), 57.1 (CH2), 58.2 (CH2), 63.6 (CH2), 122.5 (CH),
124.0 (CH), 125.9 (CH), 127.3 (CH), 129.7 (CH), 141.2 (Cq), 152.2 (Cq),
152.3 (Cq), 162.9 (Cq), 163.1 (Cq), 165.7 (Cq), 170.7 (Cq), 174.2 (Cq).
MS (ESIþ): m/z (%) 639.3 (80) [MþNa]þ, 617.3 (100) [MþH]þ. HRMS
(ESIþ) calcd for C28H36N6O10þNaþ¼639.2391, found 639.2374
(100%).
4.19. In situ preparation of the lanthanide complexes
To a solution of ligand (L1eL3) in water (2ꢀ10ꢁ3 M) was added
an equimolar amount of LnCl3$6H2O in water (2ꢀ10ꢁ3 M), con-
trolling the pH at 6.5 by simultaneous addition of diluted aqueous
NaOH solution. The reaction mixture was then allowed to stir for
24 h at rt and was then adjusted with Tris buffer (50 mM, pH 7.4) at
a final concentration of 1ꢀ10ꢁ4 M for absorption and 1ꢀ10ꢁ6 M for
emission spectroscopies.
4.19.1. Complex L1$Eu. MS (ESIꢁ): m/z (%) 630.1 (100) [(L1ꢁ3H)
EuꢁH]ꢁ. HPLC (System A): tR¼8.77 min. UV/vis (Tris buffer): lmax
(3,
L molꢁ1 cmꢁ1)¼266 (4900) nm. Luminescence (Tris buffer,
lexc¼268 nm): lem (relative intensity, corrected spectrum), 580 (4),
593 (34), 615 (100), 652 (7), 694 (85) nm.
10. For examples, see: (a) Balakrishnan, K. P.; Omar, H. A. A.; Moore, P.; Alcock, N. W.;
Pike, G. A. Dalton Trans. 1990, 2965e2969; (b) Costa, J.; Delgado, R. Inorg. Chem.
1993, 32, 5257e5265; (c) Autzen, S.; Korth, H.-G.; de Groot, H.; Sustmann, R.
Eur. J. Org. Chem. 2001, 3119e3125; (d) Herrera, A. M.; Staples, R. J.; Kryatov,
S. V.; Nazarenko, A. Y.; Rybak-Akimova, E. V. Dalton Trans. 2003, 846e856.
4.19.2. Complex L1$Tb. MS (ESIꢁ): m/z (%) 636.1 (100) [(L1ꢁ3H)
TbꢁH]ꢁ. HPLC (System A): tR¼8.73 min. UV/vis (Tris buffer): lmax
(3,
L molꢁ1 cmꢁ1)¼266 (4900) nm. Luminescence (Tris buffer,
lexc¼268 nm): lem (relative intensity, corrected spectrum), 488
(41), 544 (100), 584 (32), 620 (26) nm.
€€
11. (a) Hovinen, J.; Sillanpaa, R. Tetrahedron Lett. 2005, 46, 4387e4389; (b) Lin, Y.;
Favre-Réguillon, A.; Pellet-Rostaing, S.; Lemaire, M. Tetrahedron Lett. 2007, 48,
3463e3466.
12. For examples, see: (a) Stetter, H.; Frank, W.; Mertens, R. Tetrahedron 1981, 37,
767e772; (b) Newkome, G. R.; Pappalardo, S.; Gupta, V. K.; Fronczek, F. R. J. Org.
Chem. 1983, 48, 4848e4851; (c) Wang, T.; An, H.; Vickers, T. A.; Bharadwaj, R.;
Cook, P. D. Tetrahedron 1998, 54, 7955e7976; (d) An, H.; Cummins, L. L.; Griffey,
R. H.; Bharadwaj, R.; Haly, B. D.; Fraser, A. S.; Wilson-Lingardo, L.; Risen, L. M.;
Wyatt, J. R.; Cook, P. D. J. Am. Chem. Soc. 1997, 119, 3696e3708; (e) Aime, S.;
Botta, M.; Geninatti Crich, S.; Giovenzana, G. B.; Jommi, G.; Pagliarin, R.; Sisti, M.
Inorg. Chem. 1997, 36, 2992e3000; (f) Bazzicalupi, C.; Bencini, A.; Fusi, V.;
Giorgi, C.; Paoletti, P.; Valtancoli, B. Dalton Trans. 1999, 393e399; (g) Bazzica-
lupi, C.; Bencini, A.; Berni, E.; Bianchi, A.; Danesi, A.; Giorgi, C.; Valtancoli, B.;
Lodeiro, C.; Lima, J. C.; Pina, F.; Bernardo, M. A. Inorg. Chem. 2004, 43, 5134e5146;
(h) Dioury, F.; Ferroud, C.; Guy, A.; Port, M. Tetrahedron 2009, 65, 7573e7579.
13. Richman, J. E.; Atkins, T. J. J. Am. Chem. Soc. 1974, 96, 2268e2270.
14. (a) Aime, S.; Gianolio, E.; Corpillo, D.; Cavallotti, C.; Palmisano, G.; Sisti, M.;
Giovenzana, G. B.; Pagliarin, R. Helv. Chim. Acta 2003, 86, 615e632; (b) Hovland,
R.; Glogard, C.; Aasen, A. J.; Klaveness, J. Org. Biomol. Chem. 2003, 1, 644e647;
(c) Dioury, F.; Sambou, S.; Guéné, E.; Sabatou, M.; Ferroud, C.; Guy, A.; Port, M.
Tetrahedron 2007, 63, 204e214.
4.19.3. Complex L2$Eu. MS (ESIꢁ): m/z (%) 707.1 (100) [(L2ꢁ3H)
EuꢁH]ꢁ. HPLC (System A): tR¼7.05 min. UV/vis (Tris buffer): lmax
(3,
L molꢁ1 cmꢁ1)¼243 (7820), 305 (8950), 318sh (3450) nm. Lumi-
nescence (Tris buffer, lexc¼310 nm): lem (relative intensity, cor-
rected spectrum), 580 (2), 593 (31), 616 (100), 652 (5), 694 (72) nm.
4.19.4. Complex L2$Tb. MS (ESIꢁ): m/z (%) 713.1 (100) [(L2ꢁ3H)
TbꢁH]ꢁ. HPLC (System A): tR¼7.17 min. UV/vis (Tris buffer): lmax
(3,
L molꢁ1 cmꢁ1)¼243 (6100), 305 (7820), 318sh (4465) nm. Lumi-
nescence (Tris buffer, lexc¼310 nm): lem (relative intensity, cor-
rected spectrum), 490 (40), 544 (100), 585 (33), 621 (24) nm.
4.19.5. Complex L3$Eu. MS (ESIꢁ): m/z (%) 784.3 (100) [(L3ꢁ3H)
15. Ferroud, C.; Borderies, H.; Lasri, E.; Guy, A.; Port, M. Tetrahedron Lett. 2008, 49,
5972e5975.
EuꢁH]ꢁ. HPLC (System A): tR¼8.52 min. UV/vis (Tris buffer): lmax
(3,

8604
G. Bechara et al. / Tetrahedron 66 (2010) 8594e8604
16. (a) Pickersgill, I. F.; Rapoport, H. J. Org. Chem. 2000, 65, 4048e4057; (b) Davies,
J. S.; Al-Jamri, L. J. Peptide Sci. 2002, 8, 663e670; (c) Burdinski, D.; Lub, J.; Pik-
kemaat, J. A.; Moreno Jalón, D.; Martial, S.; Del Pozo Ochoa, C. Dalton Trans.
2008, 4138e4151.
29. (a) Weibel, N.; Charbonnière, L. J.; Guardigli, M.; Roda, A.; Ziessel, R. J. Am.
Chem. Soc. 2004, 126, 4888e4896; (b) Charbonnière, L.; Ziessel, R.; Guardigli,
M.; Roda, A.; Sabbatini, N.; Cesario, M. J. Am. Chem. Soc. 2001, 123, 2436e2437.
30. Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle, L.; de
Sousa, A. S.; Gareth Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin Trans. 2
1999, 493e503.
17. Pitt, C. G.; Bao, Y.; Thompson, J.; Wani, M. C.; Rosenkrantz, H.; Metterville, J. J.
Med. Chem. 1986, 29, 1231e1237.
18. Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis; Springer: Berlin,
1984; p 48.
19. Burdinski, D.; Pikkemaat, J. A.; Lub, J.; de Peinder, P.; Nieto Garrido, L.; Wey-
hermuller, T. Inorg. Chem. 2009, 48, 6692e6712.
31. The nonradiative temperature-dependent decay-rate constant knr(T) was
obtained by taking the difference of the reciprocal lifetimes in D2O at 298
and 77 K.
32. Laurent, S.; Vander Elst, L.; Wautier, M.; Galaup, C.; Muller, R. N.; Picard, C.
20. Maier, F. -K.; Bauer, M.; Krause, W.; Speck, U.; Schuhmann-Giampiere, G.;
Muhler, A.; Balzer, T.; Press, W. -R. U.S. Patent 5,885,548, 1999; Chem. Abstr.
1999, 130, 231398.
Bioorg. Med. Chem. Lett. 2007, 17, 6230e6233.
33. Siaugue, J.-M.; Segat-Dioury, F.; Favre-Reguillon, A.; Wintgens, V.; Madic, C.;
Foos, J.; Guy, A. J. Photochem. Photobiol., A 2003, 156, 23e29.
21. Chong, H.-S.; Ma, X.; Lee, H.; Bui, P.; Song, H. A.; Birch, N. J. Med. Chem. 2008, 51,
2208e2215.
22. Galaup, C.; Carrié, M.-C.; Tisnès, P.; Picard, C. Eur. J. Org. Chem. 2001, 2165e2175.
23. Scheytza, H.; Rademacher, O.; Reißig, H.-U. Eur. J. Org. Chem. 1999,
2373e2381.
34. (a) Cross, J. P.; Dadabhoy, A.; Sammes, P. G. J. Lumin. 2004, 110, 113e124; (b)
Ulrich, G.; Bedel, S.; Picard, C. Tetrahedron Lett. 2002, 43, 8835e8837; (c)
Galaup, C.; Azéma, J.; Tisnès, P.; Picard, C.; Ramos, P.; Juanes, O.; Brunet, E.;
Rodríguez-Ubis, J. C. Helv. Chim. Acta 2002, 85, 1613e1625; (d) Prodi, L.; Maestri,
M.; Ziessel, R.; Balzani, V. Inorg. Chem. 1991, 30, 3798e3802.
24. Havas, F.; Leygue, N.; Danel, M.; Mestre, B.; Galaup, C.; Picard, C. Tetrahedron
2009, 65, 7673e7686.
25. (a) Feely, W. E.; Beavers, E. M. J. Am. Chem. Soc. 1959, 81, 4004e4007; (b)
Autiéro, H.; Bazin, H.; Mathis, G. WO Patent. 096,877, 2001; Chem. Abstr. 2001,
136, 50664.
35. Xiao, M.; Selvin, P. R. J. Am. Chem. Soc. 2001, 123, 7067e7073.
36. Werts, M. H. V.; Verhoeven, J. W.; Hofstraat, J. W. J. Chem. Soc., Perkin Trans. 2
2000, 433e439.
37. In the case of L1$Eu, the addition of 10 equiv of EDTA led after 2 days to an
increase in the luminescence lifetime of the europium species (
suggesting the formation of a ternary adduct between L1$Eu and EDTA. This
prevents the determination of Kcond for L1$Eu.
s¼1.01 ms),
26. Latva, M.; Takalo, H.; Mukkala, V.-M.; Matachescu, C.; Rodriguez-Ubis, J.-C.;
Kankare, J. J. Lumin. 1997, 75, 149e169.
€
27. Mukkala, V.-M.; Sund, C.; Kwiatkowski, M.; Pasanen, P.; Hogberg, M.; Kankare,
38. Caravan, P.;Ellison, J.J.;McMurry, T. J.;Lauffer, R. B.Chem.Rev.1999, 99, 2293e2352.
39. Haas, Y.; Stein, G. J. Phys. Chem. 1971, 75, 3668e3677.
40. Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697e2705.
41. Meech, S. R.; Phillips, D. J. Photochem. 1983, 23, 193e217.
J.; Takalo, H. Helv. Chim. Acta 1992, 75, 1621e1632.
28. Kim, W. D.; Kiefer, G. E.; Maton, F.; McMillan, K.; Muller, R. N.; Sherry, A. D.
Inorg. Chem. 1995, 34, 2233e2243.