Time-resolved luminescence in aqueous solution - New europium labels derived from macro(bi)cyclic ligands with aminocarboxylic units

Article abstract of DOI:10.1002/1099-0690(200106)2001:11<2165::AID-EJOC2165>3.0.CO;2-F

A new family of macro(bi)cyclic ligands has been designed and synthesized, with the goal of developing lanthanide labels suitable for time-resolved luminescence-based biological applications. They are constructed from diamide (3) or tetralactam (4) complexing moieties associated with an intracyclic 2,2′-bipyridine chromophore and two exocyclic carboxylic groups. The luminescence properties of their Eu³⁺ complexes were studied in aqueous solution. In this medium, noticeable enhancements in the lifetime and quantum yield were observed, in comparison with those of other bipyridine-based ligands considered interesting as luminescent labels. The effects of coordinated water molecules, LMCT states, and ligand triplet state energy on the nonradiative deactivation processes to the ground state were examined. According to these results and in view of the stability of these complexes in aqueous solutions, the [Eu(3)]⁺ complex may be considered as a promising luminescent bioprobe.

Full text of DOI:10.1002/1099-0690(200106)2001:11<2165::AID-EJOC2165>3.0.CO;2-F

FULL PAPER
Time-Resolved Luminescence in Aqueous Solution ؊ New Europium Labels
Derived from Macro(bi)cyclic Ligands with Aminocarboxylic Units
[a]
[a]
[a]
[a]
´
`
Chantal Galaup, Marie-Christine Carrie, Pierre Tisnes, and Claude Picard*
Keywords: Cryptands / Lanthanides / Luminescence spectroscopy / Macrocyclic ligands
A new family of macro(bi)cyclic ligands has been designed
were observed, in comparison with those of other bipyridine-
and synthesized, with the goal of developing lanthanide la- based ligands considered interesting as luminescent labels.
bels suitable for time-resolved luminescence-based biolo-
gical applications. They are constructed from diamide (3) or
tetralactam (4) complexing moieties associated with an intra-
cyclic 2,2Ј-bipyridine chromophore and two exocyclic carb-
oxylic groups. The luminescence properties of their Eu3+
The effects of coordinated water molecules, LMCT states,
and ligand triplet state energy on the nonradiative deactiva-
tion processes to the ground state were examined. According
to these results and in view of the stability of these complexes
in aqueous solutions, the [Eu(3)]+ complex may be consid-
complexes were studied in aqueous solution. In this medium, ered as a promising luminescent bioprobe.
noticeable enhancements in the lifetime and quantum yield
Introduction
donors, has also been reported for homogeneous bioassays.
This technique has been applied to various models repres-
enting molecular and cellular processes.[10Ϫ12]
In time-resolved luminescence spectroscopy, the emission
is recorded after an initial delay after excitation, thus al-
lowing short-lived fluorescence background signals and
scattered excitation radiation to dissipate to zero. A signific-
ant improvement in the signal-to-noise ratio, and thus the
sensitivity, is obtained. Suitable organic fluorophores with
long luminescence lifetimes (τ Ͼ 50 ns) being very rare,
most research work has focused on EuIII and TbIII labels,
since these two ions display luminescence lifetimes greater
than 0.1 ms in aqueous solution, under ambient condi-
tions.[1] However, for practical bioanalytical purposes, or-
ganocomplexes of Eu3ϩ and Tb3ϩ ions are necessary for
labelling the analyte and for overcoming the low lumines-
cence efficiency of these aqueous ions, due to their weak
molar absorptivities.[2] In order to populate the lanthanide
excited state significantly, energy transfer from a sensitizer
is required. In this phenomenon (termed the ‘‘antenna
effect"), UV/Vis light energy is collected by allowed, chrom-
ophore-centered transitions (ε Ͼ 10000 Ϫ1·cmϪ1), fol-
lowed by intramolecular energy transfer from ligand to
LnIII, resulting in metal-centered luminescence.[3]
In recent years, a considerable research effort has been
directed towards two main classes of photoactive lanthan-
ide receptors: macrocyclic ligands such as cryptands or
branched macrocycles,[3,13Ϫ17] and aminopolycarboxylic
chelates such as diethylenetriaminepentaacetic acid (DTPA)
[18Ϫ20]
or bis(imino-diacetic acid)[21,22] derivatives. Two
structures, 1 and 2, of bipyridine-based compounds used
as luminescent labels for biological applications[23Ϫ25] and
representative of these classes of ligands are shown in
Scheme 1. Our interest in the properties of lanthanide
complexes[26Ϫ29] prompted us to investigate the preparation
of photoactive ligands presenting in the same structure
these two features: a macrocyclic framework and aminocar-
boxylic groups. We found only a few reports about lanthan-
ides bound by macrocyclic compounds incorporating both
an intracyclic chromophoric unit and pendant carboxylic
groups. Sherry has reported some luminescent properties of
Eu3ϩ and Tb3ϩ complexes with polyazamacrocyclic ligands
containing a pyridine moiety as part of the macrocyclic ring
and pendant acetate groups.[30] Sasamoto’s approach con-
sisted of incorporating two phenanthroline [or 4,7-bis(chlo-
rosulfonylphenyl)phenanthroline] moieties into an aza-
crown framework containing two pendant carboxylate
groups.[31] In his patent, he reported the use of the corres-
ponding luminescent europium complexes for the immuno-
assay technology developed by Diamandis and co-
workers.[7]
Under these conditions, luminescent Eu3ϩ and Tb3ϩ
complexes have many useful applications, especially in ana-
lytical and clinical chemistry: the biological analyte may be
a nucleic acid fragment, an enzyme or an enzyme substrate
or cofactor, or an antigen.[4Ϫ8] A detection limit of
5 ϫ 10Ϫ14  in aqueous solution has been reported with
sensitized EuIII ion, this lanthanide ion giving the highest
sensitivity.[9] Luminescence resonance energy transfer
(LRET), based on sensitized Eu3ϩ and Tb3ϩ ions as
We report here our results concerning the synthesis and
the luminescence properties of europium complexes derived
from new macrocyclic and macrobicyclic ligands 3 and 4,
respectively (Scheme 1). These incorporate a diamide 3 or
a tetralactam 4 complexing moiety in association with an
intracyclic 2,2Ј-bipyridine (bpy) chromophore and two ex-
ocyclic carboxylic groups. As well as this, to furnish better
[a]
`
´
´ ˆ
Synthese et Physicochimie de Molecules dЈInteret Biologique,
´
UMR CNRS 5068, Universite Paul Sabatier,
118 route de Narbonne, 31062 Toulouse cedex 04, France
Fax: (internat.) ϩ33Ϫ5 61 55 60 11
E-mail: picard@iris.ups-tlse.fr
Eur. J. Org. Chem. 2001, 2165Ϫ2175
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0611Ϫ2165 $ 17.50ϩ.50/0
2165

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C. Galaup, M.-C. Carrie, P. Tisnes, C. Picard
FULL PAPER
Scheme 1. Representative ligands from the literature (1, 2) and new
compounds (3, 4)
Scheme 2. Synthetic strategies towards ligands 3 and 4
understanding of the role played by the various factors that
determine the luminescence properties of this new lanthan-
ide receptor family, we studied the Gd3ϩ and Tb3ϩ com-
plexes of the macrocyclic ligand 3. Preliminary results from
this work have been published previously.[32]
benzylethylenediamine in a 2:1 molar ratio, gave the di-
amide diacid 10 in high yield.[33] Esterification of 10 with
ethylchloroformate according to the procedure reported by
S. Kim et al.,[34] followed by CF3COOH cleavage of the Boc
protecting group, gave the building block 13, although only
in 22% yield. The preparation of 13 was achieved more eas-
ily by using acetyl chloride-alcohol as reagent,[35] allowing
carboxylic acid esterification and N-tBoc deprotection of
10 in a single-step procedure. Thus, treatment of 10 with an
excess of acetyl chloride in ethanol solution at room tem-
Results and Discussion
Synthesis
The crucial step in the preparation of ligands 3 and 4 was perature afforded 13 in 55% yield. Condensation of 6,6Ј-
the macro(bi)cyclization reaction. Two general strategies for bis(bromomethyl)-2,2Ј-bipyridine with the secondary di-
these ring-closure reactions may be considered (Scheme 2): amine 13 in refluxing CH3CN and in the presence of
(1) acylation of diamine 5 or diazatetralactam 6 with activ- Na2CO3 as a base gave the NaBr complex of the macrocycle
ated diacid 8 and (2) alkylation of secondary diamine 9 with 15, which was purified by column chromatography. The
bis(bromomethyl)-2,2Ј-bipyridine (7). In process (1), pro- high yield of the macrocycle formation (67% yield of
duction of the macrocyclic product generally requires the [Na(15)]ϩ) obtained without the use of high-dilution tech-
use of high-dilution conditions ([reactants] Ͻ 2 mmol·LϪ1
)
niques is certainly the result of a template effect of the so-
to avoid predominant formation of oligomers. In the second dium ion. In this macrocyclization reaction, it is interesting
process, generation of the cyclic product may be controlled to note that replacement of ethyl ester groups by benzyl
by the presence of a metal ‘‘template’’ ion. Because of the groups in the diamine compound (14 vs. 13) produced a
presence, in the building blocks 9, of coordinating func- better cyclization yield. As a matter of fact, treatment of
tional groups that may interact with a metal ion, we pre- dibromide 7 with the diamine 14, carried out under condi-
ferred the second strategy.
tions identical to those for the formation of [Na(15)]ϩ, gave
Our synthesis of the 18-membered ring dilactam 3 is out- [Na(16)]ϩ in 93% isolated yield. This might be the result of
lined in Scheme 3. The approach involves the use of imino- restricted conformational freedom in 14, caused by the
diacetic acid, a core unit possessing three functionalization bulkier benzyl groups, and consequently a smaller loss of
sites. As a cyclic anhydride, this starting material is self- internal entropy in the cyclization process. Similar effects
activated for initial functionalization, liberating its second were reported earlier, when the bulky N-tosyl groups were
functionalization site (CO2H) upon reaction. In situ closure used in the formation of poly-N-tosylaza-crowns.[36] These
of N-Boc-iminodiacetic acid to the corresponding cyclic an- results suggested that a combination of template and en-
hydride (DCC, THF), followed by treatment with N,NЈ-di- tropy effects was operative in this cyclization process.
2166
Eur. J. Org. Chem. 2001, 2165Ϫ2175

Time-Resolved Luminescence in Aqueous Solution
FULL PAPER
groups may be considered as the result of the interaction of
1
the amide carbonyl groups and the sodium ion. In the H
NMR spectrum, the principal feature was the shift to
higher field of the H-3 heteroatomic hydrogens, as com-
pared to those in the open chain analogue 7 (δ ϭ 8.05 vs.
8.38). As reported earlier by Newkome, for macrocycles
containing 2,2Ј-bipyridine moieties,[38] this suggested that
the bipyridyl unit, possessing an anti orientation in 7, ap-
proached a syn conformation in these sodium complexes.
This was consistent with the participation of the two het-
erocyclic nitrogen atoms in the complexation.
The cleavage of the ester functions in compounds 15 and
16 was achieved under alkaline conditions. Hydrolysis of
ethyl and benzyl esters bonds using K2CO3 in refluxing
MeOH/H2O (3:1) and KOH in EtOH (0.5 ) at room tem-
perature, respectively, afforded the free diacid dilactam 3 in
65Ϫ80% yield. Attempted removal of the benzyl esters of
16 by catalytic hydrogenolysis (Pd/C in MeOH) was unsuc-
cessful. Recovery of unchanged 16 indicated poisoning of
the palladium on carbon catalyst by the heterocyclic nitro-
gens. The lack of reactivity of 16 provides additional sup-
port for recent observations concerning palladium catalyst
poisoning by 2,2Ј-dipyridyl.[39] 13C NMR spectroscopy gave
clear evidence of conformational heterogeneity in 3 in
CDCl3. Several sets of signals were observed for each type
of carbon atom. The presence of three sets of benzylic CH2
signals suggested that at least two species exist in solution
(most probably with the lactam groups assuming a cis-trans
and a trans-trans configuration). In CD3OD, only one set of
Scheme 3. Synthesis of the doubly branched macrocycle 3
The formulations [Na(15)]ϩ and [Na(16)]ϩ were consist-
ent with the Elemental analyses and spectral properties. Es-
ter and amide participation in sodium binding was estab-
lished by IR spectroscopy, comparing the solid state spectra
of the complexes and those of the open chain compounds
11 and 12, in which no intramolecular H-bonding occurs.
This comparison revealed a shift of the carbonyl stretching
frequencies to lower wavenumbers: the amide stretch band
dropped by ca. 20 cmϪ1 and the ester carbonyl band by 10
cmϪ1. Similar shifts were observed in chloroform solution.
This indicates simultaneous coordination of both types of
carbonyl oxygen in the complex. Confirmation of cation
1
complexation was provided by NMR techniques. In the H
decoupled 13C NMR spectrum (CDCl3) of these complexes,
only one set of signals was observed, clearly pointing to
the presence in solution of a single, symmetrical conformer.
Several signals for the carbons on the bipyridine ring were
shifted to lower fields (∆δ Ͼ 1 ppm), compared to the open
chain analogue (7), suggesting that the heterocyclic moiety
was involved in the complexation process. On the other
hand, the 13C chemical shift of the benzylic CH2 signals is
of particular diagnostic value and was used to distinguish
between the cis and the trans configurations (benzyl group
vs. carbonyl group) of the amide linkages in macrocyclic
polylactams (δ ഠ 48 and 51, respectively).[37] In [Na(15)]ϩ
and [Na(16)]ϩ complexes, the observation of a low field sig-
nal (δ Ն 51) for benzylic carbons indicated the presence of
a conformer with both lactam groups assuming the trans
configuration. This convergent arrangement of the lactam Scheme 4. Synthesis of the doubly branched cryptand 4
Eur. J. Org. Chem. 2001, 2165Ϫ2175
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C. Galaup, M.-C. Carrie, P. Tisnes, C. Picard
FULL PAPER
signals was observed, indicating that the higher symmetry
conformation predominates in this solvent.
All the photophysical properties of solutions of the com-
plexes in aerated methanol or water remained unchanged
The same strategy was applied to the synthesis of macro- after several days at room temperature, indicating that they
bicyclic ligand 4 (Scheme 4). Diamine 19 was obtained in are kinetically inert in these solvents. Absorption, emission
three steps, in an overall yield of 50%, starting from the lifetime, and quantum yield data for [Eu(3)]ϩ and [Eu(4)]ϩ
known diazatetralactam 6.[33] Condensation of this building are reported in Table 1, with the data for the previously
block 19 with dibromide 7 was carried out in the presence reported complexes with ligands 1 and 2 included for com-
of various alkali metal carbonates M2CO3 (M ϭ Li, Na, parison.[21,23,25] The absorption spectra of [Eu(3)]ϩ and
K, Cs). Analysis of the crude materials by size exclusion [Eu(4)]ϩ are dominated by the typical feature of the πϪπ*
chromatography[29] showed that macrocyclization process transition of the bpy chromophore. This absorption band
was markedly conditioned by cation template effects. Potas- is located at λmax ഠ 306 nm and its intensity is similar to
sium ion was found to be the best promoting ion for this that of [Eu(2)]Ϫ (one bpy unit) and about three times lower
macrocyclization reaction. Thus, with use of K2CO3 and than that of [Eu(1)]3ϩ(three bpy units). Excitation at this
after chromatographic purification on silica, macrobicycle wavelength gives entirely typical and intense europium(III)
20 was isolated as its KBr complex in 33% yield. No forma- emission spectra, containing the expected sequence of
tion of dimeric product (macrotricycle) was observed under 5D0Ǟ7Fj transitions, j ϭ 0 to 4 (Figure 1). Luminescence
these conditions. The lower cyclization yield seen for the excitation spectra monitored at the metal emission closely
formation of 20, as compared to 15, may be a function of matched the absorption bands of the bpy moiety. These re-
the size of the ring to be formed (24-membered ring vs. sults are indicative of the occurrence of an energy transfer
18-membered ring). Finally, base-catalyzed hydrolysis of 20 process from the sensitizing heterocyclic chromophore to
gave the target ligand 4 in almost quantitative yield.
the metal ion.
Complexation and Luminescence Properties
The europium complexes derived from 3 and 4 were read-
ily prepared by mixing equimolar amounts of ligands and
EuCl3·6H2O in methanol/water solutions and were isolated
by precipitation with diethyl ether (77 and 82% yield, re-
spectively). The same experimental procedure was used to
prepare the terbium and gadolinium complexes derived
from 3. All of these complexes were easily soluble in protic
solvents. They were characterized by electrospray mass
spectrometry (ESϩ-MS), which showed that 1:1 complexes
had been obtained. In the ESϩ-MS mass spectra, the most
abundant ion corresponded to [(L-2 H)Ln]ϩ species (L ϭ
ligand 3, 4); the assignment was confirmed by the agree-
ment with the observed and calculated isotope patterns, and
the m/z ϭ 1 a.m.u. separation between adjacent peaks, in-
dicating a singly charged cation. No peak ascribable to the
free ligands was observed in these mass spectra and no loss
Figure 1. Emission spectra of: a) [Eu(3)]ϩ, and b) [Eu(4)]ϩ in water
(5·10Ϫ6 ). The bands arise from 5Do Ǟ 7Fj transitions; the J values
are shown on the spectra
The emission spectra of [Eu(3)]ϩ and [Eu(4)]ϩ are similar
of metal ion was detected when Vc (extraction cone voltage) (Figure 1). In both cases, a 5D0
Ǟ
7F0 transition (at
was increased. This suggests a high affinity of ligands 3 and 581 nm), although relatively weak, is observed and ഠ50%
4 for lanthanide ions.
of the total emission is centered on the 617Ϫ618 nm peak
Table 1. Absorption and luminescence properties of the Eu3ϩ complexes
Luminescence
[a]
Absorption[b]
λmax
[nm]
Lifetimes[c]
τ (CH3CN)
[ms]
Quantum yields[d]
εmax
τ (CH3OH) τ (H2O) τ (D2O) τ (D2O, 77 K) Φ (H2O)
Φ (D2O)
[ϫ 102]
[Ϫ1·cmϪ1
]
[ms]
[ms]
[ms]
[ms]
[ϫ 102]
[Eu(3)]ϩ
[Eu(4)]ϩ
306
307
10700
10200
28000
9500
1.62
1.67
Ϫ
1.14
0.96
Ϫ
0.42
0.42
0.34
0.59
2.20
2.25
1.70
Ϫ
2.39
2.36
1.70
Ϫ
7.3
7.7
2
36
39
10
12
[Eu(1)]3ϩ [e] 303
[Eu(2)]Ϫ [f] 310
Ϫ
Ϫ
5
[a] In aerated solution at 300 K, unless otherwise noted. Ϫ [b] In water solution. Ϫ [c] Measured by excitation of the lowest-energy ligand-
7
centered absorption band and recording the emitted light intensity of the most intense metal emission band (5D0 Ǟ F2, 617Ϫ618 nm);
[d]
[e]
[f]
experimental error Ͻ10%. Ϫ
Excitation in ligand-centered bands; experimental error Ͻ30%. Ϫ Data from ref.[23]
Ϫ
Data from
ref.[21]
2168
Eur. J. Org. Chem. 2001, 2165Ϫ2175

Time-Resolved Luminescence in Aqueous Solution
7
FULL PAPER
(5D0 Ǟ F2 transition). Moreover, the ratios between the is a diagnostic feature for the degree of ligand-metal inter-
5
7
intensity of the D0 Ǟ F1 transition and the intensity of action in lanthanide complexes. The observed absorption
5
the D0 Ǟ 7F2 or 5D0 Ǟ 7F4 transitions are equal for these maximum is close to that previously reported for other
5
7
two complexes. The D0 Ǟ F1 transition is magnetic di- complexes of Eu3ϩ ion with macrocyclic ligands containing
polar in character and its radiative transition probability is bpy units.[3] Further information pertaining to the cation
relatively unaffected by the ligand environment.[1] In con- binding behavior of ligands 3 and 4 was obtained with the
trast, the 5D0 Ǟ 7F2 and 5D0 Ǟ 7F4 transitions are predom- aid of solid state (KBr disc) infrared spectroscopic data.
inantly electric dipolar in character and their emission in- In the two complexes, we observed shifts of the carboxyl
tensities are sensitive to the immediate coordination envir- stretching modes to lower frequency (∆ν ϭ 120 cmϪ1), re-
˜
onment of the EuIII. This implies similar symmetries in- vealing that all carboxyl groups were deprotonated and in-
duced by the ligands 3, 4, and the solvent around the volved in the binding of the lanthanide ion. On the other
Eu3ϩ ion.
hand, unlike with [Eu(3)]ϩ, two amide carbonyl stretching
frequencies were observed in [Eu(4)]ϩ: νCϭO ϭ 1652, 1603
The lifetimes of [Eu(3)]ϩ and [Eu(4)]ϩ in 5 ϫ 10Ϫ6

solutions were determined by excitation at 306Ϫ307 nm and cmϪ1, for free and bound carbonyl groups, respectively.
recording the intensity of the emitted light of the D0 Ǟ This suggests that some amide carbonyl groups are un-
5
7F2 transition. In these two cases, the data could be fitted bound in [Eu(4)]ϩ and is consistent with the inefficiency of
to monoexponential decay curves, indicating the presence 4, with respect to 3, in terms of improving the shielding
of only a single type of Eu3ϩ environment per complex, or of the lanthanide ion from the solvent molecules. In these
more than one Eu3ϩ environment in ‘‘fast exchange’’ on the complexes the metal ion is very probably in closer proximity
5D0 excited state timescale. From the results in Table 1, we to the bipyridine group.
may consider that the [Eu(3)]ϩ and [Eu(4)]ϩ lifetime values
Ligands 1, 2, and 3 have eight possible coordinating
in each solvent studied are equal within experimental error. atoms and the same chromophoric bpy unit, but their euro-
Interestingly, these values are rather high in CH3CN and pium(III) complexes exhibit significantly different photo-
are similar to or even greater than those reported for other physical properties. The luminescence lifetime value of
Eu3ϩ complexes with encapsulating ligands containing [Eu(3)]ϩ is intermediate between those reported for
the bipyridine chromophore, studied in the same sol- [Eu(1)]3ϩ and [Eu(2)]Ϫ, owing to the different hydration
vent.[13,17,40Ϫ42] On the other hand, the excited state life- state of the Eu3ϩ ion in these complexes. The solvation
times increase from H2O to MeOH to CH3CN. In aqueous parameter for [Eu(3)]ϩ (q ϭ 2) is smaller than that reported
solution, and upon solvent deuteration, the lifetimes are for the cryptate [Eu(1)]3ϩ (q ϭ 2.5), but higher than that of
also increased by a factor of 5.3, due to the greater effici- the chelate [Eu(2)]Ϫ (q ϭ 1). The difference in luminescence
ency of energy transfer to the vibration of OϪH bonds of quantum yields between [Eu(1)]3ϩ and [Eu(3)]ϩ in H2O at
coordinated water molecules in comparison with that to 300 K cannot be interpreted only on the basis of a better
OϪD bonds.[43,44] In [Eu(3)]ϩ and [Eu(4)]ϩ, the ion is not shielding offered in 3 towards interaction of the excited
entirely protected and is thus accessible and coordinated to state of the metal ion with OϪH oscillators. As a matter of
external species (CH3OH, H2O), as well as to the binding fact, the quantum yield of [Eu(3)]ϩ is higher in D2O, in
sites provided by the ligands. The inner-sphere water coor- which the shielding effect does not play any role. We may
dination number (q) of the lanthanide ion can be calculated also note that [Eu(3)]ϩ exhibits a quantum yield in D2O
by using the conventional equation of Horrocks or the three times higher than that of [Eu(2)]Ϫ. These results sug-
more recent Parker equation, which takes into account the gest that the efficiency of the bipyridine collector for energy
contribution of unbound closely diffusing water molec- transfer to the metal is larger in [Eu(3)]ϩ. An estimation of
ules.[45,46] The q values calculated with these empirical cor- this efficiency may be obtained with the aid of the quantity
relations reveal that both [Eu(3)]ϩ and [Eu(4)]ϩ complexes ηen.tr., which represents the energy conversion efficiency
have two inner-sphere coordinated water molecules. No un- from the absorbing singlet state of the ligand to the emitting
bound molecule should be present in the second coordina- state of the metal ion.[3] Usually, and with the assumption
tion sphere of the metal ion. Although 4 provides addi- that the decay process at 77 K in deuterated water is purely
tional donor atoms (four amide carbonyls) and is poten- radiative, ηen.tr. can be calculated by Equation (1).
tially a better encapsulating structure, as compared to 3, the
lanthanide ion is not shielded more effectively from the
water molecules when complexed by this cryptand ligand.
77K
η
en.tr. ϭ Φ·τ
/τ 300K
D O H O
2 2
(1)
The estimated ηen.tr. value is 0.42 for [Eu(3)]ϩ (Table 2)
We may also note that the luminescence quantum yield of and is among the highest obtained, in aqueous solution,
[Eu(4)]ϩ (in H2O and D2O) is analogous to that of [Eu(3)]ϩ. for Eu3ϩ complexes with bipyridine-based ligands.[3,42,47] In
This indicates a similar efficiency of ligand-to-metal energy particular, we note a marked increase in this value, by a
transfer, and suggests a similar metal-bipyridine interac- factor of four, in [Eu(3)]ϩ, as compared to [Eu(1)]3ϩ. With
tion. This is in agreement with the complexation character- regard to [Eu(2)]Ϫ, direct comparison is not possible since
istics of 3 and 4 as deduced from their ultraviolet spectro- all the reported measurements for this complex were carried
scopic data. Both [Eu(3)]ϩ and [Eu(4)]ϩ display an absorp- out at 300 K.[21,25]
tion band with a maximum at 306Ϫ307 nm. This band, at-
The efficiency of ligand-to-LnIII energy transfer and the
tributed to the πϪπ* transition of the coordinated bpy unit, LnIII luminescence quantum yield are greatly dependent on
Eur. J. Org. Chem. 2001, 2165Ϫ2175
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C. Galaup, M.-C. Carrie, P. Tisnes, C. Picard
FULL PAPER
As far as knr is concerned, it appears that high frequency
vibrational CϪH modes of the ligand 3 have a modest
quenching effect, as suggested by the long luminescent life-
time of [Eu(3)]ϩ in D2O (2.2 ms), compared to 2.3Ϫ2.6 ms
Table 2. Energy transfer (ηen.tr.), decay characteristics, and number
of coordinated water molecules (q)
[a]
[b]
[c]
ηen.tr.
kr
knr (T)[d]
[sϪ1
knr (OH)[e]
[sϪ1
q[f]
[sϪ1
]
]
]
ϩ
for free ions in this solvent [knr(CH)lig ϭ (1/τ[Eu(3)] Ϫ 1/
τEuCl
)
Ͻ 100 sϪ1].[48] Quenching by OϪH vibrations is
D O
[Eu(3)]ϩ
0.42
0.10
0.53[i]
ഠ1
420
590
370
260
Ͻ 50
Ͻ 50
115
1930
2400
480
2.0
2.5
2.0
2.7
3
2
dominant; the calculated rate constant knr(OH) is 1930 sϪ1
for [Eu(3)]ϩ, and thus a contribution of ca 1000 sϪ1 per
coordinated water molecule or 500 sϪ1 per OϪH oscillator.
This value is in good agreement with the literature data.[44]
We also note that the hydration state values obtained for
[Eu(1)]3ϩ [g]
[Tb(3)]ϩ [h]
[Tb(1)]3ϩ [g]
1300
630
[a]
[b]
[c]
In water solution. Ϫ Obtained from Equation (1). Ϫ From
the lifetimes in D2O solution at 77 K, assumed to be purely radiat-
[d]
ive; kr ϭ 1/τ7D7K. Ϫ
From the lifetimes in D2O (τD) solution at [Tb(3)]ϩ and [Eu(3)]ϩ are in good agreement with one an-
300 and 77 K; knr(T) ϭ 1/τ3D00K Ϫ1/τ7D7K. Ϫ [e] From the lifetimes in
H2O (τH) and D2O (τD) solutions at 300 K; knr(OH) ϭ 1/τ3H00K Ϫ1/
other, at 2.0 in each case. The less efficient quenching by
OϪH vibrations in [Tb(3)]ϩ [knr(OH) ϭ 480 sϪ1] is the re-
sult of the larger energy gap between the lowest luminescent
excited state and the highest ground state for the Tb3ϩ ions
[f]
τ3D00K. Ϫ Number of coordinated water molecules at 300 K, as
obtained from HorrocksЈ empirical Equation in water:[45] q ϭ A(1/
τH Ϫ 1/τD), τH and τD in ms, A ϭ 1.05 and 4.2 for the Eu3ϩ and
[g]
[h]
Tb3ϩ compounds, respectively. Ϫ Data from ref.[3]. Ϫ Values
of the experimental excited state lifetimes under various conditions
are given in the Exp. Section. Ϫ [i] Calculated with the assumption
that the equilibrium between the emitting state of the Tb3ϩ ion and
the triplet excited state of the ligand does not play an important
role.
7
(5D4 Ǟ F0 gap at about 14200 cmϪ1 for Tb3ϩ compared
5
7
to D0 Ǟ F6 gap at about 12300 cmϪ1 for Eu3ϩ).[49]
The calculated knr(T) value is small (Ͻ 50 sϪ1) for
[Eu(3)]ϩ, showing that nonradiative deactivation of the
europium emitting states by ligand-to-metal charge transfer
states (LMCT states) is negligible. However, the decay of
the [Tb(3)]ϩ complex is more dependent on the temper-
ature. This suggests that back transfer from the MC level
to triplet LC level excited state may take place in [Tb(3)]ϩ,
as observed for the Tb3ϩ complexes of bpy-based li-
gands.[3,41,42,47] However, the knr(T) value estimated for
[Tb(3)]ϩ is lower than that obtained for [Tb(1)]3ϩ by one
order of magnitude (Table 2). In order to gain better insight
into the nature of the temperature-dependent quenching
processes, we examined energy matching between the triplet
state level of the ligand 3 and the resonance level of the
metal ion. The [Gd(3)]ϩ complex was used to determine the
triplet state energy of the ligand in a structure analogous to
that of the Tb3ϩ and Eu3ϩ complexes. In a rigid matrix
at 77 K, [Gd(3)]ϩ showed ligand phosphorescence emission
(Figure 2) with a maximum at 481 nm and a lifetime of 3
ms. From the highest energy phosphorescence feature of
the distance between the chromophore and the complexed
LnIII ion. However, the quenching mechanisms involving
inner-sphere coordinated water molecules or ligand-to-LnIII
charge transfer (LMCT) and the energy level of the lowest
triplet state of the ligand are also important factors contrib-
uting to the luminescence properties of a lanthanide com-
plex. In order to analyze the factors that determine the lu-
minescence intensity of LnIII complexes derived from the
promising ligand 3, we evaluated the contributions of the
different paths to the decay of the luminescent excited states
of [Eu(3)]ϩ and [Tb(3)]ϩ complexes. For [Ln(3)]ϩ complexes
in aqueous solution, the overall decay rate constant kobs
(i.e., 1/τobs) of the luminescent level may be represented by
Equation (2).
kobs ϭ kr ϩ knr(OH) ϩ knr(CH)lig ϩ knr(T)
(2)
The term kr is the natural rate constant for emission of this spectrum, the value of the zero-zero energy for the 3ππ*
photons, while the terms knr represent the rate constants for ligand level in [Ln (3)]ϩ complexes is estimated to be 22200
radiationless deexcitation processes involving coupling with cmϪ1. Such a value is about 5000 and 1800 cmϪ1 higher
high-energy vibrations (OϪH, CϪH oscillators in the first than the 5D0 europium(III) and the 5D4 terbium(III) excited
coordination sphere). The term knr(T) accounts for temper- states, respectively. From their study on the luminescent
ature-dependent, radiationless processes, which can play an properties of 41 different TbIII chelates, Latva et al. con-
important role when short-lived, upper lying excited states cluded that deactivation of the emitting level through back
are thermally accessible. Population of ligand excited states energy transfer to the ligand (Tb* Ǟ 3ππ*) occurs when the
3
(by back transfer) or population of LMCT excited states energy difference between the ligand ππ* and the metal
from the Ln3ϩ emitting state are representative of such de- 5D4 excited states is less than about 1850 cmϪ1 [21]
activating processes.
.
The en-
ergy gap measured for [Gd(3)]ϩ is close to that minimum
With the usual assumption that the quenching by XϪD difference, offering the possibility of thermally activated
vibrations can be neglected, the contributions of the various population of the low-lying ligand triplet state in [Tb(3)]ϩ.
terms expressed in Equation (2) can be evaluated by com- In comparison with [Tb(1)]3ϩ, in which the lowest bpy trip-
paring the results obtained in non-deuterated and deuter- let excited state lies at lower energy (21600 cmϪ1),[23] this
ated solvents and at low (77 K) and higher (300 K) temper- radiationless deexcitation process is one order of magnitude
ature.[3] The values of decay rate constants obtained in H2O less efficient in [Tb(3)]ϩ.
solution for [Eu(3)]ϩ and [Tb(3)]ϩ are collected in Table 2;
In conclusion, the main quenching pathway for the Eu3ϩ
the decay characteristics of [Eu(4)]ϩ are similar to those and Tb3ϩ luminescence in [Ln(3)]ϩ complexes is the prod-
of [Eu(3)]ϩ.
uct of the high-energy vibrational modes of two coordin-
2170
Eur. J. Org. Chem. 2001, 2165Ϫ2175

Time-Resolved Luminescence in Aqueous Solution
FULL PAPER
represents a very readily available prototype for luminescent
applications. The photophysical behavior of the studied
complexes is related to an efficient ligand to LnIII energy
transfer in conjunction with a close proximity of the bipyr-
idine chromophore and the metal ion. A detailed analysis
of the photophysical properties showed that the main
quenching pathway for Eu3ϩ and Tb3ϩ luminescence in
these complexes is a function of the high-energy vibrational
modes of two coordinated water molecules. Our synthetic
approach permits substantial variation of the heterocyclic
moiety of the prototype ligand 1, and we are currently in-
vestigating the introduction of more intensely absorbing
chromophoric units in this series of macrocycles with the
aim of obtaining more intense luminescence.
Figure 2. Emission spectra of [Gd(3)]ϩ (Ϫ·Ϫ·Ϫ) in a ethanol/meth-
anol 4:1 rigid matrix at 77 K, [Tb(3)]ϩ (——) and [Eu(3)]ϩ (- - -)
in H2O at 300 K
Experimental Section
ated water molecules. On the other hand, the higher lumin-
escence lifetime value [τ (H2O) ϭ 1.04 ms] and quantum
yield [Φ (H2O) ϭ 0.20] of [Tb(3)]ϩ, as compared to those
of the corresponding Eu3ϩ complex, may be explained by
less efficient quenching of the Tb3ϩ ion by coordinated
water molecules (vide supra).
General: Melting points were determined on a Kofler apparatus. Ϫ
1H magnetic resonance spectra were recorded on Bruker AC 200
or 250 spectrometers. Data are reported in the following order:
chemical shift δ in ppm, spin multiplicity, integration and assign-
ment. Ϫ 13C magnetic resonance spectra were recorded on Bruker
AC 200 or 250 spectrometers. The multiplicity of signals (n) is given
under the interval. Ϫ Infrared spectra were recorded on a
Finally, a luminescent lanthanide complex suitable for bi-
omedical applications should be characterized by good kin-
etic stability with respect to metal ion dissociation. Indeed,
any biological sample is a highly complex mixture con-
taining a wide variety of molecules and ions that might po-
tentially quench the luminescent label. As reported (vide
supra), the [Ln(3)]ϩ complexes (Ln ϭ Eu, Tb) were found
to be stable in coordinating solvents and water. The kinetic
inertness of these complexes in the presence of other com-
peting metal ions that might be involved in a cation-pro-
moted dissociation pathway was also studied. No dissoci-
ation was observed after several days when the [Ln(3)]ϩ
complexes (0.01 m) were incubated at 37 °C and physiolo-
gical pH, in the presence of an excess of calcium (126 m,
which is the concentration of Ca2ϩ in human serum), an-
other ion presenting features similar to those of europium.
Luminescence intensities and lifetimes of these complexes
also remained unchanged in the presence of other abundant
human serum cations (Mg2ϩ, 0.8 m; Zn2ϩ, 0.01 m; Naϩ,
140 m; Kϩ, 5 m).
PerkinϪElmer spectrometer [ν cmϪ1] in potassium bromide. Ϫ Pos-
˜
itive ESI-MS spectra [mass range 400Ϫ2000, m/z (relative intens-
ity%)] were recorded with a PerkinϪElmer SCIEX API 100 appar-
atus, in methanol solution. Positive FAB-MS spectra were recorded
with a Nermag R10Ϫ10C mass spectrometer in a 3-nitrobenzyl al-
cohol (NBA) matrix. Ϫ Elemental analyses were carried out by
´ ´
the ‘‘Service Commun de Microanalyse elementaire UPS-INP’’ in
Toulouse. Ϫ UV spectra [λ nm (ε Ϫ1·cmϪ1)] were measured on a
PerkinϪElmer Lambda 17 spectrophotometer. The luminescence
spectra [λ nm (relative intensity%)] were obtained with a LS-50B
PerkinϪElmer spectrofluorimeter equipped with a Hamamatsu-
R928 photomultiplier tube and with the low-temperature accessory
no. L2250136. The decay of the metal emitting state was recorded
using the same instrument and analyzed with a least-squares fitting
program. The luminescence quantum yields were obtained by the
method described by Haas and Stein[50] with the standards
[Ru(bipy)3]2ϩ (Φ ϭ 0.028 in aerated water[51]) for the Eu3ϩ complex
and quinine sulfate (Φ ϭ 0.546 in H2SO4 1 [52]) for the Tb3ϩ
complex. The measured values were corrected for the refractive in-
dices.
Starting Materials: N-tert-Butoxycarbonyliminodiacetic acid, diaz-
atetralactam 6, and diamide diacid 10 were prepared as described
in the literature.[33]
Conclusion
The results described in this paper represent an efficient
means of access to a novel class of macro(bi)cyclic ligand-
based lanthanide complexes. The introduction of two carb-
oxylic units as pendant arms in these ligands gives rise to
solubility and stability in the complexes in aqueous solu-
tions. The corresponding Eu3ϩ complexes show lumines-
cence typical of the metal and display equivalent lumines-
cent properties. They have relatively high luminescence
quantum yields (Φ ϭ 0.36 in D2O) in comparison with
complexes of other representative cryptands or polyamino-
carboxylic ligands containing the bipyridine chromophore.
6,6Ј-Bis(bromomethyl)-2,2Ј-bipyridine was prepared in five steps,
starting from 6-bromo-2-methylpyridine. 6-Bromo-2-methylpyrid-
ine was reductively coupled using the reactive reagent
Ni[P(C6H5)3]4,[53] generated in situ, to give 6,6Ј-dimethyl-2,2Ј-bi-
pyridine in 84% yield. The two methyl groups were then converted
into bismethanol groups (62% overall yield) by the procedure de-
scribed by Newkome.[54] 6,6Ј-Bis(hydroxymethyl)-2,2Ј-bipyridine
was further transformed into 6,6Ј-bis(bromomethyl)-2,2Ј-bipyrid-
ine with PBr3, using the standard method (51% yield). White solid,
m.p. 180Ϫ182 °C (ref.[55] 180Ϫ181 °C).
Diamide Diester 11: Ethyl chloroformate (860 µL, 9 mmol) was ad-
The simplest ligand 3, obtained in four high-yielding steps, ded at 0 °C to a solution of diamide diacid 10 (2 g, 3 mmol) and
Eur. J. Org. Chem. 2001, 2165Ϫ2175 2171

´
`
C. Galaup, M.-C. Carrie, P. Tisnes, C. Picard
FULL PAPER
triethylamine (1.25 mL, 9 mmol) in dichloromethane (20 mL).
added every 15 h, three times in all. After removal of the solvent,
After 10 min. of stirring at 0 °C, DMAP (0.18 g, 1.47 mmol) was the residue was dissolved in ethyl acetate (40 mL) and washed with
added and the resulting solution was stirred for 20 h at room tem-
satd. aqueous NaHCO3 solution (2 ϫ 20 mL) and water (2 ϫ
perature. The reaction mixture was washed with 1  NaOH 20 mL). The organic layer was dried over Na2SO4 and the solvent
(10 mL), 1  HCl (10 mL), and water (10 mL). The dichlorome- was removed to afford the pure product 13 (0.22 g, 55%) as a pale
thane solution was dried over Na2SO4, evaporated to dryness, and
chromatographed (silica gel, CH2Cl2/CH3OH, 98:2) to afford 11
(0.88 g, 40%) as a pale yellow oil. Ϫ 1H NMR (250 MHz,
[D6]DMSO, 25 °C): δ ϭ 1.14Ϫ1.22 (m, 6 H, CH3), 1.23Ϫ1.34 (m,
18 H, CH3), 3.31Ϫ3.37 (m, 4 H, NCH2CH2N), 3.90Ϫ4.20 (m, 12
H, NCH2), 4.40Ϫ4.53 (m, 4 H, OCH2), 7.10Ϫ7.34 (m, 10 H, CH
Ar). Ϫ 13C NMR (63 MHz, [D6]DMSO, 25 °C): δ ϭ 14.0 (CH3),
27.7 (CH3 Boc), 43.1Ϫ43.8 (m, NCH2CH2N), 48.4Ϫ50.2 (m,
NCH2), 60.3 (OCH2), 79.3, 79.4 (Cq Boc), 126.5Ϫ128.6 (m, CH
Ar), 136.9Ϫ137.8 (n Ն 4, Cq Ar), 154.5Ϫ154.9 (n Ն 3, CO Boc),
168.3Ϫ168.6 (n Ն 2, CO amide), 169.6 (CO ester). Ϫ IR (KBr):
yellow oil.
Diamine 14: The same procedure as described for 13 was followed,
using diamide diester compound 12 to yield 14 in 95% yield. White
solid, m.p. 130Ϫ132 °C. Ϫ 1H NMR (250 MHz, [D6]DMSO, 25
°C): δ ϭ 3.00Ϫ3.43 (m, 14 H, NH, NCH2), 4.45Ϫ4.55 (m, 4 H,
NCH2Ar), 5.08, 5.13 (s, 4 H, OCH2), 7.18Ϫ7.36 (m, 20 H, CH Ar).
Ϫ
13C NMR (63 MHz, [D6]DMSO, 25 °C): δ ϭ 43.1Ϫ44.3 (m,
NCH2CH2N), 47.8Ϫ50.2 (m, NCH2), 65.39, 65.41 (OCH2),
126.5Ϫ128.6 (m, CH Ar), 135.9 (Cq Ar), 137.1Ϫ137.8 (n ϭ 4, Cq
Ar), 170.4Ϫ171.0 (n ϭ 4, CO amide), 171.6 (CO ester). Ϫ IR
(KBr): ν˜ ϭ 3286 (NH), 1731 (CO ester), 1648 (CO amide) cmϪ1
.
ν ϭ 1745 (CO ester), 1698 (CO Boc), 1662 (CO amide) cmϪ1. Ϫ
˜
MS (ESϩ, CH3OH): m/z (%) ϭ 727.3 (100) [M ϩ H]ϩ. Ϫ
C38H54O10N4 (726.9): calcd. C 62.79, H 7.49, N 7.71; found C
62.66, H 7.45, N 7.29.
Ϫ C38H42O6N4 (650.8): calcd. C 70.13, H 6.51, N 8.61; found C
70.03, H 6.46, N 8.20.
Doubly Branched Dilactam 15: A mixture of 13 (0.40 g, 0.76 mmol)
and Na2CO3 (0.81 g, 7.64 mmol) in anhydrous acetonitrile
(380 mL) under argon was heated to reflux for 1.5 h. 6,6Ј-Bis(bro-
momethyl)-2,2Ј-bipyridine (0.26 g, 0.76 mmol) was then added in
one portion. The resulting suspension was refluxed, with efficient
magnetic stirring, for a further 24 h. After cooling to room temper-
ature, the insoluble solid was filtered off and the filtrate evaporated.
HPLC (silica gel, CH2Cl2, then CH2Cl2/CH3OH/Et3N: 90:10:1) af-
forded the NaBr complex of 15 (0.43 g, 67%) as a pale yellow solid,
m.p. 140Ϫ142 °C. Ϫ 1H NMR (250 MHz, CDCl3, 25 °C): δ ϭ 1.30
Diamide Diester 12: This compound was prepared in similar man-
ner to compound 11, starting from the diamide diacid 10 (1 g,
1.50 mmol), triethylamine (540 µL, 3.90 mmol), benzyl chloro-
formate (560 µL, 3.90 mmol), and DMAP (0.09 g, 0.74 mmol).
HPLC (silica gel, CH2Cl2/CH3OH, 100:0 Ǟ 98:2) gave 12 (0.88 g,
69%) as a colorless oil. Ϫ 1H NMR (250 MHz, CDCl3, 25 °C):
δ ϭ 1.32Ϫ1.39 (m, 18 H, CH3), 3.00Ϫ3.55 (m, 4 H, NCH2CH2N),
4.00Ϫ4.24 (m, 8 H, NCH2CO), 4.24Ϫ4.60 (m, 4 H, NCH2Ar),
5.10Ϫ5.15 (m, 4 H, OCH2), 7.17Ϫ7.35 (m, 20 H, CH Ar). Ϫ 13C
NMR (63 MHz, CDCl3, 25 °C): δ ϭ 28.1, 28.3 (CH3), 42.4Ϫ43.5
(m, NCH2CH2N), 48.5Ϫ50.1 (m, NCH2), 66.6 (OCH2), 80.7Ϫ80.9
(n Ն 3, Cq Boc), 126.3Ϫ129.1 (m, CH Ar), 135.6Ϫ136.3 (n Ն 4,
Cq Ar), 155.3 (CO Boc), 169.4Ϫ169.7 (n Ն 2, CO amide), 170.1 (n
Ն 2, CO ester). Ϫ IR (KBr): ν˜ ϭ 1749 (CO ester), 1704 (CO Boc),
1660 (CO amide) cmϪ1. Ϫ MS (FABϩ, NBA): m/z (%) ϭ 851 (7)
[M ϩ H]ϩ, 751 (25) [(M Ϫ Boc) ϩ 2H]ϩ, 651 (100) [(M Ϫ 2Boc)
ϩ 3H]ϩ. Ϫ C48H58O10N4 (851.0): calcd. C 67.75, H 6.87, N 6.58;
found C 67.41, H 6.80, N 6.40.
3
(t, J ϭ 7.1 Hz, 6 H, CH3), 3.43Ϫ4.50 (m, 20 H, NCH2), 4.10 (q,
3J ϭ 7.1 Hz, 4 H, OCH2), 6.73Ϫ6.75 (m, 4 H, CH Ar), 7.17Ϫ7.21
(m, 6 H, CH Ar), 7.35 (d, 3J ϭ 7.8 Hz, 2 H, CH bpy 5,5Ј), 7.86 (t,
3J ϭ 7.8 Hz, 2 H, CH bpy 4,4Ј), 8.05 (d, 3J ϭ 7.8 Hz, 2 H, CH
bpy 3,3Ј). Ϫ 13C NMR (63 MHz, CDCl3, 25 °C): δ ϭ 14.1 (CH3),
45.6 (NCH2CH2N), 51.0 (NCH2Ar), 54.7 (NCH2CO), 56.1
(NCH2COOEt), 60.7 (NCH2bpy), 61.4 (OCH2), 120.8 (CH bpy
3,3Ј), 124.9 (CH bpy 5,5Ј), 125.6Ϫ128.8 (CH Ar), 135.5 (Cq Ar),
139.1 (CH bpy 4,4Ј), 154.4 (Cq bpy 2,2Ј), 157.7 (Cq bpy 6,6Ј), 170.9
˜
(CO amide), 172.8 (CO ester). Ϫ IR (KBr): ν ϭ 1735 (CO ester),
Diamine 13: Diamide diester 11 (1.20 g, 1.65 mmol) was dissolved
in 40 mL of CH2Cl2/CF3COOH (1:1). The solution was stirred for
24 h at room temperature. The solvent and the excess of acid were
then removed under vacuum. The residue was dissolved in 80 mL
of ethyl acetate and the solution was washed with a 2  NaOH
aqueous solution and brine. The organic layer was dried over
Na2SO4 and the solvent was removed to afford the pure product
13 (0.48 g, 55%) as a pale yellow oil. Ϫ 1H NMR (250 MHz,
[D6]DMSO, 25 °C): δ ϭ 1.14, 1.19 (t, 3J ϭ 7.1 Hz, 6 H, CH3), 2.33
(s, 2 H, NH), 3.30Ϫ3.40 (m, 12 H, NCH2),4.02, 4.09 (q, 3J ϭ
7.1 Hz, 4 H, OCH2), 4.47Ϫ4.50 (m, 4 H, NCH2Ar), 7.10Ϫ7.36 (m,
10 H, CH Ar). Ϫ 13C NMR (63 MHz, [D6]DMSO, 25 °C): δ ϭ
14.0 (CH3), 42.8Ϫ44.3 (m, NCH2CH2N), 47.8Ϫ50.2 (m, NCH2),
59.8 (OCH2), 126.4Ϫ128.6 (m, CH Ar), 137.1Ϫ137.8 (n Ն 3, Cq
1642 (CO amide) cmϪ1. Ϫ MS (FABϩ, NBA): m/z (%) ϭ 707 (10)
[M ϩ H]ϩ, 729 (100) [M ϩ Na]ϩ. Ϫ C40H46O6N6·NaBr·2H2O
(706.8 ϩ 102.9 ϩ 36.0): calcd. C 56.81, H 5.96, N 9.94; found C
56.99, H 5.59, N 9.61.
Doubly Branched Dilactam 16: This compound was prepared sim-
ilarly to compound 15, starting from 14 (0.17 g, 0.26 mmol),
Na2CO3 (0.28 g, 2.64 mmol) and 6,6Ј-bis(bromomethyl)-2,2Ј-bipyr-
idine (0.09 g, 0.26 mmol). After filtration off of the insoluble prod-
ucts and evaporation of the filtrate, the product was dissolved in
dichloromethane (2 mL), then filtered again and evaporated to give
the NaBr complex of 16 (0.235 g, 93%) as a pale yellow solid, m.p.
93Ϫ95 °C. Ϫ H NMR (200 MHz, CDCl3, 25 °C): δ ϭ 3.00Ϫ4.60
(m, 20 H, NCH2), 5.17 (s, 4 H, OCH2), 6.75 (m, 4 H, CH Ar),
7.15Ϫ7.22 (m, 16 H, CH Ar), 7.34 (d, J ϭ 7.8 Hz, 2 H, CH bpy
1
Ar), 170.4Ϫ171.0 (n ϭ 4, CO amide), 171.7 (CO ester). Ϫ IR
3
Ϫ1
˜
(KBr): ν ϭ 3297 (NH), 1733 (CO ester), 1651 (CO amide) cm
.
5,5Ј), 7.86 (t, 3J ϭ 7.8 Hz, 2 H, CH bpy 4,4Ј), 8.05 (d, 3J ϭ 7.8 Hz,
2 H, CH bpy 3,3Ј). Ϫ 13C NMR (50 MHz, CDCl3, 25 °C): δ ϭ
45.7 (NCH2CH2N), 51.1 (NCH2Ar), 55.0 (NCH2CO), 56.3
(NCH2COOBn), 60.8 (NCH2bpy), 67.1 (OCH2), 120.9 (CH bpy
Ϫ MS (ESϩ, CH3OH): m/z (%) ϭ 527.2 (100) [M ϩ H]ϩ, 549.2
(48) [M ϩ Na]ϩ, 565.1 (14) [M ϩ K]ϩ. Ϫ C28H38O6N4 (526.6):
calcd. C 63.86, H 7.27, N 10.64; found C 63.69, H 7.21, N 10.37.
Procedure for the Direct Conversion of the Diamide Diacid 10 to 3,3Ј), 125.1 (CH bpy 5,5Ј), 127.6Ϫ128.9 (CH Ar), 134.9, 135.7 (Cq
Diamine 13: Acetyl chloride (250 µL, 3.52 mmol) was added at 0 Ar), 139.3 (CH bpy 4,4Ј), 154.3 (Cq bpy 2,2Ј), 157.5 (Cq bpy 6,6Ј),
˜
°C to a solution of diamide diacid 10 (0.50 g, 0.75 mmol) in ethanol 170.9 (CO amide), 172.7 (CO ester). Ϫ IR (KBr): ν ϭ 1736 (CO
(10 mL). The solution was allowed to warm to room temperature ester), 1642 (CO amide) cmϪ1. Ϫ MS (FABϩ, NBA): m/z (%) ϭ
and stirred for 4 h. Acetyl chloride (125 µL, 1.26 mmol) was then
831 (23) [M
ϩ
H]ϩ, 853 (100) [M
ϩ
Na]ϩ.
Ϫ
2172
Eur. J. Org. Chem. 2001, 2165Ϫ2175

Time-Resolved Luminescence in Aqueous Solution
FULL PAPER
C50H50O6N6·NaBr·2H2O (830.9 ϩ 102.9 ϩ 36.0): calcd. C 62.25,
H 5.22, N 8.71; found C 61.92, H 5.61, N 8.66.
Doubly Branched Tetralactam 18: This compound was prepared
similarly to compound 11, starting from the diamide diacid 17 (1 g,
0.89 mmol), triethylamine (400 µL, 2.71 mmol), ethyl chloro-
formate (260 µL, 2.71 mmol), and DMAP (0.06 g, 0.49 mmol).
HPLC (silica gel, CH2Cl2/CH3OH, 98:2) afforded 18 (0.68 g, 66%)
as a white solid, m.p. 123Ϫ124 °C. Ϫ 1H NMR (250 MHz, CDCl3,
Doubly Branched Dilactam 3. ؊ Preparation from 15: A mixture of
15 (0.35 g, 0.41 mmol) and K2CO3 (0.07 g, 0.51 mmol) in methanol
(30 mL) and water (10 mL) was heated to reflux for 24 h. After
evaporation to dryness, the residue was dissolved in water (5 mL)
and treated with 1  HCl to reach pH ϭ 4. The diacid was ex-
tracted with dichloromethane (3 ϫ 10 mL). The combined extracts
were dried over Na2SO4, the solvent was evaporated, and the res-
idue was tritured with ethyl acetate to give the pure product 3
(0.20 g, 66%) as a pale yellow solid.
3
25 °C): δ ϭ 1.21, 1.22 (t, J ϭ 7.1 Hz, 6 H, CH3), 1.33Ϫ1.44 (m,
18 H, CH3), 2.30Ϫ2.90 (m, 4 H, NCH2CH2N), 3.40Ϫ4.90 (m, 26
3
H, NCH2), 4.11 (q, J ϭ 7.1 Hz, 4 H, OCH2), 5.20Ϫ5.40 (m, 2 H,
NCH2), 7.10Ϫ7.50 (m, 20 H, CH Ar). Ϫ 13C NMR (63 MHz,
CDCl3, 25 °C): δ ϭ 14.2, 14.3 (CH3), 28.1, 28.2 (CH3), 39.7Ϫ42.0
(m, NCH2CH2N), 47.9Ϫ49.4 (m, NCH2), 60.7 (OCH2), 80.6, 80.7
(Cq Boc), 125.7Ϫ129.5 (n Ն 9, CH Ar), 135.2Ϫ135.6 (n Ն 3, Cq
Ar), 155.0, 155.2 (CO Boc), 168.7Ϫ169.4 (n Ն 5, CO amide), 170.0
Preparation from 16: A mixture of 16 (0.20 g, 0.21 mmol) and 0.5
 KOH (5 mL) in ethanol was heated to reflux for 5 h, followed by
workup analogous to the procedure described above for the hydro-
lysis of 15. The diacid 3 was obtained as a white solid (0.12 g, 78%),
m.p. 123Ϫ124 °C. Ϫ 1H NMR (200 MHz, CDCl3, 25 °C): δ ϭ 2.90
(m, 2 H, NCH2CH2N), 3.15, 3.29 (s, 2 H, NCH2CH2N), 3.60Ϫ3.74
(m, 8 H, NCH2CO), 3.93Ϫ4.09 (m, 4 H, NCH2bpy), 4.21Ϫ4.37
(m, 4 H, NCH2Ar), 6.75Ϫ7.37 (m, 10 H, CH Ar), 7.60 (d, 3J ϭ
7.8 Hz, 1 H, CH bpy 5Ј), 7.63 (d, 3J ϭ 7.8 Hz, 1 H, CH bpy 5),
7.8 (t, 3J ϭ 7.8 Hz, 1 H, CH bpy 4Ј), 7.85 (d, 3J ϭ 7.8 Hz, 1 H,
˜
(CO ester). Ϫ IR (KBr): ν ϭ 1745 (CO ester), 1700 (CO Boc), 1666
(CO amide) cmϪ1. Ϫ C62H80O14N8 (1161.4): calcd. C 64.12, H 6.94,
N 9.65; found C 63.88, H 7.05, N 9.65.
Doubly Branched Tetralactam 19: The same procedure as described
for 13 was followed, using compound 18, to yield 19 in 95% yield.
1
White solid, m.p. 118Ϫ120 °C. Ϫ H NMR (250 MHz, CDCl3, 25
°C): δ ϭ 1.26 (t, 3J ϭ 7.1 Hz, 6 H, CH3), 2.29 (s, 2 H, NH),
2.30Ϫ2.80 (m, 4 H, NCH2CH2N), 3.02, 3.04, 3.26, 3.28 (s, 8 H,
NHCH2), 3.49, 3.55 (d, 3J ϭ 3.1 Hz, 2 H, NCH2Ar), 3.95, 4.03 (d,
3J ϭ 5.6 Hz, 2 H, NCH2Ar), 4.15 (q, 3J ϭ 7.1 Hz, 4 H, OCH2),
4.40Ϫ4.80 (m, 14 H, NCH2), 5.27, 5.32 (d, 3J ϭ 5.7 Hz, 2 H,
NCH2Ar), 7.23Ϫ7.43 (m, 20 H, CH Ar). Ϫ 13C NMR (63 MHz,
3
3
CH bpy 3Ј), 7.92 (t, J ϭ 7.8 Hz, 1 H, CH bpy 4), 8.13 (d, J ϭ
7.8 Hz, 1 H, CH bpy 3). Ϫ 13C NMR (50 MHz, CD3OD, 25 °C):
δ ϭ 46.5 (NCH2CH2N), 51.7 (NCH2Ar), 55.8 (NCH2CO), 57.3
(NCH2COOH), 61.4 (NCH2bpy), 122.2 (CH bpy 3,3Ј), 125.8 (CH
bpy 5,5Ј), 126.8Ϫ130.1 (CH Ar), 137.4 (Cq Ar), 140.4 (CH bpy
4,4Ј), 155.7 (Cq bpy 2,2Ј), 159.4 (Cq bpy 6,6Ј), 172.5 (CO amide),
175.4 (CO acid). Ϫ IR (KBr): ν˜ ϭ 3431 (OH acid), 1729, 1706 (CO
acid), 1645 (CO amide) cmϪ1. Ϫ UV (H2O, pH ϭ 6.2): λmax (ε) ϭ
290 nm (13000 Ϫ1·cmϪ1). Ϫ MS (FABϩ, NBA): m/z (%) ϭ 651
(10) [M ϩ H]ϩ, 673 (100) [M ϩ Na]ϩ. Ϫ C36H38O6N6·AcOEt
(650.7 ϩ 88.1): calcd. C 65.03, H 6.28, N 11.37; found C 65.10, H
6.14, N 11.75.
CDCl3, 25 °C):
(NCH2CH2N), 48.1Ϫ49.7 (n
δ
ϭ
14.2 (CH3), 39.9, 40.4, 42.3, 42.5
4, NCH2), 61.0 (OCH2),
Ն
126.0Ϫ129.4 (n Ն 10, CH Ar), 135.2, 135.4, 135.9, 136.1 (Cq Ar),
168.9, 169.1, 169.3, 169.5 (CO amide), 170.2, 170.3, 170.8, 170.9
˜
(CO ester). Ϫ IR (CHCl3): ν ϭ 1736 (CO ester), 1663 (CO amide)
cmϪ1. Ϫ MS (FABϩ, NBA): m/z (%) ϭ 983 (25) [M ϩ Na]ϩ, 961
(100) [M ϩ H]ϩ. Ϫ C52H64O10N8·NaCl (961.1 ϩ 58.4): calcd. C
61.26, H 6.33, N 10.99; found C 61.31, H 6.40, N 10.76.
Doubly Branched Cryptand 20: This compound was prepared in a
similar way to compound 15, starting from 19 (0.29 g, 0.29 mmol),
K2CO3 (0.42 g, 3.04 mmol), and 6,6Ј-bis(bromomethyl)-2,2Ј-bipyr-
idine (0.11 g, 0.32 mmol). HPLC (silica gel, CH2Cl2/CH3OH, 98:2
Ǟ 85:15) afforded the KBr complex of 20 (0.135 g, 33%) as a white
Doubly Branched Tetralactam 17: Dicyclohexylcarbodiimide
(0.61 g, 2.96 mmol) was added under argon atmosphere to a solu-
tion of N-tert-butoxycarbonyliminodiacetic acid (0.69 g,
2.96 mmol) in 30 mL of THF (freshly distilled from sodium). The
solution was stirred for 24 h. Diazatetralactam 6 (1 g, 1.48 mmol)
was then added, and the reaction mixture was maintained at 50 °C
for 24 h. After removal of the dicyclohexylurea by filtration, the
solvent was removed under vacuum, and the crude product was
dissolved in satd. aqueous NaHCO3 solution (100 mL). The aque-
ous solution was treated with ethyl acetate (2 ϫ 50 mL), acidified
with 1  HCl (pH ϭ 2Ϫ3), and extracted with ethyl acetate (3 ϫ
50 mL), and the organic layer was dried over Na2SO4. After re-
moval of the solvent, the crude product was subjected to HPLC
purification (silica gel, CHCl3/(CH3)2CHOH/CH3COOH, 95:5:1 Ǟ
90:10:1) to give 17 (1.33 g, 80%) as a white solid, m.p. 174Ϫ175 °C.
1
solid, m.p. Ͼ 250 °C. Ϫ H NMR (250 MHz, CDCl3, 25 °C): δ ϭ
1.05Ϫ1.23 (m, 6 H, CH3), 2.20Ϫ5.80 (m, 40 H, CH2), 6.90Ϫ7.44
(m, 22 H, CH Ar, CH bpy), 7.92Ϫ8.10 (m, 4 H, CH bpy). Ϫ IR
(KBr): ν ϭ 1730 (CO ester), 1641 (CO amide) cmϪ1. Ϫ MS (FABϩ,
˜
NBA): m/z (%)
ϭ
1141.5 (100) [M
ϩ
H]ϩ.
Ϫ
C64H72O10N10·2KBr·2H2O (1141.3 ϩ 238.0 ϩ 36.0): calcd. C 54.31,
H 5.41, N 9.90; found C 54.29, H 5.34, N 9.84.
Doubly Branched Cryptand 4: This compound was prepared in a
similar way to compound 3, starting from 20 (0.107 g, 0.076 mmol),
K2CO3 (0.02 g, 0.15 mmol) in methanol (5 mL), and water (2 mL).
White solid (0.085 g, 94%), m.p. Ͼ 250 °C. Ϫ 1H NMR (250 MHz,
CDCl3, 25 °C): δ ϭ 2.20Ϫ5.80 (m, 36 H, CH2), 6.90Ϫ7.50 (m, 22
1
Ϫ H NMR (250 MHz, CDCl3, 25 °C): δ ϭ 1.37Ϫ1.45 (m, 18 H,
CH3), 2.30Ϫ3.00 (m, 4 H, NCH2CH2N), 3.00Ϫ5.00 (m, 26 H,
CH2), 5.00Ϫ5.30 (m, 2 H, NCH2), 7.26Ϫ7.41 (m, 20 H, CH Ar).
H, CH Ar, CH bpy), 7.90Ϫ8.10 (m, 4 H, CH bpy). Ϫ IR (KBr):
Ϫ
13C NMR (63 MHz, CDCl3, 25 °C): δ ϭ 28.1 (CH3), 40.7Ϫ43.1
Ϫ1
˜
ν ϭ 3435 (OH acid), 1722 (CO acid), 1664, 1641 (CO amide) cm
.
(n Ն 4, NCH2CH2N), 48.8Ϫ50.7 (m, NCH2Ar, NCH2CO), 52.8,
53.4 (NCH2COOH), 82.0, 82.6 (Cq Boc), 125.9Ϫ129.6 (m, CH Ar),
134.5Ϫ136.2 (n Ն 5, Cq Ar), 154.15, 154.24 (CO Boc), 167.9, 168.1,
168.8, 169.0 (CO amide), 170.8, 171.0 (CO amide), 172.3 (CO es-
ter). Ϫ IR (KBr): ν˜ ϭ 3442 (OH acid), 1740 (CO acid), 1706 (CO
Boc), 1664 (CO amide) cmϪ1. Ϫ MS (FABϩ, NBA): m/z (%) ϭ
Ϫ UV (H2O, pH ϭ 6.5): λmax (ε) ϭ 298 nm (12500 Ϫ1·cmϪ1). Ϫ
MS (ESϩ, CH3OH): m/z (%) ϭ 1123.5 (24) [M ϩ K]ϩ, 1145.5 (100)
[[M Ϫ H)]Na ϩ K]ϩ, 1161.4 (38) [[M Ϫ H]K ϩ K]ϩ. Ϫ
C60H64O10N10·KCl·1.5H2O (1085.2 ϩ 74.5 ϩ 27.0): calcd. C 60.72,
H 5.69, N 11.80; found C 60.88, H 5.69, N 11.30.
1105 (4) [M ϩ H]ϩ, 1005 (100) [(M Ϫ Boc) ϩ 2H]ϩ, 905 (10) [(M General Procedure for the Synthesis of Lanthanide Complexes: The
Ϫ 2Boc) ϩ 3H]ϩ. Ϫ C58H72O14N8·H2O (1105.2 ϩ 18.0): calcd. C
62.02, H 6.64, N 9.98; found C 61.82, H 6.57, N 9.77.
lanthanide chloride salt (1 equiv.) was added to a solution of the
appropriate ligand (0.020 g, 1 equiv.) in methanol (5 mL) and water
Eur. J. Org. Chem. 2001, 2165Ϫ2175
2173

´
`
C. Galaup, M.-C. Carrie, P. Tisnes, C. Picard
FULL PAPER
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references therein.
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(5 mL). The mixture was stirred at room temperature overnight.
The solution was concentrated and the pH was raised to 7.5 using
1  NaOH or KOH solution. After evaporation, the residue was
dissolved in methanol (5 mL) and diethyl ether was added, resulting
in the formation of a precipitate, which was isolated after centrifu-
gation.
[14]
[15]
˜
[Eu(3؊2H)]Cl: Yield 77%. Ϫ IR (KBr): ν ϭ 1602 (CO acid, CO
amide) cmϪ1. Ϫ UV (H2O, pH ϭ 6.7): λmax (ε) ϭ 306 nm (10700
Ϫ1·cmϪ1). Ϫ Luminescence (H2O, λex ϭ 306 nm): λem ϭ 581 (4),
593 (27), 617 (100), 652 (3), 687 (20), 696 (32), 703 sh (24) nm. Ϫ
MS (ESϩ, CH3OH): m/z (%) ϭ 859.2 (20) [M ϩ Na]ϩ, 801.2 (100)
[M Ϫ Cl]ϩ. Ϫ C36H36O6N6EuCl· NaCl·6H2O (836.1 ϩ 58.4 ϩ
108.1): calcd. C 43.12, H 4.83, N 8.38; found C 43.51, H 4.94,
N 7.95.
[16]
[17]
[18]
[19]
[20]
[21]
[Tb(3؊2H)]Cl: Yield 70%. Ϫ IR (KBr): ν˜ ϭ 1603 (CO acid, CO
amide) cmϪ1. Ϫ UV (H2O): λmax ϭ 307 nm. Ϫ Luminescence
(H2O, λex ϭ 307 nm): λem ϭ 490 (42), 545 (100), 584 (30), 588 sh
(23), 622 (20) nm; τ (H2O, 300 K) ϭ 1.04 ms; τ (D2O, 300 K) ϭ
2.08 ms; τ (D2O, 77 K) ϭ 2.73 ms. Ϫ MS (ESϩ, CH3OH): m/z
(%) ϭ 865.3 (8) [M ϩ Na]ϩ, 829.3 (12) [M Ϫ HCl ϩ Na]ϩ, 807.4
(100) [M Ϫ Cl]ϩ. Ϫ C36H36O6N6TbCl·NaCl·5H2O (843.1 ϩ 58.4
ϩ 90.1): calcd. C 43.61, H 4.68, N 8.48; found C 43.89, H 4.43,
N 8.62.
[22]
[23]
˜
[Gd(3؊2H)]Cl: Yield 63%. Ϫ IR (KBr): ν ϭ 1603 (CO acid, CO
amide) cmϪ1. Ϫ UV (H2O): λmax ϭ 306 nm. Ϫ Phosphorescence
(EtOH/MeOH, 4:1, λex ϭ 306 nm): λem ϭ 451 (75), 481 (100), 508
sh (71) nm. Ϫ MS (ESϩ, CH3OH): m/z (%) ϭ 828.3 (20) [M Ϫ HCl
[24]
[25]
ϩ Na]ϩ, 806.2 (100) [M Ϫ Cl]ϩ, 414.7 (33) [M Ϫ Cl ϩ Na]2ϩ
.
[26]
[27]
[28]
[29]
Ϫ C36H36O6N6GdCl·NaCl·6H2O (841.4 ϩ 58.4 ϩ 108.1): calcd. C
42.90, H 4.80, N 8.34; found C 42.42, H 4.45, N 8.06.
`
B. Cathala, L. Cazaux, C. Picard, P. Tisnes, Tetrahedron Lett.
1994, 35, 1863Ϫ1866.
B. Cathala, C. Picard, L. Cazaux, P. Tisnes, M. Momtchev,
`
[Eu(4؊2H)]Cl: Yield 82%. Ϫ IR (KBr): ν˜ ϭ 1652 (CO amide), 1603
(CO acid, CO amide) cmϪ1. Ϫ UV (H2O): λmax (ε) ϭ 307 nm
Tetrahedron 1995, 51, 1245Ϫ1252.
C. Galaup, C. Picard, B. Cathala, L. Cazaux, P. Tisnes, H.
`
(10200 Ϫ1·cmϪ1). Ϫ Luminescence (H2O, λex ϭ 307 nm): λem
ϭ
Autiero, D. Aspe, Helv. Chim. Acta 1999, 82, 543Ϫ560.
581 (3), 592 (28), 618 (100), 652 (4), 687 (22), 697 (35), 703 sh (22)
´
`
J. Azema, C. Galaup, C. Picard, P. Tisnes, P. Ramos, O. Juanes,
J. C. Rodriguez-Ubis, E. Brunet, Tetrahedron. 2000, 56,
2673Ϫ2681.
nm. Ϫ MS (ESϩ, CH3OH): m/z (%) ϭ 1235.6 (100) [M Ϫ Cl]ϩ,
425.0
(50)
[M
Ϫ
Cl
ϩ
H
ϩ
K]3ϩ
.
Ϫ
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W. D. Kim, G. E. Kiefer, F. Maton, K. McMillan, R. N.
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46.16, H 5.55, N 8.97; found C 45.99, H 5.15, N 8.75.
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´
C. Galaup, M.-C. Carrie, J. Azema, C. Picard, Tetrahedron
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