Engineering of Highly Luminescent Lanthanide Tags
A R T I C L E S
the sample, photobleaching, and avoidance of accurate quantita-
tive measurements.
cryptand17 or including in the design of the ligand (possibly of
macrocyclic- or podand-type structure) numerous anionic termini
(mainly carboxylate functions18 or phosphorus derivatives19) for
stabilization of the complex through electrostatic interactions.
Our previous work in this last direction showed us that the
introduction of anionic carboxylate functions directly linked in
the ortho position of a pyridyl-containing chromophore such
as 2,2′-bipyridine9,20 or pyrazolyl-pyridine21 often gave very
luminescent complexes with europium or terbium. We would
now like to show how such chromophores can be combined on
a glutamic skeleton to give ligand LH4, which forms water-
soluble, stable, and highly luminescent complexes with eu-
ropium and terbium, [LnL(H2O)]Na. The noncoordinated
glutamate residue can be transformed in an activated ester to
form [LnL*(H2O)] complexes22 that can be easily linked to
amino residues. The suitability of these complexes for labeling
will be demonstrated, together with the capacity of labeled
model biomolecules for use in TRLM.
As soon as 1978,8 luminescent lanthanide complexes appeared
as interesting labels for time-resolved applications. In the cases
of europium and terbium complexes, luminescence lifetimes up
to the millisecond can be obtained, leading to an improved
temporal resolution in the treatment of the luminescence signal
with a concomitant increase of the signal-to-noise ratio.9 In
parallel to the development of the very first lanthanide markers,10
technical progress in time-resolved luminescence microscopy
was achieved with the introduction of new devices for gated
acquisition such as mechanical choppers,11 ferro-electric liquid
crystal shutters,12 or acousto-optical modulators.5a Luminescent
lanthanide complexes were rapidly shown to be efficient labels
for cytochemistry and histochemical studies.11b
Despite the tremendous amount of work on luminescent
lanthanide complexes,13 only a few commercially available
labels have been developed up to now.14 The main reason for
this thrift is to be found in the numerous requirements to be
fulfilled by a lanthanide complex to be used in time-resolved
applications.15 In a brief survey, the complex must be stable, at
least kinetically, in water; it must be soluble in aqueous media
or in solvents suitable for performing its conjugation to
biospecific probes; it must contain chromophoric units with large
oscillator strengths at accessible wavelengths to generate the
antenna effect;16 and the intersystem crossing that leads to
population of the lanthanide centered excited state after ligand
excitation must be efficient, as well as must be the lanthanide
centered luminescence. Finally, the synthetic procedure for
obtaining the ligand must include an orthogonal function that
can be activated to trigger the grafting of the lanthanide complex
to the compounds to be tagged.
Results and Discussion
Synthesis of the Ligand. Ligand LH4 is obtained in a seven-
step synthetic protocol as described in Scheme 1.
Starting from commercially available 2-amino-6-methylpy-
ridine 1, a Sandmeyer reaction23 gave 2-bromo-6-methylpyri-
dine, which is transformed to 2-tributylstannyl-6-methylpyridine
by metalation with n-BuLi followed by reaction with Bu3SnCl.24
A Stille coupling of the stannylated compound with 2,6-
dibromopyridine catalyzed by [Pd(PPh3)4] gave 6-bromo-6′-
methyl-2,2′-bipyridine.25 A radicalar bromination with NBS/
AIBN in benzene under UV irradiation gave compound 2 in
48% yield after purification.26 Dimethyl ester glutamate, 4, was
obtained by esterification of the racemic glutamic acid hydrate
3.27 Reacting compound 2 in a 2.2-fold excess with 4 led to the
bis N-alkylation in 67% yield, affording compound 5. Due to a
carboalkoxylation process28 effected in a 50/50 (v/v) EtOH/Et3N
mixture using [Pd(PPh3)2Cl2] as catalyst under a CO atmosphere,
5 is transformed into the tetraester 6 in 60% yield. A final
saponification of all four esters followed by reacidification of
the medium allowed for isolation of LH4 in the form of its
trihydrochloride salt.
The two main strategies that have been developed so far to
obtain stable complexes consist of either encapsulating the
lanthanide cation in the well-defined macrotricyclic cavity of a
(5) (a) Hennink, E. J.; de Haas, R.; Verwoerd, N. P.; Tanke, H. J. Cytometry
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