Synthesis and luminescence properties of new red-shifted absorption lanthanide(iii) chelates suitable for peptide and protein labelling

Article abstract of DOI:10.1039/c0ob00832j

The synthesis and photo-physical properties of an original bis-pyridinylpyrazine chromophore efficiently sensitising europium(iii) and samarium(iii) are described. The corresponding lanthanide(iii) complexes display in aqueous solutions a maximum excitation wavelength which is significantly red-shifted compared to the usual terpyridine-based chelates, and a valuable luminescence brightness above 2 000 dm³ mol^-1 cm ^-1 at 345 nm was obtained with a europium(iii) derivative. Further functionalisation with three different bioconjugatable handles was also investigated and their ability to efficiently label a model hexapeptide was evaluated and compared. Finally, the best bioconjugatable europium(iii) chelate was used in representative labelling experiments involving monoclonal antibodies and the luminescence features of the corresponding bioconjugates remained satisfactory.

Full text of DOI:10.1039/c0ob00832j

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Organic &
Biomolecular
Chemistry
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PAPER
Synthesis and luminescence properties of new red-shifted absorption
lanthanide(III) chelates suitable for peptide and protein labelling†
a
a
a
a
b,c
Nicolas Maindron, S e´ verine Poupart, Maxime Hamon, Jean-Baptiste Langlois, Nelly Pl e´ ,
a,b
a,b
a,b,d
Ludovic Jean, Anthony Romieu* and Pierre-Yves Renard*
Received 5th October 2010, Accepted 23rd December 2010
DOI: 10.1039/c0ob00832j
The synthesis and photo-physical properties of an original bis-pyridinylpyrazine chromophore
efficiently sensitising europium(III) and samarium(III) are described. The corresponding lanthanide(III)
complexes display in aqueous solutions a maximum excitation wavelength which is significantly
red-shifted compared to the usual terpyridine-based chelates, and a valuable luminescence brightness
3
-1
-1
above 2 000 dm mol cm at 345 nm was obtained with a europium(III) derivative. Further
functionalisation with three different bioconjugatable handles was also investigated and their ability to
efficiently label a model hexapeptide was evaluated and compared. Finally, the best bioconjugatable
europium(III) chelate was used in representative labelling experiments involving monoclonal antibodies
and the luminescence features of the corresponding bioconjugates remained satisfactory.
Introduction
The poor absorption of a lanthanide ion can be overcome by its
insertion inside an adequate organic ligand which is able to absorb
UV–visible light and subsequently transfer it onto the metal ion
via its triplet state (a process called luminescence sensitisation or
A wide range of bio-analytical and biomedical applications
involved in various fields such as diagnostic, drug discovery and
molecular imaging (both in cellulo and in vivo contexts), require
1
6
antenna effect). This transfer leads also to an important apparent
accurate investigations of biological events and/or materials
through the use of optical bioprobes. Since the targeted analytes
are often present in minute concentrations in complex biological
media, these biomolecular tools must be as sensitive as possible.
Besides, they have to give rise to a specific and easy-to-measure
Stokes’ shift of up to about 250 nm (compared to 30–50 nm
for most organic fluorescent dyes). These unique spectroscopic
2
features combined with lanthanide(III) cation’s narrow emission
bands in the red-light region explain why lanthanide luminescent
chelates are valuable tools in a large panel of highly sensitive
2,3
signal. For more than 25 years, luminescence bioprobes based
on lanthanide complexes, have turned out to be well-suited
7
bioaffinity assays. Using these lanthanide(III) chelates, a sub-
8
picomolar detection threshold is readily achieved, whereas with
4,5
tools for such challenging applications. Indeed, these lumi-
other fluorescent probes the highest limit of detection reached is
6
nescent chelates possess remarkable photophysical properties.
2
in the picomolar range.
For instance, long-lived emission of lanthanide(III) cations can
be temporally resolved from scattered light and background
fluorescence (e.g., from biological matrix) to dramatically enhance
measurement sensitivity.
A lanthanide luminescent bioprobe must fulfil several require-
ments to be efficient: high thermodynamic stability as well as
kinetic inertness in various biological matrices and at physiological
pH, good absorption and emission properties, photostability and
water solubility (for in cellulo applications, non-cytotoxicity is also
4
needed). Finally, one of the challenges in the design of lumines-
a
Equipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038,
cent lanthanide probes dedicated to bioanalyses and bioimaging
applications, is to be able to increase the excitation wavelength
as high as possible. The long-wave UV region or visible spectrum
are preferred and targeted (especially wavelengths around or above
rue Lucien Tesni e` re, 76131 Mont-Saint-Aignan, France. E-mail: pierre-
yves.renard@univ-rouen.fr, anthony.romieu@univ-rouen.fr; Fax: +33 2-35-
5
2-29-71; Tel: +33 2-35-52-24-14 (or 24-15); Web: http://ircof.crihan.fr
b
Universit e´ de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan,
France
3
50 nm) for two intrinsic reasons: biomolecules/biopolymers (e.g.,
c
Equipe de Chimie H e´ t e´ rocyclique, COBRA-CNRS UMR 6014 & FR 3038,
DNA) are usually damaged by short- or medium-wave UV light,
and tissues’ autofluorescence excitation wavelengths are usually
below 320 nm. Three strategies are generally adopted to extend
the excitation window of Ln(III) complexes (mainly applied to
rue Lucien Tesni e` re, 76131 Mont-Saint-Aignan, France
d
Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005 Paris,
France
†
Electronic supplementary information (ESI) available: Detailed syn-
thetic procedures for compounds 16 and 25, characterisation data for
compounds 6, 8, 10, 17 and 18, ESI mass spectra of Ln(III) chelates,
bioconjugatable derivatives and luminescent labelled peptides. See DOI:
9
Eu(III) complexes) to a long-wavelength region. Diminishing the
energy gap between the lowest S
1
state and the T state of the
1
1
0.1039/c0ob00832j
antenna ligand is considered as an effective way to extend the
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excitation wavelength for the Eu(III) complex into the visible-
light region through the usual triplet energy-transfer pathway.
Another promising means of longer-wavelength sensitisation of
Eu(III) emission is through the singlet pathway, in which the
excited-state energy of a chromophore is directly transferred from
rently used for diagnostics or drug discovery purposes, generally
possess a maximum excitation wavelength below 340 nm. These
stable luminescent complexes are generally composed of a 4¢-
aryl-substituted 2,2¢:6¢,2¢¢-terpyridine unit as the light-absorbing
moiety, methylenenitrilo diacetic acids as the chelating arms, and a
bioconjugatable group for coupling to biomolecules/biopolymers
10,11
its S state to the luminescent states of the Eu(III) center (i.e.,
1
12
14
charge transfer transitions). Two- (or multi-) photon excitation,
that is, simultaneous absorption of two photons of half energy is
the third way to circumvent the use of UV light and has recently
been successfully applied to Eu(III) complexes and nanoprobes for
(Fig. 1). To date, to our knowledge, there are only two Ln(III)
chelate labels bearing a diazine ring within their chromophoric
15,16
moiety (Fig. 2).
The first one is the bis-pyrazolylpyrazine
Ln(III) chelate 4 that displays in borate buffer (pH 8.5) a moderate
13
3
-1
-1
live cell imaging applications.
luminescence brightness (e ¥ U) 250 dm mol cm at 336 nm
17 3
-1
-1
With the goal in mind to develop a new structurally simple
Ln(III) chelate bio-labelling reagent that displays UV-A or visible
absorption maximum, we thought to implement the first strategy
to Ln(III) chelates comprising a terpyridine unit as antenna, by
replacing its central pyridine ring by a pyrazine moiety. Indeed,
the commercially available Ln(III) terpyridine-based chelates cur-
for Eu(III) ion, and a good value of 6 380 dm mol cm
for Tb(III) ion thanks to the sensitiser triplet-state energy level.
Interestingly, the introduction of this pyrazine unit instead of the
central pyridine ring of the original bis-pyrazolepyridine Ln(III)
chelate, leads to a significant bathochromic shift of 15 nm of
18,19
the excitation wavelength.
The second one 5, based on a
a
Fig. 1 Examples of Ln(III) chelates based on a terpyridine or a terpyridine-like pyrazole ligand. Determined only for the chelate without the
bioconjugatable arm.
Fig. 2 Structure and luminescence properties of lanthanide(III) chelates based on a tris-azaheterocyle ligand comprising a diazine unit, and already
reported in the literature.
2
358 | Org. Biomol. Chem., 2011, 9, 2357–2370
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Fig. 3 Structures of lanthanide(III) chelates composed of a bis-pyridinylpyrazine unit as the energy-absorbing and donating ligand, studied in this work.
Fig. 4 First synthetic pathways explored for the preparation of symmetrical bis-pyridinylpyrazine ligand 10.
bis-pyrazolylpyrimidine ligand, shows better luminescence bright-
Results and discussion
3
-1
-1
ness in water for both Eu(III) and Tb(III) ions: 2 055 dm mol cm
3
-1
-1
and 11 645 dm mol cm respectively. However, the main
drawback of such chelates remains an excitation maximum at the
more energetic wavelength of 300 nm. Thus, it seems relevant
Synthesis of the symmetrical functionalised bis-pyridinylpyrazine
ligand
20
to examine the chemistry of 2,6-di(2-pyridinyl)pyrazine ligand
The bis-pyridinylpyrazine core structure was obtained via a tricky
to optimise and efficient Stille cross-coupling reaction between
2,6-dichloropyrazine and 2-(tributylstannyl)pyridine (Fig. 4). The
most frequently used synthetic method to introduce the chelating
arms onto the 6,6¢¢-positions of a terpyridine scaffold, involves
a key di-cyano derivative which is obtained through a modified
whose functionalisation with chelating arms and its subsequent
complexation with Ln(III) ions have never been explored.
Here we report the first synthesis along with the photophysical
properties of Eu(III) chelate 6 based on a bis-pyridinylpyrazine
chromophoric moiety functionalised with two methylenenitrilo
diacetic acids moieties (Fig. 3). The preparation of the non-
symmetrical derivative 8 bearing a lysine residue within one
of the two chelating arms is also described. Its potential util-
ity in the context of luminescent bio-labelling is illustrated
through its conjugation to a model hexapeptide and monoclonal
antibodies.
21
Reissert–Henze reaction. Thus, it was theoretically possible to get
the targeted 6,6¢¢-functionalised bis-pyridinylpyrazine ligand 10 by
using one of the two following synthetic strategies: (1) the nitrile
functions could be reduced with borane–tetrahydrofuran complex
to afford the corresponding primary bis-amine which would then
be twice carboxymethylated with an alkyl bromoacetate followed
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by hydrolysis of the esters to give the free methylenenitrilo diacetic
acids, or (2) the nitrile functions could be hydrolysed to carboxylic
acids, which would be esterified and subsequently reduced to
alcohols. Thereafter, the primary bis-alcohol would be converted
tert-butyldimethylsilyl (TBS) ether to afford compound 11. The
key trimethyltin derivative was efficiently generated by trapping
the lithiated intermediate with trimethyltin chloride in dry THF
◦
from -78 C to room temperature for 4 h (89% yield for the
into the corresponding bis-bromide by treatment with PBr
3
which
crude product). The Stille cross-coupling reaction between this
could undergo S 2 reaction with an iminoacetic acid ester followed
N
stannane intermediate and 2,6-dichloropyrazine was performed
22
by hydrolysis to give the desired ligand. However, introduction
of nitrile functions onto the bis-pyridinylpyrazine scaffold by
means of the Reissert–Henze reaction gave poor yields, thus we
decided to perform their incorporation prior to the Stille cross-
coupling reaction. The optimised Stille reaction between 2,6-
with Pd(PPh
3
) as a Pd(0) catalyst in an argon deaerated xylene
4
◦
solution at 110 C overnight to give the desired bis-TBS ether 12
of bis-pyridinylpyrazine in a moderate 50% yield. The removal of
TBS ethers was performed with a 1.0 M solution of TBAF in THF
and afforded the desired bis-alcohol 13. This bis-pyridinylpyrazine
derivative was isolated in a pure form by simple precipitation in a
0.1% aq. trifluoroacetic acid (TFA) and MeOH (1 : 1) mixture
(79% yield). Next, bis-bromination reaction of 13 was carried
(
tributylstannyl)pyrazine and 2-bromo-6-cyanopyridine efficiently
23
afforded the bis-nitrile derivative. Reduction with borane–
tetrahydrofuran complex failed but nitriles hydrolysis followed by
esterification was successful. The main drawback of this synthetic
out in dry DMF with PBr to give the bis-halide compound
3
scheme is that the hydride source (NaBH
reduction of esters, led to an unresolved mixture of desired bis-
alcohol and boron (or aluminium) complex salts. Consequently,
4
ou DIBAL-H), used for
14 in a quantitative yield. This crude product was then directly
engaged in the substitution step with tert-butyliminoacetate 9.
This latter reaction was performed with DIEA as base, NaI as
catalyst and in dry DMF. Finally, the removal of tert-butyl esters
the PBr -mediated bromination reaction was performed on the
3
crude reduction mixture to give the bis-bromide derivative with an
unsatisfactory 9% overall yield for the two steps. The substitution
of the bromine atoms by di-tert-butyl iminodiacetate 9 followed by
tert-butyl deprotection gave ligand 10 with a low 13% overall yield
for this two-step reaction sequence. The insignificant overall yield
for this 8-step synthetic procedure, which amounted to 0.2%, led
us to discard this approach and develop another synthetic route
able to circumvent the tedious ester reduction step.
was achieved in a mixture of TFA and CH
2
Cl (1 : 1) containing
2
2.5% of water. The symmetrical tetrakis(acetic acid) ligand 10 was
purified by flash-chromatography on a RP-C18 silica gel column.
All spectroscopic data, in particular NMR and mass spectrometry,
were in agreement with the structure assigned (see ESI†). By
comparison with the synthetic route initially explored (Fig. 1), this
alternative pathway led to the desired ligand 10 with an improved
overall yield of 7.6%.
We decided then to investigate a synthetic strategy involv-
ing the introduction of a protected 2-(hydroxymethyl) pyridine
moiety on the 2,6-dichloropyrazine via the same Stille cross-
coupling reaction (Scheme 1). The required trimethyltin deriva-
tive was easily obtained from the commercially available 2,6-
dibromopyridine in three steps and with a good 72% yield: the
first step allowed to replace one of the two bromine atoms by the
hydroxymethyl group in almost quantitative yield as described by
Synthesis of the non-symmetrical functionalised
bis-pyridinylpyrazine ligand suitable for bioconjugation studies
In order to have a suitable chemically reactive group for the bio-
conjugation of the corresponding Ln(III) chelates derived from bis-
pyridinylpyrazine ligand to various biomolecules/biopolymers,
we also explored the synthesis of the non-symmetrical ligand
18 bearing a lysine residue from the dibromide derivative 14
24
Cai et al. Thereafter, the primary alcohol was protected as a
◦
◦
◦
Scheme 1 Reagents and conditions: a) nBuLi (1.0 equiv.), THF, -70 C, 15 min then DMF (1.5 equiv.), -78 C, 15 min then MeOH, CH
3
CO
2
◦
H, -78 C
to rt then NaBH (1.0 equiv.), rt, 92%; b) TBS-Cl (1.2 equiv.), imidazole (4.0 equiv.), DMF, rt, 25 min, 88%; c) nBuLi (1.1 equiv.), THF,-78 C, 90 min
4
◦
then Me
3
SnCl (1.05 equiv.), rt, 4 h, 89%; d) 2,6-dichloropyrazine (0.75 equiv.), Pd(PPh
3
)
4
(0.05 equiv.), xylene, 110 C, 6 h, 50%; e) 1 M TBAF in THF
◦
(
2.3 equiv.), THF, rt, 2 h 30, 79%; f) PBr
3
(3.0 equiv.), DMF, 0 C to rt, 4 h, 95%; g) secondary amine 9 (2.2 equiv.), NaI (1.1 equiv.), DIEA (10 equiv.),
◦
DMF, rt, 6 h 30, 64%; h) TFA, H
2
O (50 equiv.), CH
2
Cl
2
, 0 C to rt, 5 h, 44%.
2
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(
Scheme 2). Indeed, the post-synthetic derivatisation of the e-
with a lower overall yield (2.6%) than the one obtained for ligand
amino group of this original chelating arm with an homobifunc-
tional (or trifunctional) cross-linker should enable the introduc-
tion of a bioconjugatable moiety reactive towards the primary
amines of proteins (e.g., N-hydrosuccinimidyl ester, NHS ester).
Firstly, the required fully-protected chelating arm 16 derived from
lysine was readily prepared from commercially available Fmoc-L-
Lys(Boc)-OH using a three-step method (see ESI†). The one-pot
synthesis leading to fully-protected non-symmetrical intermediate
10.
17 was a tricky step. Indeed, dibromide derivative 14 was flanked
by two equivalent reactive sites, whereas the two amines involved
in the alkylation process did not exhibit the same reactivity
(
presumably due to steric considerations, secondary amine 9 was
found to react faster than the lysine derivative 16). Consequently,
a slight excess (1.1 equiv.) of less reactive secondary amine 16
was first added to a dry DMF solution containing 14. The
mixture was stirred at room temperature for 9 h prior to the
addition of secondary amine 9 in several portions over a period of
Synthesis and characterisation of Ln(III) chelates
Bis-pyridinylpyrazine ligands 10 and 18 were converted to the
corresponding Eu(III) chelates by treatment with aq. europium(III)
chloride. The completion of these latter reactions was checked
by UV–visible spectrophotometric measurements and purification
was achieved by flash-chromatography on a RP-C18 silica gel
column. The stoichiometry [1 : 1] and structures of these Eu(III)
chelates were confirmed by ESI mass spectrometry analyses
(see ESI†). Interestingly, the Tb(III) and Sm(III) complexes of
symmetrical ligand 10 were also prepared from their respective
hexahydrate trichloride salts (for ESI mass spectra, see ESI†).
The Tb(III) chelate was found to be non luminescent whereas
4
8 h. Eventually, the desired compound 17 was isolated from the
complex reaction mixture (containing 17 and the two possible
symmetrical compounds) with a modest 18% yield after two
successive purifications: a normal phase flash-chromatography fol-
lowed by a semi-preparative RP-HPLC purification. The removal
of acid-labile protecting groups Boc and tert-butyl was carried
out with a mixture of TFA–CH
2
2
Cl (1 : 1) containing 2.5% of
water. The non-symmetrical ligand 18 was isolated in a pure form
by semi-preparative RP-HPLC. Its structure was confirmed by
NMR and ESI mass spectrometry measurements (see ESI†). This
desymmetrisation process involving a sequential alkylation of two
different secondary amines led logically to the desired ligand 18
25
Sm(III) chelate 7 exhibited a modest quantum yield of 0.5%.
Thus, we decided to focus our next comprehensive spectral and
bioconjugation studies only on the Eu(III) chelates.
Photo-physical properties of the bis-pyridinylpyrazine ligands and
the corresponding Eu(III) chelates
The photo-physical features for Eu(III) chelates 6 and 8 are pre-
sented in Table 1. The ligand triplet-state level was not measured;
nevertheless, since the Tb(III) chelate was not luminescent contrary
to the Sm(III) derivative, we can reasonably postulate an energy
-
1
level of around 20 000 cm . The UV–vis absorption spectra
in deionised water (see Fig. 5 for ligand 18) displayed for both
ligands 10 and 18 a broad band between 260 and 350 nm with two
3
-1
-1
maxima located at 293 (e = 13 600 dm mol cm ) and 310 nm
(
3
-1
-1
e = 14 000 dm mol cm ). The molar extinction coefficients
are virtually the same for each ligand. Upon complexation with
Eu(III) cation, the broad absorption band splits into two narrower
bands with a large bathochromic shift from 310 to 344 nm due
to conformational changes leading to an improvement of the
flatness of the structure which favours the p-electron delocalisation
2
6
(
Fig. 5). In this case, the red-shift was accompanied with a slight
3
-1
-1
hyperchromic effect (e = 15 500 dm mol cm ). The higher energy
absorption band of the free ligand underwent no significant shift
(
2
l = 296 nm), but its absorption coefficient decreased (e = 11
3
-1
-1
00 dm mol cm ). The corresponding emission spectra of Eu(III)
5
7
chelates 6 and 8 present the usual D
typical of the Eu(III) ion. The luminescence brightness of Eu(III)
0
→ F
J
(J = 1–4) transitions
Scheme 2 Reagents and conditions: a) secondary amine 16 (1.1 equiv.),
chelates 6 and 8 were measured in deionised water by using a
NaI (0.6 equiv.), K
2
CO
3
(15 equiv.), DMF, rt, 9 h then secondary amine 9
◦
27
28
(
1.05 equiv.), rt, 72 h, 18%; b) TFA, H
0 h, 54%.
2
O (40 equiv.), CH
2
Cl
2
, 0 C to rt,
relative method using rhodamine 6G and the Eu(TMT)AP
3
29
2
chelate 19 recently reported by us, as standards.
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◦
Table 1 Photophysical properties of Eu(III) chelates in borate buffer pH 8.5 (chelates 4 and 20) or in deionised water (chelates 6 and 8) at 25 C
3
-1
-1
c
g
Chelate
l
abs, max (nm)
e (dm mol cm )
l
em, max (nm)
U (%)
t
H2O (ms)
t
D2O (ms)
q
H2O
a
4
335 (336)
333
344
8 400 (10 900)
12 100
15 500
621
—
615
616
3.0 (13.0)
17.6
15.7 (14.2)
14.5 (13.2)
1.37 (1.30)
1.31
—
—
2.35
2.30
0.5
—
0.3 (0)
b
2
6
8
0
d
d
e
f
f
1.35
e
345
15 500
1.30
0.35 (0)
a
c
16
19
b
38
19
Data reported by Rodrigues-Ubis et al. and values inside brackets reported by Latva et al. Data reported by Mukkala et al. and Latva et al.
5
7
d
Only the most intense emission band corresponding to the D
0
→ F
2
transition is reported. Determined by using Eu(TMT)-AP
3
19 (U = 16.9% in
29
28
e
deionised water) or R6G (U = 88% in ethanol, values inside brackets) as standard (excitation at 345 nm). A solution of chelate in deionised water
f
g
-1
at 10 mM was used. A solution of chelate in heavy water at 10 mM was used. Calculated according to the following equations: qH2O = 1.05[(tH2O
) -
-1
32
-1
-1
31
(
t
D2O) ] or qH2O = 1.11[(tH2O
)
- (tD2O
)
- 0.31] (inside brackets).
◦
Fig. 5 Absorption spectra of non-symmetrical bis-pyridinylpyrazine ligand 18 ( ) and corresponding Eu(III) chelate 8 ( ) in deionised water at 25 C
(
concentration: 9.0 mM).
yield was also observed (Table 1, see entries 2–4). Interestingly, the
small size of this novel chromophoric unit also has a beneficial
effect on the water-solubility of the resulting lanthanide(III)
chelates. Indeed, 6 and 8 were found to be perfectly soluble in
water and related aq. buffers in the concentration range of 1 mM to
Under excitation at 345 nm, the luminescence brightness varied
from 2 200 to 2 500 for 6 and from 2 000 to 2 250 for 8
according to the reference used. Interestingly, the presence of a
free primary amine within the structure of chelate 8 does not
lead to a substantial quantum yield loss as previously observed
19
for the terpyridine analogue 21. As compared with chelate 4,
the replacement of the two pyrazole rings by pyridine moieties,
in spite of a lack of luminescence in the case of Tb(III), leads to
better spectroscopic properties for Eu(III) derivative. Moreover,
30
compared to terpyridine analogue 20 , the pyrazine introduction
has been revealed to be beneficial because the excitation maximum
was red-shifted (10 nm), and a slight improvement of luminescence
2
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1
mM, contrary to 4¢-aryl-substituted 2,2¢:6¢,2¢¢-terpyridine-based
hindrance. Thus, to remove the unreacted reagent, the activated-
carbonate chelate 22 was purified by RP-HPLC but premature
hydrolysis of its active carbamate moiety occurred despite the
use of a non-basic aq. mobile phase (i.e., deionised water).
Conversely, the complete amidification of the amino group of 8
with DSSEB was successfully achieved with only one equivalent
of this homobifunctional cross-linking reagent. Interestingly, RP-
HPLC analysis of the crude reaction mixture has shown that
roughly one third of the newly formed NHS ester chelate 24
underwent a further reaction with a second chelate unit to give the
corresponding dimer, chemically unreactive towards the proteins.
Thus, no chromatographic purification was required and it was
possible to use directly the crude activated chelate 24 in peptide
and protein labelling (vide infra). Finally, we have also studied
chelates such as 19.
Finally, luminescence lifetime measurements in water and
heavy water were performed to calculate, according to the two
31,32
equations reported in the literature,
the average number (q)
of water molecules in the first coordination sphere of Eu(III),
which was found to be close to 0. It clearly indicates that the
complexation provided by the nine binding sites efficiently expels
water molecules from the inner coordination sphere of the metal
ion thus avoiding strong luminescence quenching through OH
oscillators.
Selection of the best bioconjugatable group for protein labelling
with luminescent Eu(III) chelate 8
the S Ar reaction between 8 and cyanuric chloride, aimed at
getting the corresponding dichlorotriazinyl activated chelate 23,
N
To demonstrate the potential utility of Eu(III) chelate 8 as a reagent
for biopolymer labelling (especially proteins), it was necessary
to derivatise its primary amino group under mild conditions in
order to introduce a bioconjugatable moiety reactive towards
even if solely the mono-substitution of chlorine atoms by primary
33
amines is quite difficult to obtain. This reaction was performed
in a mixture of aq. NaHCO
3
buffer (0.1 M, pH 8.7) and acetone
the NH
2
groups of e-lysine residues present in selected proteins.
◦
at 0 C for 4 h and provided a mixture of target activated
chelate 23, a dimer (resulting from two chlorine substitutions)
and starting free amino chelate 8 in a ratio of 60 : 4 : 36. This
crude mixture was also used in labelling reactions without
purification.
Thus, we investigated the conversion of 8 into three different
derivatives 22–24 by reaction with N,N¢-disuccinimidyl carbonate
(
DSC), cyanuric chloride (i.e., 2,4,6-trichloro-1,3,5-triazine) and
disuccinimidyl sebacate (DSSEB) respectively (Scheme 3). The
identity of the reaction products was confirmed by ESI mass
analyses (see ESI†). Firstly, the reaction of 8 with DSC in
dry DMSO containing TEA was found to be complete only
by using an excess of this reagent, probably due to the steric
In order to select the best bioconjugatable group between the
succinimidyl sebacate and the dichlorotriazinyl moiety from the
point of view of chemoselectivity (towards NH
efficiency of labelling, we studied the reaction of activated chelates
3 and 24 with model hexapeptide Ac-Gly-Lys-Gly-Ser-Gly-Tyr-
2
groups) and
2
OH 25 under mild aq. conditions (Scheme 4). This short sequence
was chosen to get within the same peptidyl architecture the
three representative nucleophilic amino acid side-chains able to
react with the studied bioconjugatable moieties. (i.e., primary
amine, primary alcohol and phenol). These labelling reactions
were carried out in aq. NaHCO buffer (0.1 M, pH 8.5) with three
3
equivalents of activated chelates 23 or 24. As expected, the lysine
residue of 25 readily reacted on each one of the two activated
chelates, contrary to its serine. The resulting Eu(III) chelate–
peptide conjugates 26 and 27 were isolated by RP-HPLC and
their structures confirmed by ESI mass spectrometry (see ESI†).
Interestingly, LC-MS analysis of the crude labelling mixture with
activated chelate 23 (see ESI†) revealed the significant formation of
two other peptides derivatives which were identified as the mono-
-
labelled cyclo-peptide 28 (t
bis-labelled derivative 29 (t
The formation of these non-desired side-products can be inferred
R
= 13.1 min, 1428.37 [M - H] ) and
= 15.1 min, 1159.11 [M - 2H] ).
2
-
R
34
from the chemical modification of tyrosine residue. It led to a
decrease in the mono-labelling efficiency (5.3% isolated yield for
23 against 16.6% for 24). Thus, these preliminary experiments
have clearly shown the superior ability of the NHS activated
Eu(III) chelate 24 to label selectively the lysine residues within
peptides. This latter bio-labelling reagent was chosen for the fur-
ther bioconjugation experiments involving monoclonal antibodies
(
mAb).
Antibody labelling using activated chelate 24
Scheme 3 Reagents and conditions: a) DSC (4 equiv.), DMSO, TEA, rt,
2
0
4 h; b) cyanuric chloride (1.0 equiv.), acetone, aq. NaHCO
C, 4 h; c) DSSEB (1 equiv.), TEA, DMSO, rt, 6 h.
3
(pH 8.7),
The commonly used mAb 9E10 and 12CA5 that recognise the c-
myc epitope tag and infleunza hemagglutinin (HA) epitope tag
◦
35
36
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Scheme 4 Reagents and conditions: a) activated Eu(III) chelate 23 (3.0 equiv.), 0.1 M aq. NaHCO
3
(pH 8.5), rt, overnight; b) activated Eu(III) chelate 24
+
(
3.0 equiv.) under the same conditions. TEA = triethylammonium counter ion.
respectively, were chosen as the representative proteins. Anti-c-myc
and anti-HA mAb were labelled through incubation with a 70-fold
molar excess of activated Eu(III) chelate 24 in phosphate buffer
spectra of chelate–12CA5 conjugate are also presented in Fig. 6.
Monoclonal antibody 12CA5 was found to be labelled more
efficiently as inferred from an F/P ratio twice as high as the
37
(
pH 6.5). The resulting protein luminescent conjugates were ini-
one determined for mAb 9E10. Even though the difference
of reactive lysine residues between these two mAb is unknown,
the significant ratio improvement could be partly ascribed to
the lower concentration of the 9E10 mAb aliquots used in these
ꢀ
R
tially purified by size-exclusion chromatography over a Sephadex
G-25 column but a very poor resolution between unreacted
labelling reagent and labelled proteins was obtained. Thus, the
luminescent mAb had to be isolated by affinity chromatography
using a gel-immobilised protein A. Table 2 reports quantum yields
of luminescent proteins in Tris-HCl/glycine buffer, together with
the attached chelate to protein molar ratios (F/P), estimated
from the relative intensities of protein and chelate absorptions.
As an illustrative example, the absorption/excitation/emission
-
3
bioconjugation experiments (i.e., 0.7 against 1.8 mg cm ). When
compared to the non-covalently bound Eu(III) chelate 8, the
excitation/emission maxima of the luminescent protein conjugates
remain unchanged (Fig. 6). Furthermore, the quantum yield of
grafted Eu(III) chelate was slightly reduced by the same extent
with mAb 9E10 and 12CA5 (Table 2) as compared to the value
of free luminescent label (Table 1, see entries 3–4). However, the
luminescence brightness remains satisfactory and proved that the
activated Eu(III) chelate 24 is a suitable luminescent tag for various
bio-analytical applications.
Table 2 Quantum yields (excitation at 345 nm) and chelate to protein
◦
molar ratios of luminescent bioconjugates determined at 25 C
Luminescent
bioconjugate
U (%) (Eu(TMT)-AP
3
)
U (%) (R6G)
F/P
Conclusion and future work
Anti c-myc 9E10
Anti-HA 12CA5
Hexapeptide 27
10.9
10.0
13.3
9.3
8.5
10.3
3.6
7.7
1.0
In summary, we have achieved the synthesis of new lumi-
nescent water-soluble Ln(III) chelates based on an original
2
364 | Org. Biomol. Chem., 2011, 9, 2357–2370
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◦
Fig. 6 (a) Absorption spectrum of luminescent labelled mAb anti-HA 12CA5 in Tris·HCl/glycine buffer at 25 C; (b) Excitation (—) (Em. 615 nm) and
◦
emission (---) (Ex. 345 nm) spectra of luminescent labelled mAb anti-HA 12CA5 in Tris·HCl/glycine buffer at 25 C.
a general rule, location of the bioconjugatable moiety on the
pyrazine-chromophoric moiety would be beneficial. With this goal
in mind, a new synthetic strategy involving an aminopyrazine
derivative is currently under investigation.
Experimental Section
See ESI† for general remarks and details on experimental proce-
dures for peptide synthesis.
High-performance liquid chromatography separations
Several chromatographic systems were used for the analytical
experiments and the purification steps. System A: HPLC (Thermo
Hypersil GOLD C18 column, 5 mm, 4.6 ¥ 100 mm) with CH CN
3
and 0.1% aq. trifluoroacetic acid (aq. TFA, 0.1%, v/v, pH 2.2)
as eluents [100% TFA (5 min), followed by linear gradient from
-
1
0
to 80% (40 min) of CH CN] at a flow rate of 1.0 mL min .
3
Dual UV detection was achieved at 220 and 254 nm. System B:
RP-HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 4.6 ¥
1
00 mm) with MeOH and 0.1% aq. TFA, 0.1% as eluents [90%
TFA (5 min), followed by linear gradient from 10 to 90% (40 min)
-
1
of MeOH] at a flow rate of 1.0 mL min . Triple UV detection was
achieved at 220, 290 and 320 nm. System C: RP-HPLC (Thermo
Hypersil GOLD C18 column, 5 mm, 4.6 ¥ 100 mm) with MeOH
and triethylammonium acetate buffer (TEAA 25 mM, pH 7.0) as
eluents [100% TEAA (10 min), followed by linear gradient from 50
to 100% (25 min) of MeOH] at a flow rate of 1.0 mL min . Triple
UV detection was achieved at 220, 290 and 345 nm. System D: RP-
HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 4.6 ¥ 100 mm)
bis-pyridinylpyrazine ligand which was functionalised at the 6,6¢¢-
positions through an original synthetic pathway aimed at introduc-
ing the chelating arms. The ability of these tri-azaheterocyclic lig-
ands to efficiently sensitise europium(III) was also proved. Indeed,
the corresponding chelates 6 and 8 have shown good luminescence
-
1
3
-1
-1
properties in water: luminescence yield above 2000 dm mol cm
and a decay time around 1.30 ms. The most interesting feature is
the red-shift of the maximum absorption wavelength compared to
the usual terpyridine-based Ln(III) chelates. Indeed, the absorption
maximum is located at 344 nm and stays barely unaltered until
with CH
3
CN and aq. TFA, 0.1% as eluents [80% TFA (5 min),
followed by linear gradient from 20 to 100% (32 min) of CH
at a flow rate of 1.0 mL min . Triple UV detection was achieved
3
CN]
-
1
3
54 nm. Furthermore, a comprehensive bioconjugation study
at 220, 260 and 285 nm. System E: RP-HPLC (Thermo Hypersil
conducted with the non-symmetrical Eu(III) chelate 8 and a
model hexapeptide, has enabled us to select the most appropriate
bioconjugatable group (i.e., succinimidyl sebacate) for an efficient
labelling of lysine residues of proteins. Thus, Eu(III) chelates based
on this new scaffold could be potential candidates as luminescent
markers for bioaffinity assays. However, a more straightforward
synthetic pathway for the introduction of the bioconjugation arm
within the ligand scaffold would improve the global process. As
GOLD C18 column, 5 mm, 4.6 ¥ 100 mm) with CH
(25 mM, pH 7.0) as eluents [100% TEAA (2 min), followed by
linear gradient from 0 to 80% (40 min) of CH CN] at a flow rate of
1.0 mL min . Dual UV detection was achieved at 254 and 345 nm.
System F: semi-preparative RP-HPLC (Thermo Hypersil GOLD
3
CN and TEAA
3
-
1
C
18 column, 5 mm, 21.2 ¥ 250 mm) with CH
as eluents [80% TFA (5 min), followed by linear gradient from 20
to 60% (13 min) then from 60 to 100% (32 min) of CH CN] at a
3
CN and aq. TFA, 0.1%
3
This journal is © The Royal Society of Chemistry 2011
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-
1
flow rate of 16.0 mL min . Dual UV detection was achieved at
60 and 285 nm. System G: semi-preparative RP-HPLC (Thermo
J 7.7 and 0.9), 7.33 (1H, dd, J 7.7 and 0.8), 4.80 (2H, s), 0.95 (9H,
2
s), 0.11 (6H, s); d
C
(75.4 MHz, CDCl
3
) 163.3, 141.0, 139.1, 126.1,
Hypersil GOLD C18 column, 5 mm, 21.2 ¥ 250 mm) with MeOH
and aq. TFA, 0.1% as eluents [100% TFA (5 min) followed by a
linear gradient from 0 to 10% (3 min) then from 10 to 90% (40
min) of MeOH] at a flow rate of 14.0 mL min . Dual UV detection
was achieved at 260 and 285 nm. System H: semi-preparative RP-
HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 10 ¥ 100 mm)
118.7, 65.5, 26.0, 18.4, -5.3; MS (ESI+): m/z 302.07 and 304.00
[M + H] , calcd for C12H20BrNOSi: 302.29.
+
-
1
2
,6-Bis[6-((tert-butyldimethylsilyloxy)methyl)pyridin-2-
yl]pyrazine (12)
with CH
0 mM, pH 7.5) as eluents [100% TEAB (5 min) followed by
linear gradient from 0 to 80% (80 min) of CH CN] at a flow rate of
.0 mL min . Dual UV detection was achieved at 254 and 345 nm.
System I: LC-MS with RP-HPLC (Thermo Hypersil GOLD C18
column, 5 mm, 2.1 ¥ 150 mm) with CH CN and TEAB (50 mM,
pH 7.5) as eluents [95% TEAB (2 min) followed by linear gradient
3
CN and triethylammonium bicarbonate buffer (TEAB
(a) 2-Trimethyltin-6-[(tert-butyldimethylsilyloxy)methyl] pyridine:
To a solution of silyl ether 11 (2.0 g, 6.62 mmol) in dry THF
(14.0 mL) was added dropwise, under an argon atmosphere at
5
3
-
1
◦
4
-78 C, a solution of n-BuLi in hexane (1.4 M, 5.2 mL, 7.3 mmol).
◦
The dark red solution was stirred during 90 min at -78 C before
3
adding a solution of trimethyltin chloride (1.38 g, 6.95 mmol) in
dry THF (14.0 mL). The reaction mixture was left running at room
temperature for 4 h under an argon atmosphere. Thereafter, the
mixture was evaporated to dryness and the resulting yellow-brown
-
1
from 5 to 85% (40 min) of CH CN] at a flow rate of 0.25 mL min .
3
Dual UV detection was achieved at 220 and 345 nm. ESI-MS
detection in the negative mode (full scan, 150–1500 a.m.u., data
type: centroid, sheat gas flow rate: 60 arb unit, aux/sweep gas flow
paste was triturated twice with Et O and filtered. The filtrate was
2
evaporated and the stannane derivative was obtained as an orange
oil and was used in the next step without further purification
◦
rate: 20, spray voltage: 4.5 kV, capillary temp: 270 C, capillary
voltage: -10 V, tube lens offset: -50 V).
(2.40 g, yield 89%). d
(
(
H
(300 MHz, CDCl
1H, d, J 7.3), 7.29 (1H, d, J 7.2), 4.85 (2H, s), 0.96 (9H, s), 0.32
9H, s), 0.12 (6H, s).
b) Stille-coupling reaction: To a xylene solution (6 mL) deaer-
ated with argon were added Pd(PPh (87 mg, 0.08 mmol), 2,6-
3
) 7.53 (1H, t, J 7.6), 7.35
2
-Bromo-6-[(tert-butyldimethylsilyloxy)methyl]pyridine (11)
(
(
a) 2-Bromo-6-(hydroxymethyl)pyridine: To a cooled mixture of dry
)
3 4
THF (8.0 mL) and n-BuLi in hexane (1.35 M, 9.4 mL, 12.7 mmol),
a solution of 2,6-dibromopyridine (3.0 g, 12.7 mmol) in dry THF
dichloropyrazine (224 mg, 1.50 mmol) and trimethyltin derivative
(1.27 g, 3.30 mmol). The resulting reaction mixture was heated
to 110 C overnight. The desired compound as well as the
◦
(
16.0 mL) was added dropwise under an argon atmosphere, in
◦
order to keep the mixture below -70 C. The resulting reaction
monocoupling derivative were detected by NMR analyses. Further
mixture was left stirring for 15 min. and dry DMF (1.5 mL,
amounts of stannane (270 mg, 0.70 mmol) and Pd(PPh
3
4
) (12 mg,
1
9.7 mmol) was added. The reaction solution was further stirred
0.01 mmol) were therefore added and the mixture was left
◦
◦
at -78 C for 15 min, followed by addition of acetic acid (810 mL)
in methanol (12.0 mL). Afterwards, NaBH
running 24 h at 110 C. The reaction mixture was filtrated
ꢀ
R
4
(0.48 g, 12.7 mmol)
on a Celite pad. After solvent removal, a brown solid was
recovered. Purification of this crude product was achieved by
flash-chromatography on a silica gel column with a step gradient
of AcOEt (2–4%) in cyclohexane as the mobile phase to give the
bis-pyridylpyrazine derivative 12 as a white foam (520 mg, yield
was introduced in several portions. The cooling bath was removed
and the reaction mixture was warmed up to room temperature
overnight. Thereafter, the reaction mixture was carefully quenched
with sat. NH
The organic phase was washed with brine (40 mL), dried over
MgSO , filtered and concentrated under vacuum. The resulting
yellow oil was purified by flash-chromatography on a silica gel
column with a step gradient of AcOEt (20–30%) in cyclohexane
to afford the primary alcohol as a pale yellow oil (2.20 g, yield
4
Cl (40 mL), then extracted with AcOEt (2 ¥ 40 mL).
50%). Rf (cyclohexane–AcOEt, 96: 4, v/v) 0.24; d
CDCl ) 9.62 (2H, s), 8.39 (2H, d, J 7.8), 7.89 (2H, t, J 7.8),
7.59 (2H, d, J 7.7), 4.95 (4H, s), 0.99 (18H, s), 0.17 (12H, s);
(75.4 MHz, CDCl ) 161.4, 153.4, 149.7, 142.7, 137.7, 120.9,
119.7, 66.3, 26.1, 18.5, -5.2; MS (ESI+): m/z 523.33 [M + H] ,
calcd for C28 Si : 522.83.
H
(300 MHz,
4
3
d
C
3
+
9
2%). Rf (cyclohexane–AcOEt, 7: 3, v/v) 0.29; d
CDCl ) 7.55 (1 H, t, J 7.7), 7.39 (1H, d, J 7.7), 7.28 (1H, d, J 7.6),
.74 (2H, s), 3.20 (1H, bs); d (75.4 MHz, CDCl ) 161.6, 141.3,
39.3, 126.6, 119.5, 64.2; MS (ESI+): m/z 188.07 and 190.07 [M +
H
(300 MHz,
H
42
N
4
O
2
2
3
4
1
C
3
2
,6-Bis[6-(hydroxymethyl)pyridin-2-yl]pyrazine (13)
+
H] , calcd for C
b) Silylation: The alcohol (2.12 g, 11.3 mmol) was dissolved in
dry DMF (10.0 mL). Imidazole (3.08 g, 45.2 mmol) and TBS-Cl
2.04 g, 13.5 mmol) were sequentially added and the resulting
reaction mixture was stirred at room temperature for 25 min.
Thereafter, the reaction mixture was extracted with Et O (2 ¥
0 mL). The combined ethereal phases were washed with deionised
6
H
6
BrNO: 188.02.
To a solution of bis-silyl ether 12 (547 mg, 1.05 mmol) in THF
(10 mL) was added a 1.0 M solution of TBAF in THF (2.4 mL,
2.41 mmol). The resulting reaction mixture was stirred at room
temperature for 3 h and concentrated under reduced pressure to
give an orange-brown viscous oily residue. This crude product was
first triturated in cyclohexane and then in a mixture of aq. TFA
0.1% and methanol (1: 1, v/v) to give a the bis-alcohol 13 as a
white solid which was dried by lyophilisation (244 mg, yield 79%).
(
(
2
3
water (3 ¥ 5 mL), dried over MgSO , filtered and concentrated. The
4
resulting yellow oily residue was purified by flash-chromatography
on a silica gel column with a step gradient of AcOEt (1–3%) in
cyclohexane. The desired TBS ether 11 was obtained as a viscous
turbid liquid (3.01 g, yield 88%). Rf (cyclohexane–AcOEt, 99: 1,
d
H
(300 MHz, DMSO-d
(2H, t, J 7.8), 7.63 (2H, d, J 7.6), 5.58 (2H, t, J 5.8), 4.71 (4H, d, J
5.2); d (75.4 MHz, DMSO-d ) 162.2, 152.3, 149.1, 142.1, 138.2,
121.6, 119.5, 64.3; MS (ESI+): m/z 292.50 [M + H] , calcd for
: 294.31.
6
) 9.57 (2H, s), 8.45 (2H, d, J 7.7), 8.06
C
6
+
v/v) 0.15; d
H
(300 MHz, CDCl
3
) 7.57 (1H, t, J 7.7), 7.47 (1H, dd,
C
16
H
14
N
4
O
2
2
366 | Org. Biomol. Chem., 2011, 9, 2357–2370
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2
,6-Bis[6-(bromomethyl)pyridin-2-yl]pyrazine (14)
13.8 min, purity 96% (max plot 220–450 nm); lmax(recorded during
the HPLC analysis)/nm 229, 243, 283, and 310. The elemental
composition was accurately determined by UV-titration with an
PBr (0.29 ml, 3.1 mmol) was added to dry DMF (10 mL) under
an argon atmosphere and the resulting solution was cooled to
0
dry DMF (10 mL) was added to the PBr
reaction mixture was stirred at room temperature for h. After
DMF removal under high vacuum, the resulting residue was
3
aq. solution of known concentration of EuCl
expected stoichiometry [1 : 1] for the complex. An average value of
.5 molecule of TFA per molecule of ligand was found.
3
and by assuming the
◦
C. Bis-alcohol 13 (303 mg, 1.03 mmol) previously dissolved in
solution. The resulting
3
0
Eu(III) chelate (6)
diluted in CH
0 mL). The aq. phase was then re-extracted with CH
The organic phases were collected and washed with brine (40 mL),
dried over MgSO , filtered and concentrated. A clear brown solid
2
Cl
2
(50 mL) and washed with aq. sat. NaHCO
3
(2 ¥
Cl (30 mL).
3
2
2
Bis-pyridinylpyrazine ligand 10 (TFA salt, 70 mg, 48 mmol) was
dissolved in deionised water (1 mL) and NaHCO was added
3
4
to adjust the pH of the medium to approximately 7.5. Then,
was obtained. The compound was used in the next step without
further purification (410 mg, yield 95%). Rf (cyclohexane–AcOEt,
EuCl ·6H O (43 mg, 120 mmol) was added and the resulting reac-
3
2
tion mixture was stirred at room temperature for 1 h. Thereafter,
the crude Eu(III) complex was purified by flash-chromatography
on a RP-C18 silica gel column with deionised water. The product-
containing fractions were lyophilised to afford the desired Eu(III)
chelate 6 in a pure form as a white solid (Na salt, 34.3 mg,
9
7
: 1, v/v) 0.20; d
.7 and 0.8), 7.90 (2H, t, J 7.9), 7.55 (2H, dd, J 7.7 and 0.8), 4.67
(75.4 MHz, CDCl ) 156.9, 154.2, 149.1, 143.3, 138.2,
24.3, 120.7, 33.9.
H
(300 MHz, CDCl ) 9.71 (2H, s), 8.45 (2H, dd, J
3
(
4H, s); d
C
3
1
4
9.3 mmol, quantitative yield). MS (ESI-): m/z 671.20 and 673.20
-
Fully-protected symmetrical bis-pyridinylpyrazine ligand (15)
[M - H] , calcd for C24
H
21EuN
6
O
8
: 673.42; HRMS (ESI-): m/z
. HPLC (system C): t
.4 min, purity 100% (345 nm); lmax(recorded during the HPLC
671.0541 and 673.0555 for C24
H
20EuN
6
O
8
R
=
To a solution of di-brominated derivative 14 (157 mg, 0.37 mmol)
8
and NaI (61 mg, 0.41 mmol) in dry DMF (8 mL) were added DIEA
analysis)/nm 229, 295 and 344.
(
0.65 mL, 3.7 mmol) and secondary amine 9 (200 mg, 0.81 mmol).
The reaction mixture was stirred at room temperature for 6 h and
DMF was removed under high vacuum. The resulting residue was
Sm(III) chelate (7)
dissolved in CH
with an aq. sat. NaHCO
was re-extracted with CH
washed with brine, dried over MgSO
dryness. The resulting brown viscous oily residue was purified by
flash-chromatography on a silica gel column with a step gradient
2
Cl
2
(50 mL) and the organic phase was washed
solution (3 ¥ 20 mL). The aq. phase
Cl . The combined organic phases were
, filtered and evaporated to
Bis-pyridinylpyrazine 10 (TFA salt, 3.4 mg, 5.8 mmol) was
3
dissolved in 0.1 M aq. NaHCO (200 mL, pH 8.7). Thereafter,
3
2
2
a 41 mM SmCl ·6H O solution in deionised water (143 mL,
3
2
4
6.0 mmol) was added and the resulting reaction mixture was
stirred at room temperature for 1 h. Thereafter, the reaction
mixture was purified by flash-chromatography on a RP-C18 silica
gel column with deionised water. The product-containing fractions
were lyophilised to afford the desired Sm(III) chelate 7 in a pure
form as a white powder (Na salt, 3.7 mg, 5.3 mmol, yield 92%).
of MeOH (0–5%) in CH
yellow oil (199 mg, yield 64%). Rf (CH
.30; d (300 MHz, CDCl ) 9.66 (2H, s), 8.42 (2H, d, J 7.8), 7.87
2H, t, J 7.8), 7.73 (2H, d, J 7.7), 4.15 (4H, s), 3.56 (8H, s), 1.48
2
Cl
2
to afford the tert-butyl ester 15 as a
2
Cl –MeOH, 95: 5, v/v)
2
0
H
3
-
(
(
MS (ESI-): m/z 667.33, 669.33, 672.40, 674.20 [M - H] , calcd
36H, s); d
C
(75.4 MHz, CDCl
3
) 170.7, 159.3, 153.6, 149.7, 142.8,
for C H SmN O : 671.82; HPLC (system C): t = 11.3 min;
2
4
21
6
8
R
1
37.7, 124.1, 119.9, 81.2, 59.7, 55.9, 28.3; MS (ESI+): m/z 749.20
lmax(recorded during the HPLC analysis)/nm 233, 294 and
343.
+
+
+
[M + H] , 771.27 [M + Na] and 787.33 [M + K] , calcd for
C
40
H
56
N
6
8
O : 748.91.
Protected non-symmetrical bis-pyridinylpyrazine ligand (17)
Bis-pyridinylpyrazine ligand (10)
To a solution of di-brominated derivative 14 (122 mg, 0.29 mmol)
Tetrakis-tert-butyl ester 15 (200 mg, 0.27 mmol) was dissolved
in dry DMF (5 mL) were introduced NaI (25 mg, 0.17 mmol),
in CH
2
Cl
2
(10 mL) and deionised water (0.5 mL) and cooled
anhydrous K
2
CO (601 mg, 4.35 mmol) and secondary amine 16
3
◦
to 0 C. TFA (10 mL) was added and the reaction mixture was
stirred at room temperature for 5 h. Thereafter, the mixture was
evaporated to dryness and the residue was co-evaporated thrice
(133 mg, 0.32 mmol). The resulting reaction mixture was stirred
at room temperature for 9 h. Thereafter symmetrical secondary
amine 9 was added in several portions: 21.0 mg (0.09 mmol),
21.0 mg (0.09 mmol) after 27 h and 31.0 mg (0.12 mmol)
after two days. The reaction mixture was finally stirred at room
temperature for additional 24 h after the addition of the third
aliquot of 9. Thereafter DMF was evaporated and the residue
was diluted in a mixture of cyclohexane/AcOEt (1: 1, v/v)
which was filtered. The filtrate was evporated to dryness and
the resulting orange viscous oily residue was firstly purified by
flash-chromatography on a silica gel column cyclohexane/AcOEt
(6: 4, v/v). Thereafter, a second purification was achieved by
semi-preparative RP-HPLC (system F). The product-containing
fractions were lyophilised to give the desired ligand as a brown
with cyclohexane and finally triturated with Et O to give a pale
2
brown powder. This solid was diluted in deionised water and
lyophilised. A white amorphous powder was obtained (170 mg).
A batch of 100 mg was purified by flash-chromatography on a RP-
C18 silica gel column with a step gradient of MeOH (0–30%) in aq.
TFA 0.1%. The product-containing fractions were lyophilised to
afford the TFA salt of ligand 10 as a pale yellow solid (40 mg,
yield 44%). d
d, J 7.5), 8.06 (2H, t, J 7.8), 7.69 (2H, d, J 7.3), 4.17 (4H,
s), 3.64 (8H, s); d (75.4 MHz, DMSO-d ) 172.0, 152.6, 149.0,
42.2, 138.4, 124.3, 120.0, 59.0, 54.4; MS (ESI+): m/z 525.27
H
(300 MHz, DMSO-d
6
) 9.56 (2H, s), 8.48 (2H,
C
6
1
+
[M + H] , calcd for C24
H
24
N
6
O
8
: 524.48; HPLC (system B): t
R
=
foam (47 mg, yield 18%). d
H
(300 MHz, CDCl
3
) 9.67 (1H, s, H
1
or
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H
2
), 9.64 (1H, s, H
d, J 7.6, H16 or H ), 7.93 (1H, t, J 7.8, H
17 or H ), 7.81 (1H, d, J 7.6, H or H18), 7.70 (1H, d, J 7.5, H18
), 4.63 (1H, bs, NH), 4.23 (2H, s, H10), 4.20 (1H, d, J 15.2,
2
or H
1
), 8.49 (1H, d, J 7.7, H
6
or H16), 8.40 (1H,
13.7 min, purity 93% (max plot 220–450 nm); lmax(recorded during
the HPLC analysis)/nm 229, 243, 282 and 315. The elemental
composition was accurately determined by UV-titration with an
6
7
or H17), 7.90 (1H, t, J 7.8,
H
or H
7
8
8
aq. solution of known concentration of EuCl and by assuming the
3
H
H
20), 4.10 (1H, d, J 15.3, H20), 3.83 (4H, s, H11, H12), 3.54 (2H, s,
21), 3.38 (1H, t, J 7.4, H22), 3.07 (2H, bs, H28), 1.77–1.65 (2H,
expected stoichiometry [1 : 1] for the complex. An average value of
1.3 molecules of TFA per molecule of ligand was found.
m, H25), 1.58–1.30 (49 H, m, H, H26, H27, Ht-Bu); d
CDCl ) 172.3 (C23), 170.9 (C24), 168.6 (C14, C13), 159.5 (C29), 156.1
or C19), 155.7 (C19 or C ), 153.7 (C or C15), 153.0 (C15 or C ),
49.8 (C or C ), 149.4 (C or C ), 142.7 (C or C ), 142.3 (C or
), 138.5 (C or C17), 138.0 (C17 or C ), 125.1 (C or C18), 124.3
), 120.9 (C or C16), 120.1 (C16 or C ), 82.8 (C33, C34),
1.7 (C31 or C32), 81.3 (C32 or C31), 79.1 (C30), 64.4 (C22), 58.8 (C10),
C
(75.4 MHz,
3
(
C
9
9
5
5
1
3
4
4
3
1
2
2
C
1
7
7
8
(
C18 or C
8
6
6
8
5
2
8.2 (C20), 55.1 (C11, C12), 53.5 (C21), 40.4 (C28), 29.7 (C25 or C27),
9.6 (C27 or C25), 28.5 (Ct-Bu), 28.4 (Ct-Bu), 28.2 (Ct-Bu), 23.5 (C26);
+
+
MS (ESI+): m/z 920.33 [M + H] , 942.27 [M + Na] and 958.33
Non-symmetrical Eu(III) chelate (8)
+
[
2
2
M + K] calcd for C49
H
73
N
7
O
10: 920.14; HPLC (system C): t
6.7 min; lmax(recorded during the HPLC analysis)/nm 230, 243,
83 and 312.
R
=
This complex was prepared and purified by the same procedure
used for 6, and obtained as a pale brown powder (Na salt, 6.3 mg,
+
8
.2 mmol, yield 78%). MS (ESI+): m/z 744.07 and 746.13 [M + H] ;
-
MS (ESI-): m/z 742.20 and 744.13 [M - H] and 855.87 and 857.87
-
[
M + TFA - H] , calcd for C28
H
30EuN
7
O
8
: 744.55; HRMS (ESI-):
; HPLC (system
= 5.2 min (broad peak), purity 98% (345 nm); lmax(recorded
m/z 742.1276 and 744.1290 for C28
C): t
H
29EuN
7
O
8
R
during the HPLC analysis)/nm 224, 294 and 344.
Dichlorotriazine derivative (23)
Amino Eu(III) chelate 8 (1.0 mg, 1.3 mmol) was dissolved in 0.1 M
aq. NaHCO (200 mL, pH 8.7). Thereafter, a 13 mM cyanuric
3
chloride solution in HPLC-grade acetone (100 mL, 1.3 mmol)
◦
was added at 0 C. The resulting reaction mixture was stirred
at 0 C for 4 h. The reaction was checked for completion by
Non-symmetrical bis-pyridinylpyrazine ligand (18)
◦
ESI-MS. The crude activated Eu(III) chelate 23 was used in
Fully-protected ligand 17 (60 mg, 66 mmol) was dissolved in a
the next bioconjugation step without further purification. MS
mixture of CH
2
Cl (1.9 mL) and deionised water (90 mL) and
2
-
◦
(ESI-): m/z 891.07 [M - H] , calcd for C31
H
29Cl
2
EuN10
8
O : 892.50;
cooled to 0 C. Then, TFA (1.9 mL) was added and the resulting
reaction mixture was stirred at room temperature for 7 h. The
reaction was checked for completion by RP-HPLC (system B).
Further amount of TFA (0.3 mL) was added and the reaction
mixture was stirred overnight. Thereafter, the reaction mixture
was evaporated to dryness and the resulting brownish paste was
purified by semi-preparative RP-HPLC (system G). The product-
containing fractions were lyophilised to afford the TFA salt of non-
symmetrical bis-pyridinylpyrazine ligand 18 as a brown powder
HPLC (system E): t = 13.5 min; lmax(recorded during the HPLC
R
analysis)/nm 234, 295 and 344.
Succinimidyl sebacate derivative (24)
To a solution of DSSEB reagent (0.55 mg, 1.4 mmol) in DMSO
(
50 mL) was added at room temperature, every 30 min, 10 mL
of a mixture containing amino Eu(III) chelate 8 (1.0 mg, 1.3 m
mol) and TEA (2 mL) in DMSO (98 mL). After each addition,
the reaction mixture was vortexed and finally left at room
temperature for 45 min. The reaction was checked for completion
(
H
8
8
7
1
26.3 mg, yield 54%). d
or H ), 9.53 (1H, s, H
.44 (1H, d, J 7.2, H16 or H
.03 (1H, t, J 7.7, H17 or H
.68 (1H, d, J 7.6, H18 or H
5.5, H20), 4.03 (1H, d, J 15.3, H20), 3.67 (4H, s, H11, H12), 3.55
H
(300 MHz, DMSO-d
or H ), 8.47 (1H, d, J 7.2, H
), 8.05 (1H, t, J 7.7, H
), 7.73 (1H, d, J 7.7, H
6
+D
2
O) 9.55 (1H, s,
1
2
2
1
6
or H16),
or H17),
or H18),
6
7
by LC-MS (system I). The crude NHS ester 24 was stored at
◦
7
8
4
C and used in the next bioconjugation step without further
8
), 4.19 (2H, s, H10), 4.13 (1H, d, J
-
purification. MS (ESI-): m/z 1023.31 and 1025.29 [M - H] , calcd
for C42
H
49EuN
8
O
13: 1025.85. HPLC (system C): t
R
= 16.2 min;
(
1
2H, s, H21), 3.39 (1H, t, J 7.3, H22), 2.77–2.69 (2H, m, H28), 1.74–
.64 (2H, m, H25), 1.56–1.43 (4H, m, H26, H27); d (75.4 MHz,
DMSO-d +D O) 174.1 (C24), 172.7 (C23), 172.1 (C13, C14), 159.8
19), 158.5 (C ), 152.6 (C or C15), 152.5 (C15 or C ), 149.1 (C
or C ), 149.0 (C or C ), 142.1 (C , C ), 138.3 (C or C17), 138.2
), 124.3 (C ), 123.9 (C18), 119.9 (C or C16), 119.8 (C16
), 63.2 (C22), 59.0 (C10), 57.4 (C20), 54.4 (C11, C12), 52.6 (C21),
l
max(recorded during the HPLC analysis)/nm 234, 294 and 343.
C
6
2
Luminescent labelling of hexapeptide 25 with activated Eu(III)
chelates 23 and 24
(
C
9
5
5
3
4
4
3
1
2
7
(
C17 or C
7
8
6
(a) Labelling with 23: Hexapeptide 26 was dissolved in 0.1 M
or C
6
aqueous NaHCO pH 8.7 (22.13 mM, 9.8 mL, 217 nmol) and mixed
3
3
8.7 (C28), 29.0 (C25), 26.8 (C27), 22.8 (C26); MS (ESI+): m/z 596.27
with the crude activated Eu(III) chelate 23 (150 mL, 650 nmol,
the activation of amino chelate was assumed to be quantitative).
+
[M + H] , calcd for C28
H
33
N
7
O
8
: 595.60; HPLC (system B): t
R
=
2
368 | Org. Biomol. Chem., 2011, 9, 2357–2370
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The resulting reaction mixture was stirred at room temperature
overnight. The completion of the reaction was checked by HPLC
Luminescent measurements
Fluorescence spectroscopic studies were performed with a Varian
Cary Eclipse spectrophotometer using either a semi-micro quartz
fluorescence cell (Hellma, 104F-QS, light path: 10 ¥ 4 mm,
(
system E) and purified by semi-preparative RP-HPLC (system
H). The fractions containing the luminescent labelled peptide
-
were lyophilised. MS (ESI-): m/z 1464.40 [M - H] , calcd for
1
400 mL) for ligands and Eu(III) chelates, or an ultra-micro
C
57
H
67ClEuN17
O
18: 1465.67; HPLC (system E): t
purity 95%; lmax(recorded during the HPLC analysis)/nm 229,
95, 343 nm.
R
= 12.4 min,
ꢀ
R
fluorescence cell (Hellma , 105.51-QS, light patch: 3 ¥ 3 mm,
45 mL) for labelled mAb. Emission spectra were recorded in
2
“
phosphorescence mode” with the following parameters: delay
(
b) Labelling with 24: The same procedure as described above.
-
time: 0.1 ms, gate time: 3 ms and total decay time: 10 ms, and after
excitation at 345 nm. Lifetime measurements were achieved by
using the following parameters: delay time: 0 ms, flash number: 1,
excitation slit: 5 nm, emission slit: 10 nm, total decay time: 10 ms,
cycle number: 20 and PMT (sensitivity): medium, lexc: 345 nm,
MS (ESI-): m/z 1519.67 [M - H] , calcd for C64
H
83EuN14
20
O :
1
l
520.41; HPLC (system E): t
max(recorded during the HPLC analysis)/nm 227, 295 and 343.
For both labelling reactions, the bioconjugation yield was
R
= 12.2 min, purity 97%;
determined through quantification of the labelled peptide by UV–
vis spectroscopy at the lmax of the chelate (i.e., 345 nm)
l
em: 615 nm. The luminescence quantum yields were measured in
deionised water (except for labelled mAb which were performed in
◦
a mixture of Tris-HCl and glycine buffers, vide supra) at 25 C by
27
a relative method using two standards: rhodamine 6G (U = 88%
Labelling of mAb with activated Eu(III) chelate 24
28
29
in ethanol) and Eu(TMT)-AP (U = 16.9% in deionised water)
3
All U are corrected for changes in refractive index. Excitation was
achieved at 345 nm. To minimise the inner filter effect, samples
with an absorbance below 0.1 unit were used.
(
a) Labelling of anti c-myc 9E10 mAb: An aq. solution of anti c-
-
3
myc 9E10 mAb (0.7 mg cm , 500 mL, 2.33 nmol) in phosphate
buffer (0.1 M, pH 6.5) was mixed with the crude activated Eu(III)
chelate 24 (72 equiv., the activation of amino chelate was assumed
to be quantitative). The resulting reaction mixture was left at
room temperature overnight without stirring. Thereafter, the crude
mixture was purified by affinity chromatography (vide infra).
UV–visible titrations
A solution of bis-pyridinylpyrazine ligand (estimated concentra-
tion: 10 mM) was prepared in deionised water and transferred into
a quartz UV-cell (Varian, 6610000800, cell Open Top, 10 ¥ 10 mm,
3.5 mL). Titration was performed by adding periodically aliquots
(
b) Labelling of anti HA 12CA5 mAb: The same procedure
as described above was applied to an aq. solution of anti HA
1
pH 6.5.
-
3
2CA5 mAb (1.8 mg cm , 500 mL, 6.00 nmol) in phosphate buffer
(
usually 10 mL then 2 mL when the ratio between the ligand and the
metal was closed to 1 : 1) of an exactly known concentration aq.
EuCl in deionised water ([EuCl ] = 0.48 or 0.24 mM). After each
(
c) Purification by affinity chromatography: ~ 1 mL of a gel-
3
3
immobilised protein A was used. First, the gel was rinsed with
0
addition, the solution was stirred for 5 min before recording the
.1 M borate buffer (containing 0.15 M NaCl, pH 8.5, 20 mL).
◦
corresponding UV-visible spectrum (220–450 nm) at 25 C. For
The binding step was carried out with the same buffer and the
elution of the labelled antibodies was performed with a glycine
buffer (0.1 M, pH 3.5). Each acidic fraction (~750 mL) was
neutralised by adding 50 mL of Tris-HCl buffer (0.5 M, pH 8.5).
The elution of the desired labelled antibodies was checked by UV
absorbance measurements. The luminescence features of labelled
mAb were directly determined in this mixture of buffers. After
using, the column was washed with a solution of aq. HCl (pH 1.5),
drawing the titration curves, changes of absorbance at 354 nm was
used.
Acknowledgements
This work was supported by the “Minist e` re de l¢enseignement
sup e´ rieur et de la recherche” through a young investigator grant
(
ACI-JC 4064), and a PhD grant for S e´ verine Poupart. La R e´ gion
neutralised with borate buffer and stored in PBS buffer containing
NaN
◦
Haute-Normandie via the CRUNCh program (CPER 2007–2013),
and via a shared CNRS - R e´ gion Haute-Normandie grant for
Nicolas Maindron, and Institut Universitaire de France (IUF)
are also greatly acknowledged for their financial support. We
thank Albert Marcual (COBRA-CNRS UMR 6014) for high-
resolution mass analyses and Dr Herv e´ Volland (iBiTecS, LERI,
CEA-Saclay) for kind gift of monoclonal antibodies and gel-
immobilised protein A.
3
0.01% at 4 C.
(
d) Labelling molar ratio F/P: They were measured by recording
the UV absorbance at 280 and 345 nm respectively. The absorbance
value at 345 nm enabled us to determine the concentration of
Eu(III) chelate grafted to the protein. At this wavelength, the
Eu(III) chelates were the unique absorbing-species. The mAb
concentration could be measured by subtracting from the UV
absorbance value at l = 280 nm the residual absorbance at the
same wavelength coming from grafted Eu(III) chelates (the chelate
absorbance value at 345 nm was found to be twice higher than
the one at 280 nm). The measured values were inserted into the
following equation:
Notes and references
1
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[
mAb] (mM) = (A280 - 0.5 ¥ A345)/(1.48 ¥ 0.15)
2
3
The average molecular weights of the two mAb are 150 000 Da
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E. Soini and I. Hemmil a¨ , Clin. Chem., 1979, 25, 353.
(
4 J.-C. G. B u¨ nzli, Chem. Rev., 2010, 110, 2729.
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Helv. Chim. Acta, 1997, 80, 86.
3
-1
-1
6
7
17 A value of 1 400 dm mol cm was reported by Latva et al. (ref. 19)
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(
c) E. F. Dickson Gudgin, A. Pollak and E. P. Diamandis, Pharmacol.
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9
P. R. Selvin, in Top. Fluoresc. Spectrosc., ed. J. R. Lacowicz, Kluwer
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1
1
32 W. D. Horrocks, Jr. and D. R. Sudnick, J. Am. Chem. Soc., 1979, 101,
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8
647.
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1
1
4 (a) V. M. Mukkala, M. Helenius, I. Hemmil a¨ , J. Kankare and H.
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ꢀ
R
37 As reported by Perkin Elmer for LANCE Eu-activated chelates (see
http://las.perkinelmer.com/Content/manuals/man_ad0020.pdf),
a
F/P value of 4–10 chelates per protein (whose molecular weight is
higher than 100 000) is a good labelling yield.
5 E. Brunet, O. Juanes, R. Sedano and J. C. Rodriguez-Ubis, Tetrahedron
Lett., 2007, 48, 1091.
38 V. M. Mukkala, H. Takalo, P. Liitti and I. Hemmil a¨ , J. Alloys Compd.,
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2
370 | Org. Biomol. Chem., 2011, 9, 2357–2370
This journal is © The Royal Society of Chemistry 2011