Aminopropargyl derivative of terpyridine-bis(methyl-enamine) tetraacetic acid chelate of europium (Eu (TMT)-AP3): A new reagent for fluorescent labelling of proteins and peptides

Article abstract of DOI:10.1039/b612805j

The synthesis and photophysical properties of a new terpyridine-based europium(iii) chelate (Eu (TMT)-AP₃) designed for peptide and protein labelling in aqueous solution phase is described. In order to obtain a stable, easy to handle, versatile

Full text of DOI:10.1039/b612805j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Aminopropargyl derivative of terpyridine-bis(methyl-enamine) tetraacetic acid
chelate of europium (Eu (TMT)-AP3): a new reagent for fluorescent labelling
of proteins and peptides†
Se´verine Poupart,a Ce´dric Boudou,a Philippe Peixoto,a Marc Massonneau,b Pierre-Yves Renard*a and
Anthony Romieu*a
Received 4th September 2006, Accepted 22nd September 2006
First published as an Advance Article on the web 12th October 2006
DOI: 10.1039/b612805j
The synthesis and photophysical properties of a new terpyridine-based europium(III) chelate (Eu
(TMT)-AP3) designed for peptide and protein labelling in aqueous solution phase is described. In order
to obtain a stable, easy to handle, versatile and efficient labelling agent, a reactive aminopropargyl arm
has been introduced onto the terpyridine moiety. As preliminary biochemical applications the chelate
has been 1) efficiently covalently attached onto a representative biomolecule—monoclonal
antibody—and 2) converted into iodoacetamido and aldehyde derivatives, and the photoluminescent
Eu (TMT)-AP3 was grafted onto cysteine and lysine amino acid residues respectively. These two
different solution phase labelling methods yielded original fluorogenic FRET based probes suitable for
“in vitro” detection of caspase-3 protease, a key mediator of apoptosis of mammalian cells.
Analytical Sciences. Therefore, some chelates composed of a 4ꢀ-
Introduction
aryl-substituted 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine unit as the energy-absorbing
Lanthanide chelates, due to their unique luminescence properties
and donating group, Eu(III), Sm(III) or Tb(III) as the emitting ions,
(long decay-time, large Stokes’ shift, narrow emission band
methylenenitrilo (acetic acids) as the stable chelate-forming moi-
and negligible concentration quenching), are gaining expand-
eties, and iodoacetamido or isothiocyanato groups as the activated
ing applications in a wide variety of bioanalytical assays, in
moieties for coupling to biomolecules are commercially available
diagnostics, research, drug discovery, as sensing tools and in
and routinely used in various types of time-resolved fluorometry
imaging.1 These chelates contain an organic ligand that absorbs
light (the “antenna”), a lanthanide ion that emits the light after
energy transfer from the ligand to the metal ion and a reactive
group for conjugation to target bioactive molecules (such as
proteins, peptides, oligonucleotides,· · ·) through selective reaction
with their amino or mercapto groups. In addition to these
features and with respect to energy-transfer processes and min-
imisation of non-radiative deactivation, the Ln(III) environment
in a lanthanide-containing luminescent probe must also fulfil
several other requirements: high thermodynamic stability, kinetic
inertness, and a saturated coordination sphere.2 Therefore, it is
advantageous to resort to polydentate ligands for building a
fitted coordination environment around Ln(III) ions. Among the
numerous imaginative strategies developed to insert Ln(III) ions
into functional edifices, those using functionalised terpyridine
derivatives as polydentate ligands have received considerable
attention3 especially from the research groups of GE Healthcare
(formerly Amersham-Biosciences) and Perkin Elmer Life and
(or quenching)-based assays (examples 1 and 2 in Scheme 1).4–7
The synthetic strategies for the preparation of 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine
ligands possessing functional groups directly attached to C-4ꢀ are
versatile and well established.8 However, it is amazing to note that
no general and efficient method for introducing an alkylamino
group onto these functionalised terpyridine architectures has been
reported to date. As the amino group is essential for activation
and subsequent coupling of the Ln(III) chelate to biomolecules,
several methods leading to the incorporation of a less reactive
aminophenyl moiety have been developed.6,9 They involve either
the reduction of the aromatic nitro group of the corresponding
4ꢀ-aryl-substituted 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine or the cross-coupling of
an (aminophenyl)acetylene derivative to the 4ꢀ-(4-bromophenyl)-
2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine ligand. As the aniline derivatives are often
too unstable to be easily isolated in a pure form in good yields,
these latter methods are not suitable for routine preparation of
amino-terpyridines at a small scale (∼1 mmol). Furthermore, the
luminescence of the corresponding Eu(III) chelates is quite low
because aromatic amines quench the excited state of the lanthanide
ion by an electron transfer mechanism.10 Their activation by
acylation with a bifunctionnal reagent (iodoacetic anhydride
or thiophosgene) leading to the thiol-specific iodoacetamido or
amine-reactive isothiocyanato derivative, is essential to partially
recover the luminescence.6 Thus, especially, these chelates are not
proper reagents for the labelling of proteins through traditional
techniques involving the use of non-acylating bifunctionnal cross-
linking reagents such as glutaraldehyde.11
aIRCOF/LHO, Equipe de Chimie Bio-Organique, UMR 6014 CNRS,
INSA de Rouen et Universite´ de Rouen, 1, rue Lucien Tesnie`res, FR-
76131 Mont-Saint-Aignan Cedex, France. E-mail: pierre-yves.renard@
univ-rouen.fr, anthony.romieu@univ-rouen.fr; Fax: (+33)2-35-52-29-59
bQUIDD, Technopoˆle du Madrillet – ESIGELEC, Avenue Galile´e – BP
10024, FR-76801 Saint Etienne du Rouvray, France
† Electronic supplementary information (ESI) available: Synthesis of
Eu(III) chelate dimer 18, detailed synthetic procedures and caspase-3 assay
of FRET substrate 21. See DOI: 10.1039/b612805j
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Scheme 1 Examples of commercially available Ln(III) chelates composed of a 4ꢀ-aryl-substituted 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine unit as the energy-absorbing and
donating group.
With the goal in mind to develop a new label for biochemical
applications, that displays good luminescence properties, is easy to
Results and discussion
Synthesis of the AP3-terpyridine ligand
prepare and to handle, and usable in a wide range of bioconjugate
techniques involved in solution phase protein labelling, we have
examined the chemical functionalisation of Ln(III) chelates derived
from 4ꢀ-aryl-substituted 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine ligands, with an
alkynylamino group (3-aminopropynyl or aminopropargyl, AP3).
Indeed, we thought that the structural features of this linker
(small size, rigidity and presence of a reactive and stable primary
alkylamino group) should be beneficial for the introduction of the
luminescent label onto the target biopolymers without disrupting
their original structure and properties. This AP3 arm has been
successfully used in the field of nucleic acids chemistry especially
as a key component of fluorescent labelled nucleotides currently
used in DNA sequencing.12,13
Here we report the first synthesis and luminescence properties of
the aminopropargyl derivative of terpyridine-bis(methyl-enamine)
tetraacetic acid europium chelate (Eu (TMT)-AP3) 3. The use of
this chelate for solution phase labelling of peptides and proteins
is illustrated by the preparation of two original FRET substrates
suitable for a caspase-3 assay14 and by its coupling to a monoclonal
antibody.
Terpyridine ligand was prepared in
9
3 steps from the
already published 6,6ꢀꢀ-bis(aminomethyl)-4ꢀ-(4-bromophenyl)-
2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine ligand 4 (Scheme 2).15 The introduction of
the four chelating carboxylic acid functions (by alkylation of
the free aminomethyl groups) and the reactive AP3 arm (by
Pd(0)-catalyzed Sonogashira cross-coupling16) requires the use of
reagents (bromoacetic acid and propargylamine) bearing a pro-
tected functional group (respectively CO2H or NH2). According
to the nature of protecting groups used (acid- or base-labile), two
different strategies have been tested.
We first examined the synthetic strategy which provides the full-
protected ligand 6 whose reactive functions are masked with base-
labile protecting groups (methyl ester for CO2H and trifluoroacetyl
for NH2 respectively, path a in Scheme 2). Thus, bis(aminomethyl)
terpyridine derivative 4 was carboxymethylated to the tetraester 5
by treatment with an excess of methyl bromoacetate in the presence
of DIEA and potassium iodide as catalyst in dry DMF. Thereafter,
reaction of 5 with N-propargyltrifluoroacetamide in the presence
of Pd(II) and CuI in a mixture of triethylamine and DMF
gave the desired ligand 6 in 49% yield. Finally, the removal
of the protecting groups was achieved by treatment with 1 N
sodium hydroxide in methanol. However, this treatment led to
the complete decomposition of the ligand. Furthermore, HPLC
analyses of the crude deprotection mixture have clearly shown
the formation of numerous decomposing products which were
not characterised. However, 1H NMR analyses of the crude
deprotected ligand have shownthe loss of the signal(singletaround
3.70 ppm in D2O) assigned to the methylene group of the AP3 arm.
This result suggests that the side-reaction(s) probably starts with
proton abstraction from this carbon to give a carbanion which
might decompose through different chemical pathways.
Consequently, we have explored another protection strategy
(path b in Scheme 2). Indeed, we thought that the use of
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trityl groups was achieved by treatment with a trifluoroacetic
acid solution containing 5% of water and 5% of thioanisole. The
crude AP3-terpyridine ligand 9 was isolated by precipitation in
Et2O. However, further purification by flash-chromatography on
a RP-C18 silica gel column was essential to remove hydrophobic
side-products resulting from the reaction between thioanisole and
tert-butyl or trityl cations. Thus, 9 was obtained in 41% yield and
its structure was confirmed by detailed measurements, including
ESI mass spectrometry and NMR analyses.
Synthesis and characterisation of the Eu (TMT)-AP3 chelate
The AP3-terpyridine ligand 9 was converted to the corresponding
Eu(III) chelate 3 by treatment with aqueous europium(III) chloride
(Scheme 3). Purification was achieved by flash-chromatography on
a RP–C18 silica gel column and provided 3 in quantitative yield.
The formation of this lanthanide chelate is clearly demonstrated by
UV–visible spectrophotometric titration of 9 in water by following
the variations in the UV–visible spectra of the ligand upon
Scheme 2 Synthesis of the AP3-terpyridine ligand 9.
acid-labile protecting groups (tert-butyl ester for CO2H and
trityl for NH2 respectively) which can be removed by treatment
with trifluoroacetic acid and appropriate scavengers, was more
suited to the stability of our AP3-terpyridine ligand. With this
goal in mind, the bis(aminomethyl) terpyridine derivative 4 was
carboxymethylated to the tetraester 7 by treatment with an excess
of tert-butyl bromoacetate as described above for the methyl
ester. Thereafter, reaction of 7 with N-tritylpropargylamine in
the presence of Pd(Ph3)4 and CuI in triethylamine gave the
desired ligand in moderate yield. Unlike the Sonogashira cross-
coupling reactions involving N-protected propargylamine and
less sophisticated halogeno-aryl derivatives,12 it is essential to use
a significantly higher amount of Pd catalyst to get acceptable
yields. That may be explained by partial inhibition of this catalyst
through chelation of its metal centre within the terpyridine moiety.
Furthermore, several attempts have clearly shown that more
reproducible yields in 8 were obtained when Pd(Ph3)4 was used
instead of PdCl2(Ph3)2. Finally, the removal of the tert-butyl and
Scheme 3 Synthesis of the Eu (TMT)-AP3 chelate 3 and its corresponding
iodoacetyl and aldehyde derivatives 11 and 12.amixture of reactive species
(monomeric and polymeric forms).
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addition of increasing amounts of EuCl3·6H2O salt. Addition
of aliquots of the lanthanide solution immediately results in
the displacement of the absorption maximum to lower energies,
from 290 nm for the free ligand to 298 nm for 1 equiv. of
added lanthanide salt. Upon further addition of europium the
spectra remained unchanged. This result pointed to the formation
of a single new absorbing species with a one metal to one
ligand stoichiometry. In these conditions, the apparent association
constant for the formation of the complex appears to be too
high to be determined by a single titration curve. However,
competitive binding experiments indicate that the TMT moiety
readily displaces the Eu(III) ion from the EDTA–Eu complex4 and
so that the conditional stability constant of 3 could be estimated
as superior to 1017. ESI-MS spectra of 3 were recorded in either
positive or negative modes, revealing in both cases the expected
molecular peak (Fig. 1). Indeed, the positive integration mode
displayed [(L − 3H+) + Na+ + Eu3+]+ as the major peaks with
the expected isotopic pattern distribution (823.1 (85%) and 825.1
(100%)) and the spectrum recorded in the negative mode showed
the presence of the [(L − 4H+) + Eu3+]− species at 799.2 and 801.2
m/z units (with 80% and 100% relative intensities, respectively).
Photophysical properties of the Eu (TMT)-AP3 chelate
The excitation maxima, absorption coefficient (e), emission max-
ima, mean metal luminescence lifetime (s), quantum yield (U)
and luminescence yield (eU) for the chelate 3 are presented in
Table 1. Unlike Eu(III) chelates bearing a free aromatic NH2
group,6 3 shows a strong metal luminescence in water solution
upon excitation of the terpyridine ligand. As an example, the
excitation and emission spectra of 3 are shown in Fig. 2. The
emission spectrum presents the usual 5D0
transitions typical of the Eu(III) ion. The luminescence quantum
yield of this chelate was measured by a relative method using
→
7FJ (J = 1–4)
Fig. 1 ESI mass spectra of the Eu (TMT)-AP3 chelate 3. a) positive mode, b) negative mode, calcd mass for C34H28N6O8Eu 800.60.
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under mild reducing conditions (St-Bu for cysteine and Pydec17 for
lysine) has enabled the selective introduction of the two fluorescent
labels at two different amino acid residues within the peptides, thus
obtaining FRET substrates (Eu (TMT)-AP3 chelate and Cy 5.0 dye
act as donor and acceptor respectively) through original labelling
methods. The target hexapeptides Ac-Cys(Eu (TMT)-AP3)-Asp-
Glu-Val-Asp-Lys(Cy 5.0)-NH2 16 and Ac-Lys(Cy 5.0)-Asp-Glu-
Val-Asp-Lys(Eu (TMT)-AP3)–NH2 21 contain the Asp-Glu-Val-
Asp motif which is a specific substrate for caspase-3. The cleavage
site is located after the aspartic acid residue at the C-terminal
side. Caspases are Cysteine-ASpartic-acid-ProteASES that play a
critical role as mediators for apoptotic cell death.18 Caspase-3 has
been specifically identified as being a key mediator of apoptosis
of mammalian cells19: activation of caspase-3 indicates that the
apoptotic pathway has progressed to an irreversible stage. There
is thus a growing interest in identifying caspase inhibitors to
minimise cell death in pathological conditions (neurodegenerative
diseases for instance) but also for inducing caspase activation in
cancer cells.20 In addition, caspase-3 is widely used for monitoring
apoptosis induction for general cytotoxicity screening. This inter-
est for caspase-3 resulted in the development of several assays using
a variety of formats and amenable to high throughput screening.21
Peptides 13 and 17 have been chosen as suitable targets in order
to get novel fluorogenic substrates useful for detecting apoptosis
in whole cells and for cell-based high throughput screening assays
for apoptosis inhibitors or inducers.22
Table 1 Photophysical properties of Eu (TMT)-AP3 chelate
3 in
deionised water at 25◦C
kmax, abs/nm kmax, em/nma e/L mol−1 cm−1 Ub
eUc s/msd
238, 298 616 25 000, 42 000 0.0064 210 1.41
5
7
a Only the most intense emission band corresponding to the D0 → F2
transition is presented. b Determined by using Ru(bpy)3Cl2 as a standard
(U = 0.028, according to ref. 37). c e value of 3 at k = 292 nm (32 956 L
mol−1 cm−1) corresponding to the excitation wavelength selected for the
quantum yield measurements was used for this calculation. d An aq.
solution of 3 at a concentration of 11.0 10−6 mol L−1 was used. The
measurement was also achieved in D2O under the same conditions,
s(D2O) = 2.28 ms. As expected, the hydration number (q) obtained from
the equation q = A [s(H2O)−1 − s(D2O)−1 − 0.25] (according to ref. 39),
where A = 5.0, was found to be close to zero (q = 0.10).
Firstly, the iodoacetyl moiety was introduced on the AP3 arm of
3 by acylation of its primary amino group with the commercially
available heterobifunctional cross-linker sulfosuccinimidyl(4-
iodoacetyl)aminobenzoate (i.e., Sulfo-SIAB) in borate buffer
(pH 8.1). After completion of the reaction, a further derivatisation
step of the unreacted Sulfo-SIAB reagent by treatment with a
scavenging excess of pentylamine was achieved to facilitate the
purification. Indeed, flash-chromatography on a RP-C18 silica gel
column provided the thiol-reactive luminescent Ln(III) chelate
11 in a pure form (yield 68%). Its structure was confirmed by
ESI mass spectrometry. Due to the poor stability of the couple
of fluorophores (3 and Cy 5.0) towards harsh acidic conditions
currently used for the final deprotection of synthetic peptides,
post-synthetic labelling of the peptidic backbone Ac-Cys-Asp-
Glu-Val-Asp-Lys-NH2 seems to be the best synthetic strategy to
get the target caspase-3 substrate 16 in good yield and with a
high degree of purity (Scheme 4). Thus, the crude peptide Ac-
Cys(St-Bu)-Asp-Glu-Val-Asp-Lys-NH2 13 was firstly treated with
the N-hydroxysuccinimidyl ester of Cy 5.0 dye in dry DMF in
the presence of triethylamine. Purification by RP-HPLC provided
the Cy 5.0 labelled peptide Ac-Cys(St-Bu)-Asp-Glu-Val-Asp-
Lys(Cy 5.0)-NH2 14 in 62% yield. It should be mentioned that
trifluoroacetic acid was substituted by acetic acid (or formic acid)
in the HPLC purification because prolonged exposure of Cy 5.0
dye to strong acid (such as 0.1% aq. trifluoroacetic acid solution)
was detrimental for fluorescence (although little change in the
absorption spectrum was observed).23 The removal of the tert-
butylthio protecting group from the cysteine residue was achieved
by treatment with an excess of dithiothreitol (DTT) in a mixture
of DMF and aq. sodium bicarbonate buffer (pH 8.5). Purification
of the free sulfhydryl containing peptide–Cy 5.0 conjugate 15 was
achieved by RP-HPLC. Finally, iodoacetamido activated Eu(III)
chelate 11 was attached on the free cysteine of peptide 15 by using
Fig. 2 Normalised absorption and emission spectra of 3 in deionised
water at 25 ◦C.
Ru(bpy)3Cl2 as a standard.7 The luminescence yield of 3 was found
to be comparable to values reported for some Ln(III) chelates
derived from the parent compound 10 by substitution of its 4ꢀ-
phenyl ring.6 Therefore, these photophysical properties allowed
us to consider some applications of 3 as labelling reagent of
biopolymers.
Labelling peptides in solution with the Eu (TMT)-AP3 chelate—
application to the preparation of FRET substrates of caspase-3
protease
To demonstrate the utility of the Eu (TMT)-AP3 chelate as a
reagent for biopolymer derivatisation, 3 was converted to the
corresponding iodoacetamido and aldehyde derivatives 11–12
(Scheme 3) and coupled to Cy 5.0 labelled hexapeptides bearing
either a free cysteine or a lysine amino acid residue (Scheme 4
and 5). The use of non standard protecting groups removable
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Scheme 4 Preparation of the energy-transfer substrate of caspase-3 enzyme 16.
a 2 : 1 molar excess (11 : 15) in sodium bicarbonate buffer (pH 8.5).
The caspase-3 substrate 16 was purified by RP-HPLC by using a
volatile aq. buffer (i.e., triethylammonium bicarbonate (TEAB),
50 mM, pH 7.5) and acetonitrile as eluents to keep the integrity
of the Eu(III) chelate. Due to the small differences in polarity
between the peptides 15 and 16 and iodoacetamido derivative 11,
it is essential to use a slow linear gradient of increasing acetonitrile
(0.4% min−1) in aq. TEAB buffer to get peptide 16 in a pure form
(yield 40%, purity >95%). Analysis of this fluorogenic substrate
using MALDI-TOF mass spectrometry indicated the presence
and integrity of Cy 5.0 dye and Eu(III) chelate within the peptide
architecture (Fig. 3).
Fig. 3 MALDI-TOF mass spectrum of the fluorogenic substrate of caspase-3 protease 16, in the positive mode, a-cyano-4-hydroxycinnamic acid was
used as a matrix, (M + H)+: m/z: calcd 2348.4, found 2350.7. ¶loss of Eu(III) occurred during the ionisation process.
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Scheme 5 Preparation of the energy-transfer substrate of caspase-3 enzyme 21.
In addition to this first fluorescent labelling method, we have
explored an alternative strategy for introducing the same FRET
donor–quencher pair onto the two distinct lysine residues of
hexapeptide 17 (Scheme 5). This original fluorescent labelling
method involves the use of the Pydec moiety as protecting
group of a lysine side chain. Indeed we recently designed this
amine protecting group, fully compatible with the stability of
biopolymers and fluorophores. It is of an easy synthetic access,
and removable under mild reducing conditions. This protective
group proved to be a powerful tool to efficiently differentiate
two primary amino groups of otherwise similar reactivity.17 The
conversion of the Eu (TMT)-AP3 chelate into an amine-reactive
reagent is required for its coupling to a lysine residue. Chemical
derivatisation of 3 with N,Nꢀ-disuccinimidyl carbonate (DSC)
and thiophosgene was firstly explored. However, attempts to get
the corresponding succinimidyl carbamate and isothiocyanato
derivatives failed. Indeed, the stability of the DSC reagent was
found to be poorly compatible with the neutral aq. conditions in
which 3 is fairly soluble. As far as the reaction with thiophosgene is
concerned, quantitative formation of the Eu(III) chelate dimer 18
bearing a thiourea linkage was observed (isolation by RP-HPLC
and structure confirmed by ESI mass spectrometry), even under
the experimental conditions currently used for the derivatisation
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of aromatic amines with this reagent (slow addition of the aq.
solution of 3 to a large excess of thiophosgene).
Since the dicarbonyl compounds such as glutaraldehyde are
routinely used as protein cross-linking reagents,24 we decided
to modify the free amino group of 3 with this bis-aldehyde
compound to form an activated derivative able to react with
the free lysine residue (Scheme 3). The hexapeptide Ac-Lys-
Asp-Glu-Val-Asp-Lys(Pydec)-NH2 17, synthesised by the solid-
phase method as previously described,17 was firstly treated with
the N-hydroxysuccinimidyl ester of Cy 5.0 dye in dry NMP
in the presence of DIEA. Purification by RP-HPLC provided
the Cy 5.0 labelled peptide Ac-Lys(Cy 5.0)-Asp-Glu-Val-Asp-
Lys(Pydec)-NH2 19 in 85% yield. The removal of the Pydec
protecting group from the C-terminal side lysine residue was
achieved by treatment with an excess of DTT (50 equiv.) in Tris
HCl buffer (pH 9.0). Purification of the free amine containing
peptide–Cy 5.0 conjugate 20 was achieved by RP-HPLC. Finally,
the aldehyde derivative 12, prepared immediately prior to use
by treatment of Eu (TMT)-AP3 chelate with a slight excess of
glutaraldehyde (1.5 equiv.), was used to label the free amino group
of the peptide 20 by using 2 : 1 molar excess (12 : 20) in deionised
water. After reduction of the resulting Schiff base linkages with
sodium cyanoborohydride, the caspase-3 substrate 21 was purified
by RP-HPLC (yield 50%). On the RP-HPLC elution profile of this
fluorogenic probe (see ESI†), a broad peak was observed, reflecting
the presence of a mixture of dual-labelled peptides due to the
propensity of glutaraldehyde to polymerise in aq. solution and to
react with amines by several means such as aldol condensation
or Michael-type addition.25 Thus, it was not possible to get an
easily interpretable ESI or MALDI-TOF mass spectrum of 21.
However, FRET measurements (see below) have confirmed the
presence and integrity of the two fluorophores 3 and Cy 5.0 within
the hexapeptide.
Fig. 5 Fluorescence emission time course (excitation at 298 nm) of
peptide 16 (concentration 1 lM) with recombinant human caspase-3 (3.2
10−3 U, incubation time 3 h) in caspase assay buffer (100 mM NaCl, 40 mM
HEPES, 10 mM DTT, 1 mM EDTA, 10% (w/v) sucrose and 0.1% (w/v)
CHAPS, pH 7.2, 37 ◦C) at 616 (a) and 672 nm (b).
non-radiative transfer from the donor to the acceptor according
to the theory of Fo¨rster.26 The efficiency of the energy transfer
obtained with this doubly labelled substrate is compatible with the
use of this read-out for monitoring caspase-3 activity. Enzymatic
action will result from de-quenching of donor luminescence upon
separation of the two labels as a consequence of enzymatic
peptide bond cleavage. As expected, incubation of peptide 16 with
recombinant human caspase-3 has resulted in an increase of the
fluorescence emission of Eu(III) chelate at 616 nm (Fig. 5). This
de-quenching effect of the donor is concomitant to a decrease of
Cy 5.0 acceptor emission at 672 nm due to the abolishment of
FRET resulting from the protease cleavage. After a 1 h reaction
time, the donor luminescence total recovery suggests that the
peptide bond cleavage reaction induced by caspase-3 went to
completion and is in agreement with an initial almost quantitative
transfer efficiency. Furthermore, no non-specific cleavage of this
probe was observed in a control reaction in which peptide 16
was incubated only with the caspase-3 buffer. Similar results were
obtained when peptide 21 was submitted to the same enzymatic
hydrolysis (see ESI†). These results confirmed that caspase-3
protease exhibits a high degree of substrate tolerance towards
the amino derivative coupled to the C-terminal side of the Asp-
Glu-Val-Asp motif. Indeed, a wide range of fluorophores such
as coumarin,27 phenoxazine28 and rhodamine29 derivatives have
already been linked to this tetrapeptide through their aromatic
amino groups to get flurorogenic probes suitable for cell-based
apoptosis assays.
Photophysical characterisation and in vitro cleavage by caspase-3
of the FRET substrates
When we measured the fluorescence spectrum of the probe 16 (or
21) after excitation of the donor Eu(III) chelate at 298 nm, a major
emission at 672 nm corresponding to the Cy 5.0 fluorescence was
7
observed (Fig. 4). The lack of the 5D0 → F2 transition emission
at 616 nm confirmed that the luminescence of the donor is fully
transferred to the Cy 5.0 dye which shows a significant overlapping
of its excitation spectrum with the emission spectrum of the used
Eu (TMT)-AP3 chelate. This Cy 5.0 emission occurred via the
Fig. 4 Fluorescence emission spectra (excitation at 298 nm) of peptides 16 (a) and 21 (b) in caspase-3 buffer at 37 ◦C (concentration: 1 lM). *Residual
emission at 616 nm is most likely due to an uncomplete energy transfer from the donor to the acceptor. As peptide 21 is a mixture of compounds with
various content of polymerised glutaraldehyde, this results in Eu(III) labels which are more distant from the Cy 5.0 dye than the usual Fo¨rster distance.
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Antibody labelling using the Eu (TMT)-AP3 chelate
preliminary labelling experiments at the protein level have shown
that it is possible to efficiently label antibodies with the Eu
(TMT)-AP3 chelate by means of dicarbonyl cross-linking reagents
such as glutaraldehyde. As this latter protein conjugate does not
completely maintain the photophysical properties (luminescence
lifetime and quantum yield) of the free chelate, further efforts are
in progress to improve the luminescence yield of 3 especially by
introducing a further benzene ring between the AP3 arm and the
4ꢀ-phenyl-2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine moiety. Indeed, this approach has
recently been used by Nishioka et al. to get efficient luminescent
Eu(III) chelates for DNA labelling.33
As a further illustration of the applicability of the Eu (TMT)-
AP3 chelate synthesised for biomolecule derivatisation, 3 was
used in the labelling of a monoclonal antibody (anti c-myc
peptide monoclonal antibody, mAb), with the goal in mind to
develop homogeneous TR-FRET titration assays (time-resolved
fluorescent resonance energy transfer assay) for protein–protein
interactions.30 The reaction was easily performed under the same
conditions described above for the introduction of 3 onto lysine
residues within peptide architectures. This reaction proceeded
smoothly and the protein conjugate was easily and completely
R
purified by size-exclusion chromatography over a Sephadexꢀ G-
25 column. Protein quantification by UV spectroscopy (for IgG,
1 mg mL−1 has a mean absorbance at k = 280 nm of 1.48) has
enabled determination of a high labelling level of 5.8 Eu (TMT)-
AP3/mAb. The excitation and emission spectra of this protein
conjugate are shown in Fig. 6. The main emission line of the
chelate was observed as usual at around 616 nm. The average
luminescence decay time was long (s 1.03 ms) and comparable to
those recently reported for protein conjugates of Eu(III) chelates
derived from other 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine derivatives.31 As far as
the metal luminescence quantum yield of this protein conjugate is
concerned, when measured for each lanthanide ion, it was found to
be lower than that of the free Eu (TMT)-AP3 chelate 3 (U 0.0038).
Experimental
General
Column chromatographies were performed on silica gel (40–63
lm) from SdS. Reversed-phase column flash-chromatographies
were performed on octadecyl-functionalised silica gel (mean pore
˚
size 60 A) from Aldrich or Whatmann. TLC were carried out
on Merck DC Kieselgel 60 F-254 aluminium sheets. Compounds
were visualised by one of the two following methods (or both):
(1) fluorescence quenching, (2) spray with a 0.2% (w/v) ninhydrin
solution in absolute ethanol. MeOH was freshly distilled over
˚
Mg(OMe)2 and stored over 4 A molecular sieves. DMF was
dried by distillation over BaO. DIEA and triethylamine were
distilled from CaH2 and stored over BaO. 6,6ꢀꢀ-Bis(aminomethyl)-
2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine ligand 4 was prepared from 2-acetylpyridine
and 4-bromobenzaldehyde by using the multi-steps syn-
thetic procedure developed by Hovinen and Hakala.15 N-
Propargyltrifluoroacetamide, N-tritylpropargylamine and sulfo-
cyanine dye Cy 5.0 were prepared by using literature procedures.34
Hexapeptides Ac-Cys(St-Bu)-Asp-Glu-Val-Asp-Lys-NH2 13 and
Ac-Lys-Asp-Glu-Val-Asp-Lys(Pydec)-NH2 17 were synthesised
on an automated peptide synthesizer (ABI 433A, Applied Biosys-
tems) using the stepwise solid-phase synthesis method and Fmoc
amino acids, as previously described.17 EuCl3·6H2O was purchased
from Aldrich. N-Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate
(Sulfo-SIAB) was purchased from Pierce. Recombinant human
caspase-3 enzyme (5.52 U mg−1) was purchased from Sigma. The
HPLC grade solvents (CH3CN and MeOH) were obtained from
Acros. Aqueous buffers for HPLC were prepared using water
purified with a Milli-Q system. 1.0 M triethylammonium acetate
(TEAA) and triethylammonium bicarbonate (TEAB) buffers were
prepared from distilled triethylamine and glacial acetic acid or
CO2 gas. 1H-, 13C- and 19F-NMR spectra were recorded on a
Bruker DPX 300 (Bruker, Wissembourg, France). Chemical shifts
Fig. 6 Fluorescence emission and excitation spectra of the anti c-myc
mAb labelled with the europium chelate 3 at 25 ◦C (concentration:
0.117 mg mL−1 in PBS buffer).
Conclusion
In this paper, we have described the first synthesis and photophys-
ical properties of a luminescent Eu(III) chelate containing both
the well-known nonadentate 6,6ꢀꢀ-bis(aminomethyl)-4ꢀ-(phenyl)-
2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine ligand and an unusual aminopropargyl arm
(AP3 arm). The utility and the high reactivity of this latter linker
are demonstrated through the preparation of iodoacetamido and
aldehyde derivatives 11 and 12 which have been successfully
used in solution-phase peptide labelling. Furthermore, the use
of a non-standard thiol protecting group removable under mild
reducing conditions for temporarily masking a lysine residue, has
enabled the development of an efficient method for producing
dual fluorescently-tagged peptides. We believe that this labelling
strategy is complementary to the solid-phase methods32 because
the experimental conditions are compatible with a wide range
of fluorophores showing poor or moderate chemical stability. In
addition to the preparation of fluorogenic substrates of proteases,
are expressed in parts per million (ppm) from CDCl3 (dH
=
7.26, dC = 77.36) or D2O (dH = 4.79).35 J values are in Hz.
13C substitution was determined with a JMOD pulse sequence,
differentiating signals of methyl and methine carbons pointing
“down” (−) from methylene and quaternary carbons pointing
“up” (+). Infrared (IR) spectra were recorded as thin-film on
sodium chloride plates or KBr pellets using a Perkin Elmer FT-
IR Paragon 500 spectrometer with frequencies given in reciprocal
centimeters (cm−1). UV–visible spectra were obtained on a Varian
Cary 50 scan spectrophotometer. Fluorescence spectroscopic
R
studies were performed in a semi-micro fluorescence cell (Hellmaꢀ,
104F-QS, 10 × 4 mm, 1400 lL) with a Varian Cary Eclipse
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spectrophotometer. Mass spectra were obtained with a Finnigan
LCQ Advantage MAX (ion trap) apparatus equipped with an
electrospray source. The purified peptides were characterised by
MALDI-TOF mass spectrometry on a Voyager DE PRO in the
reflector mode with CHCA as a matrix. The syntheses of Eu(III)
chelate dimer 18 and FRET substrate 21 are described in the
ESI.†
on a silica gel column (30 g, dry loading) with a step gradient
of ethyl acetate (0–40%) in cyclohexane–triethylamine (9 : 1) as
the mobile phase, giving 0.48 g (0.65 mmol) of compound 5 as
an orange oil (yield 58%). Rf 0.78 (cyclohexane–ethyl acetate–
1
Et3N 45 : 45 : 10); H-NMR (300 MHz, CDCl3): d 8.61 (s, 2H),
8.47 (d, J = 7.9 Hz, 2H), 7.82–7.52 (m, 8H), 4.11 (s, 4H, –CH2–
N(CH2–CO2CH3)2), 3.65 (s, 8H, N–CH2–CO2CH3), 3.63 (s, 12H,
N–CH2–CO2CH3).
High-performance liquid chromatography separations
Full-protected AP3-terpyridine ligand 6
Several chromatographic systems were used for the analytical
experiments and the purification steps. System A: RP-HPLC
(Zorbax Eclipse XDB-C8 column, 5 lm, 4.6 × 150 mm) with
CH3CN and 0.1% aq. trifluoroacetic acid (aq. TFA, 0.1%, v/v,
pH 2.0) as the eluents [100% TFA (5 min), linear gradient from
0 to 45% (30 min) and 45 to 90% (15 min) of CH3CN] at a flow
rate of 1 mL min−1. UV detection was achieved at 285 nm. System
B: System A with triethylammonium acetate (TEAA, 100 mM,
pH 7.0) as aqueous buffer. System C: RP-HPLC (Waters Xterra
MS C18 column, 5 lm, 7.8 × 100 mm) with CH3CN and 0.1%
aq. acetic acid (aq. AcOH, 0.1%, v/v, pH 3.8) as the eluents
[95% AcOH (5 min), linear gradient from 5 to 65% (30 min) of
CH3CN] at a flow rate of 2.5 mL min−1. UV detection was achieved
at 260 nm. System D: System C with the following gradient
[95% AcOH (5 min), linear gradient from 5 to 50% (30 min) of
CH3CN]. System E: RP-HPLC (Zorbax Eclipse XDB-C8 column,
5 lm, 4.6 × 150 mm) with CH3CN and aq. AcOH as the eluents
[100% AcOH (5 min), linear gradient from 0 to 60% (30 min)
of CH3CN] at a flow rate of 1.0 mL min−1. UV–visible detection
was achieved at 650 nm. System F: System C with the following
gradient [100% AcOH (5 min), linear gradient from 0 to 70% (70
min) of CH3CN]. System G: RP-HPLC (Waters Xterra MS C18
column, 5 lm, 7.8 × 100 mm) with CH3CN and triethylammonium
bicarbonate buffer (TEAB 50 mM, pH 7.5) as the eluents [100%
TEAB (5 min), linear gradient from 0 to 10% (10 min) and 10
to 70% (120 min) of CH3CN] at a flow rate of 2.5 mL min−1.
UV detection was achieved at 260 nm. System H: System G with
the following gradient [100% TEAB (5 min), then linear gradient
from 0 to 10% (10 min) and 10 to 60% (120 min)] at a flow rate
of 2.5 mL min−1. UV detection was achieved at 260 nm. System I:
RP-HPLC (Zorbax Eclipse XDB-C8 column, 5 lm, 4.6 × 150 mm)
with CH3CN and TEAB buffer (50 mM, pH 7.5) as the eluents
[100% TEAB (5 min), linear gradient from 0 to 60% (30 min) of
CH3CN] at a flow rate of 1.0 mL min−1. UV detection was achieved
at 260 nm.
Compound
5 (0.46 g, 0.63 mmol) and N-propargyltri-
fluoroacetamide (0.14 g, 0.95 mmol) were dissolved in a mixture
of dry DMF–triethylamine (8 : 2, 4.4 mL) and de-aerated with
argon for 10 min. CuI (19 mg, 0.1 mmol) and PdCl2(PPh3)2
(35 mg, 0.05 mmol) were added and the mixture was stirred
overnight at 65–70 ◦C (oil bath temperature). After 5.5 h, further
amounts of N-propargyltrifluoroacetamide (71 mg, 0.47 mmol),
CuI (9.5 mg, 0.05 mmol), PdCl2(PPh3)2 (17.5 mg, 0.025 mmol),
DMF (1.75 mL) and Et3N (0.46 mL) were added. The reaction was
checked for completion by TLC (cyclohexane–ethyl acetate–Et3N
55 : 35 : 10) and the mixture was evaporated to dryness. Purification
by chromatography on a silica gel column (30 g, loading with
dichloromethane) with a step gradient of ethyl acetate (0–100%)
in cyclohexane–triethylamine (9 : 1) gave 0.25 g (0.31 mmol) of
compound 6 as an orange oil (yield 49%). Rf 0.36 (cyclohexane–
1
ethyl acetate–Et3N 55 : 35 : 10); H-NMR (300 MHz, CDCl3): d
8.63 (s, 2H), 8.48 (d, J = 7.9 Hz, 2H), 7.83–7.53 (m, 8H), 4.38
(d, J = 5.2 Hz, 2H, –C≡C–CH2–NHCOCF3), 4.12 (s, 4H, –CH2–
N(CH2–CO2CH3)2), 3.65 (s, 8H, N–CH2–CO2CH3), 3.63 (s, 12H,
N–CH2–CO2CH3); 19F-NMR (282 MHz, CDCl3): d −76.15 (s, 3F,
COCF3); MS (ESI+): m/z 806.20 [M + H]+, 828.07 [M + Na]+,
calcd for C40H39F3N6O9 804.79.
Tetra(tert-butyl) ester36
7
Compound 4 (0.57 g, 0.91 mmol) was suspended in dry DMF
(4 mL). KI (0.165 g, 1.0 mmol) and DIEA (2.53 ml, 14.5 mmol)
were sequentially added and the resulting mixture was de-aerated
with argon. tert-Butyl bromoacetate (1.1 mL, 7.25 mmol) was
added and the resulting reaction mixture was stirred overnight
under an argon atmosphere. The reaction was checked for
completion by TLC (cyclohexane–ethyl acetate–Et3N 55 : 35 :
10) and the mixture was evaporated to dryness. The residue
was dissolved in dichloromethane (50 mL) and washed with
aq. 5% NaHCO3 (1 × 50 mL) and brine (1 × 50 mL), dried
over Na2SO4 and evaporated to dryness. The resulting residue
was purified by chromatography on a silica gel column (20 g,
dry loading) with a step gradient of ethyl acetate (0–20%) in
cyclohexane–triethylamine (9 : 1) as the mobile phase, giving
0.58 g (0.64 mmol) of compound 7 as an orange oil (yield 70%).
Tetramethyl ester15
5
Compound 4 (0.7 g, 1.11 mmol) was suspended in dry DMF
(5 mL). KI (0.205 g, 1.22 mmol) and DIEA (3.11 mL, 17.81 mmol)
were sequentially added and the resulting mixture was de-aerated
with argon. Methyl bromoacetate (0.85 mL, 8.90 mmol) was added
and the resulting reaction mixture was stirred overnight under
an argon atmosphere. The reaction was checked for completion
by TLC (cyclohexane–ethyl acetate–Et3N 45 : 45 : 10) and the
mixture was evaporated to dryness. The residue was dissolved in
dichloromethane (75 mL) and washed with sat. NaHCO3 (1 ×
50 mL) and brine (2 × 50 mL), dried over Na2SO4 and evaporated
to dryness. The resulting residue was purified by chromatography
1
Rf 0.96 (cyclohexane–ethyl acetate–Et3N 55 : 35 : 10); H-NMR
(300 MHz, CDCl3): d 8.62 (s, 2H), 8.47 (d, J = 7.1 Hz, 2H),
7.82–7.56 (m, 8H), 4.09 (s, 4H, –CH2–N(CH2–CO2t-Bu)2), 3.48
(s, 8H, N–CH2–CO2t-Bu), 1.39 (s, 36H, N–CH2–CO2tBu); 13C-
NMR (75.5 MHz, CDCl3): d 27.9 (12C), 55.5 (4C), 59.6 (2C),
80.8 (4C), 118.3 (2C), 119.4 (2C), 123.1 (2C), 128.7 (2C), 131.8
(2C), 137.2 (2C), 137.6 (2C), 148.5, 154.9, 155.9, 158.5 (2C), 170.4
(4C).
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Full-protected AP3-terpyridine ligand 8
After several min, further 50 lL of aq. NaHCO3 were added to
again reach pH 6.5. The reaction was checked for completion by
UV–visible spectrophotometry. The mixture was dissolved with
deionised water (∼1 mL) and purified by chromatography on a RP-
C18 silica gel column (8 g) with a step gradient of MeOH (0–16%) in
water as the mobile phase. The product-containing fractions were
lyophilised to give the compound 3 as a white amorphous powder
(38.7 mg, quantitative yield). IR (KBr): mmax 1404, 1602 (broad),
2926, 3435 (broad) cm−1; HPLC (system B): tR = 17.5 min, purity
>95%; UV–visible (water): kmax (e) = 238 (25 000 L mol−1 cm−1),
298 (42 000 L mol−1 cm−1); MS (ESI+): m/z 801.0 and 803.0 [M +
H]+, 823.1 and 825.1 [M + Na]+; MS (ESI−): m/z 799.2 and 801.2
[M − H]−, calcd for C34H28N6O8Eu 800.60.
Compound 7 (0.165 g, 0.18 mmol) and N-tritylpropargylamine
(65 mg, 0.22 mmol) were dissolved in dry triethylamine (1.6 mL)
and de-aerated with argon for 10 min. CuI (1 mg, 5.2 lmol)
and Pd(Ph3)4 (5 mg, 4.3 lmol) wer◦e sequentially added and the
resulting mixture was stirred at 80 C overnight under an argon
atmosphere. The reaction was checked for completion by TLC
(cyclohexane–ethyl acetate–Et3N 55 : 35 : 10) and the mixture was
evaporated to dryness. Purification by chromatography was made
on a silica gel column (40 g, dry loading) with a step gradient of
ethyl acetate (0–10%) in cyclohexane–triethylamine (9 : 1) as the
mobile phase. 67 mg (0.60 mmol) of compound 8 were obtained
as a yellow foam (yield 33%). Rf 0.71 (cyclohexane–ethyl acetate–
Et3N 55 : 35 : 10); IR (CH2Cl2): mmax 755, 1143, 1218, 1260, 1368,
1
1581, 1736, 2978 cm−1; H-NMR (300 MHz, CDCl3): d 8.72 (s,
Iodoacetamido derivative 11
2H), 8.54 (d, J = 7.1 Hz, 2H), 7.89–7.81 (m, 4H), 7.71 (d, J = 7.0,
2H), 7.56–7.52 (m, 8H), 7.34–7.20 (m, 9H), 4.17 (s, 4H, –CH2–
N(CH2–CO2t-Bu)2), 3.55 (s, 8H, N–CH2–CO2t-Bu), 3.24 (bd, J =
4.2 Hz, 2H, –C≡C–CH2–NHTrt), 1.46 (s, 36H, N–CH2–CO2tBu);
13C-NMR (75.5 MHz, CDCl3): d 27.9 (12C), 34.2 (1C), 55.5 (4C),
59.6 (2C), 70.8, 80.7 (4C), 118.3, 119.4, 123.0, 126.3, 126.8, 127.0,
127.6, 127.7, 128.4, 131.9, 137.2, 145.0, 148.8, 155.0, 155.8, 158.5,
170.4 (4C); MS (ESI+): m/z 1120.40 [M + H]+, 1141.60 [M + Na]+,
calcd for C69H78N6O8 1119.43.
Compound 3 (22.0 mg, 0.027 mmol) was introduced into a Reacti-
VialTM and dissolved in borate buffer (0.8 mL, 50 mM, pH 8.1).
200 lL of a solution of Sulfo-SIAB reagent in borate buffer
(19.2 mg, 0.038 mmol) were added. The reaction mixture was
protected from light and stirred at room temperature for 2 h. After
30 min, further amounts of borate buffer (1 mL) and 3 N NaOH
(12 ll) were added to again reach pH ∼8. The reaction was checked
for completion by RP-HPLC (system B). Finally, the reaction
mixture was quenched with a solution of pentylamine in borate
buffer (19.1 ll in 2.0 mL), stirred for 30 min and purified by RP-C18
silica gel column (5 g) with a step gradient of acetonitrile (0–20%)
in water. The product-containing fractions were lyophilised to give
the compound 11 as a white amorphous powder (19.9 mg, yield
68%). HPLC (system B): tR = 25.5 min, purity >95%; MS (ESI+):
m/z 1132.0 and 1134.1 [M + 2Na]+; MS (ESI−): m/z 1086.0 and
1087.9 [M − H]−, calcd for C43H34IN7O10Eu 1087.66.
AP3-Terpyridine ligand 9
A mixture of TFA–PhSCH3–H2O (90 : 5 : 5, 3 mL) was cooled to
4 ◦C and added to compound 8 (0.158 g, 0.14 mmol). The reaction
mixture was stirred for 5 h at room temperature. Thereafter,
the mixture was evaporated to dryness and co-evaporated once
with cyclohexane. The resulting oily residue was dissolved in
TFA (1 mL) and precipitated with cold Et2O (10 mL). The
resulting brown solid was collected by centrifugation and purified
by chromatography on a RP-C18 silica gel column (16 g) with
a step gradient of MeOH (0–30%) in aq. TFA as the mobile
phase. The product-containing fractions were lyophilised to give
the compound 9 as a beige amorphous powder (57 mg, yield 41%).
IR (KBr): mmax 1395, 1586, 1632 (broad), 3078, 3401 (broad) cm−1;
1H-NMR (300 MHz, D2O + 1 N NaOD): d 8.16 (s, 2H), 8.05–7.97
(m, 4H), 7.86 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 7.1 Hz, 2H), 7.53 (d,
J = 7.9 Hz, 2H), 4.05 (s, 4H, –CH2–N(CH2–CO2H)2), 3.74 (s, 2H,
–C≡C–CH2–NH2), 3.38 (s, 8H, N–CH2–CO2H); HPLC (system
A): tR = 18.1 min, purity >95%; UV–visible (recorded during the
HPLC analysis): kmax = 255, 288 nm; MS (ESI+): m/z 653.6 [M +
H]+, calcd for C34H32N6O8 652.67; Anal.: found C 48.15; H 2.91;
N 8.43. C34H32N6O8·3C2HF3O2 (TFA) requires C 48.30; H 3.55; N
8.45%.
Optical properties of Eu (TMT-AP3) chelate 3
The absorption spectrum of 3 was recorded (220–800 nm) in
deionised water (concentration: 3.0 lM) at 25 ◦C. Emission
spectra were recorded (350–800 nm) under the same conditions
(concentration: 11.0 lm) after excitation at 298 nm (excitation
and emission slit = 5 nm). The luminescence quantum yield
of 3 was measured in deionised water at 25 ◦C by a relative
method using Ru(bpy)3Cl2 as a standard (U = 0.028, according to
ref. 37). Lifetime (average luminescence decay time) 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.
Synthesis and characterisation of fluorogenic caspase-3 substrate
Ac-Cys(Eu (TMT)-AP3)-Asp-Glu-Val-Asp-Lys(Cy 5.0)-NH2 (16)
Eu (TMT)-AP3 chelate 3
Compound 9 (46.7 mg, 0.047 mmol) was introduced into a
Reacti-VialTM (Pierce, #13222) and dissolved in deionised water
(1.16 mL). 290 lL of an aq. solution of NaHCO3 (55.3 mg in
0.98 mL of deionised water) were added in order to adjust the
pH at 6.5. 150 lL of an aq. solution of EuCl3·6H2O (48.3 mg in
0.39 mL of deionised water) were added and the reaction mixture
was protected from light and stirred at room temperature for 2 h.
Preparation of Cy 5.0 carboxylic acid, succinimidyl ester. Cy
5.0 carboxylic acid (3.2 mg, 4.88 lmol) was introduced into a
Reacti-VialTM and dissolved in 85 lL of dry DMF. 40 lL of a
solution of TSTU reagent in dry DMF (1.47 mg, 4.88 lmol) and
1.35 lL of Et3N (9.88 lmol) were added and the resulting reaction
mixture was protected from light and stirred at room temperature
for 1 h.
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Synthesis of Ac-Cys(St-Bu)-Asp-Glu-Val-Asp-Lys(Cy 5.0)-NH2
(14). 5.79 mg of crude peptide Ac-Cys(St-Bu)-Asp-Glu-Val-
Asp-Lys-NH2 13 (6.09 lmol, weighed in a 1.0 mL Eppendorf
tube) was dissolved in 220 lL of dry DMF and 0.83 lL of Et3N
(6.09 lmol) was added. After complete solubilisation by vortexing,
the resulting solution was added to the crude reaction mixture
containing the succinimidyl ester of Cy 5.0 dye. Thereafter, a
further amount of Et3N (2.7 lL, 19.52 lmol) was added and the
reaction mixture was protected from light and stirred at room
temperature overnight. The reaction was checked for completion
by RP-HPLC (system C). Finally, the reaction mixture was
quenched by dilution with 1 mL of water and 1mL of aq. AcOH
0.1% and purified by RP-HPLC (system D, 2 injections). The
product-containing fractions were lyophilised to give the peptide–
Cy 5.0 conjugate 14 as a blue amorphous powder. Quantification
was achieved by UV–visible measurements at kmax = 650 nm of the
Cy 5.0 dye by using the e value 250 000 L mol−1 cm−1 (yield after
RP-HPLC purification: 62%). HPLC (system D): tR = 24.2 min;
MS (MALDI-TOF, positive mode, CHCA matrix): m/z 1476.5
[M + H]+; MS (MALDI-TOF, negative mode, CHCA matrix): m/z
1473.4 [M − H]−, calcd exact mass for C66H94N10O20S4 1475.80.
Removal of tert-butylthio group. Peptide Ac-Cys(St-Bu)-Asp-
Glu-Val-Asp-Lys(Cy 5.0)-NH2 14 (4.5 mg, 3.05 lmol) was in-
troduced into a Reacti-VialTM and dissolved in 110 lL of DMF.
340 lL of a solution of DTT (10.5 mg in 380 lL, 60.9 lmol)
in sodium bicarbonate buffer (0.1 M, pH 8.5) were added. The
reaction mixture was protected from light and stirred at room
temperature for 2 h. The reaction was checked for completion by
RP-HPLC (system E). Finally, the reaction mixture was quenched
by dilution with aq. AcOH 0.1% (2 mL) and purified by RP-
HPLC (system F, 2 injections). The product-containing fractions
were lyophilised to give the free sulfhydryl containing peptide–Cy
5.0 conjugate 15 as a blue amorphous powder. Quantification was
achieved by UV–visible measurements at kmax = 650 nm of the
Cy 5.0 dye by using the e value 250 000 L mol−1 cm−1 (yield after
RP-HPLC purification: 52%). HPLC (system E): tR = 22.5 min;
HPLC (system F): tR = 29.3 min.
Synthesis of Ac-Cys(Eu (TMT)-AP3)-Asp-Glu-Val-Asp-Lys(Cy
5.0)-NH2 (16). Peptide Ac-Cys-Asp-Glu-Val-Asp-Lys(Cy 5.0)-
NH2 15 (2.22 mg, 1.6 lmol) was introduced into a Reacti-VialTM
and dissolved in 100 lL of sodium bicarbonate buffer (0.1 M,
pH 8.5). 260 lL of a solution of iodoacetyl derivative 11 (3.54 mg,
3.25 lmol) in sodium bicarbonate buffer (0.1 M and 4% (v/v)
DMF, pH 8.5) were added. The reaction mixture was protected
from light and stirred at room temperature for 4 h. The reaction
was checked for completion by RP-HPLC (system G). Finally,
the reaction mixture was quenched by dilution with TEAB buffer
(2.65 mL) and purified by RP-HPLC (system H, 3 injections). The
product-containing fractions were twice lyophilised to give the
peptide Ac-Cys(Eu (TMT)-AP3)-Asp-Glu-Val-Asp-Lys(Cy 5.0)-
NH2 16 as a blue amorphous powder. A stock solution of
fluorogenic caspase-3 substrate 16 was prepared in HPLC grade
water and UV–visible quantification was achieved at kmax of the
Cy 5.0 dye by using the e value 250 000 L mol−1 cm−1 (yield after
RP-HPLC purification: 40%). HPLC (system G): tR = 40.8 min;
HPLC (system H): tR = 43.1 min; HPLC (system I): tR = 19.0 min,
purity >95%; UV–visible (water): kmax (e) = 297, 652 (250 000 L
mol−1 cm−1); MS (MALDI-TOF, positive mode, CHCA matrix):
m/z 2350.7 [M + H]+; MS (MALDI-TOF, negative mode, CHCA
matrix): m/z 2346.9 [M − H]−, 2771.2 [M − 6H + Na + 4HTEA]−,
calcd exact mass for C105H115N17O30S3Eu 2348.38.
in vitro peptide cleavage by recombinant human caspase-3
A 1.0 lM solution of peptide 16 (or 21) was prepared in 50 lL of
caspase-3 buffer (100 mM NaCl, 40 mM HEPES, 10 mM DTT,
1 mM EDTA, 10% (w/v) sucrose and 0.1% (w/v) CHAPS, pH 7.2)
R
and transferred into an ultra-micro fluorescence cell (Hellmaꢀ,
105.51-QS, 3 × 3 mm, 45 lL). 2 lL of human recombinant caspase-
3 (3.2 10−3 U) were added and the resulting mixture was incubated
at 37 ◦C. After excitation at 298, 308, 336 and 653 nm (excitation
slit = 5 nm), fluorescence at 616 and 672 nm (excitation slit = 5 nm)
was monitored over time with measurements recorded every 5 s.
Antibody labelling using the Eu (TMT)-AP3 chelate
7 lL of an aq. solution (0.81 mg in 250 lL of deionised water)
of chelate 3 (23.3 nmol) was added to 500 lL of an aq. solution
of anti c-myc 9E10 mAb38 (0.7 mg mL−1) in phosphate buffer
pH 6.5. The mixture was protected from light and stored for 2 h
at 4 ◦C. Thereafter, 0.9 lL of glutaraldehyde (50% in water) were
added. The resulting mixture was stored at 4 ◦C and periodically
vortexed every 30 min over 4 h. Thereafter, NaBH4 (2.0 mg) was
added. The mixture was again stored at 4 ◦C and periodically
vortexed every 15 min over 1 h. Finally, the mixture was purified
R
on a Sephadexꢀ G-25 column (7 mL of gel, elution with phosphate
buffered saline (0.01 M phosphate buffer, 0.015 M NaCl, pH 7.4)).
The labelling level was determined by UV–visible spectroscopy.
The UV absorbance at k = 325 nm (e = 22 750 L mol−1 cm−1 for
3 and no significant absorbance for mAb) enabled to determine
the concentration of Eu (TMT)-AP3 chelate within the protein
conjugate. At k = 280 nm, it was possible to determine the mAb
concentration after subtraction of the residual UV absorbance of
3 (e 17 100 L mol−1 cm−1) by using the following relation: [mAb] =
Abs (k = 280 nm)/1.48 and 150 000 Da as the average molecular
weight of mAb. The luminescence quantum yield of this conjugate
was measured in PBS buffer at 25 ◦C by a relative method using
Eu (TMT-AP3) chelate 3 as a standard.
Acknowledgements
The contributions of Laetitia Bailly (IRCOF/LCOFH),
Dr Je´roˆme Leprince (U 413 INSERM/IFRMP 23) and Co-
lette Lebrun (SCIB/LRI/CEA-Grenoble) to mass spectrome-
try measurements are greatly acknowledged. We thank Annick
Leboisselier (IRCOF) for the determination of elemental analyses
and Ce´dric Bouteiller and Guillaume Clave´ for their contribu-
tions to the total synthesis of Cy 5.0 dye. Mouse anti c-myc
monoclonal antibody was a generous gift of Dr Didier Boquet
(DRM/DSV/SPI/CEA-Saclay). QUIDD is gratefully acknowl-
edged for the generous gift of recombinant human caspase-3. This
research was supported by “Ministe`re de la Recherche et de la
Technologie” through a young researcher fellowship (ACI jeunes
chercheuses et jeunes chercheurs 2004). Re´gion Haute Normandie
financial support through PUNChORGA network is gratefully
acknowledged.
4176 | Org. Biomol. Chem., 2006, 4, 4165–4177
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