Labeling of oligopeptides with luminescent lanthanide (III) chelates on solid phase

Article abstract of DOI:10.2174/157017809790442998

Synthesis of a building block based on 6,6'-(1H-pyrazole -1, 3-diyl)bis(pyridine) (3) that allows introduction of photoluminescent lanthanide(III) chelates to the synthetic oligopeptides on solid phase using the standard Fmoc chemistry is described. Since

Full text of DOI:10.2174/157017809790442998

Letters in Organic Chemistry, 2009, 6, 659-663
659
Labeling of Oligopeptides with Luminescent Lanthanide(III) Chelates on Solid Phase
Jari Peuralahti*, Lassi Jaakkola, Harri Hakala, Veli-Matti Mukkala and Jari Hovinen
PerkinElmer Life and Analytical Sciences, Turku Site, P.O. Box 10, FI-20101 Turku, Finland
Received October 29, 2008: Revised October 29, 2009: Accepted November 04, 2009
Abstract: Synthesis of a building block based on 6,6’-(1H-pyrazole-1,3-diyl)bis(pyridine) (3) that allows introduction of
photoluminescent lanthanide(III) chelates to the synthetic oligopeptides on solid phase using the standard Fmoc chemistry
is described. Since the ligand based on (3) forms luminescent chelates with Eu3+, Sm3+, Tb3+ and Dy3+ ions, the ligand is
highly useful for multiparametric homogenous assays simplifying considerably the preparation of chelates needed in these
applications.
Keywords: Lanthanide(III) chelates, oligopeptides, solid phase synthesis, labeling.
INTRODUCTION
employed. We report here synthesis of an oligopeptide
labeling reactant based on 6,6’-(1H-pyrazole-1,3-
diyl)bis(pyridine) which extends our labeling strategy to the
preparation of synthetic oligopeptides labeled with
photoluminescent terbium, dysprosium and samarium
chelates.
The use of long life-time emitting lanthanide(III) chelate
labels or probes together with time-resolved fluorometry in
detection provides a method to generate sensitive bioaffinity
assays. Indeed, time-resolved fluorescence based on
lanthanide(III) chelates has become a successful detection
technology, and it has been used in in vitro diagnostics for
over two decades [1]. For example, time-resolved
fluorescence quenching assays based on energy transfer from
a lanthanide(III) chelate to a nonfluorescent quencher have
been applied in various assays of hydrolyzing enzymes [2-5]
as well as for nucleic acid detection [4,6,7]. The different
photochemical properties of europium, terbium, dysprosium
and samarium chelates have proved to be suitable for the
development of multiparametric homogenous assays [5].
RESULTS AND DISCUSSION
Stable luminescent lanthanide(III) chelates generally
include a ligand with a reactive group for covalent
conjugation to bioactive molecules, an aromatic structure,
which absorbs the excitation energy and transfers it to the
lanthanide ion and additional chelating groups such as
carboxylic or phosphonic acid moieties and amines. Unlike
organic chromophores, these molecules do not produce
unwanted Raman and Rayleigh scattering or suffer from
concentration quenching. Although organic chelators and
Biomolecule conjugates used in many applications, such
as homogenous quenching assays, have to be extremely pure,
because even small amounts of fluorescent impurities can
increase the luminescence background and thereby reduce
detection sensitivity. Thus, it is desirable to perform solid
phase conjugation of biomolecules because most impurities
can be removed by washing while the biomolecule is
anchored to the solid support. Once the biomolecule
conjugate is released into the solution, only a single
chromatographic purification is required.
their substituents have
a significant effect on the
photophysical properties of lanthanide(III) chelates, no
general rules for the estimation of these effects are available.
Lanthanide(III)
chelates
based
on
2,6-bis(N-
pyrazolyl)pyridine [13-18] (A; Chart (1)) and 6,6’-(1H-
pyrazole-1,3-diyl)bis(pyridine) [12,18,19] (B) are among the
best luminescent terbium(III) chelates synthesized: they have
excellent photophysical properties including high
luminescent quantum yield and relatively high energy of
their lowest triplet state. Unfortunately, biomolecule labeling
reactants based on 2,6-bis(N-pyrazolyl)pyridine have
insufficient kinetic stabilities in the presence of high
concentrations of EDTA thus making them unsuitable for
bioanalytical assays [5]. In contrast, chelates based on 6,6’-
(1H-pyrazole-1,3-diyl)bis(pyridine) have been shown to be
highly suitable to these applications [5-7].
We have already reported a solid phase method for the
labeling of oligopeptides [8,9] and oligonucleotides [10-12]
with photoluminescent lanthanide(III) chelates. The
approach involves synthesis of oligonucleotide and
oligopeptide building blocks, which can be introduced to the
biomolecule structure using commercial oligonucleotide and
oligopeptide synthesizers by phosphoramidite and Fmoc
chemistries, respectively. Upon completion of the chain
assembly, the oligomers are deprotected and finally treated
with the appropriate lanthanide(III) citrate producing the
desired biomolecule conjugates. For solid phase oligopeptide
derivatization, only europium(III) chelates have been
Synthesis of the novel oligopeptide labeling reactant is
depicted in Scheme 1. Reaction of Fmoc-glutamic acid allyl
ester with the amine (1) [17] in the presence of HATU and
DIPEA gave the protected ligand (2) in moderate yield.
Purification was easily performed on silica gel column
chromatography. Palladium-catalyzed allyl cleavage using
resin-bound sodium p-toluenesulfinate produced (3) in 2h at
RT. The work up procedure was very simple including
*Address correspondence to this author at the PerkinElmer Life and
Analytical Sciences, Turku Site, P.O. Box 10, FI-20101 Turku, Finland;
E-mail: Jari.Peuralahti@perkinelmer.com
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

660 Letters in Organic Chemistry, 2009, Vol. 6, No. 8
Peuralahti et al.
N
N
N
N
N
N
N
N
N
Ln3+
N
N
N
N
Ln3+
COO- COO-
COO- COO-
COO- COO-
COO- COO-
B
A
Chart 1.
O
O
N
O
N
N
N
NHFmoc
O
O
O
H
N
N
N
H2N
N
N
O
O
O
O
O
Fmoc-Glu-OAll
HATU/ DIPEA
N
N
O
O
N
2
O
N
O
O
O
O
1
O
O
N
N
NHFmoc
O
H
Pd(Ph3P)4
N
N
HO
N
O
O
O
O
N
O
N
3
O
O
SO2Na
O
Scheme 1. Synthesis of the oligopeptide labeling reactant (3).
removal of the resin by filtration, washing of the filtrate with
10% citric acid and drying over 4Å molecular sieves.
characterized on ESI-TOF mass spectrometry. A HPLC trace
of crude reaction mixture is shown in Fig. (1)
a
demonstrating the success of the coupling reaction. The
observed molecular weight was in accordance with the
proposed structure.
To demonstrate the applicability of the building block (3)
for oligopeptide derivatization,
a caspase-3 substrate
(DEVDK) was synthesized in 10 ꢀmol scale using standard
Fmoc chemistry and recommended protocols. The block (3)
was coupled to the amino terminus using a prolonged
coupling time (2 h instead of 30 min). HBTU/HOBt was
used as an activator and 3.5 equivalents of (3) was used
relative to the resin (the amount of standard amino acids was
5 equivalents); higher concentrations of (3) did not give any
advantages. According to fulvene-piperidine assay, coupling
efficiency of (3) was comparable to natural amino acids.
After completion of the oligopeptide synthesis, the peptide
was cleaved from the resin with TFA in the presence of
scavengers followed by precipitation with diethyl ether.
Treatment of the deprotected oligopeptide with terbium(III)
citrate converted the oligopeptide conjugate to the desired
terbium(III) chelate (Scheme 2). The oligopeptide
synthesized was purified on reversed phase column and
The excitation and emission wavelengths as well as the
decay time (ꢀ) of the oligopeptide conjugate (4) were
measured in tris-buffer containing 0.9% NaCl at pH 7.75.
The main emission line was centered as usual at 544 nm. The
excitation maximum was at 329 nm. The decay time, 2.34
ms, although slightly shorter than that of the corresponding
unconjugated terbium chelate (Table 1) was normal for a
nonadentate chelate.
Brunet et al. [17] have demonstrated that lanthanide
chelates based on 2,6-bis(N-pyrazolyl)pyridine have
reasonably good photophysical properties even with Eu3+,
Sm3+ and Dy3+. We verified that this is the case also with
chelates based on 6,6’-(1H-pyrazole-1,3-diyl)bis(pyridine)
(Table (1)). The observed emission spectra were typical for
lanthanide chelates corresponding to the 5D0 ꢁ 7Fj, 4G5/2 ꢁ

Labeling of Oligopeptides with Luminescent Lanthanide(III)
Letters in Organic Chemistry, 2009, Vol. 6, No. 8
661
Fig. (1). Reversed phase HPLC trace of the oligopeptide conjugate (4) (crude reaction mixture). The main peak is the desired product as
judged by ESI-TOF MS.
H2N
oligopeptide
synthesis using
block 3 at
NH2-terminus
followed by treatment
with piperidine
and Ac2O
O
O
O
CH C
CH2
O
CH C
CH2
O
CH C
CH CH3
CH3
O
CH C
CH2
O
CH C
CH2
NH
H
N
H
H
H
N
N
N
N
N
H
H
C
O
CH2
C
O
CH2
O
HN
O-t-Bu
C
O
O-t-Bu
CH2
O-t-Bu
CH2
NHBoc
1. TFA/scavengers
2. Tb(III) citrate
N
N
N
N
O
O
O
O
H
O
H
O
H
O
O
CH C NH2
CH2
N
O
O
NH
O
H
N
CH C
CH2
N
CH C
N
CH C
N
CH C
CH2
N
H
O
N
CH2
CH2
C
CH CH3
CH3
C
O
C
O
CH2
O
O
O
HN
OH
O
OH
CH2
O
OH
CH2
NH2
4
N
N
N
N
-O
Tb3+
-O
N
O
-O
O
N
O
HO
O
Scheme 2. Labeling of an oligopeptide in solid phase using the labeling reactant (3).

662 Letters in Organic Chemistry, 2009, Vol. 6, No. 8
Peuralahti et al.
Table 1. Emission Maxima, Luminescence Yields (ꢀꢁ) and Decay Times (ꢂ) of Lanthanide(III) Chelates Based on 6,6’-(1H-
pyrazole-1,3-diyl)bis(pyridine)
Ln
Emission/nm
ꢀꢁ
ꢂ / ms
Tb3+
Eu3+
Sm3+
Dy3+
544
616
603
575
9089
1632
45
2.80
1.05
0.014
0.012
188
5
7
4
6
6Hj, D4 ꢃ Fj and F9/2 ꢃ Hj transitions for Eu3+, Sm3+,
Tb3+ and Dy3+, respectively. The main emission lines of
chelates were centered as usual around 617 (Eu3+), 603
(Sm3+), 544 (Tb3+) and 575 nm (Dy3+). The decay times were
normal for nonadentate chelates except that of the
dysprosium chelate, which has surprisingly short decay time.
This phenomenon was found also for the corresponding
oligonucleotide conjugates [12].
was added and the mixture was stirred for 1.5 h. The mixture
was diluted with CH2Cl2 (20 mL), washed twice with 10%
citric acid and dried over Na2SO4. Purification on silica gel
(eluent 5% MeOH/CH2Cl2) gave 0.24 g (52%) of the
product. ESI-TOF-MS for C70H86N8O13 (M+H)+: calcd,
1247.64; found, 1247.64. 1H-NMR (DMSO-d6): 8.45 (s, 1H),
8.00-7.95 (m, 2H), 7.90-7.85 (m, 5H), 7.72 (d, 2H, J=7.6
Hz), 7.55 (d, 1H, J=7.7 Hz), 7.49 (d, 2H, J=8.8 Hz), 7.47 (d,
1H, J=8.8 Hz), 7.41 (t, 2H, J=7.7 Hz), 7.33 (t, 2H, J=7.4
Hz), 7.16 (d, 2H, J=8.2 Hz), 5.93-5.87 (m, 1H), 5.31 (d, 1H,
J=17.3 Hz), 5.20 (d, 1H, J=10.3 Hz), 4.60 (d, 2H, J=5.0 Hz),
4.34-4.31 (m, 1H), 4.28-4.25 (m, 1H), 4.23-4.21 (m, 1H),
4.15-4.11 (m, 1H), 4.03 (s, 2H), 3.98 (s, 2H), 3.48 (s, 4H),
3.47 (s, 4H), 3.29 (t, 1H, J=5.6 Hz), 3.19 (t, 2H, J=8.0 Hz),
2.87 (t, 2H, J=7.9 Hz), 2.42 (t, 2H, J=7.3 Hz), 2.13-2.08 (m,
1H), 1.92-1.85 (m, 1H), 1.40 (s, 18H), 1.36 (s, 18H).
MATERIALS AND METHODS
Adsorption column chromatography was performed on
columns packed with silica gel 60 (Merck). Reagents for
oligopeptide synthesis and Fmoc-Glu-Oall were purchased
from Nova Biochem. Sodium sulfinate resin (200-400 mesh,
1% DVB, 1.3 mmol g-1) was purchased from Tianjin Nankai
Hecheng Science & Technology Company Limited (China).
All dry solvents were from Merck and they were used as
received. Lanthanide chelates of 2,2’,2’’,2’’’-{[6,6’-
(pyrazole-1,3’’-diyl)bis(pyridine)-2,2’-diyl]bis(methyleneni-
trilo)}-tetrakis(acetic acid) were synthesized as described
previously [18]. The oligopeptides were assembled on an
Applied Biosystems 433A instrument, using recommended
protocols. HPLC purifications were performed using a
Shimadzu LC 10 AT instrument equipped with a diode array
detector, a fraction collector and a reversed phase column
(LiChrocart 125-3 Purospher RP-18e 5 μm). Mobile phase:
(Buffer A): 0.02 M triethylammonium acetate (pH 7.0);
(Buffer B): A in 50 % (v/v) acetonitrile. Gradient: from 0 to
1 min 95% A, from 1 to 31 min from 95% A to 100% B.
Flow rate was 0.6 mL min-1. ESI-TOF mass spectra were
recorded on an Applied Biosystems Mariner instrument.
5-{N-{4’-{2’’-{1’’’,3’’’-bis{6’’’’-[N,N-bis(tert-butoxycar-
bonylmethyl)aminomethyl]-2’’’’-pyridyl}-1H-pyrazo-
4’’’-yl}ethyl}phenyl}amino}-2-[N-(fluorenylmethoxycar-
bonyl)amino]-5-oxopentanoic acid, 3
Compound (2) (0.22 g, 0.18 mmol) was dissolved in dry
THF (2 mL), and the solution was deaerated with argon.
Pd(Ph3P)4 (13 mg, 11 ꢀmol) and sodium sulfinate resin (0.2
g) were added and the mixture was stirred overnight at RT.
The resin was filtered off, washed with THF, and the filtrate
was concentrated in vacuo. The residue was dissolved in
CH2Cl2, washed twice with 10% citric acid, dried with 4Å
molecular sieves and concentrated. Yield was 86 %. ESI-
TOF-MS for C67H82N8O13 (M+H)+: calcd. 1207.61; found,
1207.59.
Luminescence measurements were performed on
a
PerkinElmer LS-55 luminescence spectrometer equipped
with a Hamamatsu R928 red-sensitive photomultiplier tube.
Synthesis and Purification of Oligopeptide Conjugates
A model sequence (DEVDK) was synthesized on PALTM
Support for peptide amides (PE Biosystems) in 10 μmol
scale using Fmoc chemistry and recommended protocols
(coupling time 30 min for natural amino acid analogues, and
2 h for (3)) followed by treatment with piperidine and acetic
anhydride. The resin was treated with the mixture of
crystalline phenol (75 mg), ethanedithiol (25 μL), thioanisole
(50 μL), water (50 μL) and trifluoroacetic acid (1 mL) for 4
h. The resin was removed by filtration, and the solution was
concentrated in vacuo. The crude oligopeptide was
precipitated with diethyl ether. The precipitate was
redissolved in water and treated with terbium(III) citrate (5
equiv per ligand). Purification was performed on HPLC.
5-{N-{4’-{2’’-{1’’’,3’’’-bis{6’’’’-[N,N-bis(tert-butoxycar-
bonylmethyl)aminomethyl]-2’’’’-pyridyl}-1H-pyrazo-4’’’-
yl}ethyl}phenyl}amino}-2-[N-(fluorenylmethoxycar-
bonyl)amino]-5-oxopentanoic acid allyl ester, 2
Fmoc-Glu-OAll (0.15 g, 0.37 mmol), HATU (0.14 g,
0.37 mmol), and DIPEA (65 ꢀL, 0.37 mmol) were dissolved
in dry DMF (3 mL), and the mixture was stirred for 30 min
at room temperature. Tetra(tert-butyl) 2,2’,2’’,2’’’-{{6,6’-
{4’’-[2-(4-aminophenyl)ethyl]-1H-pyrazole-1’’,3’’-diyl}bis
(pyridine)-2,2’-diyl}bis(methylene-nitrilo)}tetrakis(acetate)
(1) (0.32 g, 0.37 mmol) predissolved in dry DMF (2 mL)

Labeling of Oligopeptides with Luminescent Lanthanide(III)
Letters in Organic Chemistry, 2009, Vol. 6, No. 8
663
[5]
Karvinen, J.; Elomaa, A.; Mäkinen, M.-L.; Hakala, H.; Mukkala,
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Yield was 68%. MS found: 1543.47, calcd for
C62H77N15O22Tb- 1543.45.
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We presented here a straightforward method to label
oligopeptides with a luminescent terbium(III) chelate on
solid phase using a standard oligopeptide synthesizer. Since
6,6’-(1H-pyrazole-1,3-diyl)bis(pyridine) based ligand forms
luminescent chelates with four lanthanide(III) ions, the
ligand simplifies considerably the preparation of chelates
needed in bioanalytical applications. Although the decay
times are shorter and luminescence yields much lower in the
case of samarium and dysprosium chelates than in the case
of europium and terbium chelates, these chelates are valuable
when multiparametric assays are developed.
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