Synthesis of a coumarin-based europium complex for bioanalyte labeling

Article abstract of DOI:10.1016/j.bmcl.2007.01.010

A coumarin-based europium chelate ready-to-use for analyte labeling and homogeneous time-resolved fluorescence measurements has been designed. Compound 1 displays three functional elements: an azide reactive spacer arm, a coumarin sensitizer, and a seven-coordinate europium complex. That complex can be excited at 370 nm by inexpensive UV-LEDs as a light excitation source.

Full text of DOI:10.1016/j.bmcl.2007.01.010

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1499–1503
Synthesis of a coumarin-based europium complex
for bioanalyte labeling
a
a
b
a,
*
Cl e´ mentine F e´ au, Emmanuel Klein, Paul Kerth and Luc Lebeau
a
Institut Gilbert-Laustriat, CNRS-Universit e´ Louis Pasteur, Facult e´ de Pharmacie, 74 route du Rhin, 67401 Illkirch Cedex, France
b
Preventor lTBC GmbH, Gernsheimer Strasse 46, 64319 Pfungstadt, Germany
Received 24 October 2006; revised 23 December 2006; accepted 5 January 2007
Available online 17 January 2007
Abstract—A coumarin-based europium chelate ready-to-use for analyte labeling and homogeneous time-resolved fluorescence mea-
surements has been designed. Compound 1 displays three functional elements: an azide reactive spacer arm, a coumarin sensitizer,
and a seven-coordinate europium complex. That complex can be excited at 370 nm by inexpensive UV-LEDs as a light excitation
source.
Ó 2007 Elsevier Ltd. All rights reserved.
7
Accurate, fast, affordable analysis of biological samples
is of prime interest for medicine and research. Currently,
most often sample preparation procedures involve han-
dling steps prone to introducing artifacts, and analysis
methods commonly require skilled technicians and
expensive equipment. The recent developments in micro
fluidics and miniaturized lab-on-a-chip-like devices are
extremely attractive for biomedical analysis and point-
of-care testing (POCT) has revolutionized the continu-
linked immunosorbent assay (ELISA). An additional
improvement is met with techniques based on time-re-
solved fluorescence that allow to get free from short-
lived intrinsic fluorescence of biological samples (in the
ns time range). Assays based on homogeneous time-re-
solved fluorescence (HTRF) all involve luminescent lan-
8
–10
thanide chelates.
III III
Lanthanide ions, especially Eu and Tb , display
luminescence lifetimes greater than 0.1 ms in aqueous
solution under ambient conditions. Such long decay
times result from forbidden transitions involving 4f orbi-
tals and, as a result, the molar absorption coefficients
1
,2
um of patient care process. However, monitoring of
non-treated biological samples is still challenging in
most of the cases as well as assay sensitivity. An
improvement of the latter thus results in reduced sam-
pling (i.e., blood collection, bone marrow aspiration. . .)
and consequently in enhanced comfort of the patient.
Homogeneous proximity-based immunoassay tech-
niques are good candidates for monitoring non-treated
biological samples. Early setup involving radioactive
1
1
ꢀ
1
ꢀ1
are very low (less than 10 M cm in aqueous med-
1
2
ia). Because of their weak absorption, lanthanides
are usually not directly excited and sensitizers (antennas)
are required to significantly populate the lanthanide
excited vibrational states. UV/vis light energy is collect-
ed by allowed antenna-centered absorptions, followed
by non-radiative intramolecular energy transfer from
3
,4
labels (scintillation proximity assay ) has increasingly
been replaced with fluorescent labels, mostly because
5
III
of safety issues. In these techniques, the signal of the
antenna excited states to Ln , resulting in a radiative
3,14
1
labeled component is modulated by binding and no
laborious separation steps are needed. Consequently
such assays are faster and easier to perform than heter-
ogeneous assays, for example, dissociation-enhanced
metal-centered luminescence.
Under these condi-
III
tions, luminescent Ln complexes have found many
useful applications especially in the field of bioanalytical
9
,10
chemistry.
Recently there has been a growing interest
6
lanthanide fluoroimmunoassay (DELFIA) or enzyme-
in the use of inexpensive light-emitting diodes (LEDs) as
a light source in place of expensive and cumbersome
lasers, for both steady-state and time-resolved fluores-
cence spectroscopy. However while high performance
LEDs are now available to wavelengths down to
360 nm, a rapid fall-off in their quantum efficiency
1
5
Keywords: Coumarin; Europium chelate; Probe; Homogeneous time-
resolved fluorescence; Light emitting diodes.
*
Corresponding author. Tel.: +33 3 9024 4303; fax: +33 3 9024
306; e-mail: lebeau@pharma.u-strasbg.fr
1
6
4
occurs toward shorter wavelengths.
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.010

1500
C. F e´ au et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1499–1503
In the course of our research program focused on the de-
sign of POCT devices for molecular diagnostics based
on HTRF measurements, we aimed to develop ready-
to-use specific probes to be excited at 360–370 nm, for
bioconjugation and labeling (Scheme 1). The target
compounds display 3 functional elements: (i) a chemical-
ly reactive spacer for conjugation to the bioanalyte (or
competitor), (ii) a coumarin antenna for proper excita-
tion of the lanthanide cation through non-radiative
intramolecular energy transfer, and (iii) a lanthanide
chelate moiety for HTRF detection. Methods for bio-
conjugation have been intensively investigated over the
designed the spacer arm in target structure 1 to be
hydrophilic, eliminating the solubility issue, and termi-
nated with an azide functionality. That group can be
further reduced in situ (i.e., by Staudinger reaction in
the presence of a water-soluble phosphine) prior to cou-
pling with acid compound, or directly involved in click
chemistry. The antenna has been selected so that excita-
tion can be achieved at 360–370 nm. 7-Amino-4-(trifluo-
romethyl)coumarin 5 does absorb in the blue and UV
spectral region and thus perfectly suits to the selected
2
2
LED excitation source. Furthermore, coumarin emis-
sion at ca. 490 nm in aqueous media is compatible with
Eu sensitization through a non-radiative energy trans-
1
7
III
past 2 decades. Coupling reactions between carboxylic
acids and amines (peptide chemistry) most often satis-
factorily fulfill requirements for successful labeling
experiment. Recently, new bioconjugation methods
have appeared involving the 1,3-dipolar cycloaddition
reaction of azides and alkynes (Huisgen reaction), and
are getting more and more popular under the ‘click
1
5
fer process. Connection of the coumarin antenna to
the metal chelate moiety and the spacer arm can be
achieved through acylation of the amino group without
affecting its spectroscopic properties. Finally, the choice
of the chelating motif is crucial as the complex has to be
stable in biological media which are glutted with poten-
tial metal chelating competitors (i.e., phosphoesters,
proteins, urea. . .). Indeed, partial europium ligand dis-
placement would invariably result in metal ion leaching
out of the complex with subsequent decrease of lantha-
1
8–21
chemistry’ designation.
Applying that coupling
strategy involves prior introduction of an azide or
alkyne functionality onto the bioanalyte (or competitor)
when missing in the initial structure. Consequently, we
III
nide fluorescence as Eu is not in the close vicinity of
the coumarin sensitizer any more. Examining the abun-
dant literature dedicated to lanthanide complexes (for
9
,10,23,24
0
recent reviews, see
), we selected the N,N -[2,6-
pyridinediyl bis(methylene)]bis[N-(carboxymethyl) gly-
cine] core that should provide a stable chelate in compet-
2
itive biological media.
5,26
The synthesis of compound 1 was realized using a con-
vergent approach as described in Scheme 2. The spacer
arm was prepared in 2 steps starting from commercially
available bis-1,2-(2-chloroethoxy)ethane 2.
Quantitative conversion into bis-1,2-(2-azidoethoxy)
ethane 3 and monoreduction using a Staudinger reac-
tion under biphasic conditions afforded the heterobi-
functional azido amine 4. The antenna was elaborated
2
7
starting from 7-amino-4-(trifluoromethyl) coumarin 5
that was acylated with bromoacetyl bromide. The result-
ing intermediate bromoacetamide was converted into
corresponding iodide 6 under standard conditions.
Alkylation of primary amine 4 with iodoacetamide 6
led to compound 7. The metal chelating group was pre-
pared starting from methyl isonicotinate 8. Double addi-
2
8,29
tion of hydroxymethyl radical
onto protonated
nicotinate yielded bis-2,6-hydroxymethyl compound 9
in 51% yield. Methanesulfonylation in standard condi-
tions produced a complex mixture of methanesulfony-
lated and chlorinated products. Conducting the
reaction in refluxing dichloromethane allowed quantita-
tive transformation of 9 into bis-chloride 10. The latter
compound treated with nitrilodiacetic acid di-tert-butyl
3
0
ester quantitatively afforded pentaester 11 that was
regioselectively hydrolyzed using lithium hydroxide to
yield carboxylic acid 12. Amide coupling between amino
compound 7 and acid 12 produced fully protected probe
precursor 13. Subsequent treatment with trifluoroacetic
3
1
acid yielded tetra-acid 14. which was finally converted
into lanthanide chelate 1. upon addition of europium
3
2
Scheme 1. Design of the coumarin-based europium probes.
chloride hexahydrate.

C. F e´ au et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1499–1503
1501
a
Europium chelate 1 is soluble in water at physiological
pH (pH 7.4–7.8) and exhibits strong absorbance at
Cl
Cl
2
N3
R
2
O
O
360–370 nm (Fig. 1). Excitation in the range of couma-
rin absorption band of 1 causes the well-known, struc-
2
3 : R=N3
b
3
+
4
: R=NH2
tured emission of the Eu
6
ion (ca. 596, 616, and
99 nm), showing that energy transfer from ligand-cen-
CF3
CF3
tered to metal-centered levels does take place. Fluores-
cence properties of compound 1 appeared stable in
pure water, in phosphate-buffered saline (PBS), and in
human plasma (Fig. 2). No decrease in the characteristic
europium fluorescence intensity was observed over
c
O
I
O
O
NH2
O
O
N
H
5
6
30 min incubation periods in these media. Incubation
CF3
of complex 1 in the presence of fluoride ions did not
increase fluorescence intensity suggesting that the seven
coordinate complex with the metal ion provides thermo-
dynamic stability and minimal fluorescence quenching
d
O
H
N
N3
O
O
N
O 2
13,14
H
by water.
7
As a first example of application for compound 1, cou-
pling with an AZT derivative has been achieved to offer
an alternative tracer for enzyme immunoassay of the
antiviral nucleoside. Thus, azido compound 1 was
reduced into primary amine 15 using tris(2-carboxy-eth-
O
OR
O
OMe
O
OMe
3
3
e
g
N
N
N
N
N
R
R
O
O
O
O
1
8
O
O
9
1
: R=OH
f
O
O
0 : R=Cl
1
1 : R=Me
h
1
2 : R=H
CF3
O
O
NH
O
0
i
k
500
7
N3
N
2
O
1
300
O
Wavelength (nm)
Figure 1. Absorption spectra of coumarin europium complex 1 in
water (pH 7.8).
N
N
N
OR
RO
O
O
OR RO
O
O
e
d
c
1
1
3 : R=
t-Bu
j
4 : R=H
Scheme 2. Synthesis of the europium chelate 1. Reagents and
conditions: (a) NaN (3 equiv), CH CN, NaI cat., n, 48 h, 99%; (b)
PPh (1 equiv), aq 1 N HCl, Et O/THF (9:1 v/v), rt, 2 h, 77%; (c) 1-
BrCH COBr (1.1 equiv), Et N (1.1 equiv), CH Cl
, 0 °C, 3 h. 2-NaI
1.5 equiv), acetone, n, 2 h, 98% (2 steps); (d) 4 (3.0 equiv), CH CN,
n, 2 h, 65%; (e) (NH (10 equiv), H SO cat., MeOH, n, 6 h,
1%; (f) MsCl (2.1 equiv), Et N (2.0 equiv), CH Cl , n, 15 h, 99%; (g)
HN(CH CO -t-Bu) (2.5 equiv), Na CO (2.5 equiv), NaI cat.,
CH CN, D, 15 h, 99%; (h) LiOH (1.1 equiv), H O/THF (1:6 v/v), rt,
h, 89%; (i) 12 (1.0 equiv), i-Pr NEt (3.0 equiv), BOP (1.6 equiv),
CH Cl , rt, 16 h, 50%; (j) CHCl /TFA (8:2 v/v), rt, 20 h, 99%; (k)
EuCl 6 H O (1.0 equiv), MeOH, pH 7 (NaOH 1 N), rt, 1 h, 99%.
3
3
3
2
b
a
2
3
2
2
(
3
4
)
2
S
2
O
8
2
4
5
3
2
2
2
2
2
2
3
600
700
3
2
Wavelength (nm)
8
2
Figure 2. Characteristic europium emission bands for compound 1 in
different media (kexc = 370 nm): (a) H O (pH 7.8); (b) H O, 50 mM
NaF (pH 7.8); (c) PBS (pH 6.6); (d) Standard human plasma (Dade
2
2
3
3
2
2
2
2
Behring, diluted 1/10); (e) Eu emission bands for conjugate 17 (H O,
pH 7.8).

1502
C. F e´ au et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1499–1503
3
4
5
6
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2
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NH
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H N
N
2
O
Na
2
O
8
9
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N
10. Yuan, J. L.; Wang, G. L. Tr. Anal. Chem. 2006, 25, 490.
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Rev. 1993, 123, 201.
N
N
15
EuIII
1
O
O
O
O
O
O
O
O
O
N
O
1
1
4. Dossing, A. Eur. J. Inorg. Chem. 2005, 1425.
5. Herman, P.; Maliwal, B. P.; Lin, H. J.; Lakowicz, J. R.
J. Microsc. 2001, 203, 176.
(
CH2)5
OH
N
N
H
O
b
O
16. Song, Y. K.; Nurmikko, A. V.; Gherasimova, M.; Jeon, S.
R.; Han, J. Phys. Stat. Sol. A 2004, 201, 2721.
1
OH
O
7. Hermanson, G. T. Bioconjugate Techniques; Academic
Press, 1996.
N3
16
18. Manetsch, R.; Krasinski, A.; Radic, Z.; Raushel, J.;
Taylor, P.; Sharpless, K. B.; Kolb, H. C. J. Am. Chem.
Soc. 2004, 126, 12809.
CF3
O
1
9. Wang, Q.; Chittaboina, S.; Barnhill, H. N. Lett. Org.
Chem. 2005, 2, 293.
O
NH
2
0. Sun, X. L.; Stabler, C. L.; Cazalis, C. S.; Chaikof, E. L.
Bioconjug. Chem. 2006, 17, 52.
O
O
H
N
AZT-N3-(CH2)5
N
2
O
21. Hassane, F. S.; Frisch, B.; Schuber, F. Bioconjug. Chem.
2006, 17, 849.
Na
N
O
H
O
2
2. Chakraborty, A.; Chakrabarty, D.; Hazra, P.; Seth, D.;
Sarkar, N. Chem. Phys. Lett. 2003, 382, 508.
N
23. Handl, H. L.; Gillies, R. J. Life Sci. 2005, 77, 361.
4. Yuan, J. L.; Wang, G. L. J. Fluoresc. 2005, 15, 559.
25. von Lode, P.; Rosenberg, J.; Pettersson, K.; Takalo, H.
2
N
N
1
7
EuIII
O
O
Anal. Chem. 2003, 75, 3193.
6. Hagren, V.; Crooks, S. R. H.; Elliott, C. T.; L o¨ vgren, T.;
Tuomola, M. J. Agric. Food Chem. 2004, 52, 2429.
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Eng. Data 1981, 26, 348.
O
O
2
O
O
35
2
Scheme 3. Synthesis of labeled AZT. (a) TCEP, H O; (b) DCC,
NHS.
2
8. Itokawa, H.; Inaba, T.; Haruta, R.; Kameyama, S. Chem.
Pharm. Bull. 1978, 26, 1285.
29. Scharbert, B. EP 0533131, 1992.
yl)phosphine hydrochloride in water and conjugation to
3
4
modified nucleoside 16 was realized via classical NHS
activation of the terminal carboxylic acid function
3
3
0. Achilefu, S. I. WO0152900, 2001.
1. Selected spectral data for 14. H NMR (D O, 200 MHz)
1
2
(
Scheme 3). AZT fluorescent conjugate 17 exhibits char-
acteristic lanthanide emission bands indicating that the
spectroscopic features of the label are preserved
3
5
.76 (br s, 2H, CH
.09 (m, 14H, CH
2
N
3
); 4.00 (br s, 8H, CH
N
2
CO
CH O, CH CH , NCH py); 5.18 (s,
2
H); 4.23–
2
2
2
2
3
2
2H, NCH CONH); 7.23 (s, 1H, CF C@CH); 7.71–7.88
2
3
1
3
(
Fig. 2e).
ar ar 3
(m, 1H, H ); 8.09–8.16 (m, 4H, H ). C NMR (CD OD,
5
7
0 MHz) d (ppm): 50.3; 51.6; 51.8; 56.5; 57.6; 69.5; 70.8;
1.0; 71.4; 108.3; 110.4; 115.3; 117.3; 123.2; 124.1 (q,
In summary, we have described the design, synthesis,
and preliminary evaluation of a coumarin-based europi-
um chelate for homogeneous time-resolved fluorescence
measurements with excitation by UV-LEDs at 370 nm.
Complete determination of its physical properties and
evaluation in applications for quantitative point-of-care
HTRF immunoassays are currently underway and will
be published in due course.
J = 161.0 Hz); 126.9; 142.8 (q, J = 53.0 Hz); 144.4; 151.7;
56.4; 156.7; 160.7; 168.9; 170.2; 177.7. MS (ESI) m/z 840
1
[
+
M+H] ; 862 [M+Na] .
+
3
3
2. Paul-Roth, C. O.; Lehn, J. M.; Guilhem, J.; Pascard, C.
Helv. Chim. Acta 1995, 78, 1895.
3. Brossette, T.; Klein, E.; Cr e´ minon, C.; Grassi, J.; Mios-
kowski, C.; Lebeau, L. Tetrahedron 1999, 57, 8129.
34. Brossette, T.; Le Faou, A.; Goujon, L.; Valleix, A.;
Cr e´ minon, C.; Grassi, J.; Mioskowski, C.; Lebeau, L. J.
Org. Chem. 1999, 64, 5083.
3
5. Coupling procedure: complex 1 (10.7 mg, 10.6 lmol) and
TCEP-HCl (3.5 mg, 12 lmol) are stirred for 15 h at rt in
References and notes
2
H O/MeOH 1:1 (1 mL). The reaction mixture is filtered
1
2
. Price, C. P. Brit. Med. J. 2001, 322, 1285.
. Sheehan, A. D.; Quinn, J.; Daly, S.; Dillon, P.; O’Ken-
nedy, R. Anal. Lett. 2003, 36, 511.
and solvent is removed under vacuum. Analysis for
intermediate 15: MS (ESI) m/z 960.1, 962.1 [MꢀNa] .
ꢀ

C. F e´ au et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1499–1503
1503
The residue is solubilized in H
preformed NHS-ester of AZT derivative [16 (27.9 mg,
2
O/MeCN 1:5 (6 mL) and
ed, and the residue is triturated with CH
reduced under vacuum and purified by silica gel chroma-
2
Cl
2
. The filtrate is
6
7
2 lmol), NHS (8.2 mg, 71 lmol), and DCC (14.4 mg,
1 lmol) stirred for 3 h at rt in MeCN/CH Cl 2:1 (3 mL)]
tography (CHCl /MeOH/H O 100:0:0–0:50:50) to yield 17
3
2
2
2
(5.5 mg, 37%). MS (ESI) m/z 1252.3, 1254.3 [M-azidode-
oxyribose-Na] ; 1393.3, 1395.3 [MꢀNa] .
ꢀ
ꢀ
is added. The mixture is stirred overnight at rt, evaporat-