Preparation of phosphorylated bis-pyrazolyl-pyridine nonadendate ligands and their terbium complexes. Part 2

Article abstract of DOI:10.1016/j.tetlet.2012.04.106

A series of ligands has been constructed from a central bis-pyrazolyl-pyridine core and various deprotonable chelating pockets based on mixed carboxylate/phosphate or phosphate anionic functions and a central flexible pendent arm for subsequent grafting to biomaterials. For some of these ligands, the sequence of reactions incorporates an additional step introducing a substituted diethynylphenyl residue to increase significantly their solubility in polar organic solvents. The terbium(III) complexes of some of these ligands display outstanding spectroscopic properties with lifetimes ranging from 2.7 to 3.2 ms and quantum yields from 16% to 26% in water.

Full text of DOI:10.1016/j.tetlet.2012.04.106

Tetrahedron Letters 53 (2012) 3717–3721
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Preparation of phosphorylated bis-pyrazolyl–pyridine nonadendate ligands
and their terbium complexes. Part 2
⇑
Raymond Ziessel , Alexandra Sutter, Matthieu Starck
Laboratoire de Chimie Organique et Spectroscopies Avancées (LCOSA), UMR 7515 au CNRS, ECPM, 25 rue Becquerel, 67087 Strasbourg, Cedex 02, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ligands has been constructed from a central bis-pyrazolyl–pyridine core and various deprot-
onable chelating pockets based on mixed carboxylate/phosphate or phosphate anionic functions and a
central flexible pendent arm for subsequent grafting to biomaterials. For some of these ligands, the
sequence of reactions incorporates an additional step introducing a substituted diethynylphenyl residue
to increase significantly their solubility in polar organic solvents. The terbium(III) complexes of some of
these ligands display outstanding spectroscopic properties with lifetimes ranging from 2.7 to 3.2 ms and
quantum yields from 16% to 26% in water.
Received 13 March 2012
Revised 19 April 2012
Accepted 23 April 2012
Available online 7 May 2012
Keywords:
Bis-pyrazolyl–pyridine
Glyphosate
Ó 2012 Elsevier Ltd. All rights reserved.
Phosphonate
Phosphate
6-Heptynoic acid
Biotin
Terbium
Luminescence
Current luminescence imaging techniques employing highly
sensitive detection methods and sophisticated probes have been
successively exploited for the development of analytical tech-
niques. These include multiplexing assays for the simultaneous
determination of an analyte in several samples and/or different
analytes in the same sample.1 Luminescence imaging has also been
extensively used to access the spatial distribution of a given target
molecule in chemical or biochemical processes in macro- and
microsamples, and to conduct the in vivo evaluation of biological
and pathological processes.1,2
All such studies depend fundamentally on the design and molec-
ular architecture of the luminescence probe used to tackle the prob-
lem. It is generally considered that lanthanide (Ln) binding provides
the underpinning for luminescent labels in fluoroimmunoassays.3
The workhorses in the field are europium(III) and terbium(III)
complexes which are luminescent in aqueous solution if certain
conditions are fulfilled: (i) the ligand should include a highly
absorbing chromophore (since f?f transitions of simple solvated
lanthanide ions have extremely small absorption coefficients), (ii)
energy transfer from the ligand-centred excited states to the Ln
centre should be fast and efficient, (iii) water must be largely ex-
cluded from the first coordination sphere, since detrimental non-
radiative deactivation pathways for the excited Ln atoms are pro-
moted by the vibrational modes of any bound solvent molecules.4
The antenna effect,5 represented by a combination of requirements
(i) and (ii), makes the ligand design crucial for the photophysical
properties of the resulting complexes. Amongst the first systems
satisfying these criteria to be investigated were the europium(III)
and terbium(III) complexes of oligopyridine-derived ligands includ-
ing cryptands, branched macrocycles and podands.6
If luminescent lanthanide chelates are to be used as tags in
more advanced technologies such as homogeneous assays, fluores-
cence imaging, immuno-histochemistry, or in situ hybridization
techniques, additional strict requirements arise: (iv) high thermo-
dynamic and kinetic stability, (v) hydrophilicity, (vi) very efficient
cation emission and high absorption at suitable wavelengths, (vii)
a chemical structure allowing covalent linkage of the label to the
target biomolecule. As a consequence of requirements (i) to (vi)
for the optimal stability and luminescence properties, only a few
viable labels have to date been developed and tested in biomedical
analysis protocols.
It is usually considered that lanthanide coordination occurs pre-
dominantly via ionic bonding interactions,7 leading to a strong
preference for negatively charged donor groups. The advantages
of using negatively charged donor groups are well-illustrated in
the use of the tetraanionic ligand DOTA to form Gd(III) complexes
for nuclear magnetic resonance imaging, this tetrakis(carboxyl-
atomethyl) derivative of a macrocyclic tetramine providing not
only a complex of the required kinetic and thermodynamic stabil-
ity but also engendering hydrophilicity due to the overall negative
charge of that complex. However, the synthesis of this kind of
⇑
Corresponding author.
E-mail address: ziessel@unistra.fr (R. Ziessel).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.04.106

3718
R. Ziessel et al. / Tetrahedron Letters 53 (2012) 3717–3721
40000
30000
20000
10000
40000
30000
20000
10000
0
0
220 270 320 370 420 470 520 570 620 670 720
220
270
320
370
420
470
520
570
620
670
720
Wavelength (nm)
Wavelength (nm)
1
1
τ
χ
= 3.18 ms
2 = 1.072
τ
χ
= 2.65 ms
2 = 1.039
0
0
0
0
2
4
6
8
10
1
2
3
4
5
6
7
8
9
10
Time (ms)
Time (ms)
Figure 1. Top: absorption (blue line), emission (red line) excitation (plum-red line)
of complex 18 (c = 6.9 ꢀ 10ꢁ5 M in buffer tris/HCl, 0.01 M at pH 7.2). Excitation
wavelength 320 nm for the emission spectrum, and emission wavelength at 543 nm
for the excitation wavelength. Bottom: deactivation decay of the Tb-excited state
lifetime after excitation at 320 nm.
Figure 2. Top: absorption (blue line), emission (red line) excitation (plum-red line)
of complex 19 (c = 5.77 ꢀ 10ꢁ5 M in buffer tris/HCl, 0.01 M at pH 7.2). Excitation
wavelength 320 nm for the emission spectrum, and emission wavelength at 543 nm
for the excitation wavelength. Bottom: Deactivation decay of the Tb-excited state
lifetime after excitation at 320 nm.
numerous biomolecules suggested to us that the phosphate entity
or its derivatives would be a desirable substituent of ligands
designed for lanthanide ion exploitation in aqueous media. We
have pursued this opinion through the synthesis of multiply
molecule is challenging and often requires linear multi-step proto-
cols,8–10 so that a facile method for the introduction of charged
substituents continues to be an attractive objective. The impor-
tance of phosphates for the stabilisation and solubilisation of
Br
N
N
N
2
N
N
EtOOC
COOEt
N
N
Br
(EtO)2(O)P
P(O)(OEt)2
Br
N
i)
COOEt
N
N
N
N
N
HN
HN
P(O)(OH)2
P(O)(OH)2
P(O)(OEt)2
HN
(HO)2(O)P
(HO)2(O)P
P(O)(OH)2
P(O)(OH)2
N
N
N
N
N
N
4
ii)
1
P(O)(OEt)2
P(O)(OEt)2
Br
Br
ii)
Br
N
N
N
3
N
N
(EtO)2(O)P
(EtO)2(O)P
P(O)(OEt)2
P(O)(OEt)2
N
N
Scheme 1. Key: (i) anhydrous acetonitrile, anhydrous K2CO3, 60 °C overnight, 59%; (ii) anhydrous acetonitrile, anhydrous K2CO3, 80 °C, overnight, 65% yield for 3 and 60% for 4.

R. Ziessel et al. / Tetrahedron Letters 53 (2012) 3717–3721
3719
phosphonated ligands, specifically avoiding an approach based on
the functionalization of less readily accessible macrocyclic species.
Our synthesis began with nucleophilic substitution at both bro-
momethyl groups of 111 with either glyphosate, amino-di(meth-
ylenediethylphosphite), or amino-di(methylenephosphinic acid)
under conditions previously described for pyridine derivatives
(Scheme 1).12
Schotten-Bauman peptidic coupling reaction with
allowing to isolated the biotinylated derivatives 15–17 in acceptable
yields.
Terbium complexes of ligands 9 and 11 were prepared by first
dissolving the metal-free ligand in water at pH 7.2 (about 1 mM),
then titrating in an aqueous solution of TbCl3ꢂ6H2O (about
10 mM). Addition was stopped at the first sign of precipitate for-
mation (near 1:1 composition). After stirring the mixture for 1 h,
it was filtered and the complex was precipitated from the filtrate
by addition of methanol and diethylether.
D
-(+)-biotin
Cross-coupling of derivatives 2, 3 and 4 with ethyl 6-heptynoate
allows the linkage to the chelation pocket to a short flexible chain
carrying a protected carboxylic acid for the possible use of this
group in bioconjugation (Scheme 2). For derivatives 5 and 6, simul-
taneous hydrolysis of both the carboxylate and one of the phos-
phonate ester groups proceeds using the appropriate amount of
NaOH in a water/THF mixture leading, respectively, to compounds
8 and 9. The ligands (as the sodium salts), could be isolated by pre-
cipitation with diethylether at neutral pH. For compound 7, sapon-
ification of the ester is achieved in a methanol/water solution in
the presence of NaOH (6 equiv).
The UV–vis absorption spectra of complexes 11 and 12 are
dominated by intense p?
p⁄ and n?p⁄ transitions centred on the
pyridyl14 and pyrazolyl15 groups (Fig. 1). As would be expected
by the lack of unsaturation in the pendent arms, the absorption
spectra are not sensitive to the nature of the deprotonable donor
groups (phosphate or carboxylate). The absorption band around
261 nm is attributed to a transition located on the pyrazolyl
fragments. The high energy absorption band about 325 nm is
assigned as a pyridyl-centered transition and its intensity is
sensitive to the presence of substituents in the para position.
For both complexes (Chart 1), excitation at 320 nm resulted in
the characteristic emission pattern of Tb(III) (Fig. 1 and 2).16 The
Tb emission spectrum consists of four intense emission bands with
Along these lines we succeeded on connecting 2,20-(ethylenedi-
oxy)bis(ethylamine) under the carboamidation conditions under a
flux of CO in the para position of the central pyridine (Scheme 3).13
The resulting derivatives 12 to 13 were allowed to react under
maxima at 490, 543, 583 and 624 nm, attributed to the 5D4 ! FJ
7
Br
N
N
N
N
N
Br
X1
X1
N
N
2 X1 = COOEt ; X2 = Et
3 X1 = P(O)(OEt)2 ; X2 = Et
4 X1 = P(O)(OH)2 ; X2 = H
(X2O)2(O)P
P(O)(OX2)2
N
N
N
N
N
X1
X1
N
N
2 X1 = COOEt ; X2 = Et
3 X1 = P(O)(OEt)2 ; X2 = Et
4 X1 = P(O)(OH)2 ; X2 = H
(X2O)2(O)P
P(O)(OX2)2
O
O
i)
i)
5 X1 = COOEt ; X2 = Et
6 X1 = P(O)(OEt)2 ; X2 = Et
7 X1 = P(O)(OH)2 ; X2 = H
NH2
O
N
N
N
N
N
H
O
N
O
X1
X1
N
N
12 X1 = COOEt ; X2 = Et
13 X1 = P(O)(OEt)2 ; X2 = Et
14 X1 = P(O)(OH)2 ; X2 = H
(X2O)2(O)P
P(O)(OX2)2
N
N
N
N
N
O
OH
X1
N
(X2O)2(O)P
X1
ii) iii)
N
O
P(O)(OX2)2
HN
H
NH
ii)
H
S
O
NH
O
N
N
N
N
H
O
N
N
O
X1
X1
N
N
15 X1 = COOEt ; X2 = Et
16 X1 = P(O)(OEt)2 ; X2 = Et
17 X1 = P(O)(OH)2 ; X2 = H
(X2'O)(X2O)(O)P
P(O)(OX2)(OX2')
N
N
N
N
N
8 X1 = COONa ; X2 = Et ; X2' = Na
9 X1 = COONa ; X2 = X2' = Na
10 X1 = P(O)(OEt)(ONa) ; X2 = Et ; X2' = Na
11 X1 = P(O)(ONa)2 ; X2 = X2' = Na
X1
X1
P(O)(OX2)2
N
N
(X2O)2(O)P
Scheme 2. Key: (i) ethyl 6-heptynoate (1.2 equiv), THF/TEA, [Pd(PPh3)4] (6 mol %),
CuI (10 mol %), 60 °C, 74% for 5, 78% for 6 and 69% for 7; (ii) THF, NaOH (9 equiv), rt,
Scheme 3. Key: (i) 2,20-(ethylenedioxy)bis(ethylamine) (2 equiv), [Pd(PPh3)2Cl2]
(12 mol %), benzene, TEA, CO flux at 1 atm, 70 °C, one night, 62% for 5, 58% for 6 and
overnight except for
9 for which key (iii) applies; (iii) TMSBr in anhydrous
dichloromethane and anhydrous 2,6-lutidine, rt followed by THF/water, NaOH
(excess), rt, overnight. In all cases, isolated yields were around 85–92%.
55% for 7; (ii)
and 65% for 10.
D-(+)-biotin (1.5 equiv), THF, EDCI, DMAP, 2h., rt, 75% for 8, 71% for 9

3720
R. Ziessel et al. / Tetrahedron Letters 53 (2012) 3717–3721
ONa
O
tion sphere of the complex and distorts the pyrazole-terbium
interaction in such a way that the energy transfer from the pyrazole
part is not as efficient as from the pyridine part. For both complexes,
the excited state decay can be fitted to a single exponential decay,
with exceptionally long excited state lifetimes of 2.7 and 3.2 ms,
respectively, for complexes 11 and 12. The luminescence quantum
yields are rather good at 26% and 16% in aqueous solution.
Again for both terbium complexes, the hydration number was
estimated to be close to zero by determining the excited state life-
times in D2O and thus is indicative of a saturated coordination
sphere lacking any water molecules in the first coordination sphere
of the terbium. This is in keeping with a perfect shielding of the
metal by the nonadentate ligand. The quantum yields increased
to 35% and 30% in D2O for complexes 18 and 19, respectively.
Some difficulties emerged when we tried to prepare the succi-
nic ester from the pendent carboxylic function. These complexes
are not soluble in organic solvents or in mixtures of water and
DMF or N-methyl-2-pyrrolidone (NMP). Despite the fact that the
activation of carboxylic acids in water with NHS has been re-
ported,19 we were unable to prepare the targets under standard
conditions.
O
ONa
N
N
N
N
N
N
N
N
N
N
Tb3+
Tb3+
N
N
N
N
O-
NaO
O-
ONa
O-
P
O-
O-
P
O-
O-
P
O-
ONa
P
P
O
O
P
O
ONa
O
NaO
O
NaO
O
O
O
18
19
Chart 1. Chemical formula of the terbium complex highlighting the complexation
pocket and the hanging side-arm.
transitions with J = 6–3, respectively,17 and weaker bands between
643 and 681 nm corresponding to J = 2–0. As seen from the
corresponding excitation spectra, the excitation in the UV domain
can be directly correlated with the absorption spectra of the com-
plexes as a result of an efficient ligand to metal energy transfer.18
Note that the excitation matches the first absorption at 325 nm
but not the second around 261 nm. This may indicate that in the ex-
cited state the pyridine–terbium interaction flattens the coordina-
We therefore, decided to construct a ligand with a phen-
ylpolyoxoethylene chain spanning the pyridine bis-pyrazole com-
plexation pocket and the anchoring function. The idea was to
enhance the lipophilicity of the molecule by introducing a substi-
tuted diethynylphenyl module, thereby increasing the solubility
of the complexes in DMF/water for the activation procedure. This
is illustrated in Scheme 4.
O
HO
O
Br
O
ii)
i)
iii)
O
O
O
O
O
OH
Br
20
21
Br
N
O
Br
O
O
Br
O
iv)
O
O
N
N
N
N
O
O
+
X
X
H
N
N
OH
22
23
(EtO)2(O)P
P(O)(OEt)2
2 X = COOEt
3 X = P(O)(OEt)2
O
ONa
O
O
v)
Br
O
O
O
O
O
O
O
O
O
O
vi)
vii)
viii)
O
O
N
N
N
N
N
X
N
X
N
N
N
N
N
N
N
N
N
N
N
X
X
X
N
X
(EtO)2(O)P
P(O)(OEt)2
N
N
N
P(O)(ONa)2 (NaO)2(O)P
28 X = COONa
P(O)(OEt)2 (EtO)2(O)P
24 X = COOEt
25 X = P(O)(OEt)2
26 X = COOEt
27 X = P(O)(OEt)2
29 X = P(O)(ONa)2
Scheme 4. Keys: (i) CH3OCH2CH2OTs, K2CO3, CH3CN, 80 °C, overnight, 67%; (ii) Br2, CCl4, reflux, overnight, 64%; (iii) 2-methyl-3-butyn-2-ol (1.1 equiv), THF/TEA, [Pd(PPh3)4]
(6 mol %), CuI (10 mol %), 60 °C, 40%; (iv) NaOH (10 equiv), THF/methanol, rt, 93%; (v) THF/TEA, [Pd(PPh3)4] (6 mol %), 80 °C, 74%; (vi) ethyl 6-heptynoate (1.2 equiv), THF/TEA,
[Pd(PPh3)2] (6 mol %), CuI (10 mol %), 50 °C, 80% for compound 18 and 75% for compound 19; (vii) TMSBr, anhydrous dichloromethane and anhydrous 2,6-lutidine; (viii) THF,
methanol, NaOH (8 equiv), 50 °C.

R. Ziessel et al. / Tetrahedron Letters 53 (2012) 3717–3721
3721
Alkylation of hydroquinone with a tosylate salt under basic
References and notes
conditions provides derivative 20 in 67% yield. Bromination in
CCl4 under reflux provided 21 in 64% isolated yield. The use of 2-
methyl-3-butyn-2-ol in 1:1 stoichiometry enabled the preparation
of derivative 22 in 40% yield through a statistical cross-coupling
reaction promoted by Pd(0) generated in situ with Cu(I) salts.
Chromatographic separation from the starting material and di-
substituted derivative was facilitated by the presence of the polar
alcohol function. After deprotection, the terminal alkyne 23 was
cross-coupled to the preformed platforms 2 and 3 under our stan-
dard conditions, leading to derivatives 24 and 25 in acceptable
yields. Grafting of ethyl 6-heptynoate generates ligands 26 and
27 containing pockets with at least 9 donor atoms (5N + 4O). A
two step hydrolysis of the phosphonate with TMSBr and the ethyl-
carboxylate with NaOH provided the anionic ligands 28 and 29 in
fair yields. Interestingly, these ligands as expected are soluble in
water and partially soluble in organic solvents like DMF, NMP
and DMSO. This solubility is sufficient for production of an acti-
vated N-hydroxysuccinimide (NHS) ester.
In summary, the present investigation describes the preparation
of novel phosphonylated nonadentate ligands constructed around
a bis-pyrazolyl–pyridine framework. One of their particular attri-
butes, in addition to the generation of water soluble lanthanide
ion complexes, is the exclusion of water from the coordination
sphere of Tb(III) in aqueous media. The efficient and relatively sim-
ple syntheses offer many opportunities for subtle variations in the
ligand structure which might be exploited for practical applica-
tions of their complexes. Full description of the spectroscopic prop-
erties and application of the complexes in bioanalysis will be
disclosed in forthcoming papers.
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We thank the Centre National de la Recherche Scientifique
(CNRS) and the European Commission for financial support of MS
through a grant for Specific Targeted Research Project (POC4Life-
LSHB-CT-2007-037933). Professor Jack Harrowfield (ISIS in
Strasbourg) is warmly acknowledged for a critical reading of this
manuscript prior to publication and for providing key references
to this contribution.