Synthesis of an activated phosphonated bifunctional chelate with potential for PET imaging and radiotherapy

Article abstract of DOI:10.1039/c2ob26452h

The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate function. The stability of L* in aqueous solutions at different pH values was studied by mass spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH. The reactivity of L* towards amine functions was tested using ethylamine under different conditions of pH and concentrations, and by the labeling of two reference peptides containing both an N-terminal amino function and a ε-amino group of a lysine residue in the backbone, and a supplementary thiol group of a cysteine residue for one of these two peptides. The results showed the coupling to be efficient at pH 8.0, with a total selectivity for the terminal amine function with respect to lysine and cysteine. The labeling was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C protein in human cancer. The labeled antibody was characterized by means of mass spectrometry and spectrofluorimetry, unraveling a labeling ratio of one chelate per antibody. Finally, the affinity of the labeled antibody towards its target was controlled by immunofluorescence staining experiments on human colon cancer biopsies, confirming the affinity of the labeled peptide for tenascin-C.

Full text of DOI:10.1039/c2ob26452h

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PAPER
Synthesis of an activated phosphonated bifunctional chelate with potential for
PET imaging and radiotherapy†
Câline Christine,*a Michaelle Koubemba,a Shakir Shakir,a,b Séverine Clavier,b Laurence Ehret-Sabatier,b
Falk Saupe,c Gertraud Orendc and Loïc J. Charbonnière*a
Received 25th July 2012, Accepted 2nd October 2012
DOI: 10.1039/c2ob26452h
The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is
described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-
methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate
function. The stability of L* in aqueous solutions at different pH values was studied by mass
spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH. The reactivity
of L* towards amine functions was tested using ethylamine under different conditions of pH and
concentrations, and by the labeling of two reference peptides containing both an N-terminal amino
function and a ε-amino group of a lysine residue in the backbone, and a supplementary thiol group of a
cysteine residue for one of these two peptides. The results showed the coupling to be efficient at pH 8.0,
with a total selectivity for the terminal amine function with respect to lysine and cysteine. The labeling
was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C
protein in human cancer. The labeled antibody was characterized by means of mass spectrometry and
spectrofluorimetry, unraveling a labeling ratio of one chelate per antibody. Finally, the affinity of the
labeled antibody towards its target was controlled by immunofluorescence staining experiments on human
colon cancer biopsies, confirming the affinity of the labeled peptide for tenascin-C.
So far, most stable chelates for Cu(II) complexation have been
based on polyaza macrocyclic ligands such as cyclen (1,4,7,10-
tetraazacyclododecane) and cyclam (1,4,8,11-tetraazacyclotetra-
Introduction
Owing to its favorable physical properties, 64Cu (T1/2 = 12.7 h;
β+: 0.656 MeV, 17.8%; β−: 0.573 MeV, 38.4%)1 is considered as
decane) derivatives bearing coordinating acetate pendant groups
like DOTA and TETA.2–8 While these ligands appeared theoreti-
a promising radionuclide for medicinal purposes in diagnostic
imaging by positron emission tomography (PET) and as a surro-
gate to 67Cu for radiotherapy.2–6 For such applications, 64Cu has
to be delivered within a living organism as a stable Cu complex
that is anchored to a biological target (protein, antibody, peptide
or small molecule), which requires the development of bifunc-
tional chelates (BFCs) characterized by the presence of a coordi-
nation site, able to rapidly and selectively form a stable complex,
and of a chemically reactive function allowing conjugation to the
target biomolecule.
cally attractive for Cu(II) coordination regarding their thermodyn-
amic stability constants, their application to PET imaging often
remains difficult as a result of slow Cu(II) complexation kinetics9
or in vivo instability for long term biomedical applications.10,11
In order to provide enhanced stability, several macrocyclic
ligands incorporating methylenephosphonate pendant arms have
been developed.12–17 Indeed, replacement of carboxylic acid
groups by phosphonic acid often resulted in an increased ther-
modynamic stability of the complexes, partly due to the higher
electrostatic interactions of phosphonate groups compared to car-
boxylate ones.18 While radiometal chelates bearing phosphonate
functions have been described, some of them being grafted on
peptides12 or NHS-folate,19 to our knowledge, no publication
has been reported relating to the introduction of an activated
chemical function that allows the coupling to biomolecules
without the need for supplementary chemical coupling agents
such as DCC or EDCI.
aLaboratoire d’Ingénierie Moléculaire Appliquée à l’Analyse, IPHC,
UMR 7178 CNRS-Université de Strasbourg, ECPM, 25 rue Becquerel,
67087 Strasbourg Cedex, France. E-mail: l.charbonn@unistra.fr,
caline.christine@unistra.fr
bLaboratoire de Spectrométrie de Masse Bio-Organique, IPHC, UMR
7178 CNRS-Université de Strasbourg, ECPM, 25 rue Becquerel, 67087
Strasbourg Cedex, France
cINSERM Unit 682, 3 avenue Molière, 67200 Strasbourg, France
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26452h
In our quest toward the development of BFCs for Cu(II), we
recently communicated the synthesis of a novel acyclic ligand L
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resulting trifluoroacetate salt 8 was converted into the NHS car-
bamate 9 by treatment with N,N′-disuccinimidyl carbonate and
triethylamine in MeCN (65% for the two steps). Finally, a
crucial step of the synthesis allowed the selective hydrolysis of
phosphonic esters in the presence of the active NHS carbamate
using an excess of TMSBr in the presence of 2,6-lutidine to
provide, after methanolysis, the desired ligand L* as a lutidinium
salt in 66% yield.
Scheme 1
Stability of L* and coupling with amines
based on a pyridine scaffold and featuring two bis(methanephos-
phonate)aminomethyl groups (Scheme 1).20,21 At room temp-
erature, L rapidly forms highly stable Cu(II) complexes (log
Stability of L* in aqueous solutions. Considering the reactiv-
ity of NHS esters and carbamate functions,29,30 the stability of
L* in aqueous solutions at different pH values was checked by
electrospray ionisation mass spectrometry. In a typical exper-
iment, L* was dissolved in an aqueous solution at fixed pH con-
taining volatile buffers and its stability was monitored in time by
following the intensity of the MS signals in the positive mode.
At pH 4.0 (acidified with formic acid), the half-life of L* was
measured to be 90 min, with a gradual loss in the peak of L* at
728 m/z units (Fig. S1, ESI†) and the appearance of a single new
peak at 587 m/z units, attributed to the formation of the amine
resulting from the hydrolysis of L* followed by decarboxylation
of the formed carbamic acid (R–NH–C(O)OH). When the pH is
raised to 6.8 (50 mM AcONH4 buffer), the half-life was con-
siderably shortened to 14 min and the decomposition products
were observed to be a mixture of the previously observed free
amine and of a second compound, with a peak at 630 m/z units,
attributed to the carbamic acid. At pH 8.6 (50 mM (NH4)2CO3),
the hydrolysis is faster than the measuring conditions (less than
one minute), essentially resulting in the saponification of the car-
bamate ester to the carbamic acid.
K
CuL = 22.7) over a broad pH domain.
As expected, the introduction of phosphonated groups resulted
in a large increase of the stability of the formed complexes rela-
tive to their carboxylated analogue (Δlog KCuL = 7.0).20,22
L
appeared to be a very efficient ligand for the complexation of
Cu(II) at low concentrations of the metal and under physiological
pH (pCuII = 15.5 at pH = 7.4, [L]tot = 10−8 M and [Cu]tot
=
10−9 M) and the complexation was shown to be very selective
compared to those of other nearby transition metal cations such
as Ni(II), Co(II), Zn(II) and also vs. Ga(III).21 Considering the
required properties for Cu PET radiotracers, such as high thermo-
dynamic stability and kinetic inertness, fast complexation and
selectivity for Cu(II), L can be considered as a very promising
chelating ligand for 64Cu, and may also find applications for the
complexation of other hard cations such as lanthanides.23
These findings prompted us to elaborate a synthetic route to
introduce a reactive function for further conjugation to targeted
biomaterials. Herein, we disclose the preparation of a novel
phosphonated bifunctional chelate L* derived from ligand L
(Scheme 1) and containing a lateral chain with a reactive NHS
carbamate group which is to our knowledge the first example of
a phosphonated BFC activated with a reactive functional group.
The stability of the BFC at various pH will be presented,
together with labeling experiments with ethylamine and two
reference peptides to define the optimum conditions for labeling.
Finally, the labeling of B28-13, a mouse monoclonal antibody
specifically recognizing tenascin-C protein in human cancer,24
will be performed and characterized.
Coupling with amines. Regarding the fast kinetics for
hydrolysis of L* at neutral and basic pH values, it was decided
to verify first that the reactivity towards primary amine functions
should be competitive for an efficient labeling. For that purpose,
L* was incubated in solutions containing 40 equiv. of ethyl-
amine at different pH values and the formation of the corres-
ponding ethyl urea was monitored by ESI/MS by calculation of
the intensity ratio R between the urea compound and the product
of hydrolysis. Under these conditions, it was satisfactorily
observed that R values of 0.80, 0.35 and 0.09 can be obtained at
pH values of respectively 9.9, 9.5 and 8.6. Lower R values at
low pH can be accounted for by the protonation of the amine at
Results and discussion
such pH (pKEtNH = 10.7), but when the pH is raised, the for-
2
Synthesis of L*
mation of the urea is highly competitive with regard to hydroly-
sis. A very large excess of ethylamine at pH 9.9 did not bring
significant improvements in the R value.
The synthetic strategy for the preparation of L* is depicted in
Scheme 2. Diethyl chelidamate 125 and tert-butyl (3-hydroxypro-
pyl)carbamate 226 reacted under Mitsunobu conditions to
produce the key intermediate 3 in 83% yield. Reduction of the
diester 3 with sodium borohydride in EtOH afforded the diol 4
(81%) which was converted in 85% yield into the bis-tosylated
derivative 5 by treatment with a mixture of tosyl chloride and
sodium hydroxide.27 Under basic conditions, N-alkylation reac-
tion between ditosylate 5 and tetra-ethyl iminobis(methanephos-
phonate) 628 furnished the tetra-diethylester phosphonated
intermediate 7 in 57% yield. Selective removal of the Boc pro-
tecting group was carried out quantitatively with TFA and the
The coupling reaction was then studied with two model pep-
tides: SIINFEKL and KLTPLCVSL. The first peptide presents
two potential amine residues, the one of the terminal amine of
the serine (denoted S) and the lateral n-butylamine of the lysine
group (denoted K). The best conditions for coupling of L* to the
peptide SIINFEKL were a pH of 8.0 and a ratio of 12 : 1 (L*–
peptide). The coupling product was observed at m/z 1575.5
(MH+) in agreement with the calculated mass for a monolabeled
peptide (m/z 1575.6). The labeled peptide was separated from
the unlabeled one by reverse phase HPLC (Fig. S2, ESI†). The
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Scheme 2 Synthetic protocol for the preparation of L*. (i) DIAD, PPh3, THF, 70 °C, 83%; (ii) NaBH4, EtOH, 78 °C, 81%; (iii) TsCl, NaOH, THF/
H2O, rt, 85%; (iv) HN(CH2PO(OEt)2)2 6, K2CO3, KI, MeCN, 70 °C, 57%; (v) CF3CO2H, CH2Cl2, rt, then (vi) DSC, NEt3, MeCN, rt, 65% for the
two steps; (vii) TMSBr, 2,6-lutidine, CH2Cl2, rt, then MeOH, 66%.
Scheme 3 Labeling scheme for peptide SIINFEKL and the products possibly obtained after digestion with Glu-C enzyme.
labeling site of coupling (terminal NH2 or Lys ε-NH2) was deter-
mined after digestion with Staphylococcus aureus protease V8
(Glu-C enzyme), which specifically cleaves a peptidic bond after
glutamic acid residues (Scheme 3). In the mass spectrum, the
observation of a peak at m/z 1334.4 corresponding to the labeled
N-terminal fragment *SIINFE indicated that the coupling
occurred on this site. No peak corresponding to the labeling of
the lysine residue was observed, in agreement with the higher
pKa of Lys ε-NH2 (pK = 10.8) versus terminal NH2 of serine
(pK = 9.2).
Finally, the complexation of Cu(II) by the labeled peptide was
further confirmed by a fluorescence quenching experiment in
which the fluorescence spectra of the labeled peptide were moni-
tored as a function of the added Cu(II) (Fig. 1).
Fig. 1 Fluorescence titration of the labeled SIINFEKL peptide upon
addition of 0 (black), 0.25 (red), 0.5 (blue), 0.75 (green) and excess
(pink and black) equivalent of CuCl2 (λexc = 270 nm).
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In the absence of added Cu(II), excitation at 270 nm in the
π → π* transitions of the pyridyl moiety31 of the BFC gave rise
to a broad emission centered at around 355 nm. The addition of
the Cu(II) salts resulted in a gradual quenching of this emission
band, attributed to an intramolecular metal to ligand electron
transfer process, as often observed with Cu(II).32 Finally, the Cu
complex was also detected by MALDI/MS spectrometry at m/z
1636.9 in agreement with the calculated mass (Fig. S3, ESI†).
The coupling of L* with the second model peptide was
studied to check the selectivity of the reaction with an amine
versus a thiol function. The cysteine containing peptide
KLTPLCVSL was labeled by L* at pH 8.0 and the resulting
peptide (m/z 1585.8, MH+) was then treated with iodoacetamide,
known to specifically react with cysteine residues. Mass spec-
trometry analysis revealed that the labeled peptide could be
totally carbamidomethylated (m/z 1643.0, MH+, Δ = 57 Da).
This confirmed that the cysteine could still react with iodoaceta-
mide and that it has not been labeled by L*, as anticipated for an
active NHS carbamate.
labeled by L* for potential future applications in tumor imaging
by PET.
Characterization of B28-13 and its labeled version. Prior to
labeling, a MALDI/MS analysis was carried out on the B28-13
antibody which was found to have a molecular mass of 148 000 Da
(Fig. 2). The labeled and purified antibody was also analyzed by
MALDI/MS. The mass spectrum shows a shift of the mono-
charged peak [M + H]+ to the higher masses due to the addition
of the ligand L*. At the maximum, this shift is of about 800 Da
which would roughly correspond to one ligand per antibody.
Immunofluorescence staining. In order to verify that the label-
ing procedure has not perturbed the affinity of the antibody
towards its antigen, immunofluorescence staining experiments
were performed in which the affinities of B28-13 and BFC
labeled B28-13 were checked using tissue sections of patient
biopsies of human colorectal cancers (Fig. 3). The presence of
the antibodies was revealed by counterstaining with an anti-
mouse Cy3-labeled secondary antibody. Control experiments
were also performed using biopsies of normal colon tissue and
for the non-specific affinity of the secondary antibody. As evi-
denced in Fig. 3A and B, comparison of the labeled antibody
and the non-modified B28-13 showed the specific TNC
expression in the stroma of human colorectal cancer and at the
basal lamina of normal human colon. This indicates intact tenas-
cin-C antigen recognition after labeling with L*.
Antibody labeling
Labeling of B28-13. Tenascin-C (TNC) is a glycoprotein
highly expressed during embryogenesis but largely restricted or
absent in healthy adult tissues. Its expression is regained in
several pathological situations33 including many human cancer
types.34 Many antibodies against TNC have been developed, and
some of them are currently used in preclinical or clinical trials.35
The B28-13 antibody is a mouse monoclonal antibody specifi-
cally recognizing the C-terminal FN type III repeats 13–15 of
human TNC,24 a motif which is present in all TNC splice
variants. B28-13 was thus chosen as a prototype antibody to be
Another series of immunofluorescent staining experiments
were achieved on tissue sections of patient biopsies of human
colorectal cancer to verify the affinity of the Cu(II) complex of
the labeled antibody (Cu(II)-BFC labeled B28-13). Results also
confirmed tenascin-C antigen recognition after labeling B28-13
with Cu(II)–L* (Fig. S4, ESI†).
Fig. 2 (A) Superimposed MALDI/MS spectra of B28-13 (blue) and B28-13 labeled with L* (red). (B) Zoom on the m/z region from 135 000 to
160 000 showing the 800 units shift in mass due to the added chelate.
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Experimental section
Synthesis of the compounds
General methods. Column chromatography and flash column
chromatography were performed respectively on silica
(0.063–0.200 mm, Merck) and silica gel (40–63 μm, Merck).
Solvent mixtures used for TLC and flash column chromato-
1
graphy are reported in v/v ratios. H and 13C NMR spectra were
recorded on an Avance 300 spectrometer at 300 MHz for proton
frequency and 75 MHz for carbon frequency. 31P NMR spectra
(161.9 MHz) were recorded on an Avance 400 apparatus. Chemi-
cal shifts are given in parts per million, relative to the residual
protic solvent peak.37 IR spectra were recorded on a Nicolet 380
FT-IR spectrometer (Thermo Scientific). Mass spectra (MS) of
synthesized compounds and elemental analysis were obtained by
the Service Commun d’Analyse de l’Université de Strasbourg.
THF was distilled over sodium and benzophenone under a
nitrogen atmosphere. Other solvents were used as purchased. Tri-
ethylamine was distilled over potassium hydroxide prior to use
and other commercially available reagents were used without
further purification.
Diethyl chelidamate 1,25 tert-butyl (3-hydroxypropyl)carba-
mate 226 and tetra-ethyliminobis-(methanephosphonate) 628 were
synthesized according to the literature procedures.
Fig. 3 Immunofluorescence staining on tissue sections of patient bio-
psies of human colorectal cancer (left column) or normal colon (right
column) using BFC labeled B28-13 (A) or non-modified B28-13 (B)
(scale bar = 100 µm, red = Cy3, blue = DAPI). Panel (C) shows the
absence of a non-specific signal from the secondary antibody alone.
Diethyl 4-(3-[(tert-butoxycarbonyl)amino]propoxy)pyridine-
2,6-dicarboxylate 3. To a solution of diethyl chelidamate 1
(3.27 g, 13.70 mmol) in THF (325 mL) were added under nitro-
gen, triphenylphosphine (7.20 g, 27.46 mmol) and tert-butyl
(3-hydroxypropyl)carbamate 2 (4.80 g, 27.52 mmol) diluted in
THF. DIAD (5.43 mL, 27.4 mmol) was added dropwise and the
mixture was stirred at 70 °C for 12 h before being cooled to rt.
The solvent was removed under reduced pressure and the oily
residue was purified by two flash chromatographies (respectively
with petroleum ether/EtOAc 70/30 and with CH2Cl2/MeOH
100/0 to 98.5/1.5) yielding ether 3 (4.5 g, 83%) as an off-white
solid. Rf = 0.63 (CH2Cl2/MeOH 96/4). 1H NMR (CDCl3,
300 MHz): δ 1.44 (s, 9H), 1.45 (t, J = 7.1 Hz, 6H), 2.05 (qt, J =
6.3 Hz, 2H), 3.34 (q, J = 6.3 Hz, 2H), 4.20 (t, J = 12.6 Hz, 2H),
4.47 (q, J = 7.1 Hz, 4H), 4.81 (s broad, 1H), 7.74 (s, 2H). 13C
NMR (CDCl3, 75 MHz): δ 14.2, 28.4, 29.4, 37.5, 62.4, 66.6,
79.4, 114.3, 150.2, 156.0, 164.7, 166.8. Found: C, 57.35; H,
7.02; N, 6.93. Calc. for C19H28N2O7: C, 57.56; H, 7.12; N,
7.07%. ESI+/MS: m/z 397.2 ([3 + H]+, 100%). IR (cm−1, ATR):
ν 3411, 3250, 2980, 1737, 1724, 1703, 1509, 1338, 1252, 1238,
1165, 1107, 1029, 784.
Conclusion
Following our previous findings on the efficient complexation of
Cu(II) by ligand L, the present work demonstrates the feasibility
to introduce a reactive N-hydroxysuccinimide carbamate func-
tion into the skeleton of the ligand L, providing a chemically
activated bifunctional chelate L* ready for the labeling of bio-
molecules. Although L* is relatively sensitive to hydrolysis in
aqueous solutions at neutral or basic pH, it can easily be conju-
gated in an aqueous solution to primary amines, small peptides
and even large biomolecules such as the B28-13 antibody. As
expected for such an activated function, the conjugation was
selective toward terminal amino residues of the peptides and was
not reactive towards thiol functions of cysteines. Labeling of
B28-13 could be readily achieved under conventional conditions
and the labeled antibody was characterized by mass spectrometry
and spectrofluorimetry. Immunofluorescence staining exper-
iments showed the labeled antibody to retain its affinity towards
tenascin-C in human colorectal cancer.
In conclusion, we have developed the first example of an acti-
vated phosphonated bifunctional chelate and we demonstrated its
ability to label biomolecules. Our efforts are now directed
towards the use of such chelates for in vivo imaging applications
with 64Cu PET imaging, with a particular emphasis on the
determination of the in vivo stability of the radiolabelled com-
pound, but also for other potential metal based imaging tech-
niques such as single photon emission computed tomography
with 111In or radiotherapeutic applications with the 67Cu
radioisotope.36
tert-Butyl (3-[(2,6-bis(hydroxymethyl)pyridin-4-yl)oxy]propyl)-
carbamate 4. To diester 3 (4.2 g, 10.5 mmol) in ethanol
(210 mL) was added in portions NaBH4 (2.0 g, 52.92 mmol).
The mixture was refluxed for 2 h before being cooled to rt.
Water was added and then solvents were removed in vacuo. The
residue was partitioned between water and CH2Cl2 and the
aqueous layer was extracted with CH2Cl2 (4 × 50 mL). The com-
bined organic layers were washed with a saturated aqueous sol-
ution of NH4Cl followed by water, dried with Na2SO4, filtered
and concentrated under vacuum. The resultant white solid 4 was
collected and used without purification (2.66 g, 81%). Rf = 0.36
1
(CH2Cl2/MeOH 90/10). H NMR (CDCl3, 300 MHz): δ 1.44
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(s, 9H), 2.01 (qt, J = 6.3 Hz, 2H), 3.33 (q, J = 6.3 Hz, 2H), 4.09
(t, J = 6.0 Hz, 2H), 4.71 (s, 4H), 6.71 (s, 2H). 13C NMR
(CDCl3, 75 MHz): δ 28.5, 29.4, 37.6, 64.4, 65.7, 79.4, 105.6,
156.3, 161.2, 166.5. Found: C, 56.10; H, 7.72; N, 8.80. Calc. for
C15H24N2O5: C, 57.68; H, 7.74; N, 8.97%. ESI+/MS: m/z 313.2
([4 + H]+, 100%), 355.2 (33%). IR (cm−1, ATR): ν 3372, 2974,
1685, 1603, 1573, 1520, 1273, 1146, 1049, 1009, 844.
8 was dried under vacuum to give an oil which was used directly
in the next step. Rf = 0.10 (CH2Cl2/MeOH 88/12). 1H NMR
(CDCl3, 300 MHz): δ 1.31 (t, J = 7.1 Hz, 24H), 2.29 (m, 2H),
3.30 (m, 10H), 4.13 (m, 16H), 4.32 (s broad, 4H), 4.46 (m, 2H),
7.39 (s, 2H), 7.92 (s broad, 2H). 13C NMR (CDCl3, 100 MHz):
δ 16.2 (m), 26.1, 37.1, 50.4 (dd, J = 158.9 Hz, J = 6.1 Hz),
57.6 (t, J = 5.8 Hz), 63.5 (t, J = 3.4 Hz), 67.7, 110.7, 115.5 (q,
J = 289.0 Hz, CF3CO2H), 155.7, 160.2 (q, J = 39.7 Hz,
CF3CO2H), 171.8. 31P NMR (CDCl3, 161.9 MHz): δ 24.05.
ESI+/MS: m/z 406.2 ([8 + 2H]2+, 100%), 811.3 ([8 + H]+, 32%).
IR (cm−1, ATR): ν 2989, 1774, 1634, 1202, 1160, 1023, 975,
797, 704.
To a solution of salt 8 (1.33 mmol) in MeCN (30 mL) under
nitrogen, triethylamine (0.33 mL, 2.39 mmol) and DSC
(612.2 mg, 2.39 mmol) in MeCN were added. The reaction
mixture was stirred at rt for 5 h 45. The solvent was evaporated
to dryness and the residue dissolved in dichloromethane. The
organic layer was washed with a saturated aqueous solution of
NH4Cl until TLC analysis showed complete disappearance of
the DSC residue. The organic phase was dried over Na2SO4,
filtered and concentrated under reduced pressure to yield NHS
carbamate 9 as a pale brown oil (821.0 mg, 65% for the two
steps). Rf = 0.60 (CH2Cl2/MeOH 88/12). 1H NMR (CDCl3,
300 MHz): δ 1.27 (t, J = 7.1 Hz, 24H), 2.02 (m, 2H), 2.76 (s,
4H), 3.17 (d, J = 9.8 Hz, 8H), 3.36 (m, 2H), 4.09 (m, 20H), 4.27
(m, 2H), 7.25 (s broad, 2H), 7.57 (s broad, 1H). 13C NMR
(CDCl3, 75 MHz): δ 16.5 (m), 25.5, 28.5, 38.7, 50.6 (dd,
J = 160.0 Hz, J = 8.0 Hz), 61.4, 62.2 (m), 65.8, 108.9, 152.0,
158.9, 169.9. 31P NMR (CDCl3, 161.9 MHz): δ 24.13. ESI+/
MS: m/z 869.3 (77%), 952.3 ([9 + H]+, 100%). IR (cm−1, ATR):
ν 3224, 2983, 1780, 1737, 1599, 1208, 1096, 1019, 961,
776, 731.
(4-(3[(tert-Butoxycarbonyl)amino]propoxy)pyridine-2,6-diyl)-
bis(methylene)bis(4-methylbenzenesulfonate) 5. A solution of
TsCl (3.55 g, 18.64 mmol) in THF (67 mL) was added dropwise
to a 0 °C cooled mixture of diol 4 (1.45 g, 4.66 mmol) and
NaOH (1.12 g, 27.96 mmol) in THF/H2O (1:1, 34 mL). The
mixture was warmed to rt and stirred for 5–6 h. The two layers
were decanted and the aqueous layer was extracted with CH2Cl2.
The combined organic extracts were washed with a 5% aqueous
solution of NaHCO3 followed by brine, dried with Na2SO4,
filtered and evaporated under vacuum. The crude product was
purified by flash chromatography (CH2Cl2/MeOH 99/1 to 98/2)
to afford bis-tosylated compound 5 (2.44 g), as a white solid, in
85% yield. Rf = 0.28 (CH2Cl2/MeOH 98.5/1.5). 1H NMR
(CDCl3, 300 MHz): δ 1.46 (s, 9H), 1.99 (qt, J = 8.3 Hz, 2H),
2.44 (s, 6H), 3.31 (q, J = 6.6 Hz, 2H), 4.04 (t, J = 6.1 Hz, 2H),
4.70 (s broad, 1H), 4.98 (s, 4H), 6.81 (s, 2H), 7.34 (d, J = 8.1 Hz,
4H), 7.81 (d, J = 8.3 Hz, 4H). 13C NMR (CDCl3, 75 MHz):
δ 21.8, 28.5, 29.4, 37.6, 66.1, 71.3, 79.6, 107.7, 128.2, 130.1,
132.8, 145.3, 155.3, 156.1, 166.7. Found: C, 55.49; H, 5.94; N,
4.36. Calc. for C29H36N2O9S2: C, 56.11; H, 5.85; N, 4.51%.
ESI+/MS: m/z 643.2 ([5 + Na]+, 100%). IR (cm−1, ATR): ν
3357, 2926, 1677, 1367, 1172, 1034, 955, 840, 810, 670.
tert-Butyl (3-[(2,6-bis[(bis[(diethoxyphosphoryl)methyl]amino)-
methyl]pyridin-4-yl)oxy]propyl)carbamate 7. To
of tetra-ethyliminobis(methanephosphonate)
a
solution
6
(350.2 mg,
1.10 mmol) in MeCN (22 mL), under a nitrogen atmosphere,
freshly flame-dried K2CO3 (400 mg, 2.88 mmol) was added.
The mixture was stirred for 20 min and compound 5 (300.0 mg,
0.48 mmol) in MeCN (50 mL) and freshly flame-dried KI
(167.0 mg, 1.00 mmol) were added. The reaction mixture was
heated at 70 °C for 40 h. After filtration, the solvent was
removed in vacuo and the crude oil was purified by flash chrom-
atography (CH2Cl2/MeOH 100/0 to 90/10). Compound 7 was
obtained as an oily light yellow product (249.0 mg, 57%). Rf =
0.33 (CH2Cl2/MeOH 92/8). 1H NMR (CDCl3, 300 MHz): δ
1.23 (t, J = 6.4 Hz, 24H), 1.35 (s, 9H), 1.90 (m, 2H), 3.17 (m,
10H), 4.03 (m, 22H), 5.09 (s broad, 1H), 6.99 (s, 2H). 13C NMR
(CDCl3, 75 MHz): δ 16.4 (d, J = 6.2 Hz), 28.4, 29.2, 37.5, 50.3
(dd, J = 158 Hz, J = 8.6 Hz), 62.0 (d, J = 6.4 Hz), 62.4 (t, J =
8.2 Hz), 65.5, 79.0, 108.3, 156.0, 159.4, 166.3. 31P NMR
(CDCl3, 161.9 MHz): δ 24.60. Found: C, 45.64; H, 7.92; N,
6.15. Calc. for C35H70N4O15P4: C, 46.15; H, 7.75; N, 6.15.
ESI+/MS: m/z 933.4 ([7 + Na]+, 100%). IR (cm−1, ATR): ν
3303, 2980, 1709, 1597, 1250, 1231, 1160, 1019, 959, 777.
({[(4-(3-({[(2,5-Dioxopyrrolidin-1-yl)oxy]carbonyl}amino)-
propoxy)pyridine-2,6-diyl)bis(methylene)]bis(azanetriyl)}tetra-
kis(methylene))tetraphosphonic acid L*. To a solution of NHS
carbamate 9 (51.0 mg, 0.053 mmol) in CH2Cl2 (3 mL), under
nitrogen, 2,6-lutidine (0.32 mL, 2.75 mmol) then TMSBr
(0.27 mL, 2.04 mmol) were added and the resulting mixture was
stirred at room temperature for 15 h. The solvents were evapor-
ated. The crude was coevaporated with CH2Cl2 then methanol
(5 mL) was added to the residue and was evaporated under
reduced pressure. The last procedure was realized twice again.
The resulting solid was centrifugated in CH2Cl2 and washed
several times with CH2Cl2 and methanol to afford final ligand
1
L* as a pale brown lutidinium salt (36.0 mg, 66%). H NMR
(D2O, 300 MHz): δ 2.08 (m, 2H), 2.72 (s, 6H, lutidine), 2.91 (s,
4H), 3.45 (m, 2H), 3.63 (d, J = 12.1 Hz, 8H), 4.27 (m, 2H), 4.91
(s, 4H), 7.13 (s, 2H), 7.63 (d, J = 8.1 Hz, 2H, lutidine), 8.28 (t,
J = 8.0 Hz, 1H, lutidine). 13C NMR (D2O, 100 MHz): δ 18.7
(lutidine), 25.3, 27.6, 38.2, 52.4 (d, J = 139.5 Hz), 59.0, 66.6,
110.8, 124.6 (lutidine), 146.0 (lutidine), 151.2 (lutidine), 153.0,
153.3, 167.9, 173.9. 31P NMR (D2O, 161.9 MHz): δ 7.88.
2,5-Dioxopyrrolidin-1-yl-(3-[(2,6-bis[(bis[(diethoxyphosphoryl)-
methyl]amino)methyl]pyridin-4-yl)oxy]propyl)carbamate 9. To
a solution of amine 7 (1.21 g, 1.33 mmol) in 15 mL of dichloro-
methane was added dropwise at 0 °C, under nitrogen, TFA
(1.97 mL, 26.6 mmol). The mixture was stirred at rt for 9 h. The
solvent was evaporated to dryness. The crude trifluoroacetate salt
Found: C, 30.50; H, 4.61;
N
8.32. Calc. for
C19H33N5O17P4·C7H9N·2HBr·H2O: C, 30.79; H, 4.57; N, 8.28.
ESI+/MS (H2O/MeCN/formic acid): m/z 728.1 ([L* + H]+,
100%). IR (cm−1, ATR): ν 3203, 2959, 1773, 1730, 1627, 1609,
1206, 1156, 1047, 920, 797.
9188 | Org. Biomol. Chem., 2012, 10, 9183–9190
This journal is © The Royal Society of Chemistry 2012

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Labeling of model peptides and B28-13 antibody
Labeling of the KLTPLCVSL model peptide. Mentioned
quantity for the ligand L* has been calculated taking into
account the estimated proportion (by 1H NMR) of L* alone
(25%) in the crude luditinium salt mixture. 17 µL of a 100 mM
NH4HCO3 solution, 3 µL of a 10 µg µL−1 solution of
KLTPLCVSL (30 nmol, 1 equiv.) and 0.16 mg of ligand L*
(0.22 µmol, 7.45 equiv.) were successively added in an eppen-
dorf. The pH of the reaction medium was adjusted to 8 with a
100 mM solution of NH4HCO3 and the reaction mixture was left
overnight at room temperature.
Stability of L* in aqueous solutions. Ligand solutions were
extemporaneously prepared at different pH values: pH = 4.0
(adjusted with formic acid); pH = 6.8 (50 mM AcONH4) and
pH = 8.6 (50 mM (NH4)2CO3) and infused directly into the
ESI/MS instrument (MicrOTOF-Q, Bruker Daltonics). The infu-
sion flow was 180 µL h−1 and the spectra were acquired over
1 min across the m/z range of 50–1500. The mass spectrometer
was calibrated using an ESI-L Low Concentration Tuning Mix
solution (Agilent Technologies). The intensities of peaks at 587,
630 and 728 m/z units were monitored at different times.
MALDI/MS analysis of the labeled KLTPLCVSL
peptide. 10 µL of the reaction medium were acidified by a 10%
(v/v) TFA solution. 0.6 µL of the reaction medium was then
spotted on the target with 0.6 µL of the THAP matrix solution
as described before.
Labeling of the SIINFEKL model peptide. Mentioned quan-
tity for the ligand L* has been calculated taking into account the
1
estimated proportion (by H NMR) of L* alone (25%) in the
crude luditinium salt mixture. 500 µL of a 100 mM NH4HCO3
solution, 20.5 µL of a 10 µg µL−1 solution of the SIINFEKL
peptide (0.21 µmol, 1 equiv.) and 1.18 mg of ligand L*
(1.62 µmol, 7.75 equiv.) were successively added in an eppen-
dorf. The pH of the medium was adjusted to 8 with a 100 mM
NH4HCO3 solution and the reaction mixture was left overnight
at room temperature.
Cysteine alkylation. 5 µL of the KLTPLCVSL labeling
medium (3 µg of the peptide, 3 nmol, 1 equiv.), 5 µL of a
50 mM (NH4)2CO3 solution (pH = 8.6) and 8 µL of a 1 µg µL−1
solution of iodoacetamide (40 nmol, 13 equiv.) were added in
an eppendorf. The reaction medium was left for 1 h at room
temperature in the dark, then acidified by a 10% (v/v) TFA solu-
tion and analyzed by MALDI/MS with THAP as a matrix.
MALDI/MS analysis of the labeled SIINFEKL peptide. 10 µL
of the reaction medium were acidified by a 10% (v/v) TFA
solution. 0.6 µL was then spotted on a polished steel MTP 384
target plate (Bruker Daltonics) with 0.6 µL of α-cyano-4-hydro-
xycinnamic acid (CHCA) matrix solution using the dried droplet
method. The mass spectrometer (Ultraflex, Bruker Daltonics)
was operated in positive reflectron mode.
Preparation of B28-13 antibody. The B28-13 antibody was
obtained by affinity purification using a 1 mL HiTrap Protein G
HP column (17-0404-01, GE Healthcare) connected to the
AKTAPrime™ purification system (GE Healthcare). 100 mL of
the supernatant collected from hybridoma cell cultures secreting
B28-1324 were used and purification performed according to the
manufacturer’s instructions. The obtained antibody eluate was
dialyzed against PBS and stored in aliquots at −20 °C.
SDS-PAGE followed by Coomassie staining was performed to
verify purity of the antibody preparation.
Purification of the labeled SIINFEKL peptide. The reaction
medium was purified by RP-HPLC using a Poroshell 120
EC-C18 column (2.1 × 100 mm, 2.7 µm). The labeled peptide
was eluted at 14.5 min (flow rate 0.8 mL min−1) using a gradient
of 10 to 45% of solvent B over 30 min (solvent A: 0.1% (v/v)
TFA in ultrapure water, solvent B: 0.1% (v/v) TFA in MeCN).
MALDI/MS analysis of the antibody B28-13. Prior to analy-
sis, both native and labeled antibody solutions were desalted on
a C18 ZipTip (Millipore). 70 pmol of the antibody was loaded
and then eluted in 5 µL H2O/MeCN/formic acid 20/75/5 (v/v/v).
0.6 µL of the eluate was then spotted on the target plate with
0.6 µL of sinapinic acid (2 mg mL−1 in 50% MeCN). The mass
spectrometer (Autoflex, Bruker Daltonics) was operated in posi-
tive linear mode, with BSA as a calibrant.
Digestion of the labeled SIINFEKL peptide by Glu-C
enzyme. 0.5 µg of endoproteinase Glu-C (Staphylococcus
aureus Protease V8) were added to 5 µg of the purified labeled
SIINFEKL peptide in a 100 mM NH4HCO3 solution (pH = 7.8)
and the reaction medium was left for 3 h at room temperature.
After acidification using a 10% TFA solution, the medium was
analyzed by MALDI/MS using THAP (2,4,6-trihydroxyaceto-
phenone) as a matrix (10 mg mL−1 in 50% MeCN/50 mg mL−1
ammonium citrate solution in a ratio of 9 : 1 (v/v)). A control
digestion experiment was performed on a native SIINFEKL
peptide.
Labeling of B28-13. Mentioned quantity for the ligand L* has
been calculated taking into account the estimated proportion (by
1H NMR) of L* alone (26%) in the crude luditinium salt
mixture. The labeling experiment was carried out on 350 µg
(500 µL of a 0.7 mg mL−1 solution in PBS, 1.55 nmol) of the
antibody B28-13 to which 0.22 mg of ligand L* (0.3 µmol) was
added, corresponding to 200 equiv. of the ligand for each anti-
body (a higher ligand/protein ratio was used in the labeling of
B28-13 to take into account the higher number of lysine residues
present in an antibody and to be sure of having a good labeling
ratio; this means that the reaction medium needed to be purified
to eliminate the excess of L*). The reaction medium was
adjusted to a pH of 7.1 with PBS buffer and was stirred for 1 h
at room temperature. The reaction medium was purified repeat-
edly to eliminate the excess of ligand with 10 mM Tris-HCl
Fluorescence study of the labeled SIINFEKL peptide. Fluori-
metric titration was performed on 400 µL (5 × 10−5 M) of the
purified labeled SIINFEKL peptide. The measurements were
performed on a Horiba Jobin Yvon spectrofluorimeter. The emis-
sion was measured at the maximum λ = 355 nm and the exci-
tation wavelength was λ = 270 nm. The emission value was
recorded for the labeled product first then by each successive
addition of a 2.5 × 10−4 M Cu(II) solution (0.25 equiv. per
addition).
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9183–9190 | 9189

View Article Online
11 S. Ait-Mohand, P. Fournier, V. Dumulon-Perreault, G. E. Kiefer, P. Jurek,
L. Ferreira, F. Bénard and B. Guérin, Bioconjugate Chem., 2011, 22,
1729.
pH 7.4 using Vivaspin500 (Sartorius) modules with a 30 kDa
cut-off (500 µL of Tris-HCl per cycle; 20 cycles).
12 Y. Guo, R. Ferdani and C. J. Anderson, Bioconjugate Chem., 2012, 23,
1470.
13 C. A. Boswell, C. A. S. Regino, K. E. Baidoo, K. J. Wong,
D. E. Milenic, J. A. Kelley, C. C. Lai and M. W. Brechbiel, Bioorg. Med.
Chem., 2009, 17, 548.
14 D. J. Stigers, R. Ferdani, G. R. Weisman, E. H. Wong, C. J. Anderson,
J. A. Golen, C. Moore and A. L. Rheingold, Dalton Trans., 2010, 39,
1699.
15 R. Ferdani, D. J. Stigers, A. L. Fiamengo, L. Wei, B. T. Y. Li,
J. A. Golen, A. L. Rheingold, G. R. Weisman, E. H. Wong and
C. J. Anderson, Dalton Trans., 2012, 41, 1938.
16 I. Svobodova, J. Havlickova, J. Plutnar, P. Lubal, J. Kotek and
P. Hermann, Eur. J. Inorg. Chem., 2009, 3577.
17 X. Sun, M. Wuest, Z. Kovacs, A. D. Sherry, R. Motekaitis, Z. Wang,
A. E. Martell, M. J. Welch and C. J. Anderson, J. Biol. Inorg. Chem.,
2003, 8, 217.
18 (a) I. Lukeš, J. Kotek, P. Vojtíšek and P. Hermann, Coord. Chem. Rev.,
2001, 216–217, 287; (b) K. Nchimi Nono, A. Lecointre, M. Regueiro-
Figueroa, C. Platas-Iglesias and L. J. Charbonnière, Inorg. Chem., 2011,
50, 1689.
19 A. K. Mishra, M. Chopra and V. Jain, Chem. Lett., 2005, 34, 1098.
20 S. Abada, A. Lecointre, M. Elhabiri and L. J. Charbonnière, Dalton
Trans., 2010, 39, 9055.
Immunostaining experiment. Histological analysis was per-
formed by immunofluorescence staining on 7 µm sections of
fresh frozen tissue biopsies of human colorectal cancer or
normal colon. For experiments to verify affinity of BFC labeled
B28-13, tissue sections were incubated for 45 min with 5%
normal goat serum (NGS)/PBS and coupled or non-coupled
B28-13 antibody preparations were diluted 1 : 50 in 5% NGS/
PBS. For experiments to verify affinity of Cu(II)-BFC labeled
B28-13, tissue sections were incubated for 45 min with 5%
normal donkey serum (NDS)/PBS and coupled, copper coupled
and non-coupled B28-13 antibody preparations were diluted in
5% NGS/PBS in the ratios of 1 : 800, 1 : 400 and 1 : 400 respect-
ively. Then sections were incubated in a humidified chamber at
4 °C overnight followed by a Cy3-labeled anti-mouse secondary
antibody (1 : 1600; 715-165-150, Jackson ImmunoResearch) for
1.5 h at room temperature. Cell nuclei were stained with 4′,6-di-
amidino-2-phenylindole (DAPI) and microscopic analysis per-
formed on a Zeiss Imager.Z2 microscope. Tissue samples had
been acquired upon written consent according to conventional
ethical standards.
21 S. Abada, A. Lecointre, I. Déchamps-Olivier, C. Platas-Iglesias,
C. Christine, M. Elhabiri and L. Charbonnière, Radiochim. Acta, 2011,
99, 663.
22 L. Pellegatti, J. Zhang, B. Drahos, S. Villette, F. Suzenet, G. Guillaumet,
S. Petoud and E. Tóth, Chem. Commun., 2008, 6591.
23 S. Abada, A. Lecointre, M. Elhabiri, D. Esteban-Gomez, C. Platas-
Iglesias, G. Tallec, M. Mazzanti and L. J. Charbonnière, Chem.
Commun., 2012, 48, 4085.
Acknowledgements
This work was supported by the Centre National de la Recherche
Scientifique and the Université de Strasbourg (UMR 7178
CNRS-Université de Strasbourg). FS was supported by Fonda-
tion des Treilles. GO gratefully acknowledges the INCa, the
ANR, and the Contrat d’interface with the Hospital of Haute-
pierre (Strasbourg, France) for financial support.
24 S. Schenk, J. Muser, G. Vollmer and R. Chiquet-Ehrismann,
Int. J. Cancer, 1995, 61, 443.
25 C. S. Bonnet, L. Pellegatti, F. Buron, C. M. Shade, S. Villette,
V. Kubícěk, G. Guillaumet, F. Suzenet, S. Petoud and E. Tóth, Chem.
Commun., 2010, 46, 124.
26 T. Vilaivan, Tetrahedron Lett., 2006, 47, 6739.
27 T. Wang, H. An, B. D. Haly and P. D. Cook, J. Heterocycl. Chem., 2000,
37, 687.
28 S. Aime, M. Botta, E. Garino, S. Geninatti Crich, G. Giovenzana,
R. Pagliarin, G. Palmisano and M. Sisti, Chem.–Eur. J., 2000, 6, 2609.
29 M. Morpurgo, E. A. Bayer and M. Wilchek, J. Biochem. Biophys.
Methods, 1999, 38, 17.
30 E. J. M. Tournier, J. Wallach and P. Blond, Anal. Chim. Acta, 1998, 361,
33.
References
1 S. M. Qaim, T. Bisinger, K. Hilgers, D. Nayak and H. H. Coenen, Radio-
chim. Acta, 2007, 95, 67.
2 B. M. Zeglis and J. S. Lewis, Dalton Trans., 2011, 40, 6168.
3 S. Bhattacharyya and M. Dixit, Dalton Trans., 2011, 40, 6112.
4 T. J. Wadas, E. H. Wong, G. R. Weisman and C. J. Anderson, Chem.
Rev., 2010, 110, 2858.
5 M. Shokeen and C. J. Anderson, Acc. Chem. Res., 2009, 42, 832.
6 T. J. Wadas, E. H. Wong, G. R. Weisman and C. J. Anderson, Curr.
Pharm. Des., 2007, 13, 3.
7 S. Liu, Adv. Drug Delivery Rev., 2008, 60, 1347.
8 L. M. P. Lima, D. Esteban-Gomez, R. Delgado, C. Platas-Iglesias and
R. Tripier, Inorg. Chem., 2012, 51, 6916.
9 N. Bernier, J. Costa, R. Delgado, V. Félix, G. Royal and R. Tripier,
Dalton Trans., 2011, 40, 4514.
31 M. Regueiro-Figueroa, B. Bensenane, E. Ruscsak, D. Esteban-Gomez,
L. J. Charbonnière, G. Tirsco, I. Toth, A. De Blas, T. Rodriguez-Blas and
C. Platas-Iglesias, Inorg. Chem., 2011, 50, 4125.
32 A. P. de Silva, H. Q. N. Gunaratne, T. Gnnlaugsson, A. J. M. Huxley,
C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97,
1515.
33 R. Chiquet-Ehrismann and R. P. Tucker, Persp. Biol., 2011, 3, a004960.
34 G. Orend and R. Chiquet-Ehrismann, Cancer Lett., 2006, 244, 143.
35 K. S. Midwood, T. Hussenet, B. Langlois and G. Orend, Cell. Mol. Life
Sci., 2011, 68, 3175.
36 P.-M. Smith-Jones, R. Fridrich, T. A. Kaden, I. Novak-Hofer, K. Siebold,
D. Tschudin and H. R. Maecke, Bioconjugate Chem., 1991, 2, 415.
37 H. E. Gottlieb, V. Kotlyar and A. Nudelman, J. Org. Chem., 1997, 62,
7512.
10 L. A. Bass, M. Wang, M. J. Welch and C. J. Anderson, Bioconjugate
Chem., 2000, 11, 527.
9190 | Org. Biomol. Chem., 2012, 10, 9183–9190
This journal is © The Royal Society of Chemistry 2012