Solid-phase organic tagging resins for labeling biomolecules by 1,3-dipolar cycloaddition: Application to the synthesis of a fluorescent non-peptidic vasopressin receptor ligand

Article abstract of DOI:10.1002/chem.200800273

Two novel solid-phase organic tagging (SPOrT) resins were synthesized to facilitate the labeling of peptides and small organic compounds with a fluorescent probe. Both resins were obtained from the commercially available backbone amide linker (BAL) resin. Following the solid-phase synthesis of model compounds, a tripeptide and benzazepine, the fluorescent probe derived from Lissamine Rhodamine B was incorporated through Cu¹-catalyzed 1,3-dipolar cycloaddition. Final cleavage in acidic media enabled access to both types of molecules in good yield with high purity. The SPOrT resin was successfully applied to the preparation of the first non-peptidic fluorescent compound with a nanomolar affinity for the human vasopressin V₂ receptor (V₂R) subtype. This molecule will find application in binding assays that use polarization or fluorescence resonance energy-transfer (FRET) techniques. The SPOrT resins are also well suited for other tags and the parallel synthesis of a fluorescently tagged library for protein screening.

Full text of DOI:10.1002/chem.200800273

FULL PAPER
DOI: 10.1002/chem.200800273
Solid-Phase Organic Tagging Resins for Labeling Biomolecules by 1,3-
Dipolar Cycloaddition: Application to the Synthesis of a Fluorescent Non-
Peptidic Vasopressin Receptor Ligand
Dominique Bonnet,*[a] StØphanie RichØ,[a] StØphanie Loison,[a] Rania Dagher,[a]
Marie-CØline Frantz,[a] Laure Boudier,[b] Rita Rahmeh,[b] Bernard Mouillac,[b]
Jacques Haiech,[a] and Marcel Hibert[a]
Abstract: Two novel solid-phase organ-
ic tagging (SPOrT) resins were synthe-
sized to facilitate the labeling of pep-
tides and small organic compounds
with a fluorescent probe. Both resins
were obtained from the commercially
available backbone amide linker
(BAL) resin. Following the solid-phase
synthesis of model compounds, a tri-
peptide and benzazepine, the fluores-
cent probe derived from Lissamine
through CuI-catalyzed 1,3-dipolar cy-
cloaddition. Final cleavage in acidic
media enabled access to both types of
molecules in good yield with high
purity. The SPOrT resin was successful-
ly applied to the preparation of the
first non-peptidic fluorescent com-
pound with a nanomolar affinity for
the human vasopressin V2 receptor
(V2R) subtype. This molecule will find
application in binding assays that use
polarization or fluorescence resonance
energy-transfer (FRET) techniques.
The SPOrT resins are also well suited
for other tags and the parallel synthesis
of a fluorescently tagged library for
protein screening.
Keywords: click chemistry · cyclo-
addition
· fluorescent probes ·
receptors · solid-phase synthesis
Rhodamine B
was
incorporated
Introduction
lecular association processes both in vitro and in vivo.[2] The
fluorescence polarization technique is also a very useful and
convenient tool for binding studies and can advantageously
be used for high-throughput screening of a fluorescently
tagged library.[3] More generally, fluorescent-based tech-
niques are especially appealing because of the potential sen-
sitivity and specificity that can be achieved. In addition, flu-
orescent probes represent a safe and versatile alternative to
radioligands, thus eliminating issues related to the handling
and disposal of radioactive materials.
Fluorescent techniques have proven to be extremely power-
ful tools to probe the structure and function of proteins. Nu-
merous applications exist that range from biophysical char-
acterization to fluorescent imaging of membrane proteins in
living cells.[1] Fluorescence resonance energy-transfer
(FRET) between a fluorescent donor–acceptor pair repre-
sents a convenient method to investigate intra- and intermo-
Nevertheless, the prerequisite for the application of such
techniques is to design and synthesize fluorescent peptides
or small-molecule-based ligands that retain the pharmaco-
logical profile of the nonlabeled probes. Several methods
are currently available to label biomolecules. However, they
generally suffer from limitations, such as the need for pro-
tecting groups and the separation of the product from
excess reagent and by-products, which hamper the synthesis
of fluorescent probes in high yield and purity. Therefore,
there is still a definite need for a generic and highly flexible
strategy to access to fluorescent molecules.
[a] Dr. D. Bonnet, S. RichØ, S. Loison, R. Dagher, M.-C. Frantz,
Prof. Dr. J. Haiech, Prof. Dr. M. Hibert
DØpartement de Pharmacochimie de la Communication Cellulaire
UniversitØ Strasbourg 1, Institut Gilbert Laustriat
CNRS UMR 7175, FacultØ de Pharmacie
74 route du Rhin, BP 60024, 67401 Illkirch (France)
Fax : (+33)390-244-310
E-mail: dominique.bonnet@pharma.u-strasbg.fr
[b] L. Boudier, R. Rahmeh, Dr. B. Mouillac
Institut de GØnomique Fonctionnelle
DØpartement de Pharmacologie MolØculaire
CNRS UMR 5203, INSERM U661
Herein, we describe a convenient and straightforward
solid-phase approach to prepare both fluorescent peptides
UniversitØ Montpellier I et II
141 rue de la Cardonille, 34094 Montpellier (France)
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6247

and fluorescent small organic compounds by using so-called
solid-phase organic tagging (SPOrT) resins. This method
combines the advantages of solid-phase chemistry with
those of the CuI-catalyzed 1,3-dipolar cycloaddition, referred
to as “click” chemistry.[4] To highlight the efficacy of the two
novel resins, fluorescent 2,3,4,5-tetrahydro-1H-benzo[b]aze-
pine (1-benzazepine) and a tripeptide were synthesized as
model compounds in high yield and purity. In our program
aimed at the design and synthesis of novel antagonists of ar-
ginine–vasopressin (AVP) receptors by using FRET based-
assay, the SPOrT resin was successfully applied to the prepa-
ration of the first fluorescent non-peptidic ligand of the
human vasopressin V2 receptor (V2R) subtype (Scheme 1).
through the solid-phase synthesis and enables the incorpora-
tion of the probe at the very last stage.
À
À
The preparation of CO2H and NH2 SPOrT resins 2 and 3:
We decided to immobilize the alkyne moiety on a solid sup-
port[5] to avoid the potential risk of the cross-coupling of al-
kynes in solution, such as the Glaser or Strauss coupling.
The commercially available 4-(4-formyl-3-methoxyphenoxy)-
butyryl (FMPB) resin was treated with propargylamine ac-
cording to the protocol developed by Barany and co-work-
ers, namely, using NaBH3CN in DMF/AcOH (99:1) at room
temperature.[6] However, following these conditions, the re-
action was not complete, as shown by the positive result to a
test with 2,4-dinitrophenylhydrazide (DNPH).[7] To acceler-
ate the kinetics of the reductive amination, the solubility of
the reagents was improved by adding MeOH (19%) to the
resin, and the resulting mixture was heated to 608C. After
16 h, the negative result to a test with chloranil was indica-
tive of a complete, successful reaction.[8]
To access resins 2 and 3, supported propargylamine 1 was
À
treated in presence of either succinimic anhydride or Fmoc
À
Gly OH in presence of a coupling agent. Opening of the
anhydride was efficiently performed in the presence of pyri-
dine and 4-DMAP to provide resin 2. The presence of the
carboxylic acid group was confirmed by using the malachite
green test (Scheme 2).[9] The N-Fmoc-protected glycine
moiety was successfully introduced by activation with
PyBOP in situ in CH2Cl2/DMF (1:1) to give resin 3.
Scheme 1. Fluorescent 2,3,4,5-tetrahydro-1H-benzo[b]azepine derivatives
synthesized using the SPOrT resin.
Results and Discussion
Based on the recent results obtained with the muscarinic
M1 receptor,[2b] we devised a strategy that enabled the rapid
solid-phase assembly of a fluorescent probe derived from
Lissamine Rhodamine B, linked with either peptides or
Scheme 2. Preparation of the SPOrT resins 2 and 3. i) Propargylamine
(10 equiv), NaBH3CN (10 equiv), DMF/MeOH/AcOH (80:19:1), 608C,
overnight; ii) succinic anhydride (25 equiv), 4-DMAP (1 equiv), pyridine
(4 equiv), DMF, RT, overnight; iii) Fmoc-Gly-OH (4 equiv), PyBOP
(4 equiv), DIEA (12 equiv), NMP, RT, overnight. 4-DMAP=4-dimethyl-
aminopyridine, Fmoc=9-fluorenylmethoxycarbonyl, NMP=1-methyl-2-
pyrrolidone, PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate.
small molecules through
a polyethylene glycol (PEG)
spacer. This latter species should be long enough to limit the
perturbation of ligand affinity. In addition, it should increase
the solubility of fluorescent molecules in aqueous media.
Our strategy entailed three parts: 1) the preparation of the
SPOrT resins consisted of the solid-phase incorporation of
an alkyne moiety on a backbone amide linker (BAL) resin,
followed by the introduction of either an amine or carboxyl-
À
À
ic acid group to provide CO2H and NH2 SPOrT resins 2
and 3 (Scheme 2); 2) the subsequent solid-phase elongation
of peptides or small organic compounds; 3) conjugation of
the fluorescent probe performed through a solid-phase CuI-
catalyzed 1,3-dipolar cycloaddition by using a PEG—azido
spacer bearing Lissamine Rhodamine B, a fluorescence dye
with a high molar extinction coefficient (Scheme 3 and
Scheme 4). The alkyne group is supposed to be stable
Solid-phase synthesis of model biomolecules: With resins 2
and 3 in hand, their potential to provide rapid access to fluo-
rescent biomolecules (small organic compounds and pep-
tides) was investigated by considering two types of model
compounds: a fluorescent 1-benzazepine scaffold extensively
studied by us[10] and a tripeptide VFK, which contained two
basic sites susceptible to hampering the final cleavage from
the BAL resin.
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Chem. Eur. J. 2008, 14, 6247 – 6254

Solid-Phase Organic Tagging Resins for Labeling Biomolecules
FULL PAPER
Resin-bound 1-benzazepine 6
was prepared in two steps from
À
CO2H SPOrT
resin
2
(Scheme 3). The 2,3,4,5-tetrahy-
dro-1H-benzo[b]azepin-5-amine
scaffold was obtained in a six-
step process starting from the
commercially available methyl-
2-aminobenzoate.[10] This scaf-
fold was then anchored to resin
2 through an amide linkage by
activation with PyBOP in situ.
After 16 h at room tempera-
ture, a negative result to a test
with malachite green was
found. The endocyclic nitrogen
atom of the resin-bound benza-
zepine 4 was deprotected in the
presence of TMSOTf and trie-
thylamine (resin 5) and subse-
quently acylated with benzoyl
À
Scheme 3. Solid-phase synthesis of the model compound benzazepine using CO2H SPOrT resin 2. i) Amino-
benzazepine (3 equiv), PyBOP (3 equiv), DIEA (4 equiv), DMF; ii) TMSOTf (3 equiv), Et3N (1.5 equiv),
CH2Cl2, RT, 15 min; iii) benzoyl chloride (5 equiv), pyridine (10 equiv), 4-DMAP (0.05 equiv), CH2Cl2, RT,
overnight; iv) TFA/H20 (95:5), RT, 3 h; v) 11 (3 equiv), CuI (5 equiv), piperidine/DMF (1:5), 308C, 6 h. Boc=
tert-butyloxycarbonyl, TFA=trifluoroacetic acid, TMSOTf=trimethylsilyltriflate.
chloride to provide resin
6
(Scheme 3).
Starting
from
À
NH2 SPOrT resin 3, the resin-
bound model compound tripep-
À
tide VKF OH 9 was synthe-
sized by following a classical
Fmoc/tert-butyl strategy and ac-
tivation by HBTU (Scheme 4).
To evaluate the efficacy of
the solid-phase reactions, sam-
ples of resin 6 and 9 were treat-
ed in TFA/H2O (95:5) for 3 h to
provide alkyne–benzazepine 7
and tripeptide 10 in 61 and
38% yield, respectively (91%
purity for both, as estimated by
LC-MS at l=224 nm). In an at-
tempt to increase the yield of
the alkynes, the importance of
the time of cleavage and the
À
Scheme 4. Solid-phase synthesis of the model tripeptide compound using NH2 SPOrT resin 3. i) Piperidine/
DMF (1:5), RT, 2”20 min; ii) amino acid (4 equiv), HBTU (4 equiv), DIEA (12 equiv), NMP, RT, 2 h;
iii) TFA/H20 (95:5), RT, 3 h; iv) 11 (3 equiv), CuI (5 equiv), piperidine/DMF (1:5), 308C, 6 h. HBTU=2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
Table 1. Influence of the nature of the BAL resin and the time of cleav-
nature of the BAL resin linker were investigated (Table 1).
The more acid-sensitive 2-(3,5-dimethoxy-4-formylphenoxy)-
ethyl polystyrene (DFPE) resin gave better yields and
higher purities than the FMPB resin. Whereas the cleavage
step was complete after 16 hours in TFA/water (95:5) with
the FMPB resin (Table 1, entry 2), only 3 h was necessary
for the DFPE resin. Alkyne–benzazepine 7 and alkyne–tri-
peptide 10 were obtained in 95 and 57% yield (96 and 94%
purity), respectively (Table 1, entry 3).
It is of note that Lissamine Rhodamine B was found to be
highly stable under the conditions for acidic cleavage, thus
showing the suitability of this probe for the solid-phase syn-
thesis of fluorescent compounds. Alkyne–peptide 10 was ob-
tained in a lower yield than benzazepine 7. This result can
be ascribed to the loading of the Fmoc–Gly–DFPE resin 3.
Indeed, Fmoc quantification[11] gave a loading of 0.77 versus
age on alkynes preparation.
Entry Resin(s) Cleavage[a]
Yield[b] [%]/Purity[c] [%]
Alkynes
Lissamine derivatives[d]
10
time
[h]
7
8a,b
11a,b
1
2
3
4
FMPB
FMPB
DFPE
DFPE
3
16
3
61/91 38/91 23/82
89/86 50/98 51/85
95/96 57/94 84/89
90/95 56/92 78/88
10/77
26/82
50/91
49/90
16
[a] Performed in TFA/water (95:5). [b] Determined by weight of the
crude products based on the initial BAL resin loading. [c] Determined by
RP-HPLC analysis of the crude product at l=254 nm. [d] Obtained as a
mixture of ortho and para isomers.
0.92 mmolgÀ1 for the commercially available DFPE resin.
Despite monitoring by colorimetric tests, the reductive ami-
nation/acylation reactions were not complete (83% yield
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6249

D. Bonnet et al.
based on the loading of the DFPE resin). With resin-bound
alkynes 6 and 9 in hand, we investigated the solid-phase in-
corporation of the fluorescent probe through click chemis-
try.
detection: l=254 nm) and 1H NMR spectroscopic analy-
sis.[21]
Starting from recently described solid-phase click chemis-
try conditions,[16] azido 12 was treated with both alkyne-
bound resins 5 and 9 in the presence of an excess of CuI in
DMF/piperidine (4:1, v/v). Optimization of the reaction
demonstrated that the best conversion was obtained with
three equivalents of 12 after 6 h at 308C. It is of note that
the addition of ascorbic acid or tris(carboxyethyl)phosphine,
generally used to stabilize the CuI species, did not permit an
increase in the yield of the reaction. The influence of the
time of cleavage and the nature of the BAL linker (FMDP
versus DFPE) on both the yield and purity was also investi-
gated (Table 1).
As previously shown for alkynes 7 and 10, the best results
were obtained by treating the fluorescent compound bound
DFPE resin in TFA/H2O (95:5) for 3 h (Table 1, entry 3).
Under these conditions, the fluorescent benzazepine 8 was
obtained in 84% yield (89% purity) as a mixture of ortho
and para isomers 8a,b in a 1:3 ratio. Following a six-step
solid-phase process, the fluorescent tripeptide 11 was ob-
tained in 50% yield (91% purity) as a mixture of ortho and
para isomers 11a,b. The RP-HPLC profile of the crude mix-
ture highlights the efficacy of the process (Figure 1). The al-
kynes and Lissamine-(PEG)3-azido 12 were not detected by
LC-MS analysis of the crude product, thus showing both the
completion of the solid-phase 1,3-cycloaddition reaction and
the efficient removal of the starting material azido 12 from
the resin prior to the cleavage. Owing to the efficacy of the
solid-phase click process and the excellent purity of the
crude mixture, fluorescent compounds prepared according
to this method could be used directly for biological evalua-
tion without any additional purification.
Solid-phase click chemistry: The generation of [1,2,3]tria-
zoles by the 1,3-dipolar cycloaddition of alkynes to azides
proceeds at room temperature in the presence of CuI as a
catalyst. This transformation has been successfully used for
the fluorescent solution-phase labeling of virus capsid pro-
teins,[12] the cell surface of Escherichia coli,[13] and activity-
based protein profiling.[14] We used it for the labeling of the
muscarinic M1 antagonist pirenzepine for the FRET-based
binding assay.[15] It has been shown that the 1,4-disubstituted
1,2,3-triazoles can serve as transoid amide-bond mimics in
natural compounds without compromising biological activi-
ty.[16] The solid-phase version of click chemistry was recently
described for the synthesis of peptidomimetics,[17] multiple-
labeled carbohydrate oligonucleotides,[18] and functionalized
resins.[19] However, the incorporation of fluorescent probes
on resins by click chemistry has been much less studied. To
the best of our knowledge, only one example to date has re-
lated the surface functionalization of resins (Wang and Mer-
rifield) with dyes using a Huisgen [2+3] cycloaddition reac-
tion, performed at 708C under nitrogen.[20]
Our approach relies on the solid-phase incorporation of a
Lissamine Rhodamine B derivative by employing the cata-
lytic version of the Huisgen [2+3] cycloaddition. To achieve
our goal, Lissamine-(PEG)3-azido 12 was synthesized by
treating Lissamine Rhodamine B sulfonyl chloride, commer-
cially available as a mixture of ortho and para isomers, with
11-azido-3,6,9-trioxaundecan-1-amine in CH2Cl2/DMF (4:1)
in the presence of triethylamine and a catalytic amount of 4-
DMAP (Scheme 5). The PEG chain was selected to increase
the solubility of the Lissamine, and thus favor both the reac-
tion and the elimination of excess Lissamine from the solid
support. Compound 12 was isolated in 69% yield as a 1:3
mixture of ortho and para isomers, as determined by re-
verse-phase-HPLC (RP-HPLC; column: C18, eluent: water,
À
Application of the CO2H SPOrT resin to the synthesis of a
novel fluorescent V2R ligand: FRET-based assays have been
Figure 1. RP-HPLC chromatogram and positive ESI-MS spectrum of the
crude model compound tripeptide 11a,b. Chromatographic conditions:
C18 symmetry shield column (4.6”150 mm). Flow rate: 1 mLminÀ1, buf-
fer A: 0.1% aqueous TFA, buffer B: 0.1% TFA in CH3CN, gradient: 0–
100% B over 60 min, detection: l=220 nm.
Scheme 5. Preparation of lissamine-(PEG)3-azido 12. i) 11-Azido-3,6,9-tri-
oxaundecan-1-amine (1 equiv), Et3N (3 equiv), 4-DMAP (0.1 equiv),
CH2Cl2/DMF (1:1), RT, overnight (69%).
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Solid-Phase Organic Tagging Resins for Labeling Biomolecules
FULL PAPER
extensively used by us to study G protein-coupled receptors
(GPCRs).[2a–c] In a program aimed at accelerating the dis-
covery of original ligands of vasopressin and oxytocin recep-
tors, we were interested in preparing a fluorescent probe
with good binding affinity for V2R.
Click chemistry in presence of Lissamine-(PEG)3-azido
derivative 12 followed by treatment of the resin with TFA
gave access to Lissamine benzazepine 17 as a mixture of
ortho and para isomers 17a,b (1:1.6, as shown by LC-MS
analysis). RP-HPLC purification on a C18 column enabled
isolation of both isomers in 16 and 28% yield for the ortho
and para isomers, respectively (nine steps). The retention
times of both isomers indicate that the para isomer is more
lipophilic than the ortho isomer (Table 2). The absorption
spectra and molar extinction coefficients e were recorded in
a 50 mm 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid (HEPES) buffer containing a final concentration of
0.5% dimethyl sulfoxide (DMSO; Table 2). Both isomers
have similar maximum absorptions (l=572 and 574 nm for
the ortho and para isomers, respectively) and very close
molar extinction coefficients (e=65000 and 69000mÀ1 cmÀ1
for the ortho and para isomers, respectively).
Starting from 3-methyl-4’-
[(2,3,4,5-tetrahydro-1H-1-yl)car-
bonyl]benzanilide (13), which
was reported to bind to the V2
receptor with a nanomolar af-
finity (Ki=7.2 nm on rabbit
kidney
membranes),[22]
the
solid-phase preparation of the
corresponding fluorescent com-
pound was investigated with
À
CO2H SPOrT resin 2 (Scheme 6). After the incorporation
of the aminobenzazepine scaffold on the DFPE solid sup-
port (i.e., resin 4), the endocyclic secondary amine group
Affinities for the V2 receptor:
The affinity of compound 13
and isomers 17a,b for the
human V2 receptor were deter-
mined by competition experi-
ments
(AVP =arginine vasopressin),
as described
previously[24]
against
[3H]-AVP
(Table 2). The affinity of 13 for
the human receptor was found
to be close to that determined
for rabbit kidney membranes
(Ki =2.68 versus 7.2 nm). The
À
Scheme 6. Solid-phase synthesis of fluorescent benzazepine 17a,b using the CO2H SPOrT resin. i) TMSOTf
(3 equiv), Et3N (1.5 equiv), CH2Cl2, RT, 15 min; ii) 4-nitrobenzoyl chloride (5 equiv), pyridine (10 equiv), 4-
DMAP (0.1 equiv), CH2Cl2, RT, overnight; iii) SnCl2·2H2O, DMF, RT, overnight; iv) ortho-toluyl chloride
(5 equiv), DIEA (10 equiv), 4-DMAP (1 equiv), CH2Cl2, RT, overnight; v) click chemistry; vi) TFA/H20 (95:5),
RT, 3 h.
ortho isomer 17a exhibits
a
Ki value that is threefold higher
than the para isomer 17b (187
was deprotected and acylated with para-nitrobenzoyl chlo-
ride in the presence of pyridine and 4-DMAP. After the re-
action mixture was stirred overnight, a negative result to the
colorimetric test with Chloranil was indicative of a quantita-
tive reaction (i.e., resin 14). Reduction of the nitro group
was then investigated by using tin chloride in DMF at
608C.[23]
Table 2. Characterization and properties of fluorescent compounds
17a,b.
[b]
Entry
Compound
tR
A
lmax
elmax
[103 mÀ1 cmÀ1
Ki
[min]
[nm]
]
[nm][c]
1
2
3
4
[3H]AVP
13
–
–
3.91
4.03
–
–
572
574
–
–
65
69
1.36Æ0.45
2.68Æ0.38
187.7Æ24.7
54.3Æ6.6
17a[a]
17b[a]
Under these conditions, the expected resin-bound amine
15 was obtained together with an unidentified by-product.
Further optimization showed that the side reaction could be
avoided by performing the reduction reaction at room tem-
perature. N-Acylation of the weakly nucleophilic amine
with the ortho-toluyl acyl moiety was also found to be a crit-
ical step in the synthesis. Various coupling agents, such as
PyBOP, HATU, PyBrOP, and tetramethylfluoroformamidi-
nium hexafluorophosphate (TFFH) in presence of ortho-
toluyl acid failed to provide a complete reaction. Ultimately,
the use of the corresponding acyl chloride with 4-DMAP,
the Hünig base, in CH2Cl2 was found to provide the best
conditions under which to access the benzazepine-bound
resin 17.
[a] Absorption parameters measured in a 50 nm HEPES, pH 7.5 buffer
containing a final concentration of 0.5% DMSO (v/v). [b] Retention
times were obtained on a Chromolith SpeedROD column (50”4.6 mm,
C18) under the experimental conditions described in the Experimental
Section. [c] The inhibition constants Ki of fluorescent compounds of the
human vasopressin V2 were determined on CHO cell membranes by
competition binding assays (displacement of radioactive [3H]AVP); the
results are expressed as meanÆSEM of three separate experiments per-
formed in triplicate (SEM=standard error of the mean).
versus 54 nm). The presence of the PEG spacer and the Liss-
amine moiety disturbs binding to the V2 receptor. In addi-
tion, the position of the link between the fluorophore and
the benzazepine core (ortho versus para isomers) affects the
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6251

D. Bonnet et al.
100% buffer B over 5 min; detection: l=220/254 nm. The retention
times tR from analytical RP-HPLC are reported in minutes.
affinity of the ligand for the receptor. Nevertheless, the
ligand derivatized with Lissamine at the para position still
displays an affinity that is compatible with fluorescence-
based assays by using either polarization or FRET tech-
niques.[25] It is of note that this molecule represents the first
fluorescent non-peptidic ligand for V2R described to date.
N-[10-[4 (and 2)-{[(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethyl)amino]-
sulfonyl}-2 (and 4)-(methylsulfonyl)phenyl]-7-(diethylamino)anthracen-2-
AHCTRE(GNU 9H)-ylidene]-N-ethylethanaminium (12): 11-Azido-3,6,9-trioxaundecan-
1-amine (200 mL, 1 mmol) was dissolved in CH2Cl2/DMF (4:1) and Et3N
(421 mL, 3 mmol) in the presence of 4-DMAP (12.2 mg, 0.1 mmol) in an
argon atmosphere. The resulting mixture was stirred at 08C for 15 min.
Lissamine Rhodamine B sulfonyl chloride (577 mg, 1 mmol) was added
in portions over 20 min. The solution was allowed to warm up to room
temperature and stirred overnight. Crude reaction mixture was concen-
trated under reduced pressure. Compound 12 was isolated by flash chro-
matography with a step gradient from 5 to 10% MeOH in CH2Cl2 and
obtained as a red-dark solid (526 mg, 69%, ortho and para isomers, 1:1.8
ratio, respectively). Rf=0.32 (para isomer) and 0.28 (ortho isomer)
(CH2Cl2/MeOH 9:1); 1H NMR (CDCl3/CD3OD 9:1, 300 MHz): d=8.56
(d, J=1.8 Hz, 0.63H), 8.43 (d, J=1.8 Hz, 0.37H), 8.10 (dd, J=7.8,
1.8 Hz, 0.37H), 7.88 (dd, J=7.8, 1.8 Hz, 0.63H), 7.15 (dd, J=7.8, 2.8 Hz,
0.37H), 7.01 (dd, J=9.5, 3.4 Hz, 0.37H), 7.02–6.98 (m, 2H), 6.79–6.68 (m,
2H), 6.63–6.59 (m, 2H), 3.55–3.19 (m, 20H), 3.08 (t, J=5.3 Hz, 1.26H),
2.91 (t, J=5.3 Hz, 0.74H) 1.21–1.13 ppm (m, 12H); 13C NMR (CDCl3/
CD3OD 9:1, 75 MHz): d=157.6, 157.5, 157.2, 155.4, 155.3, 148.4, 146.5,
142.0, 133.4, 132.6, 132.0, 131.1, 130.5, 130.0, 129.5, 127.3, 126.6,
125.7 ppm; RP-HPLC purity: >95%; HRMS: calcd for C35H47N6O9S2
759.2840; found: 759.2850.
Conclusion
Fluorescent peptides or small-molecule-based ligands have
proven to be extremely powerful tools with which to investi-
gate receptor function and signal-transduction processes, as
well as for applications in the field of screening for novel
therapeutic compounds. Herein, we have described two
novel SPOrT resins to facilitate the synthesis of such mole-
cules. The method is rapid and straightforward and enables
the chemoselective and quantitative labeling of both pep-
tides and small organic compounds by using click chemistry.
The final products were obtained in good yield with high
purity. This strategy was successfully applied to the prepara-
tion of the first non-peptidic fluorescent compound with
nanomolar affinity for the human vasopressin V2 receptor.
This molecule will find application in structural studies of
receptors and the discovery of original ligands by using fluo-
rescence polarization techniques. In addition, SPOrT resins
are particularly appealing for the parallel synthesis of fluo-
rescent chemical libraries. Such a program is in progress to
identify GPCR orphan ligands using FRET-based assays,
and the results will be reported in due course.
Standard washing protocol: Resin washings were performed with DMF
(3”1 min), MeOH (3”1 min), and CH2Cl2 (3”1 min).
Synthesis of resin-bound alkyne 1: Propargylamine (158 mL, 2.3 mmol) in
DMF/MeOH/AcOH (2.5 mL; 80:19:1, v/v/v) and NaBH3CN (144.5 mg,
2.3 mmol) were added to DFPE resin (210 mg, 0.92 gmolÀ1, 0.23 mmol).
The reaction mixture was heated at 608C overnight, allowed to cool to
room temperature, and filtered. The resin was washed following the stan-
dard washing procedure and dried in vacuo. Complete loading of propar-
gylamine onto the DFPE resin was verified by a negative result of a test
with DNPH.[7]
À
Synthesis of CO2H SPOrT resin 2: A solution of succinic anhydride
(575.4 mg, 57.5 mmol) in DMF (1.55 mL), pyridine (74 mL, 0.92 mmol),
and 4-DMAP (28.1 mg, 0.23 mmol) were added to the resin-bound
alkyne 1 (0.23 mmol) preswollen in DMF. The resulting mixture was
shaken overnight at room temperature. The resin was filtered, washed,
and dried in vacuo to give resin 2. Complete reaction was verified by a
negative result of a test with Chloranil, and the presence of CO2H group
by the positive result to a test with Malachite green.
Experimental Section
General: 4-(4-Formyl-3-methoxyphenoxy)butyryl aminomethylated poly-
styrene resin (FMPB AM; 100—200 mesh, 0.74 mmolgÀ1) and 2-(3,5-di-
methoxy-4-formylphenoxy)ethyl polystyrene resin (DFPE; 100—
200 mesh, 0.92 mmolgÀ1; BAL-type resin) were purchased from Novabio-
chem; 11-azido-3,6,9-trioxaundecan-1-amine was obtained from Fluka;
(Æ)-5-amino-1-tert-butoxycarbonyl-2,3,4,5-tetrahydro-1H-benzo[b]aze-
pine (1-benzazepine) was obtained as previously described.[10] Solid-
phase reactions conducted at room temperature were performed in poly-
propylene tubes equipped with polyethylene frits and polypropylene caps
using an orbital-agitator shaking device. Solid-phase reactions at 608C
were conducted in sealed glassware tubes using the Chemflex rotating
oven from Robbins Scientific as the shaking device.
1H NMR spectra were recorded at 200, 300, and 500 MHz on a Bruker
Advance spectrometer. Chemical shifts are reported in parts per million
(ppm), and the coupling constants J are reported in hertz (Hz). LC-MS
spectra were obtained on a ZQ (Z quadripole) Waters/Micromass spec-
trometer equipped with an X-Terra C18 column (4.6”50 mm, 3.5 mm)
using the electrospray ionization mode (ESI). HRMS spectra were ob-
tained on a MicroTof mass spectrometer from Bruker using the ESI
mode and a time-of-flight analyzer (TOF). Thin-layer chromatography
was performed on silica gel 60 F254 plates from Merck. Flash chromatog-
raphy was performed on silica gel 60 (230–400 Mesh ASTM) from
Merck. Analytical HPLC analyses were performed on a Chromolith
SpeedROD column (50”4.6 mm, C18) from Merck under the following
conditions: flow rate: 7 mLminÀ1; buffer A: 0.1% aqueous TFA, buf-
fer B: 0.1% TFA in CH3CN; gradient: 0% buffer B for 1 min then 0–
Synthesis of Fmoc-NH-SPOrT resin 3: Fmoc-Gly-OH (219 mg,
0.73 mmol) dissolved in NMP (1:1, 1.74 mL), PyBOP (383 mg,
0.73 mmol), and the Hünig base (385 mL, 2.2 mmol) were added to the
resin-bound alkyne 1 (0.184 mmol) preswollen in NMP (1:1, v/v). After
the reaction mixture had been stirred overnight at room temperature, a
negative result to a test with chloranil was indicative of the completion
of the reaction. The resin was washed and dried in vacuo.
À
Procedure for the loading of the 1-benzazepine scaffold onto CO2H
SPOrT resin 2: A solution of 1-benzazepine (144.8 mg, 0.552 mmol) in
DMF (1 mL), PyBOP (287.2 mg, 0.552 mmol), and the Hünig base
(128.2 mL, 0.736 mmol) were added to DFPE resin (0.184 mmol). After
the reaction mixture was shaken overnight at room temperature, the
resin was washed. A negative result for a test with Malachite showed the
completion of the coupling. Resin 4 was dried in vacuo.
General procedure for selective 1-benzazepine Boc deprotection:
A
freshly prepared solution of TMSOTf (109 mL, 0.552 mmol) and Et3N
(38.8 mL, 0.276 mmol) in anhydrous CH2Cl2 (4 mL) were added to resin-
bound N-Boc benzazepine
4 (0.184 mmol) preswollen in anhydrous
CH2Cl2. The reaction mixture was shaken at room temperature for
15 min, filtered, washed (CH2Cl2 (3”), MeOH (3”), and CH2Cl2), and
dried in vacuo to give resin 5.
Synthesis of resin-bound benzazepine 6: A solution of benzoyl chloride
(39.7 mL, 342.1 mmol) in CH2Cl2 (924 mL), 4-DMAP (0.5m in CH2Cl2,
7 mL), and pyridine (55 mL, 684 mmol) were added to resin-bound benza-
6252
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 6247 – 6254

Solid-Phase Organic Tagging Resins for Labeling Biomolecules
FULL PAPER
zepine 5 (68.4 mmol) preswollen in CH2Cl2. The reaction mixture was
shaken for one night at room temperature, washed, and dried in vacuo.
(bt, J=4.5 Hz, 4H), 3.86 (bs, 8H), 3.74 (bt, J=3.5 Hz, 2H), 3.62- 3.42
(m, 8H), 3.21 (bs, 2H), 3.11 (t, J=5.5 Hz, 2H), 2.92 (bt, J=11.5 Hz,
1H), 2.53 (bd, J=6.0 Hz, 2H), 2.46 (bd, J=6.0 Hz, 2H), 1.96–1.84 (m,
2H), 1.60–1.44 (m, 2H) 1.17 ppm (m, 12H); 13C NMR (CDCl3,
125 MHz): d=172.8, 172.1, 170.3, 157.6, 156.8, 155.3, 146.0, 142.0, 140.0,
138.4, 135.2, 133.5, 132.5, 130.1, 129.5, 128.4, 128.0, 127.9, 127.5, 127.3,
127.1, 126.4, 123.9, 123.8, 123.7, 113.8, 113.3, 95.4, 70.1, 69.9, 69.4, 68.9,
50.2, 50.1, 50.0, 46.4, 45.6, 42.7, 34.4, 31.1, 31.0, 30.9, 27.9, 24.9, 12.3 ppm;
RP-HPLC purity: >95%; HRMS: calcd for C59H72N9O12S2 1162.4736;
found: 1162.4718.
General procedure for final deprotection and cleavage steps: The resin
was treated in the presence of TFA/H2O (95:5 v/v) for 3 h at room tem-
perature, filtered, washed with CH2Cl2 (2”) and MeOH (2”). The com-
bined filtrates were mixed, and evaporated to dryness in vacuo. The
crude residue was dissolved in acetonitrile/H2O (1:1, v/v) and lyophilized.
Synthesis of N-(1-benzoyl-2,3,4,5-tetrahydro-1H-benzo[b]azepin-5-yl)-N’-
prop-2-ynyl-succinamide (7): Resin 6 (22.0 mmol) was treated with TFA/
H2O (95:5 v/v). Compound 7 was isolated by RP-HPLC on a C18 sym-
Synthesis of fluorescent peptide 11a,b: Resin 9 (12.1 mmol) was submit-
ted to the click chemistry procedure. Following deprotection and cleav-
age in TFA/H20 (95:5), triazoles 11a (ortho isomer) and 11b (para
isomer) were isolated by RP-HPLC on a C18 symmetry shield column as
a red solid. 11a (1.7 mg, 10%): tR =3.39 min; RP-HPLC purity: >95%.
11b (5.0 mg, 28%): RP-HPLC purity: >95%; HRMS: calcd for
C60H85N12O132S2 1245.5795; found: 1245.5817.
metry shield column as
a white solid (6.3 mg, 71%). tR =3.03 min;
1H NMR (CDCl3, 200 MHz): d=7.35 (dd, J=7.4, 1.8 Hz, 2H), 7.20–7.08
(m, 5H), 6.89 (td, J=7.4, 1.8 Hz, 1H), 6.57 (d, J=7.4 Hz, 1H), 5.33 (bdd,
J=11.0, 3.4 Hz, 1H), 4.64–4.46 (m, 1H), 3.98 (bt, J=2.2 Hz, 2H), 3.0–
2.96 (m, 1H), 2.70–2.44 (m, 5H), 2.18 (t, J=2.6 Hz, 1H), 2.10–1.88 (m,
1H), 1.76–1.56 ppm (m, 2H); 13C NMR (CDCl3, 75 MHz): d=172.6,
172.2, 170.4, 140.7, 138.5, 135.7, 129.9, 128.9, 128.7, 128.4, 127.9, 127.7,
127.6, 124.1, 71.5, 66.0, 50.7, 49.6, 49.2, 48.7, 46.7, 31.7, 29.3 ppm; RP-
HPLC purity: >95%; HRMS: calcd for C24H26N3O3 404.1969; found:
404.1970.
Synthesis of fluorescent V2R ligand 17a,b: 4-Nitrobenzoyl chloride
(85 mg, 460 mmol) in CH2Cl2 (1.25 mL), pyridine (74 mL, 920 mmol), and
4-DMAP (0.5m in CH2Cl2, 9.2 mL) were added to resin 5 (92 mmol). The
reaction mixture was shaken for one night at room temperature. The
completion of the reaction was verified by a negative result to a test with
chloranil. Following the washing procedure, resin 14 was preswollen in
DMF. A solution of SnCl2·2H2O (623 mg, 2.76 mmol) in DMF (1.5 mL)
was added to the resin. Following shaking for one night at room tempera-
ture, the resin was washed with DMF/AcOH (3:1, v/v; 3”), DMF/Et3N
(9:1, v/v; 3”), DMF (3”), CH2Cl2 (3”), and MeOH (3”) and dried with
Et2O. Resin 15 was preswollen in CH2Cl2. O-Toluyl chloride (60 mL) in
CH2Cl2 (1.35 mL) was added to the resin in presence of the Hünig base
(160 mL, 920 mmol) and DMAP (11.2 mg, 92 mmol). The reaction mixture
was shaken for one night at room temperature, after which the resin was
filtered and washed. To ensure the completion of the reaction, the acyla-
tion step was repeated over 6 h, the resin was washed and dried in vacuo.
The dried resin 16 (46 mmol) was submitted to the click chemistry proto-
col with azido 12 (106 mg, 139 mmol). A subsequent deprotection and
cleavage in TFA/H2O (95:5) gave compound 17a,b. Both ortho 17a and
para isomers 17b were isolated by RP-HPLC on a C18 symmetry shield
column. 17a (9.3 mg, 16%): tR =3.92 min; 1H NMR (CDCl3/CD3OD 9:1,
300 MHz): d=8.53 (d, J=1.5 Hz, 1H), 8.12 (dd, J=8.1, 1.5 Hz, 1H), 7.75
(bs, 1H), 7.39 (d, J=8.1 Hz, 2H), 7.32–6.96 (m, 11H), 6.83 (bt, J=
7.5 Hz, 1H), 6.76 (dd, J=9.3 et 2.4 Hz, 2H), 6.64 (bs, 2H), 6.50 (d, J=
7.5 Hz, 1H), 5.25 (bdd, J=11.0, 3.0 Hz, 1H), 4.46 (bs, 1H), 4.36 (bs,
2H), 4.29 (bt, J=5.0 Hz, 2H), 3.96–3.80 (m, 8H), 3.69 (t, J=4.8 Hz,
2H), 3.60–3.36 (m, 9H), 3.23 (bs, 3H), 2.95 (t, J=5,1 Hz, 1H), 2.63–2.48
(bs, 4H), 2.3 (s, 3H), 2.0–1.80 (bs, 2H), 1.40–1.60 (bs, 2H), 1.25 ppm (m,
12H); 13C NMR (CDCl3/CD3OD 9:1, 75 MHz): d=173.0, 172.6, 170,0,
169.4, 157.7, 156.1, 155.6, 148.5, 141.0, 140.6, 140.1, 138.6, 136.4, 135.9,
132.1, 131.5, 130.9, 130.7, 130.0, 129.7, 129.5, 128.3, 127.6, 127.5, 126.8,
125.8, 125.6, 124.2, 123.9, 118.8, 114.0, 95.9, 70.3, 70.1, 70.0, 69.5, 69.1,
50.5, 50.1, 48.0, 47.9, 42.9, 34.7, 31.4, 31.00, 25.2, 19.4, 12.4 ppm; RP-
HPLC purity: >95%; 17b (17 mg, 28%): tR =4.04 min; 1H NMR
(CDCl3/CD3OD 9:1, 400 MHz): d=8.63 (bs, 1H), 7.91 (bd, J=8.1 Hz,
1H), 7.74 (bs, 1H), 7.38 (bs, 2H), 7.32–6.96 (m, 11H), 6.83 (b6t, J=
7.5 Hz, 1H), 6.71 (bd, J=9.3, 2H), 6.60 (bs, 2H), 6.50 (d, J=7.5 Hz,
1H), 5.14 (bd, J=11.0 Hz, 1H), 4.48–4.24 (m, 5H), 3.84–3.68 (m, 10H),
3.58–3.36 (m, 9H), 3.23 (bs, 1H), 3.01 (bs, 1H), 2.91 (bt, J=5,1 Hz, 1H),
2.89 (bs, 1H), 2.58–2.38 (m, 4H), 2.3 (s, 3H), 1.88–1.80 (bs, 2H), 1.58–
1.44 (bs, 2H), 1.18 ppm (m, 12H); 13C NMR (CDCl3/CD3OD 9:1,
100 MHz): d=176.4, 175.9, 173.4, 172.7, 172.13, 161.2, 160.7, 158.9, 150.1,
145.8, 144.0, 143.5, 142.0, 139.8, 139.3, 137.1, 136.2, 134.2, 134.1, 133.7,
132.9, 131.6, 131.0, 130.9, 130.2, 130.1, 159.0, 127.6, 12.2, 117.5, 117.0,
99.1, 73.7, 73.5, 73.0, 72.5, 53.9, 53.6, 50.0, 49.6, 49.2, 46.3, 38.1, 34.9, 34.3,
28.6, 22.7, 15.7, 11.8 ppm; RP-HPLC purity: >95%; HRMS calcd for
C67H78N10O13S2 1295.5264; found: 1295.5269.
Synthesis of resin-bound protected peptide 9: The model peptide com-
pound was elaborated on the Fmoc-NH-SPOrT resin 3 (184 mmol) by
using a classical Fmoc/tert-butyl strategy. Removal of the Fmoc group
was performed with DMF/piperidine (4:1, v/v) for 20 min. Fmoc amino
acids (Fmoc-l-Phe-OH, Fmoc-l-LysCAHTRE(UNG Boc)-OH, and Boc-l-Val-OH;
730 mmol) were coupled by the addition of HBTU (279 mg, 730 mmol),
N,N-diisopropylethylamine (DIEA; 385 mL, 2.2 mmol), and NMP
(1.74 mL) for 2 h at room temperature. Completion of the reaction was
monitored by using trinitrobenzenesulfonic acid (TNBS) as a test.[26] The
coupling of the amino acids and deprotection of the Fmoc group were
followed by washing with DMF (3”) and CH2Cl2 (3”).
Synthesis of H-VKFG-NHCH2-CH 10: Resin 9 (23.7 mmol) was treated
with TFA/H2O (95:5 v/v) for 3 h at room temperature. Following a
freeze/drying step, peptide 10 was isolated by RP-HPLC on a C18 sym-
metry shield column as a white powder (6.4 mg, 38%). tR =2.43 min;
HRMS: calcd for C25H39N6O4 487.3027; found: 487.3041; RP-HPLC
purity: >95%.
General procedure for the solid-phase click chemistry: Resin-bound
alkyne (46 mmol) was preswollen in DMF/piperidine (4:1, v/v). In two
separate vials, CuI (44.3 mg, 230 mmol) and azido 12 (105.8 mg,
139 mmol) were dissolved in DMF/piperidine (825 mL; 4:1, v/v) using an
ultrasonic bath. Both solutions were mixed altogether and placed for a
further 5 min in an ultrasonic bath. The resulting mixture (1.65 mL) was
added to the preswollen resin-bound alkyne. After the reaction mixture
had been shaken for 6 h at 308C, the resin was washed successively with
DMF/piperidine (4:1, v/v; 3”), DMF (3”), CH2Cl2 (3”), CH2Cl2/MeOH
(3”), MeOH (3”), and CH2Cl2 (3”) and dried in vacuo.
Synthesis of fluorescent 1-benzazepine 8a,b: Resin 6 (12.1 mmol) was
submitted to click chemistry following the procedure described above.
Following deprotection and cleavage in TFA/H20 (95:5), triazoles 8a
(ortho isomer) and 8b (para isomer) were isolated by RP-HPLC on a
C18 symmetry shield column as a red solid. 8a (1.9 mg, 13%): tR =
1.90 min; 1H NMR (CDCl3/CD3OD 9:1, 500 MHz): d=8.57 (bs, 1H),
8.17 (d, J=8.1 Hz, 1H), 7.78 (bs, 1H), 7.32–7.04 (m, 10H), 6.82 (bt, J=
10.0 Hz, 3H), 6.67 (bs, 2H), 6.49 (d, J=7.4 Hz, 1H), 5.25 (bdd, J=11.0,
3.4 Hz, 1H), 4.50 (bs, 1H), 4.41 (bs, 2H), 4.37 (bt, J=5.0 Hz, 2H), 3.96–
3.72 (m, 8H), 3.60–3.44 (m, 8H), 3.41 (t, J=5.5 Hz, 2H), 3.27 (bs, 2H),
3.0 (t, J=5.5 Hz, 1H), 2.63 (bt, J=7.0 Hz, 2H), 2.55 (bt, J=7.0 Hz, 2H),
2.08–1.96 (bs, 2H), 1.68–56 (bs, 2H), 1.25 ppm (m, 12H); 13C NMR
(CDCl3/CD3OD 9:1, 75 MHz): d=172.9, 172.3, 170.4, 157.6, 156.1, 155.5,
148.4, 140.9, 138.5, 135.4, 132.0, 131.4, 131.3, 130.7, 129.6, 129.5, 128.2,
128.0, 127.6, 127.5, 127.2, 125.7, 124.1, 113.9, 95.8, 70.2, 70.0, 69.9, 69.4,
69.0, 50.2, 50.0, 46.5, 45.8, 42.8, 34.6, 31.3, 25.1, 12.2 ppm; RP-HPLC
purity: >95%; 8b (4.8 mg, 33%): tR =2.017; 1H NMR (CDCl3/CD3OD
9:1, 500 MHz): d=8.60 (bs, 1H), 7.92 (dd, J=8.1, 1.1 Hz, 1H), 7.73 (bs,
1H), 7.22 (bd, J=7.5 Hz, 2H), 7.17 (d, J=7.5 Hz, 2H), 7.16–6.96 (m,
6H), 6.77 (t, J=7.5 Hz, 1H), 6.71 (td, J=7.5, 2.0 Hz, 2H), 6.61 (s, 2H),
6.44 (d, 7.50 Hz, 1H), 5.16 (dd, J=11.0, 3.4 Hz, 1H), 4.43 (bs, 1H), 4.35
Radioligand binding assays: Binding assays were performed at 308C
using [3H]AVP as the radioligand and Chinese hamster ovary (CHO)
cell-membrane proteins (5 mg). Briefly, membranes prepared from CHO
cells stably expressing the human V2 AVP receptor were incubated in
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6253

D. Bonnet et al.
50 mm 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride (Tris—
HCl) pH 7.4, 5 mm MgCl2, 1 mgmLÀ1 bovine serum albumin (BSA; bind-
ing buffer), and radiolabeled, displacing ligands for 30 min. The affinitiy
Kd of [3H]AVP to the V2 human receptor has already been described ear-
lier, and Kd =(1.36Æ0.45) nm.[24] Affinities Ki of ligands 13, 17a, and 17b
were determined by competition experiments using [3H]AVP (1–2 nm)
and varying the concentrations of the unlabeled ligands from 100 pm to
100 mm. The nonspecific binding was determined by adding unlabeled
AVP (10 mm). Bound and free radioactive ligands were separated by fil-
tration over Whatman GF/C filters presoaked in a 10 mgmLÀ1 BSA solu-
tion for 3–4 h. The ligand-binding data were analyzed by nonlinear least-
squares regression using the computer program Ligand. All assays were
performed in triplicate on at least three separate batches of cell mem-
branes.
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Acknowledgements
This study was supported by the Centre National de la Recherche Scien-
tifique, the UniversitØ Louis Pasteur, the European Communityꢁs Sixth
Framework Program (grant LSHB-CT-2003–503337), the Institut Nation-
al de la SantØ et de la Recherche MØdicale, and the UniversitØ Montpel-
lier I and II. We thank Pascale Buisine, Patrick Werhung (IFR85) for the
ES-MS analysis, and Cyril Antheaume (IFR85) for NMR spectroscopic
experiments.
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Received: February 13, 2008
Revised: March 28, 2008
Published online: June 2, 2008
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