Template Assembled Cyclopeptides as Multimeric System for Integrin Targeting and Endocytosis

Article abstract of DOI:10.1021/ja049926n

The α_vβ₃ integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. c[-RGDfV-] peptide represents a selective α_vβ₃integrin ligand that has been extensively used for research, t

Full text of DOI:10.1021/ja049926n

Published on Web 04/10/2004
Template Assembled Cyclopeptides as Multimeric System for
Integrin Targeting and Endocytosis
Didier Boturyn,† Jean-Luc Coll,‡ Elisabeth Garanger,†,‡ Marie-Christine Favrot,‡ and
Pascal Dumy*,†
Contribution from the LEDSS, UMR CNRS 5616 and ICMG, FR-2607,
UniVersite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France, and GRCP,
INSERM U578, IFR-73, Domaine de la Merci, 38706 La Tronche, France
Received January 6, 2004; E-mail: Pascal.Dumy@ujf-grenoble.fr
Abstract: The RVâ3 integrin receptor plays an important role in human metastasis and tumor-induced
angiogenesis. c[-RGDfV-] peptide represents a selective RVâ3 integrin ligand that has been extensively
used for research, therapy, and diagnosis of neoangiogenesis. We report here the modular synthesis and
biological characterization of template assembled cyclopeptides as a multimeric system for targeting and
endocytosis of cells expressing RVâ3 integrin. c[-RGDfK-] was cleanly assembled in a multivalent mode by
chemoselective oxime bond formation to a cyclodecapeptides template labeled by different reporter groups.
Binding propensity to the RVâ3 receptor and the associated good uptake property displayed by the multivalent
molecules demonstrated the interest in the RAFT molecule to design new multimeric system with hitherto
unreported properties. These compounds offer an interesting perspective for the reevaluation of integrins
as angiogenesis regulators (Hynes, R. O. Nature Med. 2003, 9, 918-921) as well as for the design of
more sophisticated systems such as molecular conjugate vectors.
Introduction
as selective RVâ3 and RVâ5 integrin ligands using phage display
peptide libraries.6 These highly selective RGD-containing pep-
Integrins are heterodimeric transmembrane cell surface recep-
tors that play a central role in cell-cell and cell-extracellular
matrix (ECM) adhesion processes.1 The RVâ3-RVâ5 integrin
subclass has received special attention as it is expressed in
various cell types such as endothelial cells, platelets, osteoclasts,
melanoma, and smooth muscle cells. They have a pivotal
involvement in pathologies as diverse as osteoporosis, arthritis,
retinopathy, tumor-induced angiogenesis, and metastasis forma-
tion.2 Cells expressing integrins RVâ3-RVâ5 interact to the ECM
bearing a wide variety of ligands such as fibronectin and
vitronectin mainly through the recognition of the ubiquitous triad
sequence RGD (Arg-Gly-Asp) that has served as basis for the
development of high and selective integrin-peptide ligands.
Rational screening of RGD-containing cyclopeptides has led
Kessler’s group to the discovery of the highly active first-
generation peptide c[-RGDfV-] 1 (Figure 1)3 whose N-alkylated
analogue c[-RGDf(NMe)V-]4 is currently investigated in clinical
phase II as an angiogenesis inhibitor (Cilengitide, code EMD
12974 Merck KGaA).5 Interestingly, other conformationally
constrained RGD-containing peptides have been selected in vivo
tides represent key compounds for targeting and tracing RVâ3-
expressing tumor cells as well as endothelial cells during
neoangiogenesis in the fields of research, therapy, and diagnosis
as underlined by the abundant corresponding literature.7-10
Very recently, multivalent RVâ3 integrin ligands with an
improved affinity have been developed by scaffolding multiple
copies of an RGD-containing peptide to a polymer,11 an artificial
membrane,12 as well as a protein13 or a peptide.14 Because the
natural functional mode of integrins interaction involves mul-
tivalent interactions, this approach could provide not only more
effective binding molecules but also systems that could improve
the cell targeting and promote cellular uptake through the
(5) National Cancer Institute clinical trials database online. See http://
cancertrials.nci.nih.gov.
(6) Pasqualini, R.; Koivunen, E.; Ruoslahti, E. Nat. Biotechnol. 1997, 15, 542-
546.
(7) Lode, H. N.; Moehler, T.; Xiang, R.; Jonczyk, A.; Gillies, S. D.; Cheresh,
D. A.; Reisfeld R. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1591-1596.
(8) (a) Hood, J. D.; Bednarski, M.; Frausto, R.; Guccione, S.; Reisfeld, R. A.;
Xiang, R.; Cheresh, D. A. Science 2002, 296, 2404-2407. (b) Colin, M.;
Maurice, M.; Trugnan, G.; Kornprobst, M.; Harbottle, R. P.; Knight, A.;
Cooper, R. G.; Miller, A. D.; Capeau, J.; Coutelle, C.; Brahimi-Horn, M.
C. Gene Ther. 2000, 7, 139-152.
† Universite´ Joseph Fourier.
‡ Domaine de la Merci.
(9) (a) Arap, W.; Pasqualine, R.; Ruoslahti, E. Science 1998, 279, 377-380.
(b) De Groot, F. M. H.; Broxterman, H. J.; Adams, H. P. H. M.; Van Vliet,
A.; Tesser, G. I.; Elderkamp, Y. W.; Schraa, A. J.; Kok, R. J.; Molema,
G.; Pinedo, H. M.; Scheeren, H. W. Mol. Cancer Ther. 2002, 1, 901-911.
(10) (a) DeNardo, S. J.; Burke, P. A.; Leigh, B. R.; O’Donnell, R. T.; Miers, L.
A.; Kroger, L. A.; Goodman, S. L.; Matzku, S.; Jonczyk, A.; Lamborn, K.
R.; DeNardo, G. L. Cancer Biother. Radiopharm. 2000, 15, 71-79. (b)
Bitan, G.; Scheibler, L.; Greenberg, Z.; Rosenblatt, M.; Chorev, M.
Biochemistry 1999, 38, 3414-3420.
(1) Hynes, R. D. Cell 1992, 69, 11-25.
(2) (a) Brooks, P. C.; Clark, R. A. F.; Cheresh, D. A. Science 1994, 264, 569-
571. (b) Giannis, A.; Ru¨bsam, F. Angew. Chem., Int. Ed. Engl. 1997, 36,
588-590. (c) For a review, see: Haubner, R.; Finsinger, D.; Kessler, H.
Angew. Chem., Int. Ed. Engl. 1997, 36, 1375-1389.
(3) Haubner, R.; Gratias, R.; Diefenbach, B.; Goodman, S. L.; Jonczyk, A.;
Kessler, H. J. Am. Chem. Soc. 1996, 118, 7461-7472.
(4) Dechantsreiter, M. A.; Planker, E.; Matha¨, B.; Lohof, E.; Ho¨lzemann, G.;
Jonczyk, A.; Goodman, S. L.; Kessler, H. J. Med. Chem. 1999, 42, 3033-
3040.
(11) Maynard, H. D.; Okada, S. Y.; Grubbs, R. H. J. Am. Chem. Soc. 2001,
123, 1275-1279.
9
5730
J. AM. CHEM. SOC. 2004, 126, 5730-5739
10.1021/ja049926n CCC: $27.50 © 2004 American Chemical Society

Multimeric System for Integrin Targeting and Endocytosis
A R T I C L E S
Figure 1. RGD-assembled peptides derivatives 1-8 (f ) D-Phe).
integrin-dependent endocytosis pathway thus offering new
therapeutic avenues.15-17
possibility to characterize the binding interactions to cells
expressing the RVâ3 integrin as well as the endocytosis process.
For instance, biotinylated compounds 2 and 3 were designed
to form stable complexes with avidin or streptavidin protein
which are commercially available for applications such as
surface functionalization or labeling. Fluorescein-containing
molecules 4-6 represent useful compounds ready for use in
experiments such as fluorescent or confocal miscroscopy.
Compounds 7 and 8 were designed for radiolabeling with 125I
and are currently used in radiobinding assays as well as in vivo
imaging in mice.18 These molecules represent the first instance
of a cyclopeptide presenting an array of cyclopeptides which
emphasizes the efficiency of our approach to construct such
new architectures for recognition. In addition, the usefulness
of the chemical strategy presented here is currently extended
to more complex systems presenting elements for cell recogni-
tion as well as cytotoxic elements for therapeutic evaluation.19
It is expected that these bifunctional systems might be useful
in the near future as reagents to study and modulate physiologi-
cally important integrin-extracellular matrix protein interactions
and as drugs for disease-related applications as well as new
nonviral targeting systems for drug delivery. In addition, it is
worth noting that our approach is not limited to integrin targeting
or detection applications. By adapting the corresponding domain
on the template, it may be conceptually easily exploited to target
different cells or tissues due to the increasing number of selective
ligands selected in vivo, and the range of applications may be
greatly extended by using the vast repertoire of available organic
compounds.
There is however still a need for developing more appropriate
chemical methods for the preparation of well-defined multivalent
ligand architectures that may also incorporate effector molecules
such as label groups as tools for biological evaluation or
subsequently cytotoxic molecules for therapeutic applications.
In this context, we describe herein the modular synthesis of
compounds 2-8 based on a cyclic decapeptide scaffold (RAFT)
that incorporates and presents in a spatially controlled manner
two independent functional domains: a clustered ligand domain
for integrin recognition and cell targeting and a labeling domain
for detection and characterization of this binding. Preliminary
study on the biological activity of the (biotin)2RAFT(c[-RGDfK-
])4, 2, (fluorescein)2RAFT(c[-RGDfK-])4, 4, and (fluorescein)-
RAFT(c[-RGDfK-])4, 6, have shown that these multivalent
systems fully conserved the recognition and selectivity properties
of the cognate monovalent cyclopentapeptide c[-RGDfK-] to
cells expressing RVâ3 integrin receptors. Most notably, these
systems were found to penetrate cells expressing RVâ3 integrins
by endocytosis in a receptor-dependent manner more effectively
than the cognate monovalent ligand. These results underline the
effectiveness of this general strategy to provide biological active
systems for selectively targeting cells expressing the RVâ3
integrin receptors. These molecules offer a wide range of
(12) (a) Marchi-Artzner, V.; Lorz, B.; Hellerer, U.; Kantlehner, M.; Kessler,
H.; Sackmann, E. Chem.sEur. J. 2001, 7, 1095-1101. (b) Hu, B.;
Finsinger, D.; Peter, K.; Guttenberg, Z.; Ba¨rmann, M.; Kessler, H.;
Escherich, A.; Moroder, L.; Bo¨hm, J.; Baumeister, W.; Sui, S.-F.;
Sackmann, E. Biochemistry 2000, 39, 12284-12294.
(13) Kok, R. J.; Schraa, A. J.; Bos, E. J.; Moorlag, H. E.; Asgeirsdottir, S. A.;
Everts, M.; Meijer, D. K. F.; Molema, G. Bioconjugate Chem. 2002, 13,
128-135.
(14) Thumshirn, G.; Hersel, U.; Goodman, S. L.; Kessler, H. Chem.sEur. J.
2003, 9, 2717-2725.
(15) For a review of multivalent interaction, see: Mammen, M.; Choi, S. K.;
Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754-2794.
(16) Castel, S.; Pagan, R.; Mitjans, F.; Piulats, J.; Goodman, S.; Jonczyk, A.;
Huber, F.; Vilaro, S.; Reina, M. Lab. InVest. 2001, 81, 1615-1626.
(17) Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L.
G. J. Cell Sci. 2000, 113, 1677-1686.
Results
Design. We reasoned that a controlled presentation of RGD
moieties in a clustered format may result in more efficient
(18) Sancey, L.; Boturyn, D.; Vuillez, J.-P.; Dumy, P.; Fagret, D. Work in
progress.
(19) Dumy, P.; Boturyn, D.; Coll, J. L.; Favrot, M. C. FR/02/11614, PCT/FR03/
02773.
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A R T I C L E S
Boturyn et al.
clustering of the ligand-bound integrin compared to the same
ligand presented individually. The recognition module consists
of the c[-RGDfK-] ligand since it is known that an exchange
of valine residue in the cognate cyclopentapeptide for lysine
has no significant influence on biological activity3. c[-RGDfK-
] can be readily functionalized through the chemistry of its lysine
ꢀ-amino group as emphasized by various applications such as
tumor targeting9 or imaging20 and cell adhesion stimulation.21
In addition, we reported that a lysine side chain functionalized
by aminooxy or glyoxylyl groups22 represents a powerful way
to conjugation to a diversity of molecules by chemoselective
oxime bond formation.23 This ligation technique is highly
efficient, compatible with a wide variety of chemical functions,
and allows the oxime bond formation between unprotected
fragments without any coupling reagent and with minimal
chemical manipulation.24 Moreover, the oxime linkage presents
the further advantage of being stable in vitro and in vivo.25 We
selected the regioselectively addressable functionalized template
(RAFT) as a suitable scaffold to direct independently and
separately the cyclopentapeptide ligands as well as reporter
groups (Figure 1). RAFT molecules represent topological cyclic
decapeptides containing orthogonally protected attachment sites
pointing to opposite faces of the template backbone that are
chemically accessible at gram scale.26 These addressable systems
can readily exhibit various levels of regioselectivity thanks to
an appropriate choice of the residues’ side chain protecting
groups encompassing the Pro-Gly sequence. They display
homogeneous conformation control27 that have been exploited
successfully for protein mimic28 or surface functionalization.29
In addition, we recently reported successfully the chemoselective
oxime assembly of aminooxy-carbohydrates to the RAFT
molecule for the presentation of carbohydrate recognition motifs
clusters.30 The modular functionalization with c[-RGDfK-] as
an RVâ3 integrin ligand or c[-RâADfK-] as a nonsense peptide,
as well as fluorescein, biotin, or tyrosine molecules (Figure 1),
thus provides series of molecules required for integrin targeting
studies.
molecules formed the pivotal intermediate: (1) the solid phase
peptide synthesis of linear protected peptide fragments, their
cyclization in solution to provide the corresponding templates
and cyclopentapeptides; (2) the sequential deprotection-func-
tionalization of a lysine side chain with label molecules and
subsequently four aminooxy groups on the template’s face as
well as the functionalization of the lysine side chain on the
cyclopentapeptides by a glyoxylyl group; (3) the final oxime
ligation of deprotected aminooxy-containing RAFT molecules
and Nꢀ-glyoxylyl-lysyl cyclopentapeptides to provide the desired
compounds.
RAFT Molecules. The RAFT with two functional domains
formed the central intermediate required for a multivalent
presentation with an integrin ligand as well as the chemical
derivatization with labeling molecules. The convenient choice
of lysine side chain protections and their relative positioning
within the peptide primary sequence provide cyclodecapeptides
9 and 19 exhibiting two attachment faces (Scheme 1). The Boc
and Alloc groups were selected because they are well-
documented orthogonal protections and the corresponding
protected lysine residues are commercially available. The linear
precursor-containing side chain protected peptides, namely
H-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys(Alloc)-
Lys(Boc)-Pro-Gly-OH and H-Lys(Boc)-Ala-Lys(Boc)-Pro-Gly-
Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-OH, were obtained us-
ing a standard Fmoc/tBu solid-phase chemistry on an acid-labile
Sasrin resin by adapting the previously described procedures.26,28b
It is worth noting that the mild acid conditions used for the
release of the linear protected peptide from this support (e.g.,
1% TFA/CH2Cl2, 2 min) was required to be compatible with
the Boc side chain protection stability. Glycine as the C-terminal
end was essential to secure the subsequent cyclization step with
the N-terminal lysine R-amino group from epimerization. The
corresponding head-to-tail cyclization was performed in DMF
under high dilution with PyBOP reagent as reported26 to provide
the corresponding RAFT molecules 9 and 19 in quantitative
yields. Removal of the Alloc group using the well-established
Pd0/PhSiH3 procedure31 afforded the desired derivatives 10 and
20 containing one and two free amino groups, respectively, in
excellent yields. Acylation of the fluorescein or biotin groups
to the latter amine functions was accomplished directly with
FITC (isomer I) or PyBOP coupling conditions, respectively.
The corresponding labeled derivatives (biotin)2RAFT(Boc)4 11,
(fluorescein)2RAFT(Boc)4 12, and (fluorescein)RAFT(Boc)4 21
were isolated after ether precipitation in sufficient purity to carry
out the subsequent step. Compounds 11, 12, and 21 were
smoothly deprotected with 50% trifluoroacetic acid at room
temperature in dichloromethane for 1 h and acylated with the
succinimide ester of N-Boc-O-(carboxymethyl)hydroxylamine32
to yield functionalized templates 15, 16, and 23, respectively.
Removal of the Boc groups was achieved by treatment with
TFA containing triisopropylsilane (TIS) and water in CH2Cl2
(50/5/5/40) for 1 h. RP-HPLC purification provided the key
aminooxy intermediates (biotin)2RAFT(COCH2ONH2)4 17,
(fluorescein)2RAFT(COCH2ONH2)4 18, and (fluorescein)RAFT-
(COCH2ONH2)4 24 in excellent overall yields, 42%, 70%, and
36%, respectively, for four steps (Scheme 1). To introduce a
The ligand and technique for its chemical manipulation in
hand, a strategy in three steps was devised, whereby the RAFT
(20) Haubner, R.; Wester, H. J.; Weber, W. A.; Mang, C.; Ziegler, S. I.;
Goodman, S. L.; Senekowitsch-Schmidtke, R.; Kessler, H.; Schwaiger, M.
Cancer Res. 2001, 61, 1781-1785.
(21) Kantlehner, M.; Finsinger, D.; Meyer, J.; Schaffner, P.; Jonczyk, A.;
Diefenbach, B.; Nies, B.; Kessler, H. Angew. Chem., Int. Ed. 1999, 38,
560-562.
(22) Boturyn, D.; Dumy, P. Tetrahedron Lett. 2001, 42, 2787-2790.
(23) Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.; Dumy, P. Chem.s
Eur. J. 2001, 7, 3976-3984.
(24) (a) Canne, L. E.; Botti, P.; Simon, R. J.; Chen, Y.; Dennis, E. A.; Kent, S.
B. H. J. Am. Chem. Soc. 1999, 121, 8720-8727. (b) Miao, Z.; Tam, J. P.
J. Am. Chem. Soc. 2000, 122, 4253-4260. (c) Zhang, L.; Torgerson, T.
R.; Liu, X.-Y.; Timmons, S.; Colosia, A. D.; Hawiger, J.; Tam, J. P. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 9184-9189. (d) Rose, K. J. Am. Chem.
Soc. 1994, 116, 30-33.
(25) Nardin, E. H.; Calvo-Calle, J. M.; Oliveira, G. A.; Clavijo, P.; Nussenzweig,
R.; Simon, R.; Zeng, W.; Rose, K. Vaccine 1998, 16, 590-600.
(26) Dumy, P.; Eggleston I., Servigni, S.; Sila, U.; Sun, X.; Mutter, M.
Tetrahedron Lett. 1995, 36, 1255-1258.
(27) (a) Peluso, S.; Ruckle, T.; Lehmann, C.; Mutter, M.; Peggion, C.; Crisma,
M. ChemBioChem 2001, 2, 432-437 (b) Dumy, P.; Eggleston, I. M.;
Esposito, G.; Nicula, S.; Mutter, M. Biopolymers 1996, 39, 297-308.
(28) (a) Mutter, M.; Dumy, P.; Garrouste, P.; Lehmann, C.; Mathieu, M.;
Peggion, C.; Peluso, S.; Razaname, A.; Tuchscherer, G. Angew. Chem.
1996, 108, 1587-1591; Angew. Chem., Int. Ed. Engl. 1996, 35, 11482-
1485. (b) Peluso, S.; Dumy, P.; Nkubana, C.; Yokokawa, Y.; Mutter, M.
J. Org. Chem. 1999, 64, 7114-7120 and references therein.
(29) (a) Scheibler, L.; Dumy, P.; Stamou, D.; Duschl, C.; Vogel, H.; Mutter,
M. Tetrahedron 1998, 54, 3725. (b) Scheibler, L.; Dumy, P.; Boncheva,
M.; Leufgen, K.; Mathieu, H. J.; Mutter, M.; Vogel, H. Angew. Chem.,
Int. Ed. 1999, 38, 696-699.
(31) Thieriet, N.; Alsina, J.; Giralt, E.; Guibe´, F.; Albericio, F. Tetrahedron
Lett. 1997, 38, 7275-7278.
(32) Ide, H.; Akamatsu, K.; Kimura, Y.; Michiue, K.; Makino, K.; Asaeda, A.;
Takamori, Y.; Kubo, K. Biochemistry 1993, 32, 8276-8283.
(30) Renaudet, O., Dumy, P. Org. Lett. 2003, 5, 243-246.
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Multimeric System for Integrin Targeting and Endocytosis
A R T I C L E S
Scheme 1. Synthesis of Aminooxy-Containing RAFT 17, 18, 24, and 29a
a (a) (PPh3)4Pd, PhSiH3, CH2Cl2; (b) PyBOP, biotin, DIEA, DMF; (c) FITC (isomer I); (d) TFA/CH2Cl2 1:1; (e) BocNHOCH2COOSu, DIEA, DMF; (f)
PyBOP, BocY(tBu)OH, DIEA, DMF; (g) TFA/TIS/H2O/CH2Cl2 50:5:5:40.
Scheme 2. Structure of the Monovalent Biotinylated-RGD Ligands
Containing â-Alanine Spacers 34-39
tyrosine residue, a reverse functionalization strategy was
required to satisfy the use of BocTyr(OtBu)OH: first, removal
of the Boc groups of 19 followed by a subsequent acylation
with N-Boc-O-(carboxymethyl)hydroxylamine succinimide ester
afforded compound 26. Then, removal of the Alloc group and
PyBOP acylation with BocTyr(OtBu)OH provided Boc/tBu
protected template 28. A total deprotection was accomplished
using a concentrated TFA solution containing p-cresol, TIS, and
water as scavengers providing compound 29 in 33% overall
yield (Scheme 1).
periodate was readily accomplished by RP-HPLC purification
to give the pure compounds ready for oxime ligation.
Access to monovalent RGD-control peptides bearing a biotin
on the lysine side chain, c[-RGDfK([âA]n-biotin)-] 34-39 (n
) 0-5, Scheme 2) was achieved by a combination of SPPS
and solution strategies.22 Incorporation of â-alanine (n ) 0-5)
units as a spacer was used to optimize the separation between
the ligand and biotin group required for the interaction with
the integrin receptor as well as with streptavidin (vide supra).
Fluorescent cyclopentapeptide c[-RGDfK(fluorescein)-] 41 was
obtained by oxime ligation between the Nꢀ-glyoxylyl-lysyl
peptide 32 and fluorescein containing oxyamine group33 40 in
acetate solution at pH 4.6 in 77% yield. Acylation at the lysine
side chain of protected cyclopentapeptide by succinic anhydride
Cyclopeptide Ligand and Control Peptide. The exchange
of glycine residue for â-alanine in cyclic pentapeptide
c[-RGDfK-] is known to totally abolish recognition of the
peptide by RVâ3 integrin.3 Therefore, c[-RâADfK-] was used
as a negative control in our biological assays. The cyclic
pentapeptide c[-RGDfK(S)-] 30 as well as c[-RâADfK(S)-] 31
derivatives acylated on the lysine side chain with a serine residue
were prepared by two-dimensional solid-phase synthesis as
described earlier.22 Oxidative cleavage with sodium periodate
of the amino-alcohol moiety of serine containing cyclopen-
tapeptides afforded the desired glyoxylyl compounds c[-RGDfK-
(COCHO)-] 32 and c[-RâADfK(COCHO)-] 33 in quantitative
yields. Removal of formaldehyde byproduct and unreacted
(33) Tre´visiol, E.; Defrancq, E.; Lhomme, J.; Laayoun, A.; Cros, P. Tetrahedron
2000, 56, 6501-6510.
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Boturyn et al.
Scheme 3. Synthesis of Compounds 2-8 by Oxime Ligation of
Glyoxylyl Cyclopentapeptides to Aminooxy-RAFT Molecules 32
and 33
Figure 2. HPLC profiles of (a) a reaction between 17 and 1 equiv of 32
after 1 h and (b) a reaction between 17 and 6 equiv of 32 after 6 h.
compound to provide eventually the HPLC signal corresponding
to the desired product. It is important to note that attempts to
assemble the cyclopentapeptide by conventional amide forma-
tion using protected compounds 42 and template molecule 13
were totally unsuccessful35 thus emphasizing the efficacy of the
oxime ligation.
Biological Data. First, oxime-containing compounds 2-8
were incubated in phosphate buffer at pH 7.0 to assess on their
stability. Neither hydrolysis nor degradation phenomenon was
observed by HPLC even after several days (data not shown)
which indicates that these compounds remain fully stable under
the biological experimental conditions used in this study.
A. Cell Adhesion Assays. Binding studies of RGD deriva-
tives were first realized on different cell lines (data not shown).36
Among all cell lines investigated, CHO3a was selected as the
model for our in vitro experiments because it is a stable
transfectant of the chinese hamster ovary (CHO) cell line
expressing a good level of RVâ3 integrin. To test the functional
integrity of the c[-RGDfK-]-containing peptides, we studied first
the adhesion of CHO3a cells on a surface coated by biotinylated
peptides 2 and 34-39 and second the inhibition of CHO3a
attachment to vitronectin-coated wells by the same peptides.
Previously, we investigated the importance of the linker length
between the control RGD ligand and the biotin moiety.
Biotinylated-peptides 34-39 were loaded onto streptavidin-
coated 96-well plates. CHO3a cells were then incubated for 45
min at 37 °C, and their ability to adhere on these surfaces was
measured after staining (Figure 3). As expected the negative
control peptide 3 containing the RâAD sequence did not allow
cell adhesion, while the cells adhered normally on a fibronectin-
coated surface (FN). Derivatives 34 and 35 containing shorter
linkers between RGD and biotin motifs were not able to bridge
the streptavidin surface and the cells. Increasing gradually the
length of the linker from two up to five, â-alanine (respectively,
followed by acid-mediated deprotection provided c[-RGDfK-
(COCH2CH2CO2H)-] 42, as the key compound for attempting
direct RGD-peptide anchorage to the template molecule by
conventional amide bond formation (vide supra).
Template Presenting Tetravalent Cyclopeptides. Com-
pounds 17, 18, 24, and 29 exhibiting four aminooxy functions
provide the key intermediates to assemble by chemoselective
oxime formation an array of cyclopentapeptides on the template.
(biotin)2RAFT(c[-RGDfK-])4 2, (biotin)2RAFT(c[-RâADfK-])4
3, (fluorescein)2RAFT(c[-RGDfK-])4 4, (fluorescein)2RAFT-
(c[-RâADfK-])4 5, (fluorescein)RAFT(c[-RGDfK-])4 6, (fluo-
rescein)RAFT(c[-RâADfK-])4 7, and (tyrosine)RAFT(c[-RGDfK-
])4 8 were easily obtained by oxime ligation of the appropriate
cyclopentapeptide-containing aldehyde, namely c[-RGDfK-
(COCHO)-] 32 and c[-RâADfK(COCHO)-] 33, to free ami-
nooxy-containing RAFTs 17, 18, 24, and 29 under classical
conditions at pH 4.6 (Scheme 3). This provided labeled
molecules with a clustered presentation of the integrin RGD
ligand 2, 4, 6, and 8 as well as comparable molecules as negative
controls 3, 5, and 7. As inferred by HPLC, the ligation step
proceeded cleanly and the reactions were generally complete
after 6 h by using a slight 1.5-fold excess of aldehyde
compounds relative to the aminooxy-containing template. The
yields of the reactions were satisfactory ranging from 51 to 95%
after purification steps. In addition, a reaction was carried out
using an equimolar ratio of compounds 17 and 32 to probe the
putative intermediates formed along the oxime coupling reaction.
The HPLC profile (Figure 2) obtained after a 1 h reaction time
with these conditions showed the presence of intermediate
products.34 They disappeared on further addition of aldehyde
(35) This is attributed to tedious solubility problems and presumed steric
hindrance imposed by the presence of protecting groups required during
the coupling reaction.
(36) HEL, PMA-induced HEL, CHOT, K562, HeLa, and CHO3a were used
for preliminary experiments. K562 devoid of RVâ3 integrin was used along
this work as control.
(34) Intermediates were identified and studied by LC-MS technique to be the
mono, bis, and tri ligated compounds.
9
5734 J. AM. CHEM. SOC. VOL. 126, NO. 18, 2004

Multimeric System for Integrin Targeting and Endocytosis
A R T I C L E S
particularly internalized by the CHO3a cells than the control
peptide 49. At 4 °C, cellular uptake was inhibited, the peptides
being located outside the cell membrane thus suggesting a
typical endocytic pathway (Figure 4b). For molecules 2, 4, and
6, similar internalization results were obtained demonstrating
that the detection domain does not interfere with the recognition
domain. The streptavidin-2 complex was also internalized
pointing out the ability of our system to bring selectively the
exogenous protein into cells expressing the RVâ3 integrin. The
same experiments were also performed on primary human
umbilical vein endothelial cells (HUVEC) which naturally
express the RVâ3 integrin and the fluorescence observed using
a biphoton confocal microscope (see experimental part). The
results also indicate a massive uptake of the multimeric RGD-
containing peptides compared to the monovalent cyclopentapep-
tide. In addition, neither cell binding nor internalization of the
multivalent peptides was observed with K562 cells devoid of
RVâ3 integrin. Internalization experiments were also done on
CHO3a cells using peptides 4 and 5 containing two fluorescein
residues. The same tendency was broadly observed, namely a
higher uptake of the multivalent RGD-containing peptide 4. It
is worthy to note that the use of compound 4 containing two
fluorophores is invaluable because this latter ensures directly a
sufficient level of fluorescence to allow the observations without
method of amplification.
Figure 3. Adhesion assay. Streptavidin-coated 96-well dishes were loaded
with 1 µM 2, 3, and 34-39 peptides. Fibronectin (FN) was directly coated
on MaxiSorp 96-well plates at a concentration of 0.5 µg/well. CHO3a cells
(100 000) were then incubated for 45 min at 37 °C in PBS containing 1
mM MgCl2 and rinsed 3 times, and the remaining adherent cells were fixed,
stained with methylene blue, and resuspended in 100 µL HCl 0.1 N. The
OD at 630 nm was measured. The mean ( SEM (n ) 8 for each peptides)
of one representative experiment repeated 3 times is presented.
peptides 36, 37, 38, and 39) was associated with better cell
adhesion processes probably due to an increased accessibility
of the RGD motifs. Remarkably, a surface coated with the
multivalent compound 2 exhibited an adhesion pattern compa-
rable to those displayed by the optimized monovalent RGD
ligand.
Discussion
Next, we compared the multivalent compound’s efficiency
to compete with vitronectin toward CHO3a adhesion. A
suspension of cells was mixed with different concentrations of
peptides and allowed to adhere on vitronectin for 45 min at 37
°C before being rinsed 3 times in PBS, fixed, and stained. The
inhibitory ability of each peptide was quantified by the
measuring of its IC50 (concentration of peptide necessary to
inhibit 50% of the cell attachment). As expected the negative
control peptide 3 had no effect on vitronectin-mediated adhesion.
In contrast, the monovalent compound 38 as well as the
multivalent compound 2 inhibited strongly the attachment of
CHO3a on vitronectin-coated wells. An IC50 of 0.5 ( 0.05 µM
was calculated for compound 2, while a lower IC50 value around
2.5 µM was found for 38.
B. Internalization Assays. Internalization assays were per-
formed directly using fluorescent peptides 4-7 and 41 or after
the formation of an FITC-labeled streptavidin complex with the
biotinylated peptides 2 and 3. After a 15 min incubation at 37
°C in the presence of 10 µM peptides, the CHO3a cells were
washed and fixed, and the fluorescence detected directly in the
case of 2 or 3 or after permeabilization and amplification. As
shown in Figure 4, RGD-containing compound 6 is more
Our approach aims at developing multivalent ligands of RVâ3
integrins for improving cell targeting. Indeed, the selectivity
and efficiency of c[RGDfK] for targeting as well as its capacity
to penetrate cells expressing RVâ3 was called into question
recently thus emphasizing the need for multimeric RGD-
containing molecules.16 This is corroborated by the fact that
multivalency is a general strategy used by nature to mediate
and control recognition processes.15 Moreover, the shape of a
multivalent ligand controls its activity: its ability to cluster
surface receptors can promote a specific biological response via
signal transduction.37 Diverse scaffolds have been used to
generate multivalent ligands such as linear polymers,38 den-
drimers,39 calixarenes,40 multiple antigenic peptides (MAP),14,41
or globular proteins.42 The importance of ligand density in RGD
peptide-integrin interactions was assessed by scaffolding
several RGD recognition motifs from a polymer chain11,17 or
after conjugation from a humanized antibody protein.13 Although
these multidentate compounds were found to be more active in
competitive inhibition experiments than the cognate monovalent
ligand, the difficulties in predicting and controlling the homo-
geneity of the protein- or polymer-conjugate remain a problem
that may complicate the data interpretation and limit the clinical
Figure 4. Internalization of RGD-containing peptides: (a) RGD-containing RAFT 6 at 37 °C, (b) RGD-containing RAFT 6 at 4 °C, (c) RGD-containing
peptide 41 at 37 °C.
9
J. AM. CHEM. SOC. VOL. 126, NO. 18, 2004 5735

A R T I C L E S
Boturyn et al.
exploitation of these compounds. During the preparation of this
work, Kessler et al. reported a modular synthesis of tetravalent
RGD molecules that displayed improved binding to the isolated
integrin receptor.14 This approach, based on an MAP-lysine core
as scaffold to direct radially the c[-RGDfK-] pendants, represents
conceptual progress for controlling the ligand density. However,
an MAP system suffers from the lack of conformational control
which renders the spatial presentation difficult to manage.
Indeed, besides the importance of the ligand density, its local
spatial distribution and presentation were shown to be essential
for integrin-mediated signal transduction.17 Here, we chose a
decapeptide named RAFT as a pivotal element to assemble RVâ3
integrin-peptide ligands in a multivalent mode and different
reporter groups. A RAFT molecule having two addressable
domains allows functional components to be assembled and
directed in well-defined and controlled spatial orientations.27
Separation of each functional domain in space is required in
order for them to function in an independent manner. The
advantage of such conformational control by related template
molecules has been emphasized in the case of carbohydrate-
recognition elements and a malaria-epitopes presentation.30,43
In particular, an array of ligand molecules can be assembled
and directed on one face for the cell-receptor targeting, the other
being functionalized by reporter groups or drugs. Therefore, the
use of such a RAFT molecule capitalizes in a row the
requirement of multimeric ligand presentation to improve
ligand-receptor interaction, the presence of label groups for
detecting and tracing the corresponding biological activity, and
the guarantee of the control of their spatial presentation. In
addition, because the assembly is convergent and the domains
separated, this allows the synthesis of series of compounds that
are required for biological studies by only varying the func-
tionalization at one template face. To address regioselectively
the lysine side chains, an appropriate orthogonally protecting
group strategy has been devised. The Boc and Alloc protecting
groups used in this work are well-documented and exhibit an
excellent level of orthogonality. Successive adequate deprotec-
tion and coupling reaction cycles by using a combined solid-
phase and solution approach provided the key template com-
pounds 17, 18, 24, and 29 containing aminooxy groups (Scheme
1) ready for chemoselective oxime formation. The oxime
ligation technique between unprotected molecules functionalized
by aminooxy and aldehyde groups was selected because of its
high efficiency and chemoselectivity level as well as its in vivo
stability.25 Moreover, it requires minimal chemical manipulations
in aqueous conditions, since neither coupling reagent nor
protecting group is used. This ligation has been used to
circumvent inherent chemical incompatibility and proved very
effective for the conjugation of various molecules such as
peptides,24,43 DNA,23 carbohydrates,44 and labels.14 Recently,
it has also been adapted to the RAFT molecule for presenting
clusters of carbohydrate recognition motifs.30 In addition, a
preliminary attempt of amide coupling in solution between RGD
derivative 42 and template 13 failed in our hands.35 Herein,
chemoselective oxime ligation has proved to be very efficient
to prepare our conjugated molecules. The target compounds
were obtained cleanly without side reactions providing the
tetravalent RGD-containing RAFTs 2, 4, 6, and 8 as well as
the control peptides 3, 5, and 7 in good yields. Binding studies
of the RGD derivatives to different cell lines demonstrated that
our molecules displayed selectivity for cells expressing RVâ3
integrin.36 An adhesion assay with CHO3a cells (Figure 3)
shows the importance of an appropriate linker length required
between the RGD-binding motif and the biotin for the monova-
lent RGD peptides: the increase of the spacer length, namely
from three to five â-alanine residues, improves the binding
efficiency to CHO3a cells. For molecule 2, the lysine side chains
used on the template as well as on the c[-RGDfK] peptide
provide a suitable linker length to promote efficient surface and
cell attachment. Together, these observations correlate well with
those reported on the adhesion of osteoblasts to a surface
functionalized with c[-RGDfK] where a minimum linker
distance of 3.5 nm was reported.45 However, these experiments
do not make it possible to conclude from the improvement
brought by the multivalent compound compared with the
monovalent one. This is certainly due to a ligand density
problem; beyond a critical value exhibited on the surface, similar
cell attachments occur preventing the differentiation of the
compounds. To circumvent this problem, we studied the
inhibition of CHO3a adhesion to a vitronectin-coated surface
by our multimeric compound 2. The inhibitory effects of
compounds 2 and 38 were similar at high concentration (10
µM). At lower concentrations (C < 1 µM), a significant
difference was observed, showing that the binding affinity is
increased when several copies of RGD peptide are presented
to the integrin receptor (IC50 of 0.5 ( 0.05 µM for 2 and 2.5
µM for 38). This observation is consistent with those obtained
with other multivalent c[-RGDfK]-containing systems.13,14
Together, these experiments demonstrate the functional activity
of our construct. Although the IC50 value of 2 corrected for the
number of RGD elements (i.e., 2 µM) is close to that of 38,
this is certainly due to an overvaluation of the effective
concentration for inhibition. In fact, it is important to note that
a part of the multivalent compound is internalized by the RVâ3
expressing cells during the incubation period.46 Indeed, inter-
nalization of multivalent RGD-containing peptides 2, 4, and 6
were visualized at 37 °C in CHO3a and HUVEC cells but very
few for the peptide 41 (Figure 4c). No internalization was
detected at 4 °C suggesting an energy-dependent process. In
addition, neither cell binding nor internalization of the multi-
valent peptides was observed with cells devoid of the RVâ3
integrin. These observations are consistent with an RVâ3 integrin
receptor-mediated endocytosis pathway that might have been
triggered by integrin clustering resulting from the binding with
our multimeric RGD ligands.19 In addition, these results also
suggest that the proximity in space between the c[-RGDfK]
moieties is a prerequisite for such biological effects. Interest-
ingly, since the same observations were obtained with com-
(37) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L.
L. J. Am. Chem. Soc. 2002, 124, 14922-14933.
(38) Maynar, H. D.; Okada, S. Y.; Grubbs, R. H. Macromolecules 2000, 33,
6239-6248.
(39) Perec, C.; Cho, W. D.; Ungar, G. J. Am. Chem. Soc. 2000, 122, 10273-
10281.
(40) Gutsche, C. D.; Iqbal, M.; Steward, D. J. Org. Chem. 1986, 51, 742-745.
(41) Tam, J. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3879-3883.
(42) Arnon, R.; Vanregenmortel, M. H. V. FASEB J. 1992, 6, 3265-3274.
(43) Rose, K.; Zeng, W.; Brown, L. E.; Jackson, D. C. Molecular Immunology
1995, 32, 1031-1037.
(45) Kantlehner, M.; Schaffner, P.; Finsinger, D.; Meyer, J.; Jonczyk, A.;
Diefenbach, B.; Nies, B.; Ho¨lzemann, G.; Goodman, S. L.; Kessler H.
ChemBiochem 2000, 1, 107-114.
(44) Forget, D.; Renaudet, O.; Defrancq, E.; Dumy, P. Tetrahedron Lett. 2001,
42, 7829-7832.
(46) Endocytosis of compound 2 occurred after 5 min at 37 °C.
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Multimeric System for Integrin Targeting and Endocytosis
A R T I C L E S
manually in a glass reaction vessel fitted with a sintered glass frit or
automatically on a synthesizer (348 Ω synthesizer, Advance ChemTech).
Coupling reactions were performed using, relative to the resin loading,
1.5-2 equiv of N-R-Fmoc-protected amino acid activated in situ with
1.5-2 equiv of PyBOP and 3-4 equiv of DIEA in DMF (10 mL/g
resin) for 30 min. Manual syntheses were controlled by Kaiser and/or
TNBS tests. N-R-Fmoc protecting groups were removed by treatment
with a piperidine/DMF solution (1:4) (10 mL/g resin) for 10 min. The
process was repeated 3 times, and the completeness of deprotection
was verified by the UV absorption of the piperidine washings at 299
nm.
Synthetic linear peptides were recovered directly upon acid cleavage
(1% TFA in CH2Cl2). The resins were treated for 3 min repeatedly
until the resin beads became dark purple. The combined washings were
concentrated under reduced pressure, and white solid peptides were
obtained by precipitation from ether. They were analyzed by RP-HPLC
and, if necessary, purified on a preparative column.
General Procedure for Cyclization Reactions. All linear peptides
(0.5 mM) were dissolved in DMF, and the pH was adjusted to 8-9 by
addition of DIEA. PyBOP (1.2 equiv) was added, and the solution was
stirred at room temperature for 1 h as described.22,26 Solvent was
removed under reduced pressure, and the residue dissolved in the
minimum of CH2Cl2. Ether was added to precipitate the peptide. Then
it was triturated and washed 3 times with ether to yield crude material
without further purification.
c[Lys(Boc)-Lys-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys-Lys(Boc)-Pro-
Gly] 10. The linear peptide H-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-
Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-OH was assembled on Sasrin
resin (1 g) affording 1.068 g (0.6 mmol) of a white solid powder. The
cyclization reaction was carried out using linear peptide (490 mg, 0.3
mmol) as described above. Alloc groups were removed using cyclic
peptide 9 (450 mg, 0.27 mmol) dissolved in 30 mL of dry DCM under
argon by adding phenylsilane (2.0 g, 18.5 mmol) for 3 min and then
Pd(PPh3)4 (62.4 mg, 54 µmol) for 1 h at room temperature.31 The solvent
was removed under reduced pressure. The crude product was dissolved
in the minimum of a solution containing a mixture of CH2Cl2 and CH3-
OH (1:1). Ether was added to precipitate the crude product. Then it
was triturated and washed 3 times with ether. The materials were
dissolved in a solution containing 50% CH3CN in water and lyophilized
to afford the product 10 as a white powder (366 mg, 0.25 mmol, 92%).
Mass spectrum (ES-MS, positive mode) calcd 1477.9, found 1477.4.
c[Lys-Lys(Biotin)-Lys-Pro-Gly-Lys-Lys(Biotin)-Lys-Pro-Gly] 13.
Biotin (22.9 mg, 93.6 µmol), PyBOP (48.7 mg, 93.6 µmol), and DIEA
were added to a solution containing the compound 10 (58 mg, 39 µmol)
in 10 mL of DMF to adjust the pH at 8.0. The reaction was stirred for
1 h at room temperature and then concentrated under diminished
pressure. The crude product was triturated and washed with ether to
yield compound 11 as a white powder (33 mg, 22.3 µmol, 57%).
Removal of Boc moieties was carried out in a solution containing
50% TFA in DCM for 1 h at room temperature. The crude product
was concentrated, triturated, and washed with ether to yield compound
13 as a white powder (32 mg, 21 µmol, 94%). Mass spectrum (ES-
MS, positive mode) calcd 1530.0, found 1530.0.
pounds with different detection domains such as 2 or 4, this
strongly supports the idea that the template also serves to prevent
the detection and the binding domain to interfere together and
thus preserves the cell attachment as well as the endocytosis of
the molecules. This feature is important for the future design
of more sophisticated molecules. Therefore, the strong binding
to specific integrin receptors and the associated good internal-
ization property displayed by the multivalent molecules pre-
sented here offer interesting perspectives for in vivo studies as
well as for the design of more sophisticated systems such as
molecular conjugate vectors.19
Conclusion
All together, the preliminary biological results obtained here
validate the concept based on RAFT molecules to incorporate
and present in a spatially controlled manner two independent
functional domains: a clustered-ligand domain for integrin
recognition and cell targeting and a labeling domain for detection
and characterization of this event. The strategy described is
based on solid-phase peptide synthesis and chemoselective
oxime ligation. It is very flexible, modular, and adapted to the
synthesis of a series of compounds such as those presented here.
The multimeric RGD-containing compounds 2, 4, 6, and 8 are
currently used for studying integrin clustering as well as in vivo
experiments, and the corresponding results will be reported in
due time.47 Since the role of this integrin needs reevalution as
regulators of angiogenesis,48 it is expected that these bifunctional
systems might be useful in the near future as reagents to study
and modulate physiologically important integrin-extracellular
matrix protein interactions and as drugs for disease-related
applications as well as new nonviral targeting systems for drug
delivery.19 In addition, as demonstrated earlier,30 it is worth
noting that our approach is not limited to integrin ligand or for
detection applications. By adapting the corresponding domain
on the template, it may be conceptually exploited easily to target
different cells or tissues due to the increasing number of selective
ligands selected in vivo49 and the range of applications such as
drug delivery may be greatly extended by using the vast
repertoire of available organic compounds.
Experimental Section
Materials. Protected amino acids, Sasrin, and ChloroTrityl resins
were obtained from Advanced ChemTech Europe (Brussels, Belgium),
Bachem Biochimie SARL (Voivins-Le-Bretonneux, France), and France
Biochem S.A. (Meudon, France). PyBOP was purchased from France
Biochem, and other reagents were purchased from Aldrich (Saint
Quentin Fallavier, France) and Acros (Noisy-Le-Grand, France). RP-
HPLC analyses were performed on Waters equipment consisting of a
Waters 600 controller, a Waters 2487 Dual absorbance detector, and a
Waters in-line degasser. The analytical column (Nucleosil 120 Å 3 µm
C18 particles, 30 × 4.6 mm2) was operated at 1.3 mL/min, and the
preparative column (Delta-Pak 300 Å 15 µm C18 particles, 200 × 25
mm2), at 22 mL/min, with UV monitoring at 214 and 250 nm. Solvent
B consisted of 0.09% TFA in 90% acetonitrile, and solvent A, of 0.09%
TFA. Mass spectra were obtained by electron spray ionization (ES-
MS) on a VG Platform II (Micromass).
c[Lys(-COCH2ONHBoc)-Lys(Biotin)-Lys(-COCH2ONHBoc)-
Pro-Gly-Lys(-COCH2ONHBoc)-Lys(Biotin)-Lys(-COCH2ONHBoc)-
Pro-Gly] 15. BocNHOCH2COSu32 (26.5 mg, 92.0 µmol) and DIEA
were added to a solution of compound 13 (32 mg, 21 µmol) in 4 mL
of DMF to adjust the pH at 8.0. The reaction was stirred for 10 min at
room temperature and then concentrated under reduced pressure. The
crude product was triturated and washed with ether to yield compound
15 as a white powder (40 mg, 18 µmol, 86%). Mass spectrum (ES-
MS, positive mode) calcd 2222.7, found 2221.6.
General Procedure for Solid-Phase Peptide Synthesis. Assembly
of all protected peptides was carried out using Fmoc/tBu strategy
c[Lys(-COCH2ONH2)-Lys(Biotin)-Lys(-COCH2ONH2)-Pro-Gly-
Lys(-COCH2ONH2)-Lys(Biotin)-Lys(-COCH2ONH2)-Pro-Gly] 17.
Removal of Boc moieties from compound 15 (40 mg, 18 µmol) was
carried out in a solution containing 50% TFA/5% TIS/5% H2O in DCM
(47) Coll, J.-L.; Garanger, E.; Boturyn, D.; Dumy, P.; Favrot M. C. Work in
progress.
(48) Hynes, R. O. Nature Med. 2003, 9, 918-921.
(49) Monaci, P.; Bartoli, F.; Zenzo, G. D.; Nuzzo, M.; Urbanelli, L. Tumor
Targeting 1999, 4, 129-124 and references therein.
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A R T I C L E S
Boturyn et al.
for 1 h at room temperature. The crude product was concentrated,
triturated, and washed with ether. The product was then purified by
RP-HPLC to afford compound 17 as a white powder (30 mg, 16.3
µmol, 91%). Mass spectrum (ES-MS, positive mode) calcd 1822.2,
found 1821.8.
(0.1 mM, pH 4.0) was added the fluorescein derivative 40 (11 mg, 20
µmol) prepared as described.33 The reaction was stirred for 24 h at 25
°C. Conjugate 41 was isolated after a purification by RP-HPLC as a
yellow powder (5.4 mg, 5 µmol, 77%). Mass spectrum (ES-MS, positive
mode) calcd 1178.3, found 1177.7.
c[Arg(Pmc)-Gly-Asp(tBu)-D-Phe-Lys(-CO(CH2)2CO2H)] 42. Suc-
cinimic anhydride (10 mg, 100 µmol) and DIEA (20 µL, 115 µmol)
were added at room temperature to a solution containing the c[Arg-
(Pmc)-Gly-Asp(tBu)-D-Phe-Lys] (73 mg, 78.7 µmol)22 in 5 mL of DMF.
The reaction was stirred for 1 h, and the solvent was removed under
reduced pressure. The residue was washed with ether, and compound
42 was isolated after purification by RP-HPLC as a white powder (80
mg, 77.9 µmol, 99%). Mass spectrum (ES-MS, positive mode) calcd
1026.2, found 1026.7.
Peptide Conjugate 2. To a solution containing the derivative 17
(10 mg, 5.5 µmol) in 2 mL of sodium acetate buffer (0.1 mM, pH
4.0)/acetonitrile (1:1) was added the peptide 32 (22.5 mg, 33.5 µmol).
The reaction was stirred for 3 h at 25 °C. Conjugate 2 was isolated
after a purification by RP-HPLC as a white powder (22.6 mg, 5.1 µmol,
94%). Mass spectrum (ES-MS, positive mode) calcd 4390.0, found
4389.6.
Peptide Conjugate 3. To a solution containing the derivative 17
(3.5 mg, 1.5 µmol) in 3 mL of sodium acetate buffer (0.1 mM, pH
4.0)/acetonitrile (1:1) was added the peptide 33 (15 mg, 16.7 µmol).
The reaction was stirred for 6 h at 25 °C. Conjugate 3 was isolated
after purification by RP-HPLC as a white powder (5.5 mg, 1.1 µmol,
73%). Mass spectrum (ES-MS, positive mode) calcd 4445.1, found
4444.1.
Peptide Conjugate 4. To a solution containing the derivative 18
(21.5 mg, 10 µmol) in 10 mL of sodium acetate buffer (0.1 mM, pH
4.0) was added the peptide 32 (32 mg, 48 µmol). The reaction was
stirred for 6 h at 25 °C. Conjugate 4 was isolated after purification by
RP-HPLC as a yellow powder (24 mg, 5.1 µmol, 51%). Mass spectrum
(ES-MS, positive mode) calcd 4715.1, found 4715.3.
c[Lys(-COCH2ONH2)-Lys(Fluorescein)-Lys(-COCH2ONH2)-Pro-
Gly-Lys(-COCH2ONH2)-Lys(Fluorescein)-Lys(-COCH2ONH2)-Pro-
Gly] 18. To a solution containing compound 10 (10 mg, 6.8 µmol) in
1.5 mL of DMF was added FITC (8 mg, 18.5 µmol). The reaction was
stirred for 1 h at room temperature and then concentrated under
diminished pressure. The crude product was triturated and washed with
ether to yield compound 12 as a white powder. Removal of Boc
moieties was carried out in a solution containing 50% TFA in DCM
for 1 h at room temperature. The crude product was concentrated,
triturated, and washed with ether to yield compound 14 as a white
powder. Then, BocNHOCH2COSu32 (10.0 mg, 35.0 µmol) and DIEA
were added to a solution containing compound 14 in 2.5 mL of DMF
to adjust the pH at 8.0. The reaction was stirred for 1 h at room
temperature and then concentrated under reduced pressure. The crude
product was triturated and washed with ether to yield compound 16 as
a white powder. Removal of Boc moieties from compound 16 was
carried out in a solution containing 50% TFA/5% TIS/5% H2O in DCM
for 1 h at room temperature. The crude product was concentrated,
triturated, and washed with ether. The product was then purified by
RP-HPLC to afford compound 18 as a yellow powder (10.2 mg, 4.8
µmol, 70% overall yield). Mass spectrum (ES-MS, positive mode) calcd
2148.4, found 2148.0.
c[Lys(Boc)-Lys-Lys(Boc)-Pro-Gly-Lys(Boc)-Ala-Lys(Boc)-Pro-
Gly] 20. The linear peptide H-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-
Lys(Boc)-Ala-Lys(Boc)-Pro-Gly-OH was assembled on Sasrin resin
(300 mg) affording 255 mg (0.18 mmol) of a white solid powder. The
cyclization reaction and Alloc group removal were accomplished using
the procedure described above to yield the product 20 as a white powder
(227 mg, 0.16 mmol, 90%). Mass spectrum (ES-MS, positive mode)
calcd 1420.8, found 1420.4.
c[Lys(-COCH2ONH2)-Lys(Fluorescein)-Lys(-COCH2ONH2)-Pro-
Gly-Lys(-COCH2ONH2)-Ala-Lys(-COCH2ONH2)-Pro-Gly] 24. Start-
ing with compound 20 (138.5 mg, 97.5 µmol), compound 24 was
isolated after a purification by RP-HPLC as a yellow powder using
the procedure described to yield the product 18 (25.7 mg, 15.1 µmol,
36% overall yield). Mass spectrum (ES-MS, positive mode) calcd
1701.9, found 1702.0.
c[Lys(-COCH2ONH2)-Lys(-Tyrosin)-Lys(-COCH2ONH2)-Pro-
Gly-Lys(-COCH2ONH2)-Ala-Lys(-COCH2ONH2)-Pro-Gly] 29. Start-
ing with compound 19 (52.3 mg, 34.7 µmol), compound 29 was isolated
after a purification by RP-HPLC as a white powder using the procedure
described to yield the product 17 (17 mg, 11.5 µmol, 33% overall yield).
Mass spectrum (ES-MS, positive mode) calcd 1475.7, found 1475.2.
Peptide Derivatives 32, 34-39. The compounds 32 and 34-39 were
prepared as described in the literature by a combination of SPPS and
solution strategy.22
Peptide Derivatives 33. Using the same procedure,22 the linear
peptide H-Asp(tBu)-D-Phe-Lys[Boc-Ser(tBu)]-Arg(Pmc)-âAla-OH was
assembled on ChloroTrityl resin (150 mg) affording 72.8 mg (60.6
µmol) of a white solid powder. The cyclization reaction was carried
out following the procedures described above. Full deprotection was
carried out using a solution containing 10 mL of TFA/CH2Cl2 (95:5)
at room temperature during 2 h. The product 31 was isolated after
removal of solvents under reduced pressure and precipitation from ether.
A serine oxidation of derivative 31 (10-2 M) by an aqueous solution
containing 3 equiv of NaIO4 afforded the aldehyde component 33. The
product was directly purified by RP-HPLC to yield compound 33 as a
white powder (24 mg, 35.6 µmol, overall yield 60%). Mass spectrum
(ES-MS, positive mode) [M + H3O]+ calcd 692.8, found 692.3.
c[Arg-Gly-Asp-D-Phe-Lys(Fluorescein)] 41. To a solution contain-
ing the derivative 32 (4 mg, 6.5 µmol) in 2 mL of sodium acetate buffer
Peptide Conjugate 5. To a solution containing the derivative 18
(5.8 mg, 2.7 µmol) in 7 mL of sodium acetate buffer (0.1 mM, pH
4.0) was added the peptide 33 (14.8 mg, 22 µmol). The reaction was
stirred for 6 h at 25 °C. Conjugate 5 was isolated after purification by
RP-HPLC as a yellow powder (8.5 mg, 1.8 µmol, 75%). Mass spectrum
(ES-MS, positive mode) calcd 4771.2, found 4770.8.
Peptide Conjugate 6. To a solution containing the derivative 24
(10 mg, 5.9 µmol) in 2 mL of sodium acetate buffer (0.1 mM, pH
4.0)/acetonitrile (1:1) was added the peptide 32 (23 mg, 35 µmol). The
reaction was stirred for 2 h at 25 °C. Conjugate 6 was isolated after
purification by RP-HPLC as a yellow powder (18.8 mg, 4.4 µmol, 75%).
Mass spectrum (ES-MS, positive mode) calcd 4268.7, found 4269.0.
Peptide Conjugate 7. To a solution containing the derivative 24
(7.8 mg, 4.6 µmol) in 1 mL of sodium acetate buffer (0.1 mM, pH
4.0) was added the peptide 33 (21.7 mg, 32.2 µmol). The reaction was
stirred for 15 min at 25 °C. Conjugate 7 was isolated after purification
by RP-HPLC as a yellow powder (16.8 mg, 3.9 µmol, 95%). Mass
spectrum (ES-MS, positive mode) calcd 4324.8, found 4324.7.
Peptide Conjugate 8. To a solution containing the derivative 29
(4.4 mg, 3 µmol) in 2 mL of sodium acetate buffer (0.1 mM, pH 4.6)/
acetonitrile (5:1) was added the peptide 32 (15.7 mg, 23.8 µmol). The
reaction was stirred for 2 h at 25 °C. Conjugate 8 was isolated after
purification by RP-HPLC as a white powder (10.3 mg, 2.2 µmol, 73%).
Mass spectrum (ES-MS, positive mode) calcd 4042.4, found 4042.0.
Sample Preparation. Peptides were dissolved in phosphate buffer
saline (PBS, 140 mM NaCl, 10 mM Na2HPO4, 2 mM KCl) at a final
concentration of 1 mM.
Cell Culture. CHO3a cells (kind gift from Dr A.Duperray, Institut
Albert Bonniot, Grenoble) are stable transfectants of the chinese hamster
ovary (CHO) cell line. The clone was obtained by transfection of a
human â3 integrin encoding plasmid. Cells were cultured as adherent
monolayers in a DMEM 1640 medium supplemented with 10% (v/v)
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5738 J. AM. CHEM. SOC. VOL. 126, NO. 18, 2004

Multimeric System for Integrin Targeting and Endocytosis
A R T I C L E S
heat-inactivated fetal calf serum (FCS) and Geneticin (G418: 100 µg/
mL) at 37 °C in a humidified atmosphere of 5% CO2. All products
were purchased from Gibco (Cergy Pontoise, France). HUVEC were
cultured in medium 199 supplemented with 20% FCS, 100 µg/mL
heparin, and 50 µg/mL ECGs (gift of Dr A. Duperray).
Trypsinized CHO3a cells (105/well) were then mixed with the indicated
concentrations of peptides in PBS containing 1mM MgCl2 and incubated
for 45 min at 37 °C on the vitronectin-coated wells. The wells were
then rinsed, and cells were fixed and stained as described for the
adhesion assay. Each value represents the mean ( SEM of four
independent wells.
Adhesion Assay. Streptavidin-coated 96-well culture plates (Lab-
system, Helsinki, Finland) were coated extemporaneously by adding
100 µL of a 1 µM solution of each biotinylated peptide diluted in PBS
for 1 h at room temperature (rt). Nonspecific binding sites were saturated
with 3% bovine serum albumin in PBS for 30 min at rt. Trypsinized
CHO3a cells were resuspended at a concentration of 106 cells/mL in
PBS containing 1 mM MgCl2. 100 µL/well (105 cells) were added to
the wells and incubated subsequently for 45 min at 37 °C. Wells were
rinsed 3 times with PBS. Adherent cells were then fixed for 15 min
with ethanol and stained with methylene blue in borate buffer. Wells
were abundantly rinsed with water and dried overnight, and stained
cells were resuspended in 100 µL of 0.1 N HCl. The number of attached
cells was then evaluated by measuring the OD at 630 nm. Each value
represents the mean ( SEM of eight independent wells.
Internalization Assay. Trypsined CHO3a cells were washed in PBS
and incubated with 10 µM of FITC-labeled peptides in PBS 1mM
MgCl2 for 45 min at 37 °C or 4 °C. After being rinsed once in PBS
and fixed for 10 min with 4% paraformaldehyde, cells were incubated
for 5 min in a solution of Hoescht 33342 to stain the DNA. Cell
suspensions were mounted in Moeviol (Sigma, Saint Quentin Fallavier,
France), and slides examined using an Olympus AX 70 epifluorescence
microscope (60× lens).
Acknowledgment. We thank the Association pour la Re-
cherche contre le Cancer (ARC), the Centre National pour la
Recherche Scientifique (CNRS), the Institut National de la Sante´
et de la Recherche Me´dicale (INSERM), and the Institut
Universitaire de France (IUF) for supporting this work. We also
acknowledge La Ligue Nationale contre le Cancer for specific
financial support to E.G.
Competitive Adhesion Assay. MaxiSorp immunoplates (Nunc) were
incubated with 100 µL/well of a solution of vitronectin (Becton
Dickinson, Meylan, France) at 5 µg/mL in PBS. After 1 h at rt, the
solution was replaced by a 3% BSA solution in PBS for another 1 h.
Supporting Information Available: This material is available
free of charge via the Internet at http://pubs.acs.org.
JA049926N
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