Application of click-click chemistry to the synthesis of new multivalent RGD conjugates

Article abstract of DOI:10.1039/c0ob00070a

New multivalent RGD-containing macromolecules were designed by exploiting two orthogonal chemoselective ligations. They were next applied to a competitive cell adhesion assay and used for the non invasive optical imaging of tumour in small animals.

Full text of DOI:10.1039/c0ob00070a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Application of click–click chemistry to the synthesis of new multivalent RGD
conjugates†
Mathieu Galibert,a Lucie Sancey,b Olivier Renaudet,a Jean-Luc Coll,b Pascal Dumya and Didier Boturyn*a
Received 30th April 2010, Accepted 20th July 2010
DOI: 10.1039/c0ob00070a
New multivalent RGD-containing macromolecules were designed by exploiting two orthogonal
chemoselective ligations. They were next applied to a competitive cell adhesion assay and used for the
non invasive optical imaging of tumour in small animals.
Introduction
The design and the synthesis of targeting molecules for diagnostic
and therapeutic applications represent a major goal in cancer
medicine. To this end, peptide ligands for various targets have
been identified using combinatorial libraries1 or phage display
method.2 To attain improved activity and receptor selectivity, it
is often essential to restrict the conformational space of peptides
by using them in a cyclic form.3 In this context, cyclic peptides
encompassing RGD (Arg-Gly-Asp) sequence have served as the
basis for the development of potent peptide ligands used to
Fig. 1 Structure of clustered RGD-containing compounds.
selectively target the aVb3 integrin.4 The latter represents an
attractive target for cancer therapeutic purposes.5 Furthermore,
it is well known that the multivalent display of a ligand enhances
sense c[-RbADfK-] motifs in the ligands whose valency was lower
than four. We used a combinatory assembling strategy to explore
the binding strength of the ligand to its receptor and can promote
all possible positions of the RGD motifs on the cyclodecapeptide
receptor-mediated internalisation of the bound entity.6 Today,
scaffold. Consequently, we were unable to isolate the different
the principle of multivalency has then been recognized as an
isomers that differ in the position of cyclic RGD pentapeptides
important strategy for the design of synthetic ligands.7 The effect
onto the cyclodecapeptide scaffold.
of multivalency in ligand binding was particularly demonstrated
To overcome this problem, the formation of two successive
for glycoconjugates,8 and for peptide ligands.9 Enhancements of
chemoselective linkages (“click–click” chemistry) enables a direct
biological activity were especially obtained from multivalent RGD
access to complex structures.14 Such reactions, in particular the
(Arg-Gly-Asp) peptide ligand used to target cell surface receptors
azide-alkyne cycloaddition, can be achieved under conditions
which are fully compatible with biological environments.15 We
recently reported an orthogonal chemoselective ligation strategy
such as aVb3 integrin.10
Recently, we have shown that tetrameric RGD-containing scaf-
folds exhibit desirable biological properties for tumour imaging11
that allows access to well defined biomolecular assemblies by
and for targeted drug delivery.12 These compounds contain a
exploiting the Huisgen dipolar cycloaddition and the oxime
cluster of four copies of a cyclo[-RGDfK-] monomer grafted onto
bond formation.16 Following this strategy, herein we describe
a cyclic decapeptide scaffold (Fig. 1). Preliminary work aimed
the synthesis of new multivalent RGD compounds such as the
fluorescent glycoconjugate 1 (Scheme 1). The incorporation of
at studying the effect of the multivalency parameter in terms
of interaction between the ligand and the target receptor and
the carbohydrate moiety may provide an enhanced solubility and
examining the contribution of each c[-RGDfK-] motif. For this
clearance. With the molecules in hand, we then concentrated our
purpose, we designed an array of peptide derivatives containing
work on assessing biological activities to determine the potency of
from one to four copies of the c[-RGDfK-] monomer (Fig. 1).13
the different RGD-containing compounds.
In order to obtain ligands with similar shape, similar steric
hindrance and close molecular weights, which is essential for their
comparison in vitro, we opted to substitute c[-RGDfK-] for non
Results and discussion
Chemical assemblies
aDe´partement de Chimie Mole´culaire, UMR CNRS/UJF 5250, ICMG
FR 2607, 570 rue de la chimie, BP 53, 38041 Grenoble cedex 9, France.
Scheme 1 illustrates the approach used for the synthesis of
E-mail: didier.boturyn@ujf-grenoble.fr; Fax: + 33 4 56 52 08 05; Tel: + 33
compounds 1–2. The biomolecular assembling process implies
two chemoselective ligations (click–click chemistry): the oxime
ligation17 and the Cu(I)-catalyzed azide-alkyne cycloaddition
(CuAAC).18 To introduce suitable functions within peptide
moities, we synthesized building blocks such as compounds 3 and
4 which contain protected serine (masked aldehyde) and alkyne
4 56 52 08 32
bInstitut Albert Bonniot, INSERM U823, BP 170, 38042 Grenoble cedex 9,
France
† Electronic supplementary information (ESI) available: General proce-
dures for peptide synthesis; Synthesis of 9; RP-HPLC profiles and ESI-MS
analysis of 5, 6, 7, 8, 11, 1, 2 and 19; General biological procedures. See
DOI: 10.1039/c0ob00070a
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Scheme 1 Synthesis of compounds 1–2. a) Standard Fmoc/t-Bu Solid-Phase Peptide Synthesis; b) PyBOP (1 equiv.), DIPEA (4 equiv.), 1 h; c) TFA/H2O
(95 : 5), 2 h; d) NaIO4 (10 equiv.), 30 min; e) 10 (3 equiv.), t-BuOH/H2O–AcOH (50 : 45 : 5), 2 h then 9 (6 equiv.), Cu(0) microsize powder (0.5 mg), pH 7.0,
18 h; For X = Lys, then Cy5–OSu (1 equiv.), DMF, DIPEA (pH 8), 3 h. Compounds 3 and 4 were prepared as previously described.16,19
groups, respectively (Scheme 1). The use of building blocks during
the solid-phase peptide synthesis (SPPS) reduces the number of
steps involved for the construction of such conjugates.19
To evaluate the RGD-containing compounds for further in vivo
studies, we decided to introduce a fluorescent reporter such as
Cyanine 5 (Cy5) because its near-infra red (NIR) band (lem
=
In this context, linear peptides encompassing chemoselective
ligations were prepared following rigorously the standard Fmoc/t-
Bu SPPS procedure using PyBOP as coupling reagent. The head-
to-tail cyclizations provided the desired cyclodecapeptide scaffolds
5 and 6. Deprotection of serine residue using a concentrated
TFA solution followed by a subsequent oxidation with periodate20
afforded key intermediates 7 and 8, isolated in sufficient purity
to carry out subsequent chemoselective assemblies. In parallel,
RGD-containing cyclopentapeptide 9 bearing the prerequisite
azide function and aminooxy-carbohydrate 10 were prepared as
described.16,21 Very recently, we have shown that cyclopeptide
assemblies are possible by means of orthogonal oxime and copper-
mediated click reactions in a stepwise or in a one-pot approach.16
The latter method is much desired, as it avoids lengthy separation
process and purification of intermediates while increasing overall
chemical yield. Biomolecular ligations of azidopeptide 9 and
aminooxy-carbohydrate 10 were then performed on either molec-
ular scaffold 7 or 8. Peptides 7 and carbohydrate 10 (3 equiv.) were
applied under mild acidic conditions using a solution containing
dilute acetic acid. Rapid oxime ligation of 10 was observed
(Fig. 2). Neutralizing the pH and addition of 9 (6 equiv.) and
copper microsize powder resulted in complete disappearance of
the intermediate and the exclusive formation of the expected
compound 2 in a 64% yield.
670 nm) can penetrate tissue up to 6 cm allowing non invasive
optical imaging in small animals. Furthermore, this NIR band is
free from interfering biofluorescence. For this purpose, the inter-
mediate 11 was synthesized according to the procedure described
above. Cy5 dye was then introduced at the lysine side-chain of
11 under neutral conditions (pH 8.0) affording the fluorescent
conjugate 1 in 72% yield after RP-HPLC purification. Compounds
1 and 2 were characterized by ES-MS and the observed molecular
weights were found to be in excellent agreement with the calculated
values.
To study the contribution of each c[-RGDfK-] motif, an array
of molecules 12–19 was designed and prepared according to the
method previously described (Fig. 3).12a,16 Briefly, RGD ligands
and nonsense RbAD peptides were introduced onto the scaffold
by using respectively the Huisgen dipolar cycloaddition and
orthogonal oxime bond formation, the latter providing shorter
linker.
Biological assays
The adhesion potency of the different multivalent RGD-
containing peptides was first determined using a traditional
ELISA-type inhibition assay. In this experiment, we measured
the efficiency of peptides to compete with vitronectin, the natural
substrate of the aVb3 integrin, when binding to HEK-b3 cells
that overexpress aVb3 receptors. HEK-b3 cells were therefore
incubated with soluble compounds 2, 12–18 at 37 ◦C onto
vitronectin-coated assay plates. The IC50 values, or concentration
of compounds required to inhibit 50% of the cells from attaching
to vitronectin, are reported in Table 1. As expected the negative
control peptide 13 did not inhibit cell adhesion to vitronectin
as reported for similar compounds.10d,13 Increasing the number
of RGD motifs from 1 to 3 gradually improved the potency of
the ligand to compete with vitronectin. We reasoned that the
observed multivalent effect arises from a statistical rebinding of the
RGD-containing compound due to the high local concentration
of RGD moieties. This phenomenon was observed for dendrimer
scaffolds.8f It is worth noting that compounds 16 and 17 that
Fig. 2 One-pot chemoselective assembly of peptides 7, 9, and carbohy-
drate 10. HPLC traces are shown at 2 h and 18 h. Int = intermediate.
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Fig. 3 Structure of compounds 12–19.
differ in the position of the RGD units onto the cyclodecapeptide
scaffold show similar IC50 (respectively, 7.1 and 8.2 mM). The
position of the RGD peptides onto the cyclodecapeptide scaffold
does not improve the affinity of the molecule. Compounds that
display four RGD units (i.e. molecules 2, 12 and 14) showed
potent inhibitory effect. Nevertheless, IC50 values for compounds
2 and 14 (respectively, 3.8 and 4.1 mM) are slightly lower than
the value obtained for compound 12 (4.9 mM) encompassing
shorter oxime linkers. Surprisingly, the molecule 15 encompassing
three RGD units displayed the best IC50 (2.8 mM). We previously
showed that compounds including three or four RGD ligands
exhibit close IC50.13 We argue that the shorter oxime linker used to
graft non-sense RbAD peptide within molecule 15 generates less
steric hindrance than unbound RGD moieties within molecules
2, 12 or 13 while the RGD-containing compound binds to aVb3
receptor.
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Table 1 Competitive cell adhesion assay
part of the general trend of organic chemistry taking control of
macromolecule synthesis to produce well-defined constructs that
could likely become the rule in drug applications. The ensuing
RGD compounds were then evaluated through competitive cell
adhesion assays and in vivo experiments. The results obtained
highlight the utility of a clustered ligand, and as expected the
grafting of an additional carbohydrate enhances clearance of the
RGD-containing compound. It is worth noting that our approach
is not limited to integrin ligands, it may be conceptually exploited
to synthesize other sophisticated macromolecular conjugates.
Compounds
#
RGD unit/molecule
IC50/mMa
Standard deviation/mMa
12
13
14
15
16
17
18
2
4
0
4
3
2
2
1
4
4.85
NI b
4.10
2.80
7.13
8.22
48.81
3.77
0.19
—
0.08
0.15
0.36
0.16
0.24
0.11
a Values were determined from three separate experiments; b NI, no
inhibition was observed.
Experimental
Cyclodecapeptide scaffolds 5. Linear decapeptides were as-
R
sembled on 2-chlorotritylchlorideꢀ resin (150 mg, loading of
Then, fluorescent derivatives 1 and 19 have been comparatively
evaluated in vivo in order to pinpoint the influence of the carbohy-
drate moiety. We measured the capacity of the molecules to target
tumour in mice. Fig. 4 shows typical FRI images of nude mice
bearing an subcutaneous human TS/A-pc tumour at different
time points after intravenous (iv) injection of 10 nmol fluorescent
molecules (i.e. 1 or 19) (see also the ESI†). A strong signal is
observed in the kidneys reflecting the prominent and fast renal
excretion of RGD-containing molecules as previously shown.11
One hour postinjection, fluorescent molecules accumulate in the
tumour but the whole body is also fluorescent due to the presence
of unbound circulating molecules. The average values for the
tumour/skin ratios were found to be similar for mice treated with
1 and for mice treated with 19 (respectively 1.39 0.37 and 1.32
0.15) (see Table S1 in the ESI†). The contrast (tumour/skin ratio)
was found to be statistically better with 1 (Fig. 4D) 3 h after
iv injection while the ratio was lower at late time. In comparison,
tumour/skin ratio for mice treated with 19 reaches its maximum at
6 h, and then slowly decreases (see the ESI†). These experimental
results are in good agreement with a better clearance of the
carbohydrate-containing compound 1.
0.8 mmol g-1) using the general procedure (see the ESI†) by using
building blocks 3 and 4. The cyclization reaction were carried out
in DMF using linear peptide (172 mg, 100 mmol, 0.5 mM) and
PyBOP (1 equiv.) for 1 h at room temperature. After completion
of the reaction, the solvent was evaporated and the cyclic peptide
5 was obtained as a white solid powder after ether precipitation
(161 mg, 100 mmol, quantitative yield). Mass spectrum (ES-MS,
positive mode) calc for C79H122N16O18: 1583.95, found m/z: 1584.0.
Cyclodecapeptide scaffolds 6. Following the procedure previ-
ously described and starting with linear peptide (171 mg, 92 mmol),
cyclic peptide 5 was obtained as a white solid powder (167 mg,
96 mmol, 96% yield). Mass spectrum (ES-MS, positive mode) calc
for C87H137N17O20 1741.16, found m/z: 1740.9.
Cyclodecapeptide scaffolds 7. Full deprotection of peptide 5
(161 mg, 100 mmol) was carried out in a solution containing 10 mL
of TFA/H2O (95 : 5) for 2 h at room temperature. The product
was isolated after removal of solvents under reduced pressure and
precipitation from Et2O. A serine oxidation by an aqueous solution
containing NaIO4 (10 equiv.) afforded the peptide 7. The crude
product was directly purified by using RP-HPLC affording the
compound 7 as a white powder. (72 mg, 48 mmol, 48% yield).
Mass spectrum (ES-MS, positive mode) calc for C69H101N15O16:
1396.67, found m/z : 1396.7.
Cyclodecapeptide scaffolds 8. Following the procedure pre-
viously described and starting with cyclic peptide 6 (167 mg,
96 mmol), peptide 8 was obtained as a white powder. (60 mg,
41 mmol, 43% yield). Mass spectrum (ES-MS, positive mode) calc
for C72H108N16O16 1453.76, found m/z : 1453.8.
Carbohydrate 10. Compound 10 was prepared as previously
described.21
Fig. 4 Representative images of optical imaging of subcutaneous tu-
mour-bearing mice observed at (A–C) 1 h and (B–D) 3 h after iv injection
of (A–B) 10 nmol 19 and (C–D) 10 nmol 1.
Peptide 2. To a solution containing the cyclodecapeptide 7
(5 mg, 3.5 mmol) in 500 mL tBuOH/H2O–AcOH (50 : 45 : 5) were
added the carbohydrate 10 (3 equiv.). The reaction mixture was
stirred for 2 h at room temperature. Then, the pH was adjusted
to 8 by addition of a NaHCO3 solution (10%) and the compound
9 c[-RGDfK(COCH2N3)-] (6 equiv.) and Cu(0) microsize powder
(5 equiv.) were added. The reaction mixture was stirred overnight
at room temperature and centrifuged for 5 min. The solution
was then purified by RP-HPLC to give the desired compound
2 (9.8 mg, 2.3 mmol, yield 64%). Mass spectrum (ES-MS, positive
mode) calc for C191H280N64O53 4320.76, found m/z 4320.5.
Conclusions
We have expanded the scope of click–click chemistry by gaining
access to new RGD-containing macromolecules. For instance, we
have shown that biomolecular assembly combining carbohydrate
and peptides is possible by means of orthogonal oxime and copper-
mediated click reactions in a one-pot synthesis. This approach is
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Peptide 11. To a solution containing the cyclodecapeptide 8
(5 mg, 3.4 mmol) in 500 mL tBuOH/H2O–AcOH (50 : 45 : 5) were
added the carbohydrate 10 (3 equiv.). The reaction mixture was
stirred for 2 h at room temperature. Then, the pH was adjusted
to 8 by addition of a NaHCO3 solution (10%) and the compound
9 c[-RGDfK(COCH2N3)-] (6 equiv.) and Cu(0) microsize powder
(5 equiv.) were added. The reaction mixture was stirred overnight
at room temperature and centrifuged for 5 min. The solution
was then purified by RP-HPLC to give the desired compound
11 (8.7 mg, 2 mmol, yield 58%). Mass spectrum (ES-MS, positive
mode) calc for C194H287N65O53 4377.85, found m/z 4377.7.
Institut National du Cancer (INCA), the Nanoscience Foundation
and NanoBio (Grenoble).
Notes and references
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Peptide 1. The peptide 11 (7.0 mg, 1.59 mmol) was dissolved
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pH 9. The solution was added to CyTM 5 Mono NHS Ester (1.2 mg,
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C216H308N65O53S2 4727.39, found 4727.4.
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Competitive cell adhesion assays. Competitive assay was car-
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for 1 h at room temperature with 5 mg mL-1 vitronectin in PBS
and blocked for 30 min with 3% bovine serum albumin (BSA).
Varying amounts of peptides were added simultaneously with 105
trypsinated HEK-b3 cells to the wells and the plate was incubated
for 30 min at 37 ◦C. Wells were rinsed three times with cold PBS to
remove vitronectin-unbound cells. Attached cells were then fixed
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Acknowledgements
This work was supported by the Universite´ Joseph Fourier, the
Centre National de la Recherche Scientifique (CNRS), the Institut
National de la Sante´ et de la Recherche Me´dicale (INSERM), the
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