Synthesis and photophysical studies of a multivalent photoreactive RuII-calix[4]arene complex bearing RGD-containing cyclopentapeptides

Article abstract of DOI:10.3762/bjoc.14.150

Photoactive ruthenium-based complexes are actively studied for their biological applications as potential theragnostic agents against cancer. One major issue of these inorganic complexes is to penetrate inside cells in order to fulfil their function, either sensing the internal cell environment or exert a photocytotoxic activity. The use of lipophilic ligands allows the corresponding ruthenium complexes to passively diffuse inside cells but limits their structural and photophysical properties. Moreover, this strategy does not provide any cell selectivity. This limitation is also faced by complexes anchored on cell-penetrating peptides. In order to provide a selective cell targeting, we developed a multivalent system composed of a photoreactive ruthenium(II) complex tethered to a calix[4]arene platform bearing multiple RGD-containing cyclopentapeptides. Extensive photophysical and photochemical characterizations of this Ru(II)–calixarene conjugate as well as the study of its photoreactivity in the presence of guanosine monophosphate have been achieved. The results show that the ruthenium complex should be able to perform efficiently its photoinduced cytotoxic activity, once incorporated into targeted cancer cells thanks to the multivalent platform.

Full text of DOI:10.3762/bjoc.14.150

Synthesis and photophysical studies of a multivalent
photoreactive RuII-calix[4]arene complex bearing
RGD-containing cyclopentapeptides
Sofia Kajouj1, Lionel Marcelis*1,2, Alice Mattiuzzi3, Adrien Grassin4, Damien Dufour5,
Pierre Van Antwerpen5, Didier Boturyn4, Eric Defrancq4, Mathieu Surin6,
Julien De Winter7, Pascal Gerbaux7, Ivan Jabin*3 and Cécile Moucheron*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 1758–1768.
1Laboratoire de Chimie Organique et Photochimie, Université libre de
Bruxelles, Avenue F.D. Roosevelt 50, CP 160/08, 1050 Bruxelles,
Belgium, 2Engineering of Molecular NanoSystems, Ecole
Polytechnique de Bruxelles, Université libre de Bruxelles (ULB),
Avenue F.D. Roosevelt 50, CP165/64, B-1050 Brussels, Belgium,
3Laboratoire de Chimie Organique, Université libre de Bruxelles,
Avenue F.D. Roosevelt 50, CP 160/06, 1050 Bruxelles, Belgium,
4Université Grenoble Alpes, Département de Chimie Moléculaire
UMR CNRS 5250, CS 40700, 38058 Grenoble Cedex 09, France,
5Analytical Platform of the Faculty of Pharmacy, Université libre de
Bruxelles, Boulevard du Triomphe, Campus de la Plaine, CP205/05,
1050 Bruxelles, Belgium, 6Laboratory for Chemistry of Novel
Materials, Center for Innovation and Research in Materials and
Polymers, University of Mons – UMONS, 20, Place du Parc, B-7000
Mons, Belgium and 7Organic synthesis and Mass Spectrometry
Laboratory, University of Mons - UMONS, Place du Parc 23, B-7000
Mons, Belgium
doi:10.3762/bjoc.14.150
Received: 15 April 2018
Accepted: 21 June 2018
Published: 16 July 2018
This article is part of the thematic issue "Macrocyclic and supramolecular
chemistry".
Guest Editor: M.-X. Wang
© 2018 Kajouj et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Lionel Marcelis* - lmarceli@ulb.ac.be; Ivan Jabin* - ijabin@ulb.ac.be;
Cécile Moucheron* - cmouche@ulb.ac.be
* Corresponding author
Keywords:
anticancer drug; calixarene; cell targeting; RGD peptide; ruthenium
complex
Abstract
Photoactive ruthenium-based complexes are actively studied for their biological applications as potential theragnostic agents against
cancer. One major issue of these inorganic complexes is to penetrate inside cells in order to fulfil their function, either sensing the
internal cell environment or exert a photocytotoxic activity. The use of lipophilic ligands allows the corresponding ruthenium com-
plexes to passively diffuse inside cells but limits their structural and photophysical properties. Moreover, this strategy does not
provide any cell selectivity. This limitation is also faced by complexes anchored on cell-penetrating peptides. In order to provide a
selective cell targeting, we developed a multivalent system composed of a photoreactive ruthenium(II) complex tethered to a
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calix[4]arene platform bearing multiple RGD-containing cyclopentapeptides. Extensive photophysical and photochemical charac-
terizations of this Ru(II)–calixarene conjugate as well as the study of its photoreactivity in the presence of guanosine monophos-
phate have been achieved. The results show that the ruthenium complex should be able to perform efficiently its photoinduced cyto-
toxic activity, once incorporated into targeted cancer cells thanks to the multivalent platform.
Introduction
Long-living luminescent polyazaaromatic ruthenium(II) com- preventing their direct use in biological applications. More
plexes are intensively studied in a biological context, in particu- lipophilic ligands such as bathophenanthroline and modified
lar (i) for their ability to sense their environment and (ii) for dppz were developed and the internalization of the correspond-
their photoreactivity towards relevant biological targets [1-4]. ing RuII complexes was demonstrated [29-32]. These com-
Sensors for biological species are mostly based on complexes plexes are however not photoreactive due to the absence of
bearing the well-known dppz ligand (dppz = dipyrido[3,2- π-deficient ligands. More recently, RuII complexes bearing two
a:2’,3’-c]phenazine) and its derivatives. J. K. Barton et al. modified TAP ligands with highly lipophilic moieties were re-
demonstrated in 1990 that [Ru(bpy)2(dppz)]2+ behaves as a ported [33]. These compounds are able to enter the cells and
light-switch for DNA [5]: this complex is not luminescent photoinduce caspase-dependent and reactive-oxygen-species-
in water but upon intercalation within the DNA base pairs dependent apoptosis. Another strategy for the design of cell
stack, the complex luminescence is restored. Derivatives of penetrating photoreactive RuII complexes consists of tethering
[Ru(bpy)2(dppz)]2+ and complexes bearing similar aromatic the complex to a vector that allows a cellular uptake. In this
planar ligands were developed to probe specific sites of DNA, context, OsII, RhIII and RuII complexes were anchored to cell
such as mismatches [6-8], abasic sites [9] or G-quadruplexes penetrating peptides (CPP) such as polyarginine [34-37]. The
[10,11]. Aside photosensors, photoreactive complexes able to tethering of a photoreactive RuII complex on the transacti-
damage biological targets were also developed. These com- vating transcriptional activator (TAT) peptide was also re-
plexes are mainly used to induce damages in cancerous cells ported and it was shown that the corresponding RuII conjugate
upon light irradiation. Two types of photooxidative damages could be internalized inside HeLa cells without any modifica-
can be induced: (i) by photosensitization of singlet oxygen and tion of the photochemical properties of the complex [38].
subsequent generation of highly reactive oxygen species (ROS)
(type I photosensitization) or (ii) by direct oxidative electron It should be noted that modifications of ligands to make the
transfer to biological molecules such as DNA or amino acids resulting complexes more lipophilic or the conjugation of a
(type II photosensitization). In particular, it was shown that RuII complex to a CPP do not provide any control on the way these
complexes containing at least two highly π-deficient polyaza- complexes will be internalized by cells and prevent thus any
aromatic ligands such as 1,4,5,8-tetraazaphenanthrene (TAP) targeting of malignant cells over healthy ones. The next step in
[12-14] or 1,4,5,8,9,12-hexaazatriphenylene (HAT) [15] are the development of phototherapeutic agents based on polyaza-
able to oxidize the guanine base (G) of DNA or the tryptophan aromatic RuII complexes is thus the specific targeting of
(Trp) amino acid residue through a photoinduced electron- cancerous cells. In this regard, αvβ3 integrin represents an inter-
transfer (PET) process [16-19]. Interestingly, the two radical esting target as this membrane receptor is overexpressed in the
species generated by this PET can recombine to form a cova- endothelial cells of neoangiogenic vessels and in several human
lent photoadduct [20-22]. When this photoadduct is formed tumor cells [39,40]. It is well known that RGD-containing
with the guanine base, the activity of enzymes such as RNA oligopeptides (RGD = Arg-Gly-Asp tripeptide pattern) bind
polymerase or endonuclease is inhibited in vitro at the level of selectively to αvβ3 integrin with a high affinity and a very high
the photoadduct [23,24]. In order to target a specific DNA se- selectivity [41-43]. As multivalency enhances the binding
quence, photoreactive RuII complexes have been anchored to strength of a ligand to its receptor [44-46], clustered RGD-con-
specific antisense oligonucleotides to inhibit the expression of taining compounds were developed and were shown to exhibit
the complementary targeted genes under illumination [25,26]. attractive biological properties for the imaging of tumors [47-
This photoinduced gene-silencing strategy has been proven to 50] and for the targeted drug delivery [51-53].
be also efficient in living cells [27,28], paving the way for the
use of photoactivable RuII complexes as photocontrolled anti- In the course of designing phototherapeutic agents that could
cancer therapeutic agents.
specifically target cancerous cells, we envisaged to graft a
photoreactive RuII complex on a multivalent platform deco-
Despite their interesting photochemical properties, photoreac- rated with multiple RGD-containing cyclopentapeptides. A
tive RuII complexes have shown low cell-penetration efficiency, calix[4]arene moiety was chosen as the multivalent platform as
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this rigid macrocycle displays two distinct faces that can be azide–alkyne cycloaddition (CuAAC) [66-68] (Figure 1). It was
selectively functionalized [54-56]. It is noteworthy that the thus necessary to block the calix[4]arene skeleton in the cone
calix[4]arene skeleton has been already exploited for the devel- conformation and to functionalize separately the two distinct
opment of multivalent glyco- and peptidocalixarenes that can rims (Scheme 1). Firstly, known calixarene 2 with an appending
be recognized by cell-membrane receptors [57-59] and of carboxylate arm on the small rim was synthesized from com-
calixarene derivatives able to specifically target membrane pro- mercial p-tert-butylcalix[4]arene 1 according to a four-step se-
teins involved in the angiogenesis process [60]. Furthermore, quence [69]. Note that propyl groups were chosen for the modi-
the use of calixarenes for biological applications is the subject fication of the small rim because these groups are the smallest
of intensive researches. They are indeed exploited in various possible for blocking the oxygen-through-the-annulus rotation
areas such as surface recognition, structural mimes or mem- of the aromatic units [70]. The nitro groups of 2 were then
brane receptor inhibition [61-63], and it was also shown that reduced using SnCl2·2H2O in ethanol, affording tetra-amino
calixarenes themselves display antibacterial, antiviral, and anti- compound 3 [69] in 50% yield. Diazotation followed by nucleo-
cancer properties [64].
philic substitution with sodium azide gave the desired tetra-
azido compound 4 in 56% overall yield from 3. It is note-
Herein, we describe the synthesis of a multivalent photothera- worthy that the introduction of the azido groups on the
peutic agent designed in order to specifically target membrane calix[4]arene scaffold was clearly confirmed by the presence of
receptors involved in the angiogenesis process. The multivalent an intense band at 2108 cm−1 in the IR spectrum of 4. Phenan-
system is composed of a photoreactive [Ru(TAP)2phen]2+ throline derivative 5 was synthesized from 5-glycinamido-1,10-
complex tethered to a calix[4]arene platform bearing four phenanthroline in a two-step sequence consisting of a peptide-
c-[RGDfK] moieties [65] (Figure 1). Before studying this type coupling reaction with a Boc-protected glycine N-hydro-
conjugate in vitro, it was first mandatory to check that the succinimide ester followed by the deprotection of the amino
photochemistry of the RuII complex was not altered by the pres- group (see Supporting Information File 1) [71]. Different cou-
ence of the targeting platform. The photophysical properties of pling agents (DCC/HOBt, EDC·HCl/HOBt, PyBOP) and condi-
this RuII–calixarene conjugate were thus examined and com- tions were then tested for the peptide-type coupling reaction be-
pared to those of the reference complex [Ru(TAP)2phen]2+.
tween calix[4]arene 4 and phenanthroline derivative 5. The use
of an excess of 5 (2 equiv) in the presence of EDC·HCl and
HOBt in DMF at room temperature led to the best yield and the
easiest purification process. Under these optimal conditions, the
Results and Discussion
Synthesis of RuII-calixarene conjugate 9
For the synthesis of the target multivalent system, the desired compound 6 was isolated in a high 94% yield. Finally,
strategy relies on the anchoring i) of the photoreactive the reaction between [Ru(TAP)2(H2O)2]2+ and 6 in DMF at
[Ru(TAP)2phen]2+ complex on the calix[4]arene small rim 100 °C gave the RuII-calix[4]arene complex 7 in 95% yield
through a peptide-type coupling and ii) of the four c-[RGDfK] after C18 reversed-phase silica gel column chromatographic
moieties on the opposite rim through a copper-catalyzed purification. Complex 7 was fully characterized by 1D and
Figure 1: Targeted multivalent phototherapeutic agent and its calix[4]arene-based precursor. RGD = Arg–Gly–Asp residues, f = D-Phe residue.
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Scheme 1: Synthesis of RuII-calix[4]arene complex 7.
2D NMR spectroscopy in CD3CN at 600 MHz. In accordance cyclopeptide 8 in the presence of CuNPs and the mixture was
with the presence for the chiral Ru(TAP)2phen moiety, the heated by microwave (100 W) at 50 °C for 1 hour. The use of
1H NMR spectrum of 7 is characteristic of a C1 symmetrical CuNPs greatly facilitated the monitoring of the reaction and the
compound as all the protons belonging to the ArH, ArCH2 and work-up, as these nanomaterials being easily removed from
OPr group are differentiated. Moreover, complex 7 was also the crude mixture by simple centrifugation. To our delight,
characterized by high-resolution mass spectrometry (HRMS). [Ru(TAP)2phen]2+-calix[4]arene-[c-(RGDfK)]4 conjugate 9
The ESI mass spectrum displays two intense signals at m/z was isolated in 31% yield after purification by semi-preparative
764.736 and m/z 1642.456 that are attributed respectively to the RP-HPLC (Scheme 2). The successful synthesis and purifica-
doubly charged 72+ and singly charged [7 + CF3COO−]+ by tion of conjugate 9 was also confirmed by HRMS. Indeed, the
comparison between the experimental and theoretical isotope ESI mass spectrum features several peaks corresponding to
distributions (see Supporting Information File 1).
characteristic ions of different charge states at m/z 1421.577
(3+), 1066.434 (4+) and 853.556 (5+) that are attributed to
With RuII-calix[4]arene complex 7 in hands, we next moved [9 + H]3+, [9 + 2H]4+ and [9 + 3H]5+ by comparison between
to the introduction of the cellular targeting units on the large the experimental and theoretical isotope distributions (see Sup-
rim through copper-catalyzed azide–alkyne cycloaddition porting Information File 1).
(CuAAC). Note that the triazole moieties that would result from
such a cycloaddition are known to be stable towards hydrolysis Molecular modeling simulations were carried out to provide
and protease, which allows their use in a biological environ- insights into the size and morphology of conjugate 9. An opti-
ment [72]. For the CuAAC, the use of CuI-generated in situ mized geometry is presented in Figure 2, as issued from a mo-
from a mixture of CuSO4·5H2O and sodium ascorbate is often lecular dynamics (MD) simulations. The ruthenium complex
reported in the field of calixarene chemistry [66,73-78]. Unfor- and the RGD units are spatially well-separated thanks to their
tunately, this methodology led to poor yields and a lack of grafting on opposite faces of the rigid calixarene-based plat-
reproducibility in the case of calixarene 7 and c-[RGDfK]- form. In this conformation, the distances between the Ru atom
alkyne 8, even when a microwave heating was used. We then and each of the nearest carbon atoms of RGDfK units exceed
evaluated the use of copper nanoparticles (CuNPs), as these 30 Å. Along the MD simulations, we noticed that the Ru com-
nanomaterials are known to catalyze efficiently a wide range of plex remained far from the cyclic pentapeptides. This is due to
organic reactions and notably the azide–alkyne cycloaddition the fact that the linkers of each arm are smaller than the size of
[79]. Calixarene 7 was reacted with a slight excess (5 equiv) of the calixarene platform, preventing contacts between the Ru
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Scheme 2: Synthesis of RuII-calix[4]arene-[c-(RGDfK)]4 conjugate 9.
atoms at C=O in the linker and the hydrogen atoms of (N–H) of
arginine of a neighboring ‘arm’; (ii) H-bonds between arginine
terminal N–H and C=O of the peptide bond of phenylalanine of
an adjacent cyclic pentapeptide (see Supporting Information
File 1), yielding adjacent cyclic pentapeptides in close prox-
imity for a large set of conformations.
This separation between the Ru complex and the cyclic peptides
by the calixarene should be an advantage by preventing any
negative effect of the RGD peptidic units on the photochem-
istry of the complex and, alternatively, prevents any influence
of the complex on the affinity of the RGD patterns to interact
with the targeted integrins. However, the possible H-bonding
interactions between neighboring RGD units could be a draw-
back in view of the accessibility of the arginine groups to
interact with the integrins.
Photophysical properties of RuII-calixarene
conjugate 9
The absorption and emission spectra of Ru-calix(RGD)4 conju-
gate 9 (as its CF3COO− salt) were recorded in water at room
temperature (Figure 3). These spectro-scopic data are gathered
in Table 1 with the ones of the free [Ru(TAP)2phen]2+ complex
for comparison purpose.
Figure 2: MD snapshot showing an optimized model of conjugate 9.
RGDfK units are depicted in orange ribbons, the calixarene is in blue
and the Ru complex is colored by atom type.
complex and the RGDfK units. The global structure has an av- Conjugate 9 exhibits absorption bands at 416 and 458 nm that
erage radius of gyration Rg of 1.25 nm ± 0.1 nm. Noteworthy, corresponds to dπ(Ru)–π*(phen/TAP) metal-to-ligand charge
the distance between the RGDfK units largely varies along the transitions (MLCT) similarly to what is observed for the unteth-
MD simulations, ranging from 10 Å to 24 Å (average at 17 Å), ered [Ru(TAP)2phen]2+ complex (MLCT bands at 412 and
as estimated from the distance between equivalent carbon atoms 464 nm). The presence of the calix[4]arene platform has thus no
crossing the linker and the cyclic pentapeptides. This large vari- impact on the visible part of the spectrum. The influence of the
ation in the distance is due to the flexibility of the linkers be- calixarene moiety is however visible in the UV region of the
tween the calixarene platform and the RGDfK units, together spectrum (around 200 nm) where the absorption bands are more
with the many possibilities of H-bonding between: (i) oxygen intense. This increase is due to the contribution of the peptidic
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Photoreactivity of RuII-calixarene conjugate 9
The photoreactivity of Ru-TAP complexes is based on their
ability to induce direct oxidation of guanine upon light excita-
tion. In order to confirm that the tethering onto the calixarene
platform does not impede the conjugated complex to photoreact
with its biological target, we measured the evolution of the lu-
minescence intensity and the excited state lifetime of conjugate
9 as function of the concentration of guanosine monophosphate
(GMP, Figure 4).
Figure 3: Absorption and emission spectra of RuII-calix[4]arene-
[c-(RGDfK)]4 conjugate 9 in water.
subunits of the RGD moieties and of the aromatic units of the
calixarene. It should be noted that the absorption at wave-
lengths longer than 550 nm does not go perfectly down to zero.
This phenomenon is likely due to some light scattering caused
by the presence of some small aggregates in solution. It appears
that conjugate 9 is not completely soluble in pure water despite
the presence of the charged RuII complex and the peptidic
moieties on the calix[4]arene scaffold. Fortunately, these small
aggregates totally disappeared when only 5% of DMSO was
added to the medium [80].
Figure 4: Luminescence intensity and excited state lifetime of conju-
gate 9 in the presence of GMP measured in 10 mM Tris·HCl buffer at
pH 7.0.
The photoluminescence emission originating from the 3MLCT Stern–Volmer analyses indicate that a dynamic quenching is
state is centered at 645 nm for both conjugate 9 and reference occurring, with a quenching rate close to the diffusion limit
[Ru(TAP)2phen]2+complex. We measured the luminescence (kQ = 5.6 108 M−1s−1 in intensity and kQ = 5.3 108 M−1s−1 in
lifetime and determined the quantum yield of luminescence lifetime). This quenching of the luminescence of conjugate 9 in
under air and argon atmosphere for conjugate 9 and reference the presence of GMP reveals that a photoinduced electron
[Ru(TAP)2phen]2+ in water with 5% DMSO in order to avoid transfer can take place between the excited complex and the
any formation of aggregates. The data gathered in Table 1 guanine moiety, which could give rise to the formation of a
clearly indicate that the tethering of the [Ru(TAP)2phen]2+ photoadduct from the recombination of the monoreduced com-
complex onto the calixarene platform does not induce any mod- plex and the radical guanine generated after the photoinduced
ification of the photophysical properties of the complex. In electron transfer (PET). In order to confirm the occurrence of
order to rule out any intramolecular quenching processes, PET, transient absorption measurements with conjugate 9 were
control experiments were realized with the complex grafted performed in the absence and in the presence of GMP. The re-
onto the unmodified calixarene (conjugate 7) in the presence of corded transient absorption spectra are presented in Figure 5. In
free cyclic pentapeptide units c-[RGDfK] 8 (see Supporting absence of GMP, the transient absorption spectrum of conju-
Information File 1). No modification of the luminescence by gate 9 is dominated by the luminescence, the ground state
intermolecular quenching was observed, confirming the absence bleaching and some excited state absorption around 340 nm
of internal quenching in the conjugate 9.
whereas in the presence of GMP a positive transient signal can
Table 1: Photophysical properties of conjugate 9 and [Ru(TAP)2phen]2+ in water.
Complex
λabs (nm)
λem (nm)
ΦAir a,b
ΦAr a,b
τavAir b,c (ns)
τavAr b,c (ns)
[Ru(TAP)2phen]2+
231, 272, 412, 464
274, 416, 458
645
645
0.029
0.025
0.055
0.044
714
901
891
conjugate 9
1087
aPhotoluminescence quantum yields are determined by comparison with [Ru(bpy)3]2+. Errors on Φ estimated to <20%. bMeasurement with 5%
DMSO. cErrors on lifetime estimated to 15%.
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be observed around 500 nm on a long time scale. This transient
is specific of a monoreduced RuII-TAP•− species [81], confirm-
ing that a PET occurs.
To verify if a photoadduct can be obtained between the com-
plex anchored on the calixarene platform and a guanine base, a
continuous irradiation of a solution containing conjugate 9 and
GMP was achieved. The crude irradiation mixture was then
analyzed by MALDI mass spectrometry (HRMS, Figure 6).
Alongside the parent conjugate 9 ions, ionized species at higher
mass-to-charge ratio (m/z = 4625.9) are detected and formally
correspond to the addition of GMP minus two hydrogen atoms.
The comparison between the experimental and theoretical
isotope patterns confirms (inset Figure 6) that irradiation of
conjugate 9 and GMP efficiently yielded the desired photo-
adduct.
Conclusion
The present work validates the design strategy that consists in
using calix[4]arenes as addressable platforms for the elabo-
ration of multivalent photoreactive systems that could poten-
tially target and enter cancer cells. The selective tethering of
a photoreactive Ru-TAP complex on the small rim of a
calix[4]arene and the introduction of four c-[RGDfK] moieties
on its large rim were efficiently achieved. In good agreement
with molecular modeling simulations, it was shown that the
Figure 5: Transient absorption spectra of RuII-calix[4]arene-
[c-(RGDfK)]4 conjugate 9 (in 10 mM Tris·HCl buffer at pH 7.0)
measured 500 ns after the laser pulse (gray, top) and 1 µs after the
laser pulse in the presence of 10 mM GMP (purple, bottom).
Figure 6: MALDI–MS analysis of a solution containing conjugate 9 and GMP after continuous light irradiation. In the inset, the experimental (bottom)
and theoretical (top) isotope distributions are compared for both 9+ and [9 + GMP − 2H]+ ions.
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photophysical properties of the tethered complex 9 are not (3-(4-tert-butylphenyl)-2-methyl-2-propenylidene)malononi-
altered by the anchoring onto the calixarene platform and that trile, was prepared as a 40 mg/mL solution in chloroform. The
the cyclic pentapeptide units do not interfere with the photore- matrix solution (1 μL) was applied to a stainless-steel target and
activity of the complex. Moreover, we verified that the com- air-dried. The crude photoirradiation product was dissolved in
plex is able to photoreact with its biological target, i.e., the acetonitrile and 1 μL aliquot of this solution was applied onto
guanine content of DNA, by demonstrating the occurrence of a the target area (already bearing the matrix crystals) and then air-
photoinduced electron transfer and the formation of a covalent dried.
photoadduct between the Ru-calix(RGD)4 conjugate 9 and
GMP. In conclusion, the ruthenium complex should be able to The HPLC purification process on final compound 9 was per-
perform efficiently its photoinduced cytotoxic activity, once in- formed on a semi-preparative Infinity Agilent 1290 UHPLC
corporated into targeted cancer cells thanks to the multivalent system equipped with a binary pump, a thermostatically con-
platform. In cellulo studies are currently under investigation and trolled injection system, a thermostatically controlled column
will be reported in the near future.
compartment and a Diode Array detector. Waters C18 (Atlantis
T3) column was used and the elution conditions are described in
Supporting Information File 1.
Experimental
General: All the solvents and reagents for the syntheses were at
least reagent grade quality and were used without further purifi- Calix[4]arenes 2 and 3 were synthesized from commercial
cation. Anhydrous N,N-dimethylformamide was purchased p-tert-butylcalix[4]arene 1 according to procedures described in
from ACROS Organics. Reactions were magnetically stirred the literature [69]. The experimental procedures and characteri-
and monitored by thin-layer chromatography using Fluka silica zation data for calixarene derivatives 4, 6, 7 and 9, phenanthro-
gel or aluminium oxide on TLC-PET foils with fluorescent line derivative 5 and c-[RGDfK]-alkyne 8 are given in Support-
indicator at 254 nm. All reactions involving ruthenium(II) ing Information File 1.
were carried out in the dark. C18 reversed-phase silica gel
(230−400 mesh) was used for chromatography. 1H NMR spec- The UV–vis absorption spectra were recorded on a Perkin-
tra were recorded at ambient temperature on Bruker 300, Elmer Lambda UV–vis spectrophotometer and the emission
Variant 400 and 600 MHz spectrometers and 13C NMR spectra spectra with a Shimadzu RF-5001 PC spectrometer (detection:
were recorded at 75, 100 or 150 MHz. Traces of residual sol- Hamamatsu R-928 red-sensitive photomultiplier tube, excita-
vents were used as internal standards for 1H NMR (7.26 ppm tion source: xenon lamp 250 W). Emission quantum yields were
for CDCl3, 3.31 for CD3OD, 4.79 for D2O, 2.50 for DMSO-d6 determined by integrating the corrected emission spectra over
and 1.94 ppm for CD3CN) and 13C NMR (77.16 ppm for the frequencies. [Ru(bpy)3]2+ in water under air was chosen as
CDCl3, 49.00 for CD3OD, 39.52 for DMSO-d6 and 118.26 ppm the standard luminophore (quantum yield of 0.042 under argon).
for CD3CN) chemical shift referencing. Abbreviations: The luminescence lifetimes were measured by the time-corre-
s = singlet, d = doublet, t = triplet, q = quartet, br = broad, lated single photon counting (TC-SPC) technique with the Edin-
m = massif, mult = multiplet). 2D NMR spectra (COSY, burgh Instruments LifeSpecII Picosecond Fluorescence Life-
HSQC, HMBC, HSQC) were recorded to complete signal as- time Spectrometer equipped with a laser diode (λ = 439 nm,
signments. Melting points were recorded on a Stuart Scientific pulse = 100 ps). The samples were thermostatted at 20 ± 2 °C
Analogue SMP11 or Büchi Melting Point B-545. Infrared spec- with a Haake Model NB22 temperature controller. The data
tra were recorded on a Bruker Alpha (ATR) spectrometer.
were collected by a multichannel analyzer (2048 channels) with
a number of counts in the first channel equal to 104. The result-
High-resolution mass spectra were obtained on a Waters Synapt ing decays were deconvoluted for the instrumental response and
G2-Si spectrometer (Waters, Manchester, UK) equipped with fitted to the exponential functions using the original manufac-
an electrospray ionization used in the positive ion mode. Source turer software package (Edinburgh Instruments). The reduced
parameters were as follow: capillary voltage, 3.1 kV; sampling χ2, weighted residuals and autocorrelation function were em-
cone, 30 V; source Offset, 80 V; source temperature, 150 °C ployed to judge the quality of the fits.
and desolvation temperature, 200 °C. Matrix-assisted laser de-
sorption/ionization time-of-flight (MALDI-ToF) mass spectra For molecular modelling simulations, the initial structure of the
were recorded using a Waters QToF Premier mass spectrome- ruthenium complex was obtained from previous DFT calcula-
ter equipped with a Nd-YAG laser of 355 nm with a maximum tions using a previously reported methodology [82]. The
pulse energy of 65 μJ delivered to the sample at 50 Hz repeating geometries of the RAFTs and calixarene were built using DS
rate. Time-of-flight mass analyses were performed in the reflec- BIOVIA© software and geometry-optimized using the
tion mode at a resolution of about 10 000. The matrix, trans-2- CHARMM force-field, taking into account previous molecular
1765

Beilstein J. Org. Chem. 2018, 14, 1758–1768.
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scape of this large molecule, the conformations reported here
represent relaxed geometries showing possible intermolecular
contacts between the cyclic pentapeptides. The analysis and vi-
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Acknowledgements
S.K. thanks the Fonds pour la Formation à la Recherche dans
l’Industrie et dans l’Agriculture (FRIA-FRS, Belgium) for her
PhD grant. C.M. and M.S. thank the F.R.S.-FNRS (“Fonds
National pour la Recherche Scientifique”, Belgium) for contin-
uing support, notably through the grants 2.4530.12, 2.4615.11,
UN02715F, CDR J.0022.18, and F.4532.16. This work was also
supported by the COST action CM 1202 and a “10 km de Brux-
elles” grant. The mass spectrometry laboratory @ UMONS is
grateful to the “Fonds National pour la Recherche Scientifique”
(FNRS-Belgium) for financial support in the acquisition of the
Waters QTOF premier and the Waters Synapt G2-Si mass spec-
trometers. D.B. and E.D. acknowledge support from the French
National Research Agency (Labex program ARCANE, ANR-
11-LABX-0003-01). L.M. thanks Pr. Kristin Bartik and
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