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

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.


General information.
All the solvents and reagents for the syntheses were at least reagent grade quality and were used without further purification. Anhydrous N,N-dimethylformamide was purchased from ACROS Organics. Reactions were magnetically stirred and monitored by thin layer chromatography using Fluka Silica gel or Aluminium oxide on TLC-PET foils with fluorescent indicator at 254 nm. All reactions involving ruthenium (II) were carried out in the dark. C18 reversed phase silica gel (230−400 mesh) was used for chromatography. 1  sampling cone, 30 V; source Offset, 80 V; source temperature, 150°C and desolvation temperature, 200 °C. HPLC purification process on final compound 9 was performed on a semipreparative Infinity Agilent 1290 UHPLC equipped with a binary pump, a thermostatically controlled injection system, a thermostatically controlled column compartment and a Diode Array detector. Waters C18 (Atlantis T3) column was used and the elution conditions are described in the following table.

Ru II -calix[4]arene-[c-(RGDfK)]4 conjugate 9. Ru II -calix[4]arene complex 7 (5 mg, 3 µmol)
and c-(RGDfK)-alcyne 8 (9.7 mg, 15 µmol) were dissolved in DMF (0.5 mL) and copper nanoparticles (1.5 mg) were added. The synthesis was performed using micro-wave heating according to the following parameters: temperature = 50°C, run time = 30 sec, hold time = 60 min, medium stirring and power = 100W. The reaction mixture was concentrated under reduced pressure and the crude residue was purified by semi-preparative HPLC using an Atlantis T3 column (CH3CN/H2O/TFA; see the "General information" section for the details). The Ru IIcalix[4]arene-[c-(RGDfK)]4 conjugate 9 was obtained as a brown solid (3.9 mg, 0.9 µmol, 31%   ii) Similarly to the previously reported method [1], the linear peptide 8a was then dissolved in DMF (0.5 mM) and the pH values were adjusted to 8-9 by addition of DIPEA. PyBOP (1.3 equiv.) was added and the solution was stirred at room temperature for 1 h. Solvent was removed under reduced pressure and the residue was dissolved in a minimum of DCM. Diethyl ether was added to precipitate the cyclopeptide that was then triturated and washed three times with diethyl ether. The protected cyclopeptide was used in the next step without further purification.
iii) The protecting groups of the cyclopeptide were removed using a TFA/H2O/TIS (95:2.5:2.5) solution. After 1 h of reaction, the reaction mixture was concentrated under vacuum providing an oily residue. Pure compound 8 was obtained as a white powder after purification using preparative RP-HPLC (117 mg, 0.17 mmol, 66% yield over 3 steps).

MS (ESI+)
: m/z calculated for C32H45N9O8: 683.8, found 683.5. S16 Figure S14: UPLC analysis of compound 8.   As depicted in Figure S17, the presence of the free cyclic pentapeptide units c-[RGDfK] 8 does not have a significative influence on the luminescence intensity of the intermediate 7.
Determination of the luminescence lifetime in the same conditions, i.e. for 7 alone in MeCN under air (625 ns) and for 7 in the presence of 0.5 and 1.0 mM of c-[RGDfK] 8 (644 and 692 ns respectively), confirms that no quenching is taking place between the ruthenium complex and RGD peptidic units.
In conclusion, we can safely assert that no internal quenching process is taking place in conjugate 9 and that the photophysical and photochemical properties of the ruthenium complex anchored on the calixarene platform are conserved.