An artificial metalloenzyme that can oxidize water photocatalytically: design, synthesis, and characterization.

In nature, light-driven water oxidation (WO) catalysis is performed by photosystem II via the delicate interplay of different cofactors positioned in its protein scaffold. Artificial systems for homogeneous photocatalytic WO are based on small molecules that often have limited solubility in aqueous solutions. In this work, we alleviated this issue and present a cobalt-based WO-catalyst containing artificial metalloenzyme (ArM) that is active in light-driven, homogeneous WO catalysis in neutral-pH aqueous solutions. A haem-containing electron transfer protein, cytochrome B5 (CB5), served to host a first-row transition-metal-based WO catalyst, CoSalen (CoIISalen, where H2Salen = N,N'-bis(salicylidene)ethylenediamine), thus producing an ArM capable of driving photocatalytic WO. The CoSalen ArM formed a water-soluble pre-catalyst in the presence of [Ru(bpy)3](ClO4)2 as photosensitizer and Na2S2O8 as the sacrificial electron acceptor, with photocatalytic activity similar to that of free CoSalen. During photocatalysis, the CoSalen-protein interactions were destabilized, and the protein partially unfolded. Rather than forming tens of nanometer sized CoOx nanoparticles as free CoSalen does under photocatalytic WO conditions, the CB5 : CoSalen ArM showed limited protein cross-linking and remained soluble. We conclude that a weak, dynamic interaction between a soluble cobalt species and apoCB5 was formed, which generated a catalytically active adduct during photocatalysis. A detailed analysis was performed on protein stability and decomposition processes during the harsh oxidizing reaction conditions of WO, which will serve for the future design of WO ArMs with improved activity and stability.

Cyclic Ruthenium-Peptide Conjugates as Integrin-Targeting Phototherapeutic Prodrugs for the Treatment of Brain Tumors.

To investigate the potential of tumor-targeting photoactivated chemotherapy, a chiral ruthenium-based anticancer warhead, Λ/Δ-[Ru(Ph2phen)2(OH2)2]2+, was conjugated to the RGD-containing Ac-MRGDH-NH2 peptide by direct coordination of the M and H residues to the metal. This design afforded two diastereoisomers of a cyclic metallopeptide, Λ-[1]Cl2 and Δ-[1]Cl2. In the dark, the ruthenium-chelating peptide had a triple action. First, it prevented other biomolecules from coordinating with the metal center. Second, its hydrophilicity made [1]Cl2 amphiphilic so that it self-assembled in culture medium into nanoparticles. Third, it acted as a tumor-targeting motif by strongly binding to the integrin (Kd = 0.061 µM for the binding of Λ-[1]Cl2 to αIIbß3), which resulted in the receptor-mediated uptake of the conjugate in vitro. Phototoxicity studies in two-dimensional (2D) monolayers of A549, U87MG, and PC-3 human cancer cell lines and U87MG three-dimensional (3D) tumor spheroids showed that the two isomers of [1]Cl2 were strongly phototoxic, with photoindexes up to 17. Mechanistic studies indicated that such phototoxicity was due to a combination of photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) effects, resulting from both reactive oxygen species generation and peptide photosubstitution. Finally, in vivo studies in a subcutaneous U87MG glioblastoma mice model showed that [1]Cl2 efficiently accumulated in the tumor 12 h after injection, where green light irradiation generated a stronger tumoricidal effect than a nontargeted analogue ruthenium complex [2]Cl2. Considering the absence of systemic toxicity for the treated mice, these results demonstrate the high potential of light-sensitive integrin-targeted ruthenium-based anticancer compounds for the treatment of brain cancer in vivo.

A screening method for binding synthetic metallo-complexes to haem proteins.

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV-Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.

Cationic iron porphyrins with sodium dodecyl sulphate for micellar catalysis of cyclopropanation reactions.

Here, we report that the combination of cationic iron porphyrins with sodium dodecyl sulphate (SDS) gives rise to efficient micellar catalysis of cyclopropanation reactions of styrene derivatives, using diazoacetates as carbene precursors. This simple, yet effective approach for cyclopropanations illustrates the power of micellar catalysis.