Molecular Insights into Sequence-Specific Protein Hydrolysis by a Soluble Zirconium-Oxo Cluster Catalyst.

The development of catalysts for controlled fragmentation of proteins is a critical undertaking in modern proteomics and biotechnology. {Zr6O8}-based metal-organic frameworks (MOFs) have emerged as promising candidates for catalysis of peptide bond hydrolysis due to their high reactivity, stability, and recyclability. However, emerging evidence suggests that protein hydrolysis mainly occurs on the MOF surface, thereby questioning the need for their highly porous 3D nature. In this work, we show that the discrete and water-soluble [Zr6O4(OH)4(CH3CO2)8(H2O)2Cl3]+ (Zr6) metal-oxo cluster (MOC), which is based on the same hexamer motif found in various {Zr6O8}-based MOFs, shows excellent activity toward selective hydrolysis of equine skeletal muscle myoglobin. Compared to related Zr-MOFs, Zr6 exhibits superior reactivity, with near-complete protein hydrolysis after 24 h of incubation at 60 °C, producing seven selective fragments with a molecular weight in the range of 3-15 kDa, which are of ideal size for middle-down proteomics. The high solubility and molecular nature of Zr6 allow detailed solution-based mechanistic/interaction studies, which revealed that cluster-induced protein unfolding is a key step that facilitates hydrolysis. A combination of multinuclear nuclear magnetic resonance spectroscopy and pair distribution function analysis provided insight into the speciation of Zr6 and the ligand exchange processes occurring on the surface of the cluster, which results in the dimerization of two Zr6 clusters via bridging oxygen atoms. Considering the relevance of discrete Zr-oxo clusters as building blocks of MOFs, the molecular-level understanding reported in this work contributes to the further development of novel catalysts based on Zr-MOFs.

Supramolecular Self-Assembly of Proteins Promoted by Hybrid Polyoxometalates.

Controlling the formation of supramolecular protein assemblies and endowing them with new properties that can lead to novel functional materials is an important but challenging task. In this work, a new hybrid polyoxometalate is designed to induce controlled intermolecular bridging between biotin-binding proteins. Such bridging interactions lead to the formation of supramolecular protein assemblies incorporating metal-oxo clusters that go from several nanometers in diameter up to the micron range. Insights into the self-assembly process and the nature of the resulting biohybrid materials are obtained by a combination of Small Angle X-ray Scattering (SAXS), Transmission Electron Microscopy (TEM), and Dynamic Light Scattering (DLS), along with fluorescence, UV-vis, and Circular Dichroism (CD) spectroscopy. The formation of hybrid supramolecular assemblies is determined to be driven by biotin binding to the protein and electrostatic interactions between the anionic metal-oxo cluster and the protein, both of which also influence the stability of the resulting assemblies. As a result, the rate of formation, size, and stability of the supramolecular assemblies can be tuned by controlling the electrostatic interactions between the cluster and the protein (e.g., through varying the ionic strength of the solution), thereby paving the way toward biomaterials with tunable assembly and disassembly properties.

Cavity-Directed Synthesis of Labile Polyoxometalates for Catalysis in Confined Spaces.

The artificial microenvironments inside coordination cages have gained significant attention for performing enzyme-like catalytic reactions by facilitating the formation of labile and complex molecules through a "ship-in-a-bottle" approach. Despite many fascinating examples, this approach remains scarcely explored in the context of synthesizing metallic clusters such as polyoxometalates (POMs). The development of innovative approaches to control and influence the speciation of POMs in aqueous solutions would greatly advance their applicability and could ultimately lead to the formation of elusive clusters that cannot be synthesized by using traditional methods. In this study, we employ host-guest stabilization within a coordination cage to enable a novel cavity-directed synthesis of labile POMs in aqueous solutions under mild conditions. The elusive Lindqvist [M6O19]2- (M=Mo or W) POMs were successfully synthesized at room temperature via the condensation of molybdate or tungstate building blocks within the confined cavity of a robust and water-soluble Pt6L4(NO3)12 coordination cage. Importantly, the encapsulation of these POMs enhances their stability in water, rendering them efficient catalysts for environmentally friendly and selective sulfoxidation reactions using H2O2 as a green oxidant in a pure aqueous medium. The approach developed in this paper offers a means to synthesize and stabilize the otherwise unstable metal-oxo clusters in water, which can broaden the scope of their applications.

Fine-tuning non-covalent interactions between hybrid metal-oxo clusters and proteins.

Interactions between the protein Hen Egg White Lysozyme (HEWL) and three different hybrid Anderson-Evans polyoxometalate clusters - AE-NH2 (δ-[MnMo6O18{(OCH2)3CNH2}2]3-), AE-CH3 (δ-[MnMo6O18{(OCH2)3CCH3}2]3-) and AE-Biot (δ-[MnMo6O18{(OCH2)3CNHCOC9H15N2OS}2]3-) - were studied via tryptophan fluorescence spectroscopy and single crystal X-ray diffraction. Quenching of tryptophan fluorescence was observed in the presence of all three hybrid polyoxometalate clusters (HPOMs), but the extent of quenching and the binding affinity were greatly dependent on the nature of the organic groups attached to the cluster. Control experiments further revealed the synergistic effect of the anionic polyoxometalate core and organic ligands towards enhanced protein interactions. Furthermore, the protein was co-crystallised with each of the three HPOMs, resulting in four different crystal structures, thus allowing for the binding modes of HPOM-protein interactions to be investigated with near-atomic precision. All crystal structures displayed a unique mode of binding of the HPOMs to the protein, with both functionalisation and the pH of the crystallisation conditions influencing the interactions. From the crystal structures, it was determined that HPOM-protein non-covalent complexes formed through a combination of electrostatic attraction between the polyoxometalate cluster and positively charged surface regions of HEWL, and direct and water-mediated hydrogen bonds with both the metal-oxo inorganic core and the functional groups of the ligand, where possible. Hence, functionalisation of metal-oxo clusters shows great potential in tuning their interactions with proteins, which is of interest for several biomedical applications.

Exploiting Interactions between Polyoxometalates and Proteins for Applications in (Bio)chemistry and Medicine.

The specific interactions of anionic metal-oxo clusters, known as polyoxometalates (POMs), with proteins can be leveraged for a wide range of analytical and biomedical applications. For example, POMs have been developed as selective catalysts that can induce protein modifications and have also been shown to facilitate protein crystallization, both of which are instrumental in the structural characterization of proteins. POMs can also be used for selective protein separation and enzyme inhibition, which makes them promising therapeutic agents. Hence, understanding POM-protein interactions is essential for the development of POM-based materials and their implementation in several fields. In this Review we summarize in detail the key insights that have been gained so far on POM-protein interactions. Emphasis is also given to hybrid POMs functionalized with organic ligands to prompt further research in this direction owing to the promising recent results on tuning POM-protein interactions through POM functionalization.

Probing Dynamic Library of Metal-Oxo Building Blocks with γ-Cyclodextrin.

Formation of one of the most representative polyoxometalate (POM) archetypes, namely the Lindqvist anion M6O192- with M = MoVI or WVI, was considered as elusive in aqueous solution. Herein, we report on the host-guest stabilization of the M6O192- species with M = Mo or W from aqueous solution using γ-cyclodextrin as trapping agent. The adduct {M6O19@γ-CD}2- reveals remarkable hydrolytic stability that offers new opportunities for exploring potentialities of molybdates or tungstates in the field of biology and medicine, when combined to CDs as efficient drug carrier/delivery agents.

pH-Controlled One Pot Syntheses of Giant Mo2O2S2-Containing Seleno-Tungstate Architectures.

Self-assembly reactions of Na2WO4, SeO2/SeO32-, and [Mo2O2(µ-S)2(H2O)6]2+ give, depending on the pH value, two new large polyoxometalate complexes: i.e., [(γ-Se2W14O52)3(Mo2O2(µ-S)2(H2O)2)6]24- (1) isolated at pH 3.5 and [(γ-Se2W14O52)4(WO3(H2O))8(W2O5)2(W4O12)2(Mo2O2(µ-S)2(H2O)2)4(Mo2O2(µ-S)2(SeO3))4]36- (2) isolated at pH 2. Both compounds were characterized in the solid state by single-crystal X-ray diffraction, EDX, elemental analysis, TGA, and FT-IR and in solution by ESI-MS and NMR.

Nonconventional Three-Component Hierarchical Host-Guest Assembly Based on Mo-Blue Ring-Shaped Giant Anion, γ-Cyclodextrin, and Dawson-type Polyoxometalate.

In this communication, we report on a noteworthy hybrid supramolecular assembly built from three functional components hierarchically organized through noncovalent interactions. The one-pot synthesis procedure leads to the formation of large Mo-blue ring-shaped anion {Mo154}, which contains the supramolecular adduct based on the symmetric encapsulation of the Dawson-type [P2W18O62]6- anion by two γ-cyclodextrin units. Such a nanoscopic onion-like structure, noted [P2W18O62]@2γ-CD@{Mo154} has been characterized by single-crystal X-ray diffraction, thus demonstrating the capability of the giant inorganic torus to develop relevant supramolecular chemistry, probing the strong affinity of the inner and outer faces of the γ-CD for the polyoxometalate surfaces. Furthermore, interactions and behavior in solution have been studied by multinuclear NMR spectroscopy, which supports specific interactions between γ-CD and POM units. Finally, the formation of this three-component hybrid assembly from one-pot procedure, in water and using nearly stoichiometric conditions, is discussed in terms of the driving forces orchestrating this highly efficient multilevel recognition process.

Polyoxometalate, Cationic Cluster, and γ-Cyclodextrin: From Primary Interactions to Supramolecular Hybrid Materials.

Herein, we report on a three-component supramolecular hybrid system built from specific recognition processes involving a Dawson-type polyoxometalate (POM), [P2W18O62]6-, a cationic electron-rich cluster [Ta6Br12(H2O)6]2+, and γ-cyclodextrin (γ-CD). Such materials have been investigated using a bottom-up approach by studying the specific interactions between γ-CD and both types of inorganic units. Their ability to interact has been investigated in the solid state by single-crystal X-ray diffraction (XRD) and in solution using multinuclear NMR methods (including DOSY, EXSY, and COSY), electrospray ionization mass and UV-vis spectroscopies, electrochemistry, and isothermal titration calorimetry experiments. Single-crystal XRD analysis reveals that POM:γ-CD constitutes a highly versatile system which gives aggregates with 1:1, 1:2, and 1:3 stoichiometry. Surprisingly, these arrangements exhibit a common feature wherein the γ-CD moiety interacts with the Dawson-type POMs through its primary face. We present also the first structural model involving an octahedral-type metallic cluster with γ-CD. XRD study reveals that the cationic [Ta6Br12(H2O)6]2+ ion is closely embedded within two γ-CD units to give a supramolecular ditopic cation, suitable to be used as a linker within extended structure. Solution study demonstrates clearly that pre-associations exist in solution, for which binding constants and thermodynamic parameters have been determined, giving preliminary arguments about the chaotropic nature of the inorganic ions. Finally, both building blocks, i.e., the ditopic supramolecular cation {[Ta6Br12(H2O)6]@2CD}2+ and the Dawson-type anion, react together to give a three-component, well-ordered hybrid material derived either as a supramolecular hydrogel or single crystals. The solid-state structure shows an unprecedented helicoidal tubular chain resulting from the periodic alternation of POM and supramolecular cation, featuring short hydrogen-bonding contacts between the electron-poor POM and electron-rich cluster. The 1D tubular ionic polymer observed in the single crystals should make it possible to understand the long-range ordering observed within the hydrogel hybrid material. The supramolecular chemical complementarities between the γ-CD-based ditopic cation and POM open a wide scope for the design of hybrid materials that accumulate synergistic functionalities.

Redox-active polyoxovanadates as cofactors in the development of functional protein assemblies.

The interactions of polyoxovanadates (POVs) with proteins have increasingly attracted interest in recent years due to their potential biomedical applications. This is especially the case because of their redox and catalytic properties, which make them interesting for developing artificial metalloenzymes. Organic-inorganic hybrid hexavanadates in particular offer several advantages over all-inorganic POVs. However, they have been scarcely investigated in biological systems even though, as shown in this work, hybrid hexavanadates are highly stable in aqueous solutions up to relatively high pH. Therefore, a novel bis-biotinylated hexavanadate was synthesized and shown to selectively interact with two biotin-binding proteins, avidin and streptavidin. Bridging interactions between multiple proteins led to their self-assembly into supramolecular bio-inorganic hybrid systems that have potential as artificial enzymes with the hexavanadate core as a redox-active cofactor. Moreover, the structure and charge of the hexavanadate core were determined to enhance the binding affinity and slightly alter the secondary structure of the proteins, which affected the size and speed of formation of the assemblies. Hence, tuning the polyoxometalate (POM) core of hybrid POMs (HPOMs) with protein-binding ligands has been demonstrated to be a potential strategy for controlling the self-assembly process while also enabling the formation of novel POM-based biomaterials that could be of interest in biomedicine.

Rational synthesis of elusive organic-inorganic hybrid metal-oxo clusters: formation and post-functionalization of hexavanadates.

Paving the way towards new functional materials relies increasingly on the challenging task of forming organic-inorganic hybrid compounds. In that regard, discrete atomically-precise metal-oxo nanoclusters have received increasing attention due to the wide range of organic moieties that can be grafted onto them through functionalization reactions. The Lindqvist hexavanadate family of clusters, such as [V6O13{(OCH2)3C-R}2]2- (V6-R), is particularly interesting due to the magnetic, redox, and catalytic properties of these clusters. However, compared to other metal-oxo cluster types, V6-R clusters have been less extensively explored, which is mainly due to poorly understood synthetic challenges and the limited number of viable post-functionalization strategies. In this work, we present an in-depth investigation of the factors that influence the formation of hybrid hexavanadates (V6-R HPOMs) and leverage this knowledge to develop [V6O13{(OCH2)3CNHCOCH2Cl}2]2- (V6-Cl) as a new and tunable platform for the facile formation of discrete hybrid structures based on metal-oxo clusters in relatively high yields. Moreover, we showcase the versatility of the V6-Cl platform through its post-functionalization via nucleophilic substitution with various carboxylic acids of differing complexity and with functionalities that are relevant in multiple disciplines, such as supramolecular chemistry and biochemistry. Hence, V6-Cl was shown to be a straightforward and versatile starting point for the formation of functional supramolecular structures or other hybrid materials, thereby enabling their exploration in various fields.

Versatile post-functionalisation strategy for the formation of modular organic-inorganic polyoxometalate hybrids.

Hybrid structures incorporating different organic and inorganic constituents are emerging as a very promising class of materials since they synergistically combine the complementary and diverse properties of the individual components. Hybrid materials based on polyoxometalate clusters (POMs) are particularly interesting due to their versatile catalytic, redox, electronic, and magnetic properties, yet the controlled incorporation of different clusters into a hybrid structure is challenging and has been scarcely reported. Herein we propose a novel and general strategy for combining multiple types of metal-oxo clusters in a single hybrid molecule. Two novel hybrid POM structures (HPOMs) bis-functionalised with dipentaerythritol (R-POM1-R; R = (OCH2)3CCH2OCH2C(CH2OH)) were synthesised as building-blocks for the formation of heterometallic hybrid triads (POM2-R-POM1-R-POM2). Such a modular approach resulted in the formation of four novel heterometallic hybrids combing the Lindqvist {V6}, Anderson-Evans {XMo6} (X = Cr or Al) and trisubstituted Wells-Dawson {P2V3W15} POM structures. Their formation was confirmed by multinuclear Nuclear Magnetic Resonance (NMR), infrared (IR) and UV-Vis spectroscopy, as well as Mass Spectrometry, Diffusion Ordered Spectroscopy (DOSY) and elemental analysis. The thermal stability of the hybrids was also examined by Thermogravimetric Analysis (TGA), which showed that the HPOM triads exhibit higher thermal stability than comparable hybrid structures containing only one type of POM. The one-pot synthesis of these novel compounds was achieved in high yields in aqueous and organic media under simple reflux conditions, without the need of any additives, and could be translated to create other hybrid materials based on a variety of metal-oxo cluster building-blocks.