Reactivity of metal-oxo clusters towards biomolecules: from discrete polyoxometalates to metal-organic frameworks.

Metal-oxo clusters hold great potential in several fields such as catalysis, materials science, energy storage, medicine, and biotechnology. These nanoclusters of transition metals with oxygen-based ligands have also shown promising reactivity towards several classes of biomolecules, including proteins, nucleic acids, nucleotides, sugars, and lipids. This reactivity can be leveraged to address some of the most pressing challenges we face today, from fighting various diseases, such as cancer and viral infections, to the development of sustainable and environmentally friendly energy sources. For instance, metal-oxo clusters and related materials have been shown to be effective catalysts for biomass conversion into renewable fuels and platform chemicals. Furthermore, their reactivity towards biomolecules has also attracted interest in the development of inorganic drugs and bioanalytical tools. Additionally, the structural versatility of metal-oxo clusters allows for the efficiency and selectivity of the biomolecular reactions they promote to be readily tuned, thereby providing a pathway towards reaction optimization. The properties of the catalyst can also be improved through incorporation into solid supports or by linking metal-oxo clusters together to form Metal-Organic Frameworks (MOFs), which have been demonstrated to be powerful heterogeneous catalysts. Therefore, this review aims to provide a comprehensive and critical analysis of the state of the art on biomolecular transformations promoted by metal-oxo clusters and their applications, with a particular focus on structure-activity relationships.

Exploring the Reactivity of Polyoxometalates toward Proteins: From Interactions to Mechanistic Insights.

The latest advances in the study of the reactivity of metal-oxo clusters toward proteins showcase how fundamental insights obtained so far open new opportunities in biotechnology and medicine. In this Perspective, these studies are discussed through the lens of the reactivity of a family of soluble anionic metal-oxo nanoclusters known as polyoxometalates (POMs). POMs act as catalysts in a wide range of reactions with several different types of biomolecules and have promising therapeutic applications due to their antiviral, antibacterial, and antitumor activities. However, the lack of a detailed understanding of the mechanisms behind biochemically relevant reactions-particularly with complex biological systems such as proteins-still hinders further developments. Hence, in this Perspective, special attention is given to reactions of POMs with peptides and proteins showcasing a molecular-level understanding of the reaction mechanism. In doing so, we aim to highlight both existing limitations and promising directions of future research on the reactivity of metal-oxo clusters toward proteins and beyond.

A zirconium metal-organic framework with SOC topological net for catalytic peptide bond hydrolysis.

The discovery of nanozymes for selective fragmentation of proteins would boost the emerging areas of modern proteomics, however, the development of efficient and reusable artificial catalysts for peptide bond hydrolysis is challenging. Here we report the catalytic properties of a zirconium metal-organic framework, MIP-201, in promoting peptide bond hydrolysis in a simple dipeptide, as well as in horse-heart myoglobin (Mb) protein that consists of 153 amino acids. We demonstrate that MIP-201 features excellent catalytic activity and selectivity, good tolerance toward reaction conditions covering a wide range of pH values, and importantly, exceptional recycling ability associated with easy regeneration process. Taking into account the catalytic performance of MIP-201 and its other advantages such as 6-connected Zr6 cluster active sites, the green, scalable and cost-effective synthesis, and good chemical and architectural stability, our findings suggest that MIP-201 may be a promising and practical alternative to commercially available catalysts for peptide bond hydrolysis.

Enhancing the Catalytic Activity of MOF-808 Towards Peptide Bond Hydrolysis through Synthetic Modulations.

The performance of MOFs in catalysis is largely derived from structural features, and much work has focused on introducing structural changes such as defects or ligand functionalisation to boost the reactivity of the MOF. However, the effects of different parameters chosen for the synthesis on the catalytic reactivity of the resulting MOF remains poorly understood. Here, we evaluate the role of metal precursor on the reactivity of Zr-based MOF-808 towards hydrolysis of the peptide bond in the glycylglycine model substrate. In addition, the effect of synthesis temperature and duration has been investigated. Surprisingly, the metal precursor was found to have a large influence on the reactivity of the MOF, surpassing the effect of particle size or number of defects. Additionally, we show that by careful selection of the Zr-salt precursor and temperature used in MOF syntheses, equally active MOF catalysts could be obtained after a 20 minute synthesis compared to 24 h synthesis.

Heterogeneous nanozymatic activity of Hf oxo-clusters embedded in a metal-organic framework towards peptide bond hydrolysis.

Materials with enzyme-like activities and proteolytic potential are emerging as a robust and effective alternative to natural enzymes. Herein, a Hf6O8-based NU-1000 metal organic framework (Hf-MOF) is shown to act as a heterogeneous catalyst for the hydrolysis of peptide bonds under mild conditions. In the presence of Hf-MOF, a glycylglycine model dipeptide was hydrolysed with a rate constant of kobs = 8.33 × 10-7 s-1 (half-life (t1/2) of 231 h) at 60 °C and pD 7.4, which is significantly faster than the uncatalyzed reaction. Other Gly-X peptides (X = Ser, Asp, Ile, Ala, and His) were also smoothly hydrolysed under the same conditions with similar rates, except for the faster reactions observed for Gly-His and Gly-Ser. Moreover, the Hf6O8-based NU-1000 MOF also exhibits a high selectivity in the cleavage of a protein substrate, hen egg white lysozyme (HEWL). Our results suggest that embedding Hf6O8 oxo-clusters is an efficient strategy to conserve the hydrolytic activity while smoothing the strong substrate adsorption previously observed for a discrete Hf oxo-cluster that hindered further development of its proteolytic potential. Furthermore, comparison with isostructural Zr-NU-1000 shows that although the Hf variant afforded the same cleavage pattern towards HEWL but slightly slower reaction rates, it exhibited a larger stability window and a better recyclability profile. The results suggest that these differences originate from the intrinsic differences between HfIV and ZrIV centers, and from the lower surface area of Hf-NU-1000 in comparison to Zr-NU-1000.

The Dawn of Metal-Oxo Clusters as Artificial Proteases: From Discovery to the Present and Beyond.

The selective cleavage of peptide bonds in proteins is of paramount importance in many areas of the biological and medical sciences, playing a key role in protein structure/function/folding analysis, protein engineering, and targeted proteolytic drug design. Current applications that depend on selective protein hydrolysis largely rely on costly proteases such as trypsin, which are sensitive to the pH, ionic strength, and temperature conditions. Moreover, >95% of peptides deposited in databases are generated from trypsin digests, restricting the information within the analyzed proteomes. On the other hand, harsh and toxic chemical reagents such as BrCN are very active but cause permanent modifications of certain amino acid residues. Consequently, transition-metal complexes have emerged as smooth and selective artificial proteases owing to their ability to provide larger fragments and complementary structural information. In the past decade, our group has discovered the unique protease activity of diverse metal-oxo clusters (MOC) and pioneered a distinctive approach to the development of selective artificial proteases. In contrast to classical coordination complexes which often depend on amino acid side chains to control the regioselectivity, the selectivity profile of MOCs is determined by a complex combination of structural factors, such as the protein surface charge, metal coordination to specific side chains, and hydrogen bonding between the protein surface and the MOC scaffold.In this Account, we present a critical overview of our detailed kinetic, spectroscopic, and crystallographic studies in MOC-assisted peptide bond hydrolysis, from its origins to the current rational and detailed mechanistic understanding. To this end, reactivity trends related to the structure and properties of MOCs based on the hydrolysis of small model peptides and key structural aspects governing the selectivity of protein hydrolysis are presented. Finally, our endeavors in seeking the next generation of heterogeneous MOC-based proteases are briefly discussed by embedding MOCs in metal-organic frameworks or using them as discrete nanoclusters in the development of artificial protease-like materials (i.e., nanozymes). The deep and comprehensive understanding sought experimentally and theoretically over the years in aqueous systems with intrinsic polar and charged substrates provides a unique view of the reactivity between inorganic moieties and biomolecules, thereby broadly impacting several different fields (e.g., catalysis in biochemistry, inorganic chemistry, and organic chemistry).

Mechanism of the highly effective peptide bond hydrolysis by MOF-808 catalyst under biologically relevant conditions.

Efficient and selective hydrolysis of inert peptide bonds is of paramount importance. MOF-808, a metal-organic framework based on Zr6 nodes, can hydrolyze peptide bonds efficiently under biologically relevant conditions. However, the details of the catalyst structure and of the underlying catalytic reaction mechanism are challenging to establish. By means of DFT calculations we first investigate the speciation of the Zr6 nodes and identify the nature of ligands that bind to the Zr6O8H4-x core in aqueous conditions. The core is predicted to strongly prefer a Zr6O8H4 protonation state and to be predominantly decorated by bridging formate ligands, giving Zr6(µ3-O)4(µ3-OH)4(BTC)2(HCOO)6 and Zr6(µ3-O)4(µ3-OH)4(BTC)2(HCOO)5(OH)(H2O) as the most favorable structures at physiological pH. The GlyGly peptide can bind MOF in several different ways, with the preferred structure involving coordination through the terminal carboxylate analogously to the binding mode of formate ligand. The pre-reactive binding mode in which the amide carbonyl oxygen coordinates the metal core lies 7 kcal higher in free energy. The preferred reaction pathway is predicted to have two close-lying transition states, either of which could be the rate-determining step: nucleophilic attack on the amide carbon atom and C-N bond breaking, with calculated relative free energies of 31 and 32 kcal mol-1, respectively. Replacement of formate by water and hydroxide at the Zr6 node is predicted to be possible, but does not appear to play a role in the hydrolysis mechanism.

Selective Hydrolysis of Transferrin Promoted by Zr-Substituted Polyoxometalates.

The hydrolysis of the iron-binding blood plasma glycoprotein transferrin (Tf) has been examined at pH = 7.4 in the presence of a series of Zr-substituted polyoxometalates (Zr-POMs) including Keggin (Et2NH2)10[Zr(PW11O39)2]∙7H2O (Zr-K 1:2), (Et2NH2)8[{α-PW11O39Zr-(µ-OH) (H2O)}2]∙7H2O (Zr-K 2:2), Wells-Dawson K15H[Zr(α2-P2W17O61)2]·25H2O (Zr-WD 1:2), Na14[Zr4(α-P2W16O59)2(µ3-O)2(µ-OH)2(H2O)4]·57H2O (Zr-WD 4:2) and Lindqvist (Me4N)2[ZrW5O18(H2O)3] (Zr-L 1:1), (nBu4N)6[(ZrW5O18(µ-OH))2]∙2H2O (Zr-L 2:2)) type POMs. Incubation of transferrin with Zr-POMs resulted in formation of 13 polypeptide fragments that were observed on sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), but the hydrolysis efficiency varied depending on the nature of Zr-POMs. Molecular interactions between Zr-POMs and transferrin were investigated by using a range of complementary techniques such as tryptophan fluorescence, circular dichroism (CD), 31P-NMR spectroscopy, in order to gain better understanding of different efficiency of investigated Zr-POMs. A tryptophan fluorescence quenching study revealed that the most reactive Zr-WD species show the strongest interaction toward transferrin. The CD results demonstrated that interaction of Zr-POMs and transferrin in buffer solution result in significant secondary structure changes. The speciation of Zr-POMs has been followed by 31P-NMR spectroscopy in the presence and absence of transferrin, providing insight into stability of the catalysts under reaction condition.

Hydrolysis of Peptide Bonds in Protein Micelles Promoted by a Zirconium(IV)-Substituted Polyoxometalate as an Artificial Protease.

The development of artificial proteases is challenging, but important for many applications in modern proteomics and biotechnology. The hydrolysis of hydrophobic or unstructured proteins is particularly difficult due to their poor solubility, which often requires the presence of surfactants. Herein, it is shown that a zirconium(IV)-substituted Keggin polyoxometalate (POM), (Et2 NH2 )10 [Zr(α-PW11 O39 )2 ] (1), is able to selectively hydrolyze ß-casein, which is an intrinsically unstructured protein at pH 7.4 and 60 °C. Four surfactants (sodium dodecyl sulfate (SDS), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and polyethylene glycol tert-octylphenyl ether (TX-100)), which differ in the nature of their polar groups, were investigated for their role in influencing the selectivity and efficiency of protein hydrolysis. Under experimental conditions, ß-casein forms micellar structures in which the hydrophilic part of the protein is water accessible and able to interact with 1. Identical fragmentation patterns of ß-casein in the presence of 1 were observed through SDS poly(acrylamide) gel electrophoresis both in the presence and absence of surfactants, but the rate of hydrolysis varied, depending on the nature of surfactant. Whereas TX-100 surfactant, which has a neutral polar head, caused only a slight decrease in the hydrolysis rate, stronger inhibition was observed in the presence surfactants with charges in their polar heads (CHAPS, ZW3-12, SDS). These results were consistent with those of tryptophan fluorescencequenching studies, which showed that the binding between ß-casein and 1 decreased with increasing repulsion between the POM and the polar heads of the surfactants. In all cases, the micellar structure of ß-casein was not significantly affected by the presence of POM or surfactants, as indicated by circular dichroism spectroscopy.

Redox Activity of Ce(IV)-Substituted Polyoxometalates toward Amino Acids and Peptides.

Redox reactions between polyoxometalates (POMs) and biologically relevant molecules have been virtually unexplored but are important, considering the growing interest in the biological applications of POMs. In this work we give a detailed account on the redox behavior of CeIV-substituted polyoxometalates (CeIV-POMs) toward a range of amino acids and peptides. CeIV-POMs have been shown to act as artificial proteases that promote the selective hydrolysis of peptide bonds. In presence of a protein, a concomitant reduction of CeIV to CeIII ion is frequently observed, leading us to examine the origins of this redox reaction by first using amino acid building blocks as simple models. Among all of the examined amino acids, cysteine (Cys) showed the highest activity in reducing CeIV-POMs to CeIII-POMs, followed by the aromatic amino acids tryptophan (Trp), tyrosine (Tyr), histidine (His), and phenylalanine (Phe). While the redox reaction with Cys afforded the well-defined product cystine, no oxidation products were detected for the Trp, His, Tyr, and Phe amino acids after their reaction with CeIV-POMs, suggesting a radical pathway in which the solvent likely regenerates the amino acid. In general, the rate of redox reactions increased upon increasing the pD, temperature, and ionic strength of the reaction. Moreover, the redox reaction is highly sensitive to the type of polyoxometalate scaffold, as complexation of CeIV to a Keggin (K) or Wells-Dawson (WD) polyoxotungstate anion resulted in a large difference in the rate of redox reaction for both Cys and aromatic amino acids. The reduction of CeIVK was at least 1 order of magnitude faster in comparison to CeIVWD, in accordance with the higher redox potential of CeIVK in comparison to CeIVWD. The reaction of CeIVPOMs with a range of peptides containing redox-active amino acids revealed that the redox reaction is influenced by their coordination mode with CeIV ion, but in all examined peptides the redox reaction is favored in comparison to the hydrolytic cleavage of the peptide bond.

Ovariectomy increases RANKL protein expression in bone marrow adipocytes of C3H/HeJ mice.

Estrogen deficiency induces bone loss by increasing bone resorption, in part through upregulation of receptor activator of nuclear factor-κB ligand (RANKL). RANKL is secreted by osteoblasts and osteocytes, but more recently bone marrow (pre)adipocytes have also been shown to express RANKL. Estrogen deficiency increases bone marrow adipose tissue (BMAT). The aim of this study was to determine the effect of ovariectomy (OVX) on RANKL protein expression by bone marrow adipocytes in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice (n = 20) were randomized to sham surgery (Sham) or OVX. After 4 wk animals were euthanized. BMAT volume fraction (BMAT volume/marrow volume) was quantified by polyoxometalate-based contrast-enhanced nano-computed tomography. The percentage of RANKL-positive bone marrow adipocytes (RANKL-positive bone marrow adipocytes/total adipocytes) and the percentage of RANKL-positive osteoblasts covering the bone surface (bone surface covered in RANKL-positive osteoblasts/total bone surface) were quantified in the distal metaphysis of immunohistochemically stained sections of the left femur. The effects of OVX were analyzed by Student's t test or Mann-Whitney U test. RANKL was detected in osteoblasts, osteocytes, and bone marrow adipocytes. OVX significantly increased mean percentage of RANKL-positive bone marrow adipocytes [mean (SD): Sham 42 (18)%; OVX 64 (12)%; P = 0.029] as well as BMAT volume/marrow volume [median (interquartile range): Sham 1.4 (4.9)%; OVX 7.2 (7.3)%; P = 0.008] compared with Sham. We show that OVX increased both the percentage of RANKL-positive bone marrow adipocytes and the total BMAT volume fraction in C3H/HeJ mice. Therefore, RANKL produced by bone marrow adipocytes could be an important contributor to OVX-induced bone loss in C3H/HeJ mice.

Amphiphilic Nanoaggregates with Bimodal MRI and Optical Properties Exhibiting Magnetic Field Dependent Switching from Positive to Negative Contrast Enhancement.

Mixed micelles based on amphiphilic gadolinium(III)-DOTA and europium(III)-DTPA complexes were synthesized and evaluated for their paramagnetic and optical properties as potential bimodal contrast agents. Amphiphilic folate molecule for targeting the folate receptor protein, which is commonly expressed on the surface of many human cancer cells, was used in the self-assembly process in order to create nanoaggregates with targeting properties. Both targeted and nontargeted nanoaggregates formed monodisperse micelles having distribution maxima of 10 nm. The micelles show characteristic europium(III) emission with quantum yields of 2% and 1.1% for the nontargeted and targeted micelles, respectively. Fluorescence microscopy using excitation at 405 nm and emission at 575-675 nm was employed to visualize the nanoaggregates in cultured HeLa cells. The uptake of folate-targeted and nontargeted micelles is already visible after 5 h of incubation and was characterized with the europium(III) emission, which is clearly observable in the cytoplasm of the cells. The very fast longitudinal relaxivity r1 of ca. 26 s-1 mM-1 per gadolinium(III) ion was observed for both micelles at 60 MHz and 310 K. Upon increasing the magnetic field to 300 MHz, the nanoaggregates exhibited a large switching to transversal relaxivity with r2 value of ca. 52 s-1 mM-1 at 310 K. Theoretical fitting of the 1H NMRD profiles indicate that the efficient T1 and T2 relaxations are sustained by the favorable magnetic and electron-configuration properties of the gadolinium(III) ion, rotational correlation time, and coordinated water molecule. These nanoaggregates could have versatile application as a positive contrast agent at the currently used magnetic imaging field strengths and a negative contrast agent in higher field applications, while at the same time offering the possibility for the loading of hydrophobic therapeutics or targeting molecules.

The effect of PPARγ inhibition on bone marrow adipose tissue and bone in C3H/HeJ mice.

Bone marrow adipose tissue (BMAT) increases after menopause, and increased BMAT is associated with osteoporosis and prevalent vertebral fractures. Peroxisome proliferator-activated receptor-γ (PPARγ) activation promotes adipogenesis and inhibits osteoblastogenesis; therefore, PPARγ is a potential contributor to the postmenopausal increase in BMAT and decrease in bone mass. The aim of this study is to determine if PPARγ inhibition can prevent ovariectomy-induced BMAT increase and bone loss in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice ( n = 40) were allocated to four intervention groups: sham surgery (Sham) or ovariectomy (OVX; isoflurane anesthesia) with either vehicle (Veh) or PPARγ antagonist administration (GW9662; 1 mg·kg-1·day-1, daily intraperitoneal injections) for 3 wk. We measured BMAT volume, adipocyte size, adipocyte number. and bone structural parameters in the proximal metaphysis of the tibia using polyoxometalate-based contrast enhanced-nanocomputed topogaphy. Bone turnover was measured in the contralateral tibia using histomorphometry. The effects of surgery and treatment were analyzed by two-way ANOVA. OVX increased the BMAT volume fraction (Sham + Veh: 2.9 ± 2.7% vs. OVX + Veh: 8.1 ± 5.0%: P < 0.001), average adipocyte diameter (Sham + Veh: 19.3 ± 2.6 µm vs. OVX + Veh: 23.1 ± 3.4 µm: P = 0.001), and adipocyte number (Sham + Veh: 584 ± 337cells/µm3 vs. OVX + Veh: 824 ± 113cells/µm3: P = 0.03), while OVX decreased bone volume fraction (Sham + Veh: 15.5 ± 2.8% vs. OVX + Veh: 7.7 ± 1.9%; P < 0.001). GW9662 had no effect on BMAT, bone structural parameters, or bone turnover. In conclusion, ovariectomy increased BMAT and decreased bone volume in C3H/HeJ mice. The PPARγ antagonist GW9662 had no effect on BMAT or bone volume in C3H/HeJ mice, suggesting that BMAT accumulation is regulated independently of PPARγ in C3H/HeJ mice.

Interactions between polyoxometalates and biological systems: from drug design to artificial enzymes.

Polyoxometalates have long been studied in a variety of biological applications. Interactions between the highly charged POM molecules and biological molecules frequently occur through hydrogen-bonding and electrostatic interactions. Tellurium-centred Anderson-Evans POMs show exceptional promise as crystallization agents, while acidic and metal-substituted POMs may provide interesting alternatives to enzymes in proteomics applications. While POMs also show interesting results in a number of medicinal applications, for example as anti-amyloid agents for the treatment of Alzheimer's disease and as anti-tumoral agents, their use is often impeded by their toxicity. Many recent studies have therefore focussed on POM-functionalization to reduce toxicity and increase activity by addition of biological targeting molecules.

Na/K-ATPase as a target for anticancer metal based drugs: insights into molecular interactions with selected gold(iii) complexes.

Na/K-ATPase is emerging as an important target for a variety of anticancer metal-based drugs. The interactions of Na/K-ATPase (in its E1 state) with three representative and structurally related cytotoxic gold(iii) complexes, i.e. [Au(bipy)(OH)2][PF6], bipy = 2,2'-bipyridine; [Au(pydmb-H)(CH3COO)2], pydmb-H = deprotonated 6-(1,1-dimethylbenzyl)-pyridine and [Au(bipydmb-H)(OH)][PF6], bipyc-H = deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine, are investigated here in depth using a variety of spectroscopic methods, in combination with docking studies. Detailed information is gained on the conformational and structural changes experienced by the enzyme upon binding of these gold(iii) complexes. The quenching constants of intrinsic enzyme fluorescence, the fraction of Trp residues accessible to gold(iii) complexes and the reaction stoichiometries were determined in various cases. Specific hypotheses are made concerning the binding mode of these gold(iii) complexes to the enzyme and the likely binding sites. Differences in their binding behaviour toward Na/K-ATPase are explained on the ground of their distinctive structural features. The present results offer further support to the view that Na/K-ATPase may be a relevant biomolecular target for cytotoxic gold(iii) compounds of medicinal interest and may thus be involved in their overall mode of action.

Gallium(III)-Containing, Sandwich-Type Heteropolytungstates: Synthesis, Solution Characterization, and Hydrolytic Studies toward Phosphoester and Phosphoanhydride Bond Cleavage.

The gallium(III)-containing heteropolytungstates [Ga4(H2O)10(ß-XW9O33)2](6-) (X = As(III), 1; Sb(III), 2) were synthesized in aqueous acidic medium by reaction of Ga(3+) ions with the trilacunary, lone-pair-containing [XW9O33](9-). Polyanions 1 and 2 are isostructural and crystallized as the hydrated sodium salts Na6[Ga4(H2O)10(ß-AsW9O33)2]·28H2O (Na-1) and Na6[Ga4(H2O)10(ß-SbW9O33)2]·30H2O (Na-2) in the monoclinic space group P21/c, with unit cell parameters a = 16.0218(12) Å, b = 15.2044(10) Å, c = 20.0821(12) Å, and ß = 95.82(0)°, as well as a = 16.0912(5) Å, b = 15.2178(5) Å, c = 20.1047(5) Å, and ß = 96.2(0)°, respectively. The corresponding tellurium(IV) derivative [Ga4(H2O)10(ß-TeW9O33)2](4-) (3) was also prepared, by direct reaction of sodium tungstate, tellurium(IV) oxide, and gallium nitrate. Polyanion 3 crystallized as the mixed rubidium/sodium salt Rb2Na2[Ga4(H2O)10(ß-TeW9O33)2]·28H2O (RbNa-3) in the triclinic space group P1̅ with unit cell parameters a = 12.5629(15) Å, b = 13.2208(18) Å, c = 15.474(2) Å, α = 80.52(1)°, ß = 84.37(1)°, and γ = 65.83(1)°. All polyanions 1-3 were characterized in the solid state by single-crystal XRD, FT-IR, TGA, and elemental analysis, and polyanion 2 was also characterized in solution by (183)W NMR and UV-vis spectroscopy. Polyanion 2 was used as a homogeneous catalyst toward adenosine triphosphate (ATP) and the DNA model substrate 4-nitrophenylphosphate, monitored by (1)H and (31)P NMR spectroscopy. The encapsulated gallium(III) centers in 2 promote the Lewis acidic synergistic activation of the hydrolysis of ATP and DNA model substrates at a higher rate in near-physiological conditions. A strong interaction of 2 with the P-O bond of ATP was evidenced by changes in chemical shift values and line broadening of the (31)P nucleus in ATP upon addition of the polyanion.

Molecular Insight from DFT Computations and Kinetic Measurements into the Steric Factors Influencing Peptide Bond Hydrolysis Catalyzed by a Dimeric Zr(IV)-Substituted Keggin Type Polyoxometalate.

Peptide bond hydrolysis of several peptides with a Gly-X sequence (X = Gly, Ala, Val, Leu, Ile, Phe) catalyzed by a dimeric Zr(IV)-substituted Keggin type polyoxometalate (POM), (Et2NH2)8[{α-PW11O39Zr(µ-OH)(H2O)}2]·7H2O (1), was studied by means of kinetic experiments and (1)H NMR spectroscopy. The observed rate of peptide bond hydrolysis was found to decrease with increase of the side chain bulkiness, from 4.44 × 10(-7) s(-1) for Gly-Gly to 0.81 × 10(-7) s(-1) for Gly-Ile. A thorough DFT investigation was performed to elucidate (a) the nature of the hydrolytically active species in solution, (b) the mechanism of peptide bond hydrolysis, and (c) the influence of the aliphatic residues on the rate of hydrolysis. Formation of substrate-catalyst complexes of the dimeric POM 1 was predicted as thermodynamically unlikely. Instead, the substrates prefer to bind to the monomerization product of 1, [α-PW11O39Zr(OH)(H2O)](4-) (2), which is also present in solution. In the hydrolytically active complex two dipeptide ligands are coordinated to the Zr(IV) center of 2. The first ligand is bidentate-bound through its amino nitrogen and amide oxygen atoms, while the second ligand is monodentate-bound through a carboxylic oxygen atom. The mechanism of hydrolysis involves nucleophilic attack by a solvent water molecule on the amide carbon atom of the bidentate-bound ligand. In this process the uncoordinated carboxylic group of the same ligand acts as a general base to abstract a proton from the attacking water molecule. The decrease of the hydrolysis rate with an increase in the side chain bulkiness is mostly due to the increased ligand conformational strain in the rate-limiting transition state, which elevates the reaction activation energy. The conformational strain increases first upon substitution of Hα in Gly-Gly with the aliphatic α substituent and second with the ß branching of the α substituent.

Detailed Mechanism of Phosphoanhydride Bond Hydrolysis Promoted by a Binuclear Zr(IV)-Substituted Keggin Polyoxometalate Elucidated by a Combination of (31)P, (31)P DOSY, and (31)P EXSY NMR Spectroscopy.

A detailed reaction mechanism is proposed for the hydrolysis of the phosphoanhydride bonds in adenosine triphosphate (ATP) in the presence of the binuclear Zr(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(µ-OH)(H2O)}2]·7H2O (ZrK 2:2). The full reaction mechanism of ATP hydrolysis in the presence of ZrK 2:2 at pD 6.4 was elucidated by a combination of (31)P, (31)P DOSY, and (31)P EXSY NMR spectroscopy, demonstrating the potential of these techniques for the analysis of complex reaction mixtures involving polyoxometalates (POMs). Two possible parallel reaction pathways were proposed on the basis of the observed reaction intermediates and final products. The 1D (31)P and (31)P DOSY spectra of a mixture of 20.0 mM ATP and 3.0 mM ZrK 2:2 at pD 6.4, measured immediately after sample preparation, evidenced the formation of two types of complexes, I1A and I1B, representing different binding modes between ATP and the Zr(IV)-substituted Keggin type polyoxometalate (ZrK). Analysis of the NMR data shows that at pD 6.4 and 50 °C ATP hydrolysis in the presence of ZrK proceeds in a stepwise fashion. During the course of the hydrolytic reaction various products, including adenosine diphosphate (ADP), adenosine monophosphate (AMP), pyrophosphate (PP), and phosphate (P), were detected. In addition, intermediate species representing the complexes ADP/ZrK (I2) and PP/ZrK (I5) were identified and the potential formation of two other intermediates, AMP/ZrK (I3) and P/ZrK (I4), was demonstrated. (31)P EXSY NMR spectra evidenced slow exchange between ATP and I1A, ADP and I2, and PP and I5, thus confirming the proposed reaction pathways.

Interaction Study and Reactivity of Zr(IV) -Substituted Wells-Dawson Polyoxometalate towards Hydrolysis of Peptide Bonds in Surfactant Solutions.

The interaction between the 1:2 Zr(IV) :Wells-Dawson complex, K15 H[Zr(α2 -P2 W17 O61 )2] (1), and a range of surfactants was studied in detail with the aim of developing metal-substituted POMs as potential artificial proteases for membrane proteins. The surfactants include the positively charged cetyl(trimethyl)ammonium bromide (CTAB), the negatively charged sodium dodecyl sulfate (SDS), the neutral Triton X-100 (TX-100), and zwitterionic 3-[dodecyl(dimethyl)ammonio]-1-propanesulfonate (Zw3-13) and 3-[dimethyl(3-{[(3α,5ß,7α,12α)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)ammonio]-1-propanesulfonate (CHAPS). A combination of multinuclear (1)H, (13)C, and (31) P NMR spectroscopy, (1)H diffusion-ordered NMR spectroscopy ((1)H DOSY), and nuclear Overhauser effect spectroscopy (NOESY) was used to examine the interaction between 1 and each surfactant on the molecular level. Cationic surfactant CTAB caused precipitation of 1 due to strong electrostatic interactions, while the anionic SDS and neutral TX-100 surfactants did not exhibit any interaction at neutral pD. (1)H DOSY NMR spectroscopy indicated an interaction between 1 and zwitterionic surfactants Zw3-12 and CHAPS, which occurs via the positively charged ammonium group in the surfactant molecule. In the presence of anionic, neutral, and zwitterionic surfactants, 1 preserves its catalytic activity towards the hydrolysis of the peptide bond in the dipeptide glycyl-l-histidine (GH). The fastest hydrolysis was observed at pD 7.0 and could be rationalized by taking into account pD-dependent speciation of 1 and coordination properties of GH.

Ultrasmall Superparamagnetic Iron Oxide Nanoparticles with Europium(III) DO3A as a Bimodal Imaging Probe.

A new prototype consisting of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles decorated with europium(III) ions encapsulated in a DO3A organic scaffold was designed as a platform for further development of bimodal contrast agents for MRI and optical imaging. The USPIO nanoparticles act as negative MRI contrast agents, whereas the europium(III) ion is a luminophore that is suitable for use in optical imaging detection. The functionalized USPIO nanoparticles were characterized by TEM, DLS, XRD, FTIR, and TXRF analysis, and a full investigation of the relaxometric and optical properties was conducted. The typical luminescence emission of europium(III) was observed and the main red emission wavelength was found at 614 nm. The relaxometric study of these ultrasmall nanoparticles showed r2 values of 114.8 mM(-1) Fes(-1) at 60 MHz, which is nearly double the r2 relaxivity of Sinerem(®).