Peptide Bond Hydrolysis Catalyzed by the Wells–Dawson Zr(α₂‑P₂W₁₇O₆₁)₂ Polyoxometalate

In this paper we report the first example of peptide hydrolysis catalyzed by a polyoxometalate complex. A series of metal-substituted Wells–Dawson polyoxometalates were synthesized, and their hydrolytic activity toward the peptide bond in glycylglycine (GG) was examined. Among these, the Zr­(IV)- and Hf­(IV)-substituted ones were the most reactive. Detailed kinetic studies were performed with the Zr­(IV)-substituted Wells–Dawson type polyoxometalate K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O which was shown to act as a catalyst for the hydrolysis of the peptide bond in GG. The speciation of K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O which is highly dependent on the pD, concentration, and temperature of the solution, was fully determined with the help of ³¹P NMR spectroscopy and its influence on the GG hydrolysis rate was examined. The highest reaction rate (<i>k</i>_obs = 9.2 (±0.2) × 10^–5 min^–1) was observed at pD 5.0 and 60 °C. A 10-fold excess of GG was hydrolyzed in the presence of K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O proving the principles of catalysis. ¹³C NMR data suggested the coordination of GG to the Zr­(IV) center in K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O via its N-terminal amine group and amide carbonyl oxygen. These findings were confirmed by the inactivity of K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O toward the N-blocked analogue acetamidoglycylglycinate and the inhibitory effect of oxalic, malic, and citric acid. Triglycine, tetraglycine, and pentaglycine were also fully hydrolyzed in the presence of K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O yielding glycine as the final product of hydrolysis. K₁₅H­[Zr­(α₂-P₂W₁₇O₆₁)₂]·25H₂O also exhibited hydrolytic activity toward a series of other dipeptides

A Simple Nucleophilic Substitution as a Versatile Postfunctionalization Method for the Coupling of Nucleophiles to an Anderson-Type Polyoxometalate

A new postfunctionalization method was developed for the Anderson-type POM based on a nucleophilic substitution reaction occurring at an electrophilic sp³ hybridized carbon localized on the hybrid POM. Using this method, several types of different nucleophiles including primary and secondary amines, carboxylates, and thiolates were efficiently coupled to a chloride-functionalized Anderson-type POM in high yields and purity. The heterogeneous acetonitrile-Na₂CO₃ conditions were found to be superior over other bases and solvents for the coupling of amines and thiolates to the chloride-functionalized POM. Moreover, the addition of 1 equiv of tetra­butyl­ammonium iodide as a catalyst drastically decreased the reaction times to 24 h for the complete coupling of amines and only a couple of hours for thiolates. In the case of carboxylic acids as substrates, using tetra­butyl­ammonium hydroxide as the base for the reaction proved to be beneficial. This is because the resulting tetra­butyl­ammonium carboxylates were found to be much more reactive than the corresponding sodium carboxylates and allowed homogeneous reaction conditions. Using sodium carbonate, only 25% of <i>N</i>-acetyl glycyl­glycine could be coupled after 24 h at 80 °C, while full conversion was achieved after the same reaction time when using tetra­butyl­ammonium hydroxide as a base

Investigating Polyoxometalate–Protein Interactions at Chemically Distinct Binding Sites

In this study, a combined molecular docking (rigid and flexible) and all-atom molecular dynamics simulations technique have been employed to investigate interactions of 1:1 Zr-containing Keggin polyoxometalate (ZrK) with four chemically distinct cleavage sites [Arg114–Leu115 (site 1), Ala257–Asp258 (site 2), Lys313–Asp314 (site 3), and Cys392–Glu393 (site 4)] of human serum albumin (HSA). The ZrK–HSA complexations were analyzed using electrostatic potentials, the chemical nature of amino acid residues, binding free energies, and secondary structures as parameters. They suggested that ZrK binds in a rather distinct manner to different cleavage sites, and its association was dominated by hydrogen bonding, both direct and solvent mediated, and electrostatic interactions, as suggested experimentally. The computed binding free interaction energies (−57.5, −24.2, −50.8, and −91.2 kJ/mol for sites 1, 2, 3, and 4, respectively) predicted the existence of one major binding site (site 4) and three minor binding sites (site 1, site 2, and site 3). The strong exothermicity of the binding was also supported by isothermal calorimetry experiments. Additionally, the binding of ZrK did not alter the overall α-helical secondary structure of HSA, which was in line with experimental observation. Furthermore, hydrolysis of the peptide bonds of the substrate was found to retain its overall structure. These results have provided a deeper understanding of the complex ZrK interactions with proteins, and they will lead to the design of the next generation of catalytically active polyoxometalates with improved hydrolytic activities

Hydrolytic Activity of Vanadate toward Serine-Containing Peptides Studied by Kinetic Experiments and DFT Theory

Hydrolysis of dipeptides glycylserine (Gly-Ser), leucylserine (Leu-Ser), histidylserine (His-Ser), glycylalanine (Gly-Ala), and serylglycine (Ser-Gly) was examined in vanadate solutions by means of ¹H, ¹³C, and ⁵¹V NMR spectroscopy. In the presence of a mixture of oxovanadates, the hydrolysis of the peptide bond in Gly-Ser proceeds under the physiological pH and temperature (37 °C, pD 7.4) with a rate constant of 8.9 × 10^–8 s^–1. NMR and EPR spectra did not show evidence for the formation of paramagnetic species, excluding the possibility of V­(V) reduction to V­(IV) and indicating that the cleavage of the peptide bond is purely hydrolytic. The pD dependence of k_obs exhibits a bell-shaped profile, with the fastest hydrolysis observed at pD 7.4. Combined ¹H, ¹³C, and ⁵¹V NMR experiments revealed formation of three complexes between Gly-Ser and vanadate, of which only one complex, designated Complex 2, formed via coordination of amide oxygen and amino nitrogen to vanadate, is proposed to be hydrolytically active. Kinetic experiments at pD 7.4 performed by using a fixed amount of Gly-Ser and increasing amounts of Na₃VO₄ allowed calculation of the formation constant for the Gly-Ser/VO₄^3‑ complex (K_f = 16.1 M^–1). The structure of the hydrolytically active Complex 2 is suggested also on the basis of DFT calculations. The energy difference between Complex 2 and the major complex detected in the reaction mixture, Complex 1, is calculated to be 7.1 kcal/mol in favor of the latter. The analysis of the molecular properties of Gly-Ser and their change upon different modes of coordination to the vanadate pointed out that only in Complex 2 the amide carbon is suitable for attack by the hydroxyl group in the Ser side chain, which acts as an effective nucleophile. The origin of the hydrolytic activity of vanadate is most likely a combination of the polarization of amide oxygen in Gly-Ser due to the binding to vanadate, followed by the intramolecular attack of the Ser hydroxyl group

Detailed Mechanism of Phosphoanhydride Bond Hydrolysis Promoted by a Binuclear Zr^IV-Substituted Keggin Polyoxometalate Elucidated by a Combination of ³¹P, ³¹P DOSY, and ³¹P 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 ³¹P, ³¹P DOSY, and ³¹P 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 ³¹P and ³¹P 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. ³¹P EXSY NMR spectra evidenced slow exchange between ATP and I1A, ADP and I2, and PP and I5, thus confirming the proposed reaction pathways

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 (<b>1</b>), was studied by means of kinetic experiments and ¹H 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 <b>1</b> was predicted as thermodynamically unlikely. Instead, the substrates prefer to bind to the monomerization product of <b>1</b>, [α-PW₁₁O₃₉Zr­(OH)­(H₂O)]^4– (<b>2</b>), which is also present in solution. In the hydrolytically active complex two dipeptide ligands are coordinated to the Zr­(IV) center of <b>2</b>. 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 H_α in Gly-Gly with the aliphatic α substituent and second with the β branching of the α substituent

Reactivity of Dimeric Tetrazirconium(IV) Wells–Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach

Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium­(IV)-substituted Wells–Dawson-type polyoxometalate Na₁₄­[Zr₄­(P₂W₁₆O₅₉)₂­(μ₃-O)₂­(OH)₂­(H₂O)₄]·57H₂O (<b>1</b>) were performed by a combination of ¹H, ¹³C, and ³¹P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10^–7 s^–1, representing a significant acceleration as compared to the uncatalyzed reaction. ¹³C NMR data were indicative for coordination of Gly-Gly to <b>1</b> via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of <b>1</b> was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and <b>1</b>. To elucidate the role of <b>1</b> in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the −CO···Zr­(IV) binding

Controlled Synthesis of a Novel Heteropolymetallic Complex with Selectively Incorporated Lanthanide(III) Ions

A novel synthetic strategy toward a heteropolymetallic lanthanide complex with selectively incorporated gadolinium and europium ions is outlined. Luminescence and relaxometric measurements suggest possible applications in bimodal (magnetic resonance/optical) imaging

Phosphate Ester Bond Hydrolysis Promoted by Lanthanide-Substituted Keggin-type Polyoxometalates Studied by a Combined Experimental and Density Functional Theory Approach

Hydrolytic cleavage of 4-nitrophenyl phosphate (NPP), a commonly used DNA model substrate, was examined in the presence of series of lanthanide-substituted Keggin-type polyoxometalates (POMs) [Me₂NH₂]₁₁­[Ce^III(PW₁₁O₃₉)₂], [Me₂NH₂]₁₀­[Ce^IV(PW₁₁O₃₉)₂] (abbreviated as (Ce^IV(PW₁₁)₂), and K₄[EuPW₁₁O₃₉] by means of NMR and luminescence spectroscopies and density functional theory (DFT) calculations. Among the examined complexes, the Ce­(IV)-substituted Keggin POM (Ce^IV(PW₁₁)₂) showed the highest reactivity, and its aqueous speciation was fully determined under different conditions of pD, temperature, concentration, and ionic strength by means of ³¹P and ³¹P diffusion-ordered NMR spectroscopy. The cleavage of the phosphoester bond of NPP in the presence of (Ce^IV(PW₁₁)₂) proceeded with an observed rate constant <i>k</i>_obs = (5.31 ± 0.06) × 10^–6 s^–1 at pD 6.4 and 50 °C. The pD dependence of NPP hydrolysis exhibits a bell-shaped profile, with the fastest rate observed at pD 6.4. The formation constant (<i>K</i>_f = 127 M^–1) and catalytic rate constant (<i>k</i>_c = 19.41 × 10^–5 s^–1) for the NPP-Ce­(IV)-Keggin POM complex were calculated, and binding between Ce^IV(PW₁₁)₂ and the phosphate group of NPP was also evidenced by the change of the chemical shift of the ³¹P nucleus in NPP upon addition of the POM complex. DFT calculations revealed that binding of NPP to the parent catalyst Ce^IV(PW₁₁)₂ is thermodynamically unlikely. On the contrary, formation of complexes with the monomeric 1:1 species, Ce^IVPW₁₁, is considered to be more favorable, and the most stable complex, [Ce^IVPW₁₁(H₂O)₂(NPP-κO)₂]^7–, was found to involve two NPP ligands coordinated to the Ce^IVcenter of Ce^IVPW₁₁ in the monodentate fashion. The formation of such species is considered to be responsible for the hydrolytic activity of Ce^IV(PW₁₁)₂ toward phosphomonoesters. On the basis of these findings a principle mechanism for the hydrolysis of NPP by the POM is proposed

Superactivity of MOF-808 toward Peptide Bond Hydrolysis

MOF-808, a Zr­(IV)-based metal–organic framework, has been proven to be a very effective heterogeneous catalyst for the hydrolysis of the peptide bond in a wide range of peptides and in hen egg white lysozyme protein. The kinetic experiments with a series of Gly-X dipeptides with varying nature of amino acid side chain have shown that MOF-808 exhibits selectivity depending on the size and chemical nature of the X side chain. Dipeptides with smaller or hydrophilic residues were hydrolyzed faster than those with bulky and hydrophobic residues that lack electron rich functionalities which could engage in favorable intermolecular interactions with the btc linkers. Detailed kinetic studies performed by ¹H NMR spectroscopy revealed that the rate of glycylglycine (Gly-Gly) hydrolysis at pD 7.4 and 60 °C was 2.69 × 10^–4 s^–1 (<i>t</i>_1/2 = 0.72 h), which is more than 4 orders of magnitude faster compared to the uncatalyzed reaction. Importantly, MOF-808 can be recycled several times without significantly compromising the catalytic activity. A detailed quantum-chemical study combined with experimental data allowed to unravel the role of the {Zr₆O₈} core of MOF-808 in accelerating Gly-Gly hydrolysis. A mechanism for the hydrolysis of Gly-Gly by MOF-808 is proposed in which Gly-Gly binds to two Zr­(IV) centers of the {Zr₆O₈} core via the oxygen atom of the amide group and the N-terminus. The activity of MOF-808 was also demonstrated toward the hydrolysis of hen egg white lysozyme, a protein consisting of 129 amino acids. Selective fragmentation of the protein was observed with 55% yield after 25 h under physiological pH