Metal-Substituted Polyoxometalates as Artificial Peptidases.

Selective hydrolytic cleavage of proteins has become an important procedure in numerous biochemical applications. Several transition metal and lanthanide complexes have long been known to effect the hydrolytic cleavage of unactivated amide bonds in proteins. Because they often operate under mild reaction conditions, the development of metal-based artificial peptidases has become an increasingly important area of research over the past decades.Within our lab, polyoxometalates (POMs) have been demonstrated to exhibit hydrolytic activity towards phosphoester bonds in RNA and DNA model systems. Inspired by studies reporting the interaction between POMs and proteins, we recently designed a conceptually new approach to the development of artificial peptidases by using POMs as ligands for various metal ions. Literature reports show that negatively charged POMs effectively bind to positively charged surface domains of the protein in a non-covalent, mainly electrostatic manner. Consequently, in our approach the POM does not only act as a ligand for the active metal ion, but is also responsible for the selectivity that is necessary for a controlled fragmentation of the polyamide backbone.In our study the Wells-Dawson POM was functionalized with various transition and lanthanide metal ions and their use as an artificial peptidase was examined on the dipeptide glycylglycine (GG). From all the metals under investigation Zr(IV) was selected and a detailed analysis of the hydrolysis of GG was given. This study was further extended to dipeptides with a Gly-X and X-Gly sequence in order to evaluate the influence of the X amino acid side chain. Furthermore, longer peptides were used and the hydrolytic activity of the synthesized POM was evaluated on ovalbumin and bovine serum albumin. These protein studies show that the POM selectively hydrolyses internal amide bonds in both proteins.nrpages: 172status: publishe

Selective Hydrolysis of Oxidized Insulin Chain B by a Zr(IV)-Substituted Wells-Dawson Polyoxometalate

We report for the first time on the selective hydrolysis of a polypeptide system by a metal-substituted polyoxometalate (POM). Oxidized insulin chain B, a 30 amino acid polypeptide, was selectively cleaved by the Zr(IV)-substituted Wells–Dawson POM, K15H[Zr(α2-P2W17O61)2]·25H2O, under physiological pH and temperature conditions in aqueous solution. HPLC-ESI-MS, LC-MS/MS, MALDI-TOF and MALDI-TOF MS/MS data indicate hydrolysis at the Phe1–Val2, Gln4–His5, Leu6–Cys(SO3H)7, and Gly8–Ser9 peptide bonds. The rate of oxidized insulin chain B hydrolysis (0.45 h−1 at pH 7.0 and 60 °C) was calculated by fitting the integration values of its HPLC-UV signal to a first-order exponential decay function. 1H NMR measurements show significant line broadening and shifting of the polypeptide resonances upon addition of the Zr(IV)-POM, indicating that interaction between the Zr(IV)-POM and the polypeptide takes place in solution. Circular dichroism (CD) measurements clearly prove that the flexible unfolded nature of the polypeptide was retained in the presence of the Zr(IV)-POM. The thermal stability of the Zr(IV)-POM in the presence of the polypeptide chain during the hydrolytic reaction was confirmed by 31P NMR spectroscopy. Despite the highly negative charge of the Zr(IV)-POM, the mechanism of interaction appears to be dominated by a strong metal-directed binding between the positively charged Zr(IV) center and negatively charged amino acid side chains.status: publishe

Eu(III) luminescence and tryptophan fluorescence spectroscopy as a tool for understanding regioselective hydrolysis of hen egg white lysozyme by metal-substituted Keggin type polyoxometalates

The interaction between the lacunary Keggin K7PW11O39, the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) and the Ce(IV)-substituted Keggin [Me2NH2]10[Ce(PW11O39)2] (Ce-Keggin) polyoxometalates (POMs), and the proteins hen egg white lysozyme (HEWL) and the structurally homologous α lactalbumin (α-LA) was studied by steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of Eu-Keggin at lower concentrations ([Eu-Keggin] 250 µM) the 5L6←7F0 transition becomes the most intense peak. In the absence of protein, the number of coordinated water molecules to the Eu(III) center of Eu-Keggin is 4, indicating a 1:1 Eu(III):POM species. In the presence of phosphate buffer this number linearly decreases from 4 to 2 upon increasing phosphate buffer concentration. Upon addition of HEWL, there are no coordinated water molecules, suggesting interaction between Eu-Keggin and the protein surface. In addition, this interaction results in a more than threefold increase of the hypersensitive 5D0→7F2 transition for the Eu-Keggin/HEWL mixture. The calculated association constant amounted to 2.2×102 M-1 for the Eu-Keggin/HEWL complex. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated to be 9.1×104 M-1, 4×104 M-1 and 4.1×105 M-1for the lacunary Keggin/HEWL, the Eu-Keggin/HEWL and the Ce-Keggin/HEWL complexes, respectively. The number of bound POM molecules to HEWL was 1.04 for the lacunary Keggin POM, and 1.0 for Eu-Keggin, indicating the formation of a 1:1 POM-HEWL complex. The value of 1.38 for Ce-Keggin might indicate a transition from 1:1 to 1:2 interaction.status: publishe

Influence of the Amino Acid Side Chain on Peptide Bond Hydrolysis Catalyzed by a Dimeric Zr(IV)-Substituted Keggin Type Polyoxometalate

© The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2016. Peptide bond hydrolysis of 18 different dipeptides, divided into four groups depending on the nature of the amino acid side chain, by the 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 1H/13C NMR spectroscopy. The observed rate constants highly depend on the bulkiness and chemical nature of the X amino acid side chain. X-Ser and X-Thr dipeptides showed increased reactivity due to intramolecular nucleophilic attack of the hydroxyl group in the side chain on the amide carbon, resulting in a reactive ester intermediate. A similar effect in which the amino acid side chain acted as an internal nucleophile was observed for the hydrolysis of Gly-Asp. Interestingly, in the presence of 1 deamidation of Gly-Asn and Gly-Gln into Gly-Asp and Gly-Glu was observed. Dipeptides containing positively charged amino acid side chains were hydrolyzed at higher rates due to electrostatic interactions between the negatively charged POM surface and positive amino acid side chains.status: publishe

Understanding the regioselective hydrolysis of human serum albumin by Zr(IV)-substituted polyoxotungstates using tryptophan fluorescence spectroscopy

The interaction between human serum albumin (HSA) and a series of Zr(IV)-substituted polyoxometalates (POMs) (Lindqvist type POM ((nBu4N)6[{W5O18Zr (μ-OH)}2]•2H2O, Zr2-L2), two Keggin type POMs ((Et2NH2)10[Zr(PW11O39)2]•7H2O, Zr1-K2 and (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]•7H2O, Zr2-K2), and two Wells-Dawson type POMs (K15H[Zr(α2-P2W17O61)2]•25H2O, Zr1-WD2 and Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4]•10H2O, Zr4-WD2) was investigated by tryptophan (Trp) fluorescence spectroscopy. The fluorescence data were analyzed using the Tachiya model, ideally suited for multiple binding site analysis. The obtained quenching constants have the same order of magnitude for all the measured POM:protein complexes, ranging from 1.9 × 105 M−1 to 5.1 × 105 M−1. The number of bound POM molecules to HSA was in the range of 1.5 up to 3.5. The influence of the ionic strength was studied for the Zr1-WD2:HSA complex in the presence of NaClO4. The calculated quenching constant decreases upon increasing the ionic strength of the solution from 0.0004 M to 0.5004 M, indicating electrostatic nature of the interaction. The number of POM molecules bound to HSA increases from 1.0 to 4.8. 31P NMR spectroscopy provided evidence for the stability of all investigated POM structures during the interaction with HSA.status: publishe

ESRF Experimental Report 26-01 935 - Metal-Substituted Polyoxometalates as Artificial Peptidases

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Polyoxomolybdate promoted hydrolysis of a DNA-model phosphoester studied by NMR and EXAFS spectroscopy

Hydrolysis of (p-nitrophenyl)phosphate (NPP), a commonly used phosphatase model substrate, was examined in molybdate solutions by means of (1)H, (31)P, and (95)Mo NMR spectroscopy and Mo K-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. At 50 degrees C and pD 5.1 the cleavage of the phosphoester bond in NPP proceeds with a rate constant of 2.73 x 10(-5) s(-1) an acceleration of nearly 3 orders of magnitude as compared to the hydrolysis measured in the absence of molybdate. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed in solutions where [Mo(7)O(24)](6-) is the major species in solution. Mixing of NPP and [Mo(7)O(24)](6-) resulted in formation of these two intermediate complexes that were detected by (31)P NMR spectroscopy. Complex A was characterized by a (31)P NMR resonance at -4.27 ppm and complex B was characterized by a (31)P NMR resonance at -7.42 ppm. On the basis of the previous results from diffusion ordered NMR spectroscopy, performed with the hydrolytically inactive substrate phenylphosphonate (PhP), the structure of these two complexes was deduced to be (NPP)(2)Mo(5)O(21)(4-) (complex A) and (NPP)(2)Mo(12)O(36)(H(2)O)(6)(4-) (complex B). The pH studies point out that both complexes are hydrolytically active and lead to the hydrolysis of phosphoester bond in NPP. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), and indicating that the cleavage of the phosphomonoester bond is purely hydrolytic. The Mo K-edge XANES region also did not show any sign of Mo(VI) to Mo(V) reduction during the hydrolytic reaction. (95)Mo NMR and Mo K-edge EXAFS spectra measured during different stages of the hydrolytic reaction showed a gradual disappearance of [Mo(7)O(24)](6-) during the hydrolytic reaction and appearance of [P(2)Mo(5)O(23)](6-), which was the final complex observed at the end of hydrolytic reaction

Copper(II) 15-metallacrown-5 lanthanide(III) complexes derived from L-serine and L-threonine hydroxamic acids

Formation of pentanuclear copper(II) complexes with serine hydroxamic acid (Serha) and threonine hydroxamic acid (Thrha) was studied in solution by electrospray ionization mass spectrometry (ESI-MS), absorption spectrophotometry and proton NMR spectroscopy. The presence of lanthanide(III) ions is essential for the self-assembly of the 15-metallacrown-5 compounds. The positive mode ESI-MS spectra of the solutions containing Serha or Thrha, copper(II), and lanthanide(III) ions (Ln = La, Nd, Gd,) in the ratio 5:5:1 showed only the peaks that could be unambiguously assigned to the following intact molecular ions: {La(NO3)[15-MC-5]−}+ and {La(NO3)2[15-MC-5]}2+. The NMR spectra of the pentanuclear species revealed only one set of peaks indicating a five-fold symmetry of the complex. The UV–vis experiments revealed that upon addition of 0.2 equivalents of lanthanide(III) to the equimolar solutions containing Serha and copper(II), the self-assembly of the metallacrown structure was accomplished within 30 min.status: publishe

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

Detailed Mechanism of ATP hydrolysis promoted by a Binuclear ZrIV-Substituted Keggin Polyoxometalate Elucidated by a Combination of 31P, 31P DOSY and 31P EXSY NMR Spectroscopy

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