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 H NMR spectroscopy revealed that the rate of glycylglycine (Gly-Gly) hydrolysis at pD 7.4 and 60 °C was 2.69 × 10 s ( t = 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 {ZrO} 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 {ZrO} 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.

Hen-egg-white lysozyme (HEWL) is specifically cleaved at the Trp28-Val29 and Asn44-Arg45 peptide bonds in the presence of a Keggin-type [Ce(α-PW(11)O(39))(2)](10-) polyoxometalate (POM; 1) at pH 7.4 and 37 °C. The reactivity of 1 towards a range of dipeptides was also examined and the calculated reaction rates were comparable to those observed for the hydrolysis of HEWL. Experiments with α-lactalbumin (α-LA), a protein that is structurally highly homologous to HEWL but has a different surface potential, showed no evidence of hydrolysis, which indicates the importance of electrostatic interactions between 1 and the protein surface for the hydrolytic reaction to occur. A combination of spectroscopic techniques was used to reveal the molecular interactions between HEWL and 1 that lead to hydrolysis. NMR spectroscopy titration experiments showed that on protein addition the intensity of the (31)P NMR signal of 1 gradually decreased due to the formation of a large protein/polyoxometalate complex and completely disappeared when the HEWL/1 ratio reached 1:2. Circular dichroism (CD) measurements of HEWL indicate that addition of 1 results in a clear decrease in the signal at λ=208 nm, which is attributed to changes in the α-helical content of the protein. (15)N-(1)H heteronuclear single quantum coherence (HSQC) NMR measurements of HEWL in the presence of 1 reveal that the interaction is mainly observed for residues that are located in close proximity to the first site in the α-helical part of the structure (Trp28-Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44-Arg45), which is found in the β-strand region of the protein, might be caused by weaker metal-directed binding, compared with strong POM-directed binding at the first site.

SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp-X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site.

The effect of the protein environment on the formation and stabilization of an elusive catalytically active polyoxometalate (POM) species, K [Hf(α -P W O )] (1), is reported. In the co-crystal of hen egg-white lysozyme (HEWL) with 1, the catalytically active monomeric species is observed, originating from the dimeric 1:2 POM form, while it is intrinsically unstable under physiological pH conditions. The protein-assisted dissociation of the dimeric POM was rationalized by means of DFT calculations. The dissociation process is unfavorable in bulk water, but becomes favorable in the protein-POM complex due to the low dielectric response at the protein surface. The crystal structure shows that the monomeric form is stabilized by electrostatic and water-mediated hydrogen bonding interactions with the protein. It interacts at three distinct sites, close to the aspartate-containing hydrolysis sites, demonstrating high selectivity towards peptide bonds containing this residue.

Proteolytic activity of heterogeneous Zr-based nanozymes is a promising technology for the development of selective protein cleavage protocols which are pivotal in modern proteomics. Here, we report the hydrolytic activity of nanoporous Zr6-based UiO-66 metal-organic framework (MOF) toward peptide bonds in a series of peptides and in hen egg white lysozyme protein. The standard UiO-66 MOF featuring unsubstituted 1,4-benzenedicarboxylate linkers hydrolyzed the glycylglycine with a rate constant of 7.9 × 10 -7 s -1 , (t1/2 = 10 days), which represents >10 4 -fold acceleration compared to the uncatalyzed reaction. Further, this reactivity was compared with UiO-66 analogs synthesized using modified linkers bearing NO2 and NH2 substituents, or using trifluoracetic acid as a modulator. Although the overall crystalline structure and particle size of these UiO-66 derivatives were generally conserved, they presented distinct nanoporous structures that could be directly correlated with the reaction rates at least an order of magnitude faster than the parent UiO-66 MOF. Further, the modified nanoporous structures also provided distinct reactivities across a series of dipeptide substrates probed. We propose that these differences might arise from the distinct MOF Lewis and Brønsted acidity resulting from the structural modifications. These findings highlight the potential of further optimizing Zr6-based MOF nanozymes to achieve residue-selective hydrolytic activity.

Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the presence of the dimeric zirconium(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (1) were performed by a combination of (1)H, (13)C and (31)P NMR spectroscopy. The observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10(-7) s(-1) and 4.44 × 10(-7) s(-1) respectively, representing a significant acceleration as compared to the uncatalyzed reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile with the fastest hydrolysis observed in the pD range of 5.5-6.0. Interaction of 1 with Gly-Ser and Gly-Gly via their amine nitrogen and amide oxygen was proven by (13)C NMR spectroscopy. The effective hydrolysis of Gly-Ser in the presence of 1 is most likely a combination of the polarization of the amide oxygen due to its binding to the Zr(IV) ion in 1 and the intramolecular attack of the Ser hydroxyl group on the amide carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant was also examined. The solution structure of 1 was investigated by means of (31)P NMR spectroscopy, revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution of 1 was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence and in the absence of the dipeptides.

A recent study [Angew. Chem. Int. Ed. 2015, 54, 7391-7394] has shown that horse heart myoglobin (HHM) is selectively hydrolyzed by a range of zirconium(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study, the molecular interactions between the Zr-POM catalysts and HHM are investigated by using a range of complementary techniques, including circular dichroism (CD), UV/Vis spectroscopy, tryptophan fluorescence spectroscopy, and H and P NMR spectroscopy. A tryptophan fluorescence quenching study reveals that, among all examined Zr-POMs, the most reactive POM, 2:2 Zr -Keggin, exhibits the strongest interaction with HHM. P NMR spectroscopy studies show that this POM dissociates in solution, resulting in the formation of a monomeric 1:1 Zr -Keggin structure, which is likely to be a catalytically active species. In the presence of Zr -POMs, HHM does not undergo complete denaturation, as evidenced by CD, UV/Vis, tryptophan fluorescence, and H NMR spectroscopy. CD spectroscopy shows a gradual decrease in the α-helical content of HHM upon addition of Zr -POMs. The largest effect is observed in the presence of a large Zr -Wells-Dawson structure, whereas small Zr -Lindqvist POM has the least influence on the decrease in the α-helical content of HHM. In all cases, the Soret band at λ=409 nm is maintained in the presence of all examined Zr-POMs, which indicates that no conformational changes in the protein occur near the heme group.

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 (1) were performed by a combination of (1)H, (13)C, and (31)P 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. (13)C NMR data were indicative for coordination of Gly-Gly to 1 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 1 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 1. To elucidate the role of 1 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.