Peptide bond hydrolysis catalyzed by NU-1000 MOF revealed key structural features that influence the catalytic performance of Zr₆ based MOFs.

CONSPECTUS 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 like trypsin, which are sensitive to 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 like 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 last decade, our group has discovered the unique protease activity of diverse metal-oxo clusters (MOC), and pioneered a distinctive approach for the development of selective artificial proteases. In contrast to classical coordination complexes which often depend on amino acid side chains to control regioselectivity, the selectivity profile of MOCs is determined by a complex combination of structural factors, such as 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 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.

The selective hydrolysis of proteins by non‐enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal‐oxo cluster [Hf18O10(OH)26(SO4)13⋅(H2O)33] (Hf18), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18. X‐ray scattering and ESI‐MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.

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The selective hydrolysis of proteins by non‐enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal‐oxo cluster [Hf18O10(OH)26(SO4)13⋅(H2O)33] (Hf18), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18. X‐ray scattering and ESI‐MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.