Artificial Metalloenzymes

Artificial metalloenzymes have emerged as a promising approach to merge the attractive properties of homogeneous catalysis and biocatalysis. The activity and selectivity, including enantioselectivity, of natural metalloenzymes are due to the second coordination sphere interactions provided by the protein. Artificial metalloenzymes aim at harnessing second coordination sphere interactions to create transition metal complexes that display enzyme-like activities and selectivities. In this Review, the various approaches that can be followed for the design and optimization of an artificial metalloenzyme are discussed. An overview of the synthetic transformations that have been achieved using artificial metalloenzymes is provided, with a particular focus on recent developments. Finally, the role that the second coordination sphere plays in artificial metalloenzymes and their potential for synthetic applications are evaluated

Bacterial ligands as flexible and sensitive detectors in rapid tests for antibodies to SARS-CoV-2

Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7-97.0) and selectivity (91.7%, 82.6-100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs

Catalytic enantioselective syn hydration of enones in water using a DNA-based catalyst

The enantioselective addition of water to olefins in an aqueous environment is a common transformation in biological systems, but was beyond the ability of synthetic chemists. Here, we present the first examples of a non-enzymatic catalytic enantioselective hydration of enones, for which we used a catalyst that comprises a copper complex, based on an achiral ligand, non-covalently bound to (deoxy)ribonucleic acid, which is the only source of chirality present under the reaction conditions. The chiral β-hydroxy ketone product was obtained in up to 82% enantiomeric excess. Deuterium-labelling studies demonstrated that the reaction is diastereospecific, with only the syn hydration product formed. So far, this diastereospecific and enantioselective reaction had no equivalent in conventional homogeneous catalysis

DNA-based catalysis and micellar catalysis

In this thesis the use of the DNA molecule as a chiral scaffold for enantioselective catalysis was discussed. The intrinsic chirality of the DNA is transferred within a metal catalyzed reaction resulting in an excess of one of the enantiomers of the product. It has been shown that the assembly of DNA-based catalysts allows for excellent selectivity and enantioselectivity in several metal catalyzed reactions in water. Initially, the focus was on DNA-based catalysts from ligands of the first generation (which involve a metal binding domain, a spacer and a DNA-interacting moiety) in two reactions: the enantioselective CuII-catalyzed Diels-Alder reaction and the asymmetric CuII-catalyzed syn-hydration of enones in water, a reaction for which no equivalent in conventional catalysis exists. The role of DNA in the observed catalysis and enantioselectivity was defined for both reactions and the attempts to improve the design and the performance of the catalysts were reported. In the second part of the thesis the concept of micellar catalysis was introduced. A recent application which involved the use of an anionic surfactant in combination with a copper salt in order to achieve a dramatic acceleration of the Friedel-Craft reaction in water was presented. Lastly, the use of DNA as a promising tool to achieve control over the geometry and the location of the catalyst in space in the presence of a micellar aggregate, and to tune its activity, was described. All together, the results demonstrate the great potential of DNA in catalysis and supramolecular chemistry.

A Ligand Structure–Activity Study of DNA-Based Catalytic Asymmetric Hydration and Diels–Alder Reactions.

A structure–activity relationship study of the first generation ligands for the DNA-based asymmetric hydration of enones and Diels–Alder reaction in water is reported. The design of the ligand was optimized resulting in a maximum ee of 83% in the hydration reaction and 75% in the Diels–Alder reaction, and some guidelines for ligand design were formulated. A comparison between these two reaction classes using salmon testes DNA/Cu2+-L catalysts, showed that the enantioselectivity in the hydration reaction was not the result of selective shielding of one π face of the enone. In contrast, the structure of the ligand was suggested to be crucial to position and orient the substrate bound Cu2+ complex optimally with respect to the structured first hydration layer of the DNA. Likely, the DNA activates and directs the H2O nucleophile for attack to one preferred π face of the enone.

Dramatic micellar rate enhancement of the Cu2+ catalyzed vinologous Friedel–Crafts alkylation in water

A dramatic rate enhancement of the Cu2+ catalyzed Friedel–Crafts alkylation in water was achieved in the presence of sodium dodecyl sulfate (SDS) micelles.

A Kinetic and Structural Investigation of DNA-Based Asymmetric Catalysis Using First-Generation Ligands

The recently developed concept of DNA-based asymmetric catalysis involves the transfer of chirality from the DNA double helix in reactions using a noncovalently bound catalyst. To date, two generations of DNA-based catalysts have been reported that differ in the design of the ligand for the metal. Herein we present a study of the first generation of DNA-based catalysts, which contain ligands comprising a metal-binding domain linked through a spacer to a 9-aminoacridine moiety. Particular emphasis has been placed on determining the effect of DNA on the structure of the CuII complex and the catalyzed Diels–Alder reaction. The most important findings are that the role of DNA is limited to being a chiral scaffold; no rate acceleration was observed in the presence of DNA. Furthermore, the optimal DNA sequence for obtaining high enantioselectivities proved to contain alternating GC nucleotides. Finally, DNA has been shown to interact with the CuII complex to give a chiral structure. Comparison with the second generation of DNA-based catalysts, which bear bipyridine-type ligands, revealed marked differences, which are believed to be related to the DNA microenvironment in which the catalyst resides and where the reaction takes place.

Expression and function of P-glycoprotein and absence of multidrug resistancerelated protein in rat and beige mouse peritoneal mast cells, Histochem

Summary To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistancerelated Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT-PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules