Roelfes Group

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  • Synergistic Catalysis of Tandem Michael Addition / Enantioselective Protonation Reactions by an Artificial Enzyme
    Z. Zhou and G. Roelfes

    ACS Catalysis, 2021, 11, 9366-9369

    • Enantioselective protonation is conceptually one of the most attractive methods to generate an α-chiral center. However, enantioselective protonation presents major challenges, especially in water. Herein, we report a tandem Michael addition/enantioselective protonation reaction catalyzed by an artificial enzyme employing two abiological catalytic sites in a synergistic fashion: a genetically encoded noncanonical p-aminophenylalanine residue and a Lewis acid Cu(II) complex. The exquisite stereocontrol achieved in the protonation of the transient enamine intermediate is illustrated by up to >20:1 dr and >99% ee of the product. These results illustrate the potential of exploiting synergistic catalysis in artificial enzymes for challenging reactions
  • Repurposed and artificial heme enzymes for cyclopropanation reactions
    G. Roelfes

    Journal of Inorganic Biochemistry, 2021, accepted

    • Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed.
  • Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy
    A. Iyer, M. Baranov , A.J. Foster, S. Chordia, G. Roelfes, R. Vlijm, G. van den Bogaart, and B. Poolman

    ACS Chemical Biology, 2021, 16, 891–904

    • Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein–protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.
  • In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis
    S. Chordia, S. Narasimhan, A. Lucini Paioni, M. Baldus and G. Roelfes

    Angewandte Chemie International Edition, 2021, 60, 5913-5920
    - VIP paper

    • Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.
  • Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase
    R.B. Leveson-Gower, Z. Zhou, I. Drienovská and G. Roelfes

    ACS Catalysis, 2021, 11, 6763–6770

    • The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realise new mechanisms in biocatalysis. Here, we show that incorporation of the non-canonical amino acid para-aminophenylalanine (pAF) into the non-enzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-Crafts alkylation between alpha,beta-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates towards conjugate addition via the formation of intermediate iminium ion species, whilst the protein scaffold provides rate acceleration and enantio-induction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis-Menten kinetics of LmrR_pAF and evolved mutants reveals that new activities emerge via evolutionary pathways that diverge from one another and specialise catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many new biocatalytic transformations inspired by organocatalysis.
  • Merging whole-cell biosynthesis of styrene and transition-metal catalyzed derivatization reactions
    R.V. Maaskant, S. Chordia and G. Roelfes

    ChemCatChem, 2021, 13, 1607-1613

    • The approach of combining enzymatic and transition‐metal catalysis has been focused almost exclusively on using purified, isolated enzymes. The use of whole‐cell biocatalysis, instead of isolated enzymes, with transition‐metal catalysis, however, has been investigated only sparsely, to date. Herein we present the development of two transition‐metal catalyzed reactions used to derivatize styrene obtained from whole‐cell biosynthesis. Using a biocompatible ruthenium cross‐metathesis catalyst up to 1.5 mM stilbene could be obtained in the presence of E. coli , which simultaneously produced styrene. Using palladium catalysts and arylboronic acids, titers of up to 1 mM of several stilbene derivatives were obtained. These two transition‐metal catalyzed reactions are valuable additions to the toolbox of combined whole‐cell biocatalysis and transition‐metal catalysis, offering the possibility to supplement biosynthetic pathways with the chemical versatility of abiological transition metal catalysis.
  • Rapid and selective chemical editing of Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) via Cu(II)-catalyzed β-borylation of dehydroamino acids
    R.H. de Vries, J.H. Viel, O.P Kuipers and G. Roelfes

    Angewandte Chemie International Edition, 2021, 60, 3946-3950

    • We report the fast and selective chemical editing of ribosomally synthesized and post‐translationally modified peptides (RiPPs) by β‐borylation of dehydroalanine residues. The thiopeptide thiostrepton was modified efficiently using Cu(II)‐catalysis under mild conditions and 1D/2D NMR of the purified product showed site‐selective borylation of the terminal Dha residues. Using similar conditions, the thiopeptide nosiheptide, lanthipeptide nisin Z and protein SUMO_G98Dha were also modified efficiently. Borylated thiostrepton showed an up to 84‐fold increase in water solubility, and MIC assays showed that antimicrobial activity was maintained in thiostrepton and nosiheptide. The introduced boronic acid functionalities were shown to be valuable handles for chemical mutagenesis and in a reversible click reaction with triols for the pH‐controlled labeling of RiPPs.
  • A Water-Soluble Iridium Photocatalyst for Chemical Modification of Dehydroalanines in Peptides and Proteins
    R.C.W. van Lier, A.D. de Bruijn and G. Roelfes

    Chemistry, A European Journal, 2021, 27, 1430-1437

    • Dehydroalanine (Dha) residues are attractive non-canonical amino acids that occur naturally in ribosomally synthesised and post-translationally modified peptides (RiPPs). Dha residues are attractive targets for selective late-stage modification of these complex biomolecules. In this work, we show the selective photocatalytic modification of dehydroalanine residues in the antimicrobial peptide nisin and in the proteins Small Ubiquitin-like Modifier (SUMO) and superfolder Green Fluorescent Protein (sfGFP). For this purpose, a new water-soluble iridium(III) photoredox catalyst was used. The design and synthesis of this new photocatalyst, [Ir(dF(CF3)ppy)2(dNMe3bpy)]Cl3, is presented. In contrast to commonly used iridium photocatalysts, this complex is highly water-soluble and allows modification of peptides and proteins in water and aqueous solvents under physiologically relevant conditions and with short reaction times and low reagent and catalyst loadings. This work suggests that photoredox catalysis using this newly designed catalyst is a promising strategy to modify dehydroalanine-containing natural products and thus may have great potential for novel bioconjugation strategies.
  • 2020
    • Cofactor Binding Dynamics Influence the Catalytic Activity and Selectivity of an Artificial Metalloenzyme
      L. Villarino, S. Chordia, L. Alonso-Cotchico, E. Reddem, Z. Zhou, A.-M.W.H. Thunnissen, J.D. Maréchal and G. Roelfes

      ACS Catalysis, 2020, 10, 11783-11790

      • We present an artificial metalloenzymes based on the transcriptional regulator LmrR that exhibits dynamics involving the positioning of its abiological metal cofactor. The position of the cofactor, in turn, was found to be related to the preferred catalytic reactivity, which is either the enantioselective Friedel-Crafts alkylation of indoles with beta-substituted enones or the tandem Friedel-Crafts alkylation / enantioselective protonation of indoles with alpha-substituted enones. The artificial metalloenzyme could be specialized for one of these catalytic reactions introducing a single mutation in the protein. The relation between cofactor dynamics and activity and selectivity in catalysis has not been described for natural enzymes and, to date, appears to be particular for artificial metalloenzymes.
    • Novel modifications of nonribosomal peptides from Brevibacillus laterosporus MG64 and investigation of their mode of action
      Z. Li, R.H. de Vries, P. Chakraborty, C. Song, X. Zhao, D.-J. Scheffers, G. Roelfes, and O.P. Kuipers

      Applied and Environmental Microbiology, 2020, 86, e01981-20

      • Nonribosomal peptides (NRPs) are a class of secondary metabolites usually produced by microorganisms. They are of paramount importance in different applications, including biocontrol and pharmacy. Brevibacillus spp. are a rich source of NRPs yet have received little attention. In this study, we characterize four novel bogorol variants (bogorols I to L, cationic linear lipopeptides) and four succilins (succilins I to L, containing a succinyl group that is attached to the Orn3/Lys3 in bogorols I to L) from the biocontrol strain Brevibacillus laterosporus MG64. Further investigation revealed that the bogorol family of peptides employs an adenylation pathway for lipoinitiation, different from the usual pattern, which is based on an external ligase and coenzyme A. Moreover, the formation of valinol was proven to be mediated by a terminal reductase domain and a reductase encoded by the bogI gene. Furthermore, succinylation, which is a novel type of modification in the family of bogorols, was discovered. Its occurrence requires a high concentration of the substrate (bogorols), but its responsible enzyme remains unknown. Bogorols display potent activity against both Gram-positive and Gram-negative bacteria. Investigation of their mode of action reveals that bogorols form pores in the cell membrane of both Gram-positive and Gram-negative bacteria. The combination of bogorols and relacidines, another class of NRPs produced by B. laterosporus MG64, displays a synergistic effect on different pathogens, suggesting the great potential of both peptides as well as their producer B. laterosporus MG64 for broad applications. Our study provides a further understanding of the bogorol family of peptides as well as their applications.
    • Cu(II)-Catalyzed β-Silylation of Dehydroalanine Residues in Peptides and Proteins
      R.H. de Vries and G. Roelfes

      Chemical Communications, 2020, 56, 1058 - 11061

      • We report the efficient and selective Cu(II)-catalysed β-silylation of naturally occurring dehydroalanine (Dha) residues in various Ribosomally synthesized and Post-translationally modified Peptides (RiPPs). The method is also applicable to proteins, as was shown by the modification of a Dha residue that was chemically introduced into Small Ubiquitin-like Modifier (SUMO).
    • Unexpected catalytic activity of the regulatory protein QacR
      C. Gutiérrez de Souza, L. Alonso-Cotchico, M. Bersellini and G. Roelfes

      ChemRxiv, 2020, preprint

    • Characterization of two relacidines belonging to a novel class of circular lipopeptides that act against Gram-negative bacterial pathogens
      Z. Li, P. Chakraborty, R.H. de Vries, C. Song, X. Zhao, G. Roelfes, D.-J. Scheffers and O.P. Kuipers

      Environmental Microbiology, 2020, 22, 5125-5136

      • The development of sustainable agriculture and the increasing antibiotic resistance of human pathogens call for novel antimicrobial compounds. Here, we describe the extraction and characterization of a class of cationic circular lipopeptides, for which we propose the name relacidines, from the soil bacterium Brevibacillus laterosporus MG64. Relacidines are composed of a fatty acid side chain (4‐methylhexanoic acid) and 13 amino acid residues. A lactone ring is formed by the last five amino acid residues and three positively charged ornithines are located in the linear fragment. Relacidines selectively combat Gram‐negative pathogens, including phytopathogens and human pathogens. Further investigation of the mode of action revealed that relacidine B binds to the lipopolysaccharides (LPS) but does not form pores in the cell membrane. We also provide proof to show that relacidine B does not affect the biosynthesis of the cell wall and RNA. Instead, it affects the oxidative phosphorylation process of cells and diminishes the biosynthesis of ATP. Transcription of relacidines is induced by plant pathogens, which strengthens the potential of B. laterosporus MG64 to be used as a biocontrol agent. Thus, we identified a new group of potent antibiotic compounds for combating Gram‐negative pathogens of plants or animals.
    • A hydroxyquinoline-based unnatural amino acid for the design of novel artificial metalloenzymes
      I. Drienovská, R.A. Scheele, C. Gutiérrez de Souza and G. Roelfes

      ChemBioChem, 2020, 21, 3077-3081

      • Here we examine the potential of the non-canonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug resistance regulator (LmrR) via stop codon suppression methodology. LmrR_HQAla was shown to efficiently complex with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII bound LmrR_HQAla enzyme was shown through its ability to catalyze asymmetric Friedel-Craft alkylation and water addition reactions, whereas the ZnII coupled enzyme was shown to mimic natural Zn-hydrolase activity.
    • Impact of binding to the Multidrug Resistant Regulator protein LmrR on the photo-physics and -chemistry of photosensitizers
      S.H. Mejías, G. Roelfes and W.R. Browne

      Physical Chemistry Chemical Physics, 2020, 22, 12228-12238

      • Light activated photosensitizers generate reactive oxygen species (ROS) that interfere with cellular components and can induce cell death, e.g., in photodynamic therapy (PDT). The effect of cellular components and especially proteins on the photochemistry and photophysics of the sensitizers is a key aspect in drug design and the correlating cellular response with the generation of specific ROS species. Here, we show the complex range of effects of binding of photosensitizer to a multidrug resistance protein, produced by bacteria, on the formers reactivity. We show that recruitment of drug like molecules by LmrR (Lactococcal multidrug resistance Regulator) modifies their photophysical properties and their capacity to induce oxidative stress especially in 1O2 generation, including rose bengal (RB), protoporphyrin IX (PpIX), bodipy, eosin Y (EY), riboflavin (RBF), and rhodamine (Rh6G). The range of neutral and charged dyes with different exited redox potentials, are broadly representative of the dyes used in PDT.
    • Artificial Metalloenzymes based on TetR Proteins and Cu(II) for Enantioselective Friedel-Crafts Alkylation Reactions
      C. Gutiérrez de Souza, M. Bersellini and G. Roelfes

      ChemCatChem, 2020, 12, 3190-3194

      • The supramolecular approach is among the most convenient methodologies for creating artificial metalloenzymes (ArMs). Usually this approach involves the binding of a transition metal ion complex to a biomolecular scaffold via its ligand, which also modulates the catalytic properties of the metal ion. Herein, we report ArMs based on the proteins CgmR, RamR and QacR from the TetR family of multidrug resistance regulators (MDRs) and Cu2+ ions, assembled without the need of a ligand. These ArMs catalyze the enantioselective vinylogous Friedel‐Crafts alkylation reaction with up to 75 % ee. Competition experiments with ethidium and rhodamine 6G confirm that the reactions occur in the chiral environment of the hydrophobic pocket. It is proposed that the Cu2+‐substrate complex is bound via a combination of electrostatic and π‐stacking interactions provided by the second coordination sphere. This approach constitutes a fast and straightforward way to assemble metalloenzymes and may facilitate future optimization of the protein scaffolds via mutagenesis or directed evolution approaches.
    • Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sites
      Z. Zhou and G. Roelfes

      Nature Catalysis, 2020, 3, 289-294

      • Artificial enzymes, which are hybrids of proteins with abiological catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiological catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiological catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalysed Michael addition reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(ii) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiological catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes.
    • Expanding the enzyme universe with genetically encoded unnatural amino acids.
      I. Drienovská and G. Roelfes

      Nature Catalysis, 2020, 3, 192-202

      • The emergence of robust methods to expand the genetic code allows incorporation of non-canonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chemical functionalities in enzymes. In this Perspective, we show how this powerful methodology is used to create enzymes with improved and novel, even new-to-nature, catalytic activities. We provide an overview of the current state of the art, and discuss the potential benefits of developing and using enzymes with genetically encoded non-canonical amino acids compared with enzymes containing only canonical amino acids.
    • Cationic iron porphyrins with sodium dodecyl sulphate for micellar catalysis of cyclopropanation reactions
      R.V. Maaskant, E.A. Polanco, R.C.W. van Lier and G. Roelfes

      Organic & Biomolecular Chemistry, 2020, 18, 638-641

      • Here, we report that the combination of cationic iron porphyrins with sodium dodecyl sulphate (SDS) gives rise to efficient micellar catalysis of cyclopropanation reactions of styrene derivatives, using diazoacetates as carbene precursors. This simple, yet effective approach for cyclopropanations illustrates the power of micellar catalysis.
    • A trifunctional linker for palmitoylation and peptide and protein localization in biological membranes
      L. Syga, R.H. de Vries, H. van Oosterhout, R. Bartelds, A.J. Boersma, G. Roelfes and B. Poolman

      ChemBioChem, 2020, 21, 1320-1328

      • Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunctional linker. The linker allows in a single step facile incorporation of two different functionalities at a cysteine. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α‐helical model peptide WALP w/wo palmitoyl groups in giant unilamellar vesicles that contain a liquid‐ordered (Lo) and liquid‐disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane‐spanning peptide.
  • 2019
    • The importance of catalytic promiscuity for enzyme design and evolution
      R.B. Leveson-Gower, C. Mayer and G. Roelfes

      Nature Reviews Chemistry, 2019, 3, 687-705

      • The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms' abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new- to-nature reactions and highlight the successes of subsequent directed- evolution efforts to fine- tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.
    • Selective Modification of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs) via Diels-Alder Cycloadditions on Dehydroalanine Residues - hot paper
      R.H. de Vries, J.H. Viel, R. Oudshoorn, O.P. Kuipers and G. Roelfes

      Chemistry, A European Journal, 2019, 25, 12698-12702
      - hot paper

      • We report the late stage chemical modification of ribosomally synthesized and posttranslationally modified peptides (RIPPs) by Diels‐Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition metal free conditions. The Diels‐Alder adducts were isolated and the different site‐ and endo/exo isomers were identified by 1D/2D 1H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. MIC assays of the purified thiostrepton Diels‐Alder products against thiostrepton‐susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels‐Alder products were also subjected successfully to Inverse‐electron‐demand Diels‐Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.
    • Folic acid conjugates of a Bleomycin mimic for selective targeting of folate receptor positive cancer cells
      A. Geersing, R.H. de Vries, M.G. Rots, G. Jansen and G. Roelfes

      Bioorganic & Medicinal Chemistry Letters, 2019, 29, 1922-1927

      • A major challenge in the application of cytotoxic anti-cancer drugs is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. A particularly promising target for selective targeting are the folate receptors (FR) that are often over-expressed on cancer cells. Here, we report on a conjugate of the pentadentate nitrogen ligand N4Py to folic acid, via a cleavable disulphide linker, which shows selective cytotoxicity against folate receptor expressing cancer cells.
    • An Integrated Computational Study of the Cu-Catalyzed Hydration of Alkenes in Water Solvent and into the Context of an Artificial Metallohydratase
      L. Alonso Cotchico, G. Sciortino, P. Vidossich, J. Rodríguez-Guerra Pedregal, I. Drienovská, G. Roelfes, A. Lledos and J.-D. Maréchal

      ACS Catalysis, 2019, 5, 4615-4626

      • Despite the increasing efforts in the last few years, the identification of efficient catalysts able to perform the enantioselective addition of water to double bonds has not been achieved yet. Natural hydratases represent an interesting pool of biocatalysts to generate chiral alcohols, but modifying their substrate scope remains an issue. The use of artificial metalloenzymes (ArMs) appears as a promising solution in this field. In the last few years, Roelfes and co-workers have been designing a variety of DNA- and protein-based ArMs able to carry out the copper-mediated addition of water to conjugated alkenes with promising enantioselective levels. Still, from a mechanistic point of view, the copper-mediated hydration reaction remains unclear and a matter of debate. This lack of information greatly hampers further designs and optimizations of the LmrR-based copper hydratases in terms of substrates and/or enantioselective profiles. In this study, we aim to provide a better understanding of the copper-catalyzed hydration of alkenes occurring both in water solvent and into the context of the LmrR protein as designed by Roelfes and co-workers. For that purpose, we make use of an integrated computational protocol that combines quantum mechanics (QM) (including small and large cluster models as well as ab initio molecular dynamics (AIMD)) and force-field approaches (including proteinligand docking and classical molecular dynamics (MD) simulation). This integrative study sheds light on the general doubts around the copper-catalyzed hydration mechanism and also paves the way toward more conscious designs of ArMs able to efficiently catalyze the enantioselective addition of water to double bonds.
    • LmrR: a privileged scaffold for artificial metalloenzymes
      G. Roelfes

      Accounts of Chemical Research, 2019, 52, 545−556

      • The biotechnological revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense number of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, that is, effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compounds by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramolecular assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chemical modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed FriedelCrafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the FriedelCrafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)phenanthroline was successful in FriedelCrafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramolecular assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metabolism.
    • A "Broad Spectrum" Carbene Transferase for Synthesis of Chiral alpha-Trifluoromethylated Organoborons ("First Reactions")
      L. Alonso-Cotchico and G. Roelfes

      ACS Central Science, 2019, 5, 206-208

      • Directed evolution generated an enzyme for the enantioselective synthesis of α-trifluoromethylated organoboronspotentially attractive synthons for fluorinated compounds.
    • Directed evolution of a designer enzyme featuring an unnatural catalytic amino acid - hot paper
      C. Mayer, C. Dulson, E. Reddem, A.-M.W.H. Thunnissen and G. Roelfes

      Angewandte Chemie International Edition, 2019, 58, 2083-2087
      - hot paper

      • The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Here we show that such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, we report the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction. Consecutive rounds of directed evolution identified a number of mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat) of the designer enzyme by almost 100‐fold. Crucially, these gains result from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by >200‐fold.
    • Bio-orthogonal metalloporphyrin catalyzed selective methionine alkylation in the lanthipeptide Nisin
      R.V. Maaskant and G. Roelfes

      ChemBioChem, 2019, 20, 57-61

      • Bioorthogonal catalytic modification of ribosomally synthesized and post‐translationally modified peptides (RiPPs) is a promising approach to obtaining novel antimicrobial peptides with improved properties and/or activities. Here, we present the serendipitous discovery of a selective and rapid method for the alkylation of methionines in the lanthipeptide nisin. Using carbenes, formed from water‐soluble metalloporphyrins and diazoacetates, methionines are alkylated to obtain sulfonium ions. The formed sulfonium ions are stable, but can be further reacted to obtain functionalized methionine analogues, expanding the toolbox of chemical posttranslational modification even further.
  • 2018
    • A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue
      I. Drienovská, C. Mayer, C. Dulson and G. Roelfes

      Nature Chemistry, 2018, 10, 946-952

      • Creating designer enzymes with the ability to catalyse abiological transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
    • Chemical modification of dehydrated amino acids in natural antimicrobial peptides by photoredox catalysis
      A.D. de Bruijn and G. Roelfes

      Chemistry, A European Journal, 2018, 24, 11314-11318

      • Dehydroalanine (Dha) and dehydrobutyrine (Dhb) are remarkably versatile non‐canonical amino acids often found in antimicrobial peptides. Here, we present the selective modification of Dha and Dhb in antimicrobial peptides via photocatalytic activation of organoborates under influence of visible light. Ir(dF(CF3)ppy)2(dtbbpy)PF6 was used as photoredox catalyst in aqueous solutions for the modification of thiostrepton and nisin. The mild conditions and high selectivity for the dehydrated residues, show photoredox catalysis is a promising tool for modification of peptide derived natural products.
    • Hybrid catalysts as Lewis acid
      G. Roelfes, I. Drienovská and L. Villarino

      in Artificial Metalloenzymes and MetalloDNAzymes in Catalysis, From Design to Application , 2018, M. Diéguez, J.-E. Bäckvall and O. Pàmies (Eds.), Wiley-VCH, Weinheim, 225-251

    • Catalytic modification of dehydroalanine in peptides and proteins via palladium mediated cross coupling - hot paper
      A.D. de Bruijn and G. Roelfes

      Chemistry, A European Journal, 2018, 24, 12728-12733
      - hot paper

      • Dehydroalanine (Dha) is a remarkably versatile non‐canonical amino acid often found in antimicrobial peptides. Here we present the catalytic modification of Dha via a palladium mediated cross coupling reaction. Using Pd(EDTA)(OAc)2 as water soluble catalyst, a variety of arylboronic acids was coupled to the dehydrated residues in proteins and peptides such as nisin. The cross coupling reaction yields both the Heck product, in which the sp2‐hybridisation of the α‐carbon is retained, as well as the conjugated addition product. The reaction can be performed under mild aqueous conditions, which makes this method an attractive addition to the palette of bio‐orthogonal catalytic methods.
    • Importance of Metal Ion Exchange for the Biological Activity of Co-ordination Complexes of the Biomimetic Ligand N4Py
      A. Geersing, N. Ségaud, M.P.G. van der Wijst, M.G. Rots and G. Roelfes

      Inorganic Chemistry, 2018, 57, 7748-7756

      • Metal coordination complexes can display interesting biological activity, as illustrated by the bleomycins (BLMs), a family of natural antibiotics that when coordinated to a redox-active metal ion, show antitumor activity. Yet, which metal ion is required for the activity in cells is still subject to debate. In this study, we described how different metal ions affect the intracellular behavior and activity of the synthetic BLM-mimic N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py). Our study shows that a mixture of iron(II), copper(II), and zinc(II) complexes can be generated when N4Py is added to cell cultures but that the metal ion can also be exchanged by other metal ions present in cells. Moreover, the combination of chemical data, together with the performed biological experiments, shows that the active complex causing oxidative damage to cells is the FeII-N4Py complex and not per se the metal complex that was initially added to the cell culture medium. Finally, it is proposed that the high activity observed upon the addition of the free N4Py ligand is the result of a combination of scavenging of biologically relevant metals and oxidative damage caused by the iron(II) complex.
    • An artificial heme enzyme for cyclopropanation reactions
      L. Villarino, K.E. Splan, E. Reddem, L. Alonso-Cotchico, C. Gutiérrez de Souza, A. Lledós, J.-D. Maréchal, A.-M.W.H. Thunnissen and G. Roelfes

      Angewandte Chemie International Edition, 2018, 57, 7785-7789

      • An artificial heme enzyme was created through self‐assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre‐catalytic structures.
    • Responsive DNA G-quadruplex micelles
      L. Cozzoli, L. Gjonaj, M.C.A. Stuart, B. Poolman and G. Roelfes

      Chemical Communications, 2018, 54, 260-263

      • A novel and versatile design of DNAlipid conjugates is presented. The assembly of the DNA headgroups into G-quadruplex structures is essential for the formation of micelles and their stability. By hybridization with a complementary oligonucleotide the micelles were destabilized, resulting in cargo release. In combination with a hairpin DNA aptamer as complementary strand, the release is obtained selectively by the presence of ATP.
  • 2017
    • Artificial metalloproteins for binding and stabilization of a semiquinone radical
      N. Ségaud, I. Drienovská, J. Chen, W.R. Browne and G. Roelfes

      Inorganic Chemistry, 2017, 56, 13293-13299

      • The interaction of a number of first-row transition-metal ions with a 2,2'-bipyridyl alanine (bpyA) unit incorporated into the lactococcal multidrug resistance regulator (LmrR) scaffold is reported. The composition of the active site is shown to influence binding affinities. In the case of Fe(II), we demonstrate the need of additional ligating residues, in particular those containing carboxylate groups, in the vicinity of the binding site. Moreover, stabilization of di-tert-butylsemiquinone radical (DTB-SQ) in water was achieved by binding to the designed metalloproteins, which resulted in the radical being shielded from the aqueous environment. This allowed the first characterization of the radical semiquinone in water by resonance Raman spectroscopy.
    • Design of an enantioselective artificial metallo-hydratase enzyme containing an unnatural metal-binding amino acid
      I. Drienovská, L. Alonso-Cotchico, P. Vidossich, A. Lledós, J.-D. Maréchal and G. Roelfes

      Chemical Science, 2017, 8, 7228-7235

      • The design of artificial metalloenzymes is a challenging, yet ultimately highly rewarding objective because of the potential for accessing new-to-nature reactions. One of the main challenges is identifying catalytically active substrate-metal cofactorhost geometries. The advent of expanded genetic code methods for the in vivo incorporation of non-canonical metal-binding amino acids into proteins allow to address an important aspect of this challenge: the creation of a stable, well-defined metal-binding site. Here, we report a designed artificial metallohydratase, based on the transcriptional repressor lactococcal multidrug resistance regulator (LmrR), in which the non-canonical amino acid (2,2′-bipyridin-5yl)alanine is used to bind the catalytic Cu(II) ion. Starting from a set of empirical pre-conditions, a combination of cluster model calculations (QM), proteinligand docking and molecular dynamics simulations was used to propose metallohydratase variants, that were experimentally verified. The agreement observed between the computationally predicted and experimentally observed catalysis results demonstrates the power of the artificial metalloenzyme design approach presented here.
    • Multidrug resistance regulators (MDRs) as scaffold for the design of artificial metalloenzymes
      M. Bersellini and G. Roelfes

      Organic & Biomolecular Chemistry, 2017, 15, 3069-3073

      • The choice of protein scaffolds is an important element in the design of artificial metalloenzymes. Herein, we introduce Multidrug Resistance Regulators (MDRs) from the TetR family as a viable class of protein scaffolds for artificial metalloenzyme design. In vivo incorporation of the metal binding amino acid (2,2-bipyridin-5yl)alanine (BpyA) by stop codon suppression methods was used to create artificial metalloenzymes from three members of the TetR family of MDRs: QacR, CgmR and RamR. Excellent results were achieved with QacR Y123BpyA in the Cu(2+) catalyzed enantioselective vinylogous Friedel-Crafts alkylation reaction with ee's up to 94% of the opposite enantiomer that was achieved with other mutants and the previously reported LmrR-based artificial metalloenzymes.
    • A metal ion regulated artificial metalloenzyme
      M. Bersellini and G. Roelfes

      Dalton Transactions, 2017, 46, 4325-4330

      • Regulation of enzyme activity is essential in living cells. The rapidly increasing number of designer enzymes with new-to-nature activities makes it necessary to develop novel strategies for controlling their catalytic activity. Here we present the development of a metal ion regulated artificial metalloenzyme created by combining two anchoring strategies, covalent and supramolecular, for introducing a regulatory and a catalytic site, respectively. This artificial metalloenzyme is activated in the presence of Fe(2+) ions, but only marginally in the presence of Zn(2+)
  • 2016
    • DNA-accelerated copper catalysis of Friedel-Crafts conjugate addition / enantioselective protonation reactions in water
      A. García-Fernández, R. P. Megens, L. Villarino and G. Roelfes

      Journal of the American Chemical Society, 2016, 138, 16308-16314

      • DNA-induced rate acceleration has been identified as one of the key elements for the success of the DNA-based catalysis concept. Here we report on a novel DNA-based catalytic FriedelCrafts conjugate addition/enantioselective protonation reaction in water, which represents the first example of a reaction that critically depends on the >700- to 990-fold rate acceleration caused by the presence of a DNA scaffold. The DNA-induced rate acceleration observed is the highest reported due to the environment presented by a biomolecular scaffold for any hybrid catalyst, to date. Based on a combination of kinetics and binding studies, it is proposed that the rate acceleration is in part due to the DNA acting as a pseudophase, analogous to micelles, in which all reaction components are concentrated, resulting in a high effective molarity. The involvement of additional second coordination sphere interactions is suggested by the enantioselectivity of the product. The results presented here show convincingly that the DNA-based catalysis concept, thanks to the DNA-accelerating effect, can be an effective approach to achieving a chemically challenging reaction in water.
    • DNA-Accelerated Catalysis of Carbene Transfer Reactions by a DNA/Cationic Iron Porphyrin Hybrid
      A. Rioz-Martínez, J. Oelerich, N. Ségaud and G. Roelfes

      Angewandte Chemie International Edition, 2016, 55, 14136-14140

      • A novel DNA-based hybrid catalyst comprised of salmon testes DNA and an iron(III) complex of a cationic meso-tetrakis(N-alkylpyridyl)porphyrin was developed. When the N-methyl substituents were placed at the ortho position with respect to the porphyrin ring, high reactivity in catalytic carbene-transfer reactions was observed under mild conditions, as demonstrated in the catalytic enantioselective cyclopropanation of styrene derivatives with ethyl diazoacetate (EDA) as the carbene precursor. A remarkable feature of this catalytic system is the large DNA-induced rate acceleration observed in this reaction and the related dimerization of EDA. It is proposed that high effective molarity of all components of the reaction in or near the DNA is one of the key contributors to this unique reactivity. This study demonstrates that the concept of DNA-based asymmetric catalysis can be expanded into the realm of organometallic chemistry.
    • A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization
      J.H.M. van der Velde, J. Oelerich, J. Huang, J.H. Smit, A. Aminian Jazi, S. Galiani, K. Kolmakov, G. Guoridis, C. Eggeling, A. Herrmann, G. Roelfes and T. Cordes

      Nature Communications, 2016, 7, 10144

      • Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.
  • 2015
    • DNA-based asymmetric catalysis
      A. Rioz-Martínez and G. Roelfes

      in DNA in supramolecular chemistry and nanotechnology, 2015, E. Stulz and G.H. Clever (Eds.), John Wiley & Sons, 474-490

    • Supramolecular Assembly of Artificial Metalloenzymes Based on the Dimeric Protein LmrR as Promiscuous Scaffold
      J. Bos, W.R. Browne, A.J.M. Driessen and G. Roelfes

      Journal of the American Chemical Society, 2015, 137, 9796–9799

      • Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel–Crafts alkylation of indoles
    • Selective chemical modification of DNA with alkoxy- and benzyloxyamines
      L. Gjonaj and G. Roelfes

      Organic & Biomolecular Chemistry, 2015, 30, 6059-6065

      • A new method for the selective chemical modification of DNA at cytosine nucleobases using alkoxy- and benzyloxyamines is presented. It is shown that in particular benzyloxyamines are effective DNA modifying agents, giving rise to almost exclusive formation of the mono addition products. By using a bifunctional derivative, that is, p-azidobenzyloxyamine hydrochloride, an azide moiety, which is a convenient handle for further functionalization, could be introduced into the DNA. The azido modified DNA was then further reacted in a copper(I)-monophos catalysed 1,3-dipolar cycloaddition. These results illustrate the potential of the presented method for application in site and chemo-selective modification of DNA.
    • The power of four (News & Views)
      A.J. Boersma and G. Roelfes

      Nature Chemistry, 2015, 7, 277-279

      • Supramolecular assembly has been used to design and create new proteins capable of performing biomimetic functions in complex environments such as membranes and inside living cells.
    • Targeting Nrf2 in healthy and malignant ovarian epithelial cells: Protection versus promotion
      M.G.P van der Wijst, C. Huisman, A. Mposhi, G. Roelfes and M.G. Rots

      Mol. Oncol., 2015, 9, 1259-1273

      • Risk factors indicate the importance of oxidative stress during ovarian carcinogenesis. To tolerate oxidative stress, cells activate the transcription factor Nrf2 (Nfe2l2), the master regulator of antioxidant and cytoprotective genes. Indeed, for most cancers, hyperactivity of Nrf2 is observed, and siRNA studies assigned Nrf2 as therapeutic target. However, the cancer-protective role of Nrf2 in healthy cells highlights the requirement for an adequate therapeutic window. We engineered artificial transcription factors to assess the role of Nrf2 in healthy (OSE-C2) and malignant ovarian cells (A2780). Successful NRF2 up- and downregulation correlated with decreased, respectively increased, sensitivity toward oxidative stress. Inhibition of NRF2 reduced the colony forming potential to the same extent in wild-type and BRCA1 knockdown A2780 cells. Only in BRCA1 knockdown A2780 cells, the effect of Nrf2 inhibition could be enhanced when combined with PARP inhibitors. Therefore, we propose that this combination therapy of PARP inhibitors and Nrf2 inhibition can further improve treatment efficacy specifically in BRCA1 mutant cancer cells without acquiring the side-effects associated with previously studied Nrf2 inhibition combinations with either chemotherapy or radiation. Our findings stress the dual role of Nrf2 in carcinogenesis, while offering approaches to exploit Nrf2 as a potent therapeutic target in ovarian cancer.
    • Alkylidene malonates and alpha,beta-unsaturated alpha-hydroxyketones as practical substrates for vinylogous Friedel-Crafts alkylations in water catalysed by scandium(III) triflate / SDS
      J. Oelerich and G. Roelfes

      Org. Biomol. Chem., 2015, 13, 2793-2799

      • Alkylidene malonates and α,β-unsaturated α′-hydroxyketones are demonstrated to be efficient classes of electrophiles for the scandium(III) triflate/sodium dodecyl sulphate (SDS) catalysed vinylogous FriedelCrafts alkylation of indoles and pyrroles in water. These substrates contain an easily removable auxiliary group that increases affinity for the catalytic metal ion in such a way that they can compete with water for binding to the catalytic metal ion. Thus, alkylidene malonates and α,β-unsaturated α′-hydroxyketones are attractive substitutes for, e.g., α,β-unsaturated carboxylic acids and -esters, which in aqueous media are not reactive enough in these reactions. The combination of Lewis acid and SDS in catalysis results in considerable acceleration of the reaction in water compared to organic solvents. The method presented is attractive because the reactions are fast, experimentally straightforward and give rise to high yields of products.
    • DNA-based hybrid catalysis
      A. Rioz-Martínez and G. Roelfes

      Curr. Opin. Chem. Biol., 2015, 25, 80-87

      • In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed
    • Characterization of the Interactions between Substrate, Copper(II) Complex and DNA and their Role in Rate Acceleration in DNA-based Asymmetric Catalysis
      A. Draksharapu, A.J. Boersma, W.R. Browne and G. Roelfes

      Dalton Trans., 2015, 44, 3656-3663

      • Interactions of the azachalcone derived substrate Aza with copper(II) complexes in the presence and absence of st-DNA were studied in detail by UV/Vis absorption, EPR and Raman and (UV and vis) resonance Raman spectroscopies. The binding of Aza to the Lewis acidic copper(II) complexes, which results in activation of the substrate, was established spectroscopically. It was shown that the binding of Aza differs between CuIIdmbpy and CuIIterpy, consistent with the observed differences in catalytic asymmetric DielsAlder reactions with regard to both the rate and enantiomeric preference. Finally, it was shown that DNA has a major beneficial effect on the binding of Aza to the copper(II) complex due to the fact that both bind to the DNA. The result is a high effective molarity of both the copper complexes and the Aza substrate, which leads to a significant increase in binding of Aza to the copper(II) complex. This effect is a key reason for the observed rate acceleration in the catalyzed reactions brought about by the presence of DNA.
    • Binding of Copper(II) Polypyridyl Complexes to DNA and Consequences for DNA-based Asymmetric Catalysis
      A. Draksharapu, A.J. Boersma, M. Leising, A. Meetsma, W.R. Browne and G. Roelfes

      Dalton Trans., 2015, 44, 3647-3655

      • The interaction between salmon testes DNA (st-DNA) and a series of CuII polypyridyl complexes, i.e. [Cu(dmbpy)(NO3)2] (1) (dmbpy = 4,4′-dimethyl-2,2′-bipyridine), [Cu(bpy)(NO3)2] (2) (bpy = 2,2′-bipyridine), [Cu(phen)(NO3)2] (3) (phen = phenanthroline), [Cu(terpy)(NO3)2]H2O (4) (terpy = 2,2′:6′,2″-terpyridine), [Cu(dpq)(NO3)2] (5) (dpq = dipyrido-[3,2-d:2′,3′-f]-quinoxaline) and [Cu(dppz)(NO3)2] (6) (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was studied by UV/Vis absorption, Circular Dichroism, Linear Dichroism, EPR, Raman and (UV and vis) resonance Raman spectroscopies and viscometry. These complexes catalyse enantioselective CC bond forming reactions in water with DNA as the source of chirality. Complex 1 crystallizes as an inorganic polymer with nitrate ligands bridging the copper ions, which adopt essentially a distorted square pyramidal structure with a fifth bridging nitrate ligand at the axial position. Raman spectroscopy indicates that in solution the nitrate ligands in 1, 2, 3 and 4 are displaced by solvent (H2O). For complex 1, multiple supramolecular species are observed in the presence of st-DNA in contrast to the other complexes, which appear to interact relatively uniformly as a single species predominantly, when st-DNA is present. Overall the data suggest that complexes 1 and 2 engage primarily through groove binding with st-DNA while 5 and 6 undergo intercalation. For complexes 3 and 4 the data indicates that both groove binding and intercalation takes place, albeit primarily intercalation. Although it is tempting to conclude that the groove binders give highest ee and rate acceleration, it is proposed that the flexibility and dynamics in binding of CuII complexes to DNA are key parameters that determine the outcome of the reaction. These findings provide insight into the complex supramolecular structure of these DNA-based catalysts.
    • Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids - hot paper; highlighted in Chemistry World & Nature Chemistry
      I. Drienovská, A. Rioz-Martínez, A. Draksharapu and G. Roelfes

      Chem. Sci., 2015, 6, 770-776
      - hot paper; highlighted in Chemistry World & Nature Chemistry

      • Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric FriedelCrafts alkylation reactions and up to 83% ee for the product was achieved.
    • Artificial Metalloenzymes for Asymmetric Catalysis by Creation of Novel Active Sites in Protein and DNA Scaffolds
      I. Drienovská and G. Roelfes

      Isr. J. Chem., 2015, 55, 21-31

      • Artificial metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we give an overview of the developments in the two approaches to artificial metalloenzymes for asymmetric catalysis investigated in our group: creation of a novel active site on a peptide or protein dimer interface and using DNA as a scaffold for artificial metalloenzymes.
  • 2014
    • The power of two: covalent coupling of photostabilizers for fluorescence applications
      J.H.M. van der Velde, J. Oelerich, J. Huang, J.H. Smit, M. Hiermaier, E. Ploetz, A. Herrmann, G. Roelfes and T. Cordes

      J. Phys. Chem. Lett., 2014, 5, 3792–3798

      • Fluorescence is a versatile tool for spectroscopic investigations and imaging of dynamic processes and structures across various scientific disciplines. The photophysical performance, that is, signal stability and signal duration, of the employed fluorophores is a major limiting factor. In this Letter, we propose a general concept to covalently link molecules, which are known for their positive effect in photostabilization, to form a combined photostabilizer with new properties. The direct linkage of two (or more) photostabilizers will allow one to obtain combined or synergetic effects in fluorophore stabilization and can simplify the preparation of imaging buffers that would otherwise require a mixture of photostabilizers for optimal performance. This concept was explored by synthesizing a molecule with a reducing and oxidizing moiety that is referred to as internal ROXS or iROXS. Using single-molecule fluorescence microscopy, inter- and intramolecular healing of iROXS was observed, that is, strongly reduced blinking and increased photostability of the cyanine fluorophore Cy5. Moreover, it is shown that a covalently coupled photostabilizer can replace a mixture of molecules needed to make a functional photostabilizing ROXS buffer and might hence represent the new standard for defined and reproducible imaging conditions in single-molecule experiments. In self-healing fluorophores with intramolecular triplet-state quenching, an unprecedented photostability increase of >100-fold was obtained when using iROXS, which is even competitive with solution-based healing. Control experiments show that the oxidizing part of the iROXS molecule, an aromatic nitro group, dominates the healing process. The suggested synthetic concept and the proof-of-concept experiments represent the starting point for the quest to identify optimal combinations of linked photostabilizers for various fluorescence applications.
    • Efficient Nuclear DNA Cleavage in Human Cancer Cells by Synthetic Bleomycin Mimics
      Q. Li, M.G.P. van der Wijst, H.G. Kazemier, M.G. Rots and G. Roelfes

      ACS Chem. Biol., 2014, 9, 1044–1051

      • Iron complexes of N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)-methylamine (N4Py) have proven to be excellent synthetic mimics of the Bleomycins (BLMs), which are a family of natural antibiotics used clinically in the treatment of certain cancers. However, most investigations of DNA cleavage activity of these and related metal complexes were carried out in cell-free systems using plasmid DNA as substrate. The present study evaluated nuclear DNA cleavage activity and cell cytotoxicity of BLM and its synthetic mimics based on the ligand N4Py. The N4Py-based reagents induced nuclear DNA cleavage in living cells as efficiently as BLM and Fe(II)-BLM. Treatment of 2 cancer cell lines and 1 noncancerous cell line indicated improved cytotoxicity of N4Py when compared to BLM. Moreover, some level of selectivity was observed for N4Py on cancerous versus noncancerous cells. It was demonstrated that N4Py-based reagents and BLM induce cell death via different mechanistic pathways. BLM was shown to induce cell cycle arrest, ultimately resulting in mitotic catastrophe. In contrast, N4Py-based reagents were shown to induce apoptosis effectively. To the best of our knowledge, the present study is the first demonstration of efficient nuclear DNA cleavage activity of a synthetic BLM mimic within cells. The results presented here show that it is possible to design synthetic bioinorganic model complexes that are at least as active as the parent natural product and thereby are potentially interesting alternatives for BLM to induce antitumor activity.
    • Artificial metalloenzymes for enantioselective catalysis
      J. Bos and G. Roelfes

      Curr. Opin. Chem. Biol., 2014, 19, 135-143

      • Artificial metalloenzymes have emerged over the last decades as an attractive approach towards combining homogeneous catalysis and biocatalysis. A wide variety of catalytic transformations have been established by artificial metalloenzymes, thus establishing proof of concept. The field is now slowly transforming to take on new challenges. These include novel designs, novel catalytic reactions, some of which have no equivalent in both homogenous catalysis and biocatalysis and the incorporation of artificial metalloenzymes in chemoenzymatic cascades. Some of these developments represent promising steps towards integrating artificial metalloenzymes in biological systems. This review will focus on advances in this field and perspectives discussed.
    • A one-pot oxidation/enantioselective oxa-Michael cascade
      J.S. Willemsen, R.P. Megens, G. Roelfes, J.C.M. van Hest, F.P.J.T. Rutjes

      Eur. J. Org. Chem., 2014, 2892-2894

      • A laccase/(2,2,6,6-tetramethylpiperidin-1-yl)oxy (TEMPO) mediated oxidation was combined with an aqueous, enantioselective copper-catalyzed Michael addition reaction of water in one pot. The copper catalyst was also immobilized onto DNA to induce enantioselectivity in the reaction. Low conversions were observed when the reactions were performed simultaneously, caused by an undesired reaction of an oxidised TEMPO intermediate. We increased the conversions by using a stepwise approach. Thus, after completion of the oxidation, the first reaction was stopped by inhibiting the enzyme with HCO2K and reducing the reactive TEMPO intermediate. Next, the Michael addition reaction was started by adding the Cu catalyst. By applying this strategy, an efficient two-step one-pot sequence, proceeding with 20 % ee, was realized. The yield and ee of the second reaction were not affected by the oxidation reaction.
  • 2013
    • Mechanism of Intramolecular Photostabilization in Self- Healing Cyanine Fluorophores
      J.H.M. van der Velde, E. Ploetz, M. Hiermaier, J. Oelerich, J.W. de Vries, G. Roelfes and T. Cordes

      ChemPhysChem, 2013, 14, 4084-4093

      • Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark-states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood. In this contribution, we investigate the mechanism of intramolecular photostabilization in self-healing cyanines, in which photodamage is automatically repaired. Experimental evidence is provided to demonstrate that a single photostabilizer, that is, the vitamin E derivative Trolox, efficiently heals the cyanine fluorophore Cy5 in the absence of any photostabilizers in solution. A plausible mechanism is that Trolox interacts with the fluorophore through intramolecular quenching of triplet-related dark-states, which is a mechanism that appears to be common for both triplet-state quenchers (cyclooctatetraene) and redox-active compounds (Trolox, ascorbic acid, methylviologen). Additionally, the influence of solution-additives, such as cysteamine and procatechuic acid, on the self-healing process are studied. The results suggest the potential applicability of self-healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super-resolution. The presented data contributes to an improved understanding of the mechanism involved in intramolecular photostabilization and has high relevance for the future development of self-healing fluorophores, including their applications in various research fields.
    • An enantioselective artificial metallo-hydratase
      J. Bos, A. García-Herraiz and G. Roelfes

      Chem. Sci., 2013, 4, 3578-3582

      • Direct addition of water to alkenes to generate important chiral alcohols as key motif in a variety of natural products still remains a challenge in organic chemistry. Here, we report the first enantioselective artificial metallo-hydratase, based on the transcription factor LmrR, which catalyses the conjugate addition of water to generate chiral β-hydroxy ketones with enantioselectivities up to 84% ee. A mutagenesis study revealed that an aspartic acid and a phenylalanine located in the active site play a key role in achieving efficient catalysis and high enantioselectivities.
    • DNA-based asymmetric organometallic catalysis in water - hot paper
      J. Oelerich and G. Roelfes

      Chem. Sci., 2013, 4, 2013-2017
      - hot paper

      • Here, the first examples of DNA-based organometallic catalysis in water that give rise to high enantioselectivities are described. Copper complexes of strongly intercalating ligands were found to enable the asymmetric intramolecular cyclopropanation of α-diazo-β-keto sulfones in water. Up to 84% ee was achieved, in the presence of salmon testes DNA as the only source of chirality, using dipyrido[3,2-a:2′,3′-c]phenazine (dppz) derivatives as ligands.
    • Novel Catalyst Design using Cisplatin for Covalent Anchoring of Catalytically Active Copper Complexes to DNA
      L. Gjonaj and G. Roelfes

      ChemCatChem, 2013, 5, 1718–1721

      • Anchored to DNA: Covalent anchoring of catalytically active complexes to DNA is achieved through a tethered cisplatin moiety. The resulting DNA-based catalysts give good enantiomeric excess values in the catalyzed DielsAlder and FriedelCrafts alkylation reactions.
    • Hierarchical self-assembly of a biomimetic light harvesting antenna based on DNA G-quadruplexes
      N. Sancho Oltra, W.R. Browne and G. Roelfes

      Chem. Eur. J., 2013, 19, 2457–2461

      • A new modular approach to an artificial light-harvesting antenna system is presented. The approach involves the hierarchical self-assembly of porphyrin acceptor molecules to G-quadruplexes tethered to coumarin donor moieties.
  • 2012
    • Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface - HOT PAPER
      J. Bos, F. Fusetti, A.J.M. Driessen and G. Roelfes

      Angew. Chem. Int. Ed., 2012, 51, 7472-7475
      - HOT PAPER

      • A game of two halves: Artificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97 % ee was obtained in the benchmark CuII catalyzed DielsAlder reaction (see scheme).
    • Photo-induced oxidation of [FeII(N4Py)CH3CN] and related complexes
      A. Draksharapu, Q. Li, G. Roelfes and W. Browne

      Dalton Trans., 2012, 41, 13180-13190

      • The photochemistry of the complexes [Fe(N4Py)(CH3CN)](ClO4)2 (1), where N4Py is 1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine and [Fe(MeN4Py)(CH3CN)](ClO4)2 (2), where MeN4Py is 1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)ethanamine, in water, dichloromethane and methanol is described. Under UV or visible irradiation both 1 and 2 undergo enhancement of the rate of outer sphere electron transfer to 3O2 to yield the superoxide radical anion and the complexes in the Fe(III) redox state. Addition of ascorbic acid to the photoproduct leads to a recovery of the initial UV/Vis spectrum of 1 and 2, indicating that ligand oxidation does not occur. The results are discussed within the context of the recent report of the enhancement of the oxidative DNA cleavage activity of 1 under UV and visible irradiation (Inorg. Chem. 2010, 49, 11009).
    • DNA-based catalytic enantioselective intermolecular oxa-Michael addition reactions
      R. P. Megens and G. Roelfes

      Chem. Commun., 2012, 48, 6366-6368

      • Using the DNA-based catalysis concept, a novel Cu(II) catalyzed enantioselective oxa-Michael addition of alcohols to enones is reported. Enantioselectivities of up to 86% were obtained. The presence of water is important for the reactivity, possibly by reverting unwanted side reactions such as 1,2-additions.
    • DNA-Based Metal Catalysis
      J. Oelerich and G. Roelfes

      in Progress in Inorganic Chemistry, 2012, vol 57, K.D. Karlin (Ed.), John Wiley & Sons, Inc., Hoboken, New Jersey, 353-393

    • The role of ligand exchange and spin state equilibriums in the activation of Molecular Oxygen by FeII(N4Py) and related complexes in aqueous media
      A. Draksharapu, Q. Li, H. Logtenberg, T.A. van den Berg, A. Meetsma, J.S. Killeen, B.L. Feringa, R. Hage, G. Roelfes and W.R. Browne

      Inorg. Chem., 2012, 51, 900-913

      • We report the characterization and solution chemistry of a series of FeII complexes based on the pentadentate ligands N4Py (1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine), MeN4Py (1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)ethanamine), and the tetradentate ligand Bn-N3Py (N-benzyl-1,1-di(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine) ligands, i.e., [Fe(N4Py)(CH3CN)](ClO4)2 (1), [Fe(MeN4Py)(CH3CN)](ClO4)2 (2), and [Fe(Bn-N3Py)(CH3CN)2](ClO4)2 (3), respectively. Complexes 2 and 3 are characterized by X-ray crystallography, which indicates that they are low-spin FeII complexes in the solid state. The solution properties of 13 are investigated using 1H NMR, UV/vis absorption, and resonance Raman spectroscopies, cyclic voltammetry, and ESI-MS. These data confirm that in acetonitrile the complexes retain their solid-state structure, but in water immediate ligand exchange of the CH3CN ligand(s) for hydroxide or aqua ligands occurs with full dissociation of the polypyridyl ligand at low (<3) and high (>9) pH. pH jumping experiments confirm that over at least several minutes the ligand dissociation observed is fully reversible for complexes 1 and 2. In the pH range between 5 and 8, complexes 1 and 2 show an equilibrium between two different species. Furthermore, the aquated complexes show a spin equilibrium between low- and high-spin states with the equilibrium favoring the high-spin state for 1 but favoring the low-spin state for 2. Complex 3 forms only one species over the pH range 48, outside of which ligand dissociation occurs. The speciation analysis and the observation of an equilibrium between spin states in aqueous solution is proposed to be the origin of the effectiveness of complex 1 in cleaving DNA in water with 3O2 as terminal oxidant.
    • Enantioselective catalysis at the DNA scaffold
      A. García-Fernàndez and G. Roelfes

      in Met. Ions Life Sci., 2012, 10, , 249-268

    • Ligand denticity controls enantiomeric preference in DNA-based asymmetric catalysis
      A.J. Boersma, B. de Bruin, B.L. Feringa and G. Roelfes

      Chem. Commun., 2012, 48, 2394

      • DNA-based catalysis can be used to control the enantioselectivity of copper-catalysed DielsAlder and FriedelCrafts reactions to produce either enantiomer of the product by changing the denticity of the ligand coordinated to the Cu(II) ion, even though the DNA adopts a right handed helical conformation only.
  • 2011
    • DNA Cleavage Activity of Fe(II)N4Py under Photo Irradiatiion in the Presence of 1,8-Naphthalimide and 9-Aminoacridine: Unexpected Effects of ROS Scavengers
      Q.Li, W.R. Browne and G. Roelfes

      Inorg. Chem., 2011, 50, 8318-8325

      • The DNA cleavage activity of the iron(II) complex of the ligand N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py) was investigated in the presence of the chromophores 1,8-naphthalimide (NI) and 9-aminoacridine (AA) under photo irradiation at 355 and 400.8 nm and compared to the activity of the complex without the chromophores. Whereas in most cases no synergistic effect of the added chromophores on DNA cleavage efficiency was observed, it was found that for Fe(II)N4Py, in combination with NI under irradiation at 355 nm, the DNA cleavage activity was increased. Surprisingly, it was found that the addition of reactive oxygen species (ROS) scavengers gave rise to significantly increased DNA cleavage efficiency, which is a highly counterintuitive observation since ROS are needed to achieve DNA cleavage. A hypothesis is put forward to explain, at least partly, these results. It is proposed that the addition of scavengers inhibits quenching of 3NI*, thus making photo-induced electron transfer between 3NI* and Fe(III)N4Py more efficient. This results in reduction of Fe(III)N4Py to Fe(II)N4Py, which can then react with ROS giving rise to DNA cleavage. Hence the role of the scavengers is to maintain a close to optimal concentration of ROS. The present study serves as an illustration of the care that needs to be exercised in interpreting the results of experiments using standard ROS scavengers, since especially in complex systems such as presented here they can give rise to unexpected phenomena. In the presence of 1,8-naphthalimide or 9-aminoacridine, ROS scavengers can increase the DNA cleavage efficiency of Fe(II)N4Py complex under photo irradiation.
    • Asymmetric Catalysis with Helical Polymers.
      R.P. Megens and G. Roelfes

      Chem. Eur. J., 2011, 17, 8514-8523

      • Inspired by nature, the use of helical biopolymer catalysts has emerged over the last years as a new approach to asymmetric catalysis. In this Concept article the various approaches and designs and their application in asymmetric catalysis will be discussed.
    • Design and Synthesis of ATP-Based Nucleotide Analogues and Profiling of Nucleotide Binding Proteins
      J.C. Wolters, G. Roelfes and B. Poolman

      Bioconj. Chem., 2011, 22, 1345-1353

      • Two nucleotide-based probes were designed and synthesized in order to enrich samples for specific classes of proteins by affinity-based protein profiling. We focused on the profiling of adenine nucleotide-binding proteins. Two properties were considered in the design of the probes: the bait needs to bind adenine nucleotide-binding proteins with high affinity and carry a second functional group suitable and easily accessible for coupling to a chromatography resin. For this purpose, we synthesized p-biotinyl amidobenzoic acid-ATP (p-BABA-ATP) and p-biotinyl aminomethylbenzoic acid-ATP (p-BAMBA-ATP). p-BABA-ATP and p-BAMBA-ATP both bind to ATP-binding cassette (ABC) proteins with at least 10-fold higher affinity than ATP. Several ABC transporters could be enriched using p-BABA-ATP or p-BAMBA-ATP.
    • A ligand structure-activity study of DNA-based catalytic asymmetric hydration and Diels-Alder reactions.
      F. Rosati and G. Roelfes

      ChemCatChem, 2011, 3, 973-977

      • A structureactivity relationship study of the first generation ligands for the DNA-based asymmetric hydration of enones and DielsAlder 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 DielsAlder 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.
    • Enantioselective Cu(II)-Catalyzed Diels-Alder and Michael Addition Reactions in Water using Bio-Inspired Triazacyclophane-Based Ligands
      H.B. Albada, F. Rosati, D. Coquière, G. Roelfes and R.M.J. Liskamp

      Eur. J. Org. Chem., 2011, 17, 1714-1720

      • A triazacyclophane (TAC) scaffold decorated with three histidine amino acid residues was used as a tridentate ligand in asymmetric copper(II)-catalysed DielsAlder and Michael addition reactions in water. Enantiomeric excesses up to 55 % were obtained in DielsAlder reactions using ligands in which the histidine residues were directly attached to the TAC scaffold. Additional amino acid residues on the N-termini of the histidine residues or positioned between the histidine residues and the TAC scaffold, resulted in almost complete loss of enantioselectivity. Modelling studies of the coordination complex of the most specific ligand indicated the presence of a substrate binding pocket in proximity to the catalytically active centre.
    • Metallopeptides for Enantioselective Catalysis
      G. Roelfes

      ChemCatChem, 2011, 3, 647-648.

      • Helix the cat: Metalloenzymes are the benchmark catalysts for efficient enantioselective chemical transformations and, as such, a source of inspiration for catalyst design. Novel metallopeptides, comprising a dirhodium tetracarboxylate active site embedded in a helical peptide dimer, have been shown to catalyze highly enantioselective Si[BOND]H insertion reactions.
  • 2010
    • Photoenhanced Oxidative DNA cleavage with Non-Heme Iron(II) Complexes
      Q. Li, W.R. Browne and G. Roelfes

      Inorg. Chem., 2010, 49, 11009-11017

      • The DNA cleavage activity of iron(II) complexes of a series of monotopic pentadentate N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py)-derived ligands (1−5) was investigated under laser irradiation at 473, 400.8, and 355 nm in the absence of a reducing agent and compared to that under ambient lighting. A significant increase in activity was observed under laser irradiation, which is dependent on the structural characteristics of the complexes and the wavelength and power of irradiation. Under photoirradiation at 355 nm, direct double-stand DNA cleavage activity was observed with FeII-1 and FeII-3−5, and a 56-fold increase in the single-strand cleavage activity was observed with FeII-2. Mechanistic investigations revealed that O2−, 1O2, and OH contribute to the photoenhanced DNA cleavage activity, and that their relative contribution is dependent on the wavelength. It is proposed that the origin of the increase in activity is the photoenhanced formation of an FeIIIOOH intermediate as the active species or precursor.
    • Control over Enzymatic Activity by DNA-directed Split Enzyme Reassembly
      N. Sancho Oltra, J. Bos and G. Roelfes

      ChemBioChem, 2010, 11, 2255-2258

      • A novel and versatile approach toward DNA-controlled enzymatic activity is presented. The approach was based on the DNA-conjugated split enzyme, murine dihydrofolate reductase (mDHFR), which can be reassembled into a catalytically active conformation by hybridization of the protein fragment conjugated oligonucleotides with a DNA template strand.
    • Catalytic Enantioselective Syn-Hydration of Enones in Water using a DNA-based Catalyst
      A.J. Boersma, D. Coquère, D. Geerdink, F. Rosati, B.L. Feringa and G. Roelfes

      Nature Chem., 2010, 2, 991-995

      • 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.
    • Dramatic Micellar Rate Enhancement of the Cu2+ Catalyzed Vinologous Friedel-Crafts Alkylation in Water
      F. Rosati, J. Oelerich and G. Roelfes

      Chem. Commun., 2010, 46, 7804-7806

      • A dramatic rate enhancement of the Cu2+ catalyzed FriedelCrafts alkylation in water was achieved in the presence of sodium dodecyl sulfate (SDS) micelles.
    • Mononuclear Fe(II)-N4Py Complexes in Oxidative DNA Cleavage: Structure, Activity and Mechanism
      Q. Li, T.A. van den Berg, B.L. Feringa and G. Roelfes

      Dalton Trans., 2010, 39, 8012-8021

      • A series of monotopic N4Py (N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine, 1) derived ligands have been prepared and evaluated in the iron catalyzed oxidative cleavage of pUC18 DNA, in the presence and absence of external reducing agent DTT. The mononuclear iron(II) complexes induce efficient DNA cleavage in air with a low catalyst loading. It was demonstrated that covalent attachment of 9-aminoacridine, ammonium group or 1,8-naphthalimide leads to increased DNA cleavage activity in the presence of a reductant. Also some complexes displayed a small degree of double-strand DNA cleavage activity. In contrast, in the absence of reducing agent, no beneficial effect of the covalently attached DNA binding moieties was observed, which was attributed to the reduction from Fe(III) to Fe(II), which is required for oxygen activation, becoming rate limiting. Mechanistic investigations revealed an important role for superoxide radicals. A proposed mechanism involves the formation of an Fe(III)OOH intermediate as the active species or precursor.
    • Artificial Metalloenzymes
      F. Rosati and G. Roelfes

      ChemCatChem, 2010, 2, 916-927

      • 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.
    • On the Role of DNA in DNA-based Catalytic Enantioselective Conjugate Addition Reactions
      E.W. Dijk, A.J. Boersma, B.L. Feringa and G. Roelfes

      Org. Biomol. Chem., 2010, 8, 3868-3873

      • A kinetic study of DNA-based catalytic enantioselective FriedelCrafts alkylation and Michael addition reactions showed that DNA affects the rate of these reactions significantly. Whereas in the presence of DNA, a large acceleration was found for the FriedelCrafts alkylation and a modest acceleration in the Michael addition of dimethyl malonate, a deceleration was observed when using nitromethane as nucleophile. Also, the enantioselectivities proved to be dependent on the DNA sequence. In comparison with the previously reported DielsAlder reaction, the results presented here suggest that DNA plays a similar role in both cycloaddition and conjugate addition reactions.
    • DNA-based Asymmetric Catalysis.
      A.J. Boersma, R.P. Megens, B.L. Feringa and G. Roelfes

      Chem. Soc. Rev, 2010, 39, 2083-2092

      • The unique chiral structure of DNA has been a source of inspiration for the development of a new class of bio-inspired catalysts. The novel concept of DNA-based asymmetric catalysis, which was introduced only five years ago, has been applied successfully in a variety of catalytic enantioselective reactions. In this tutorial review, the ideas behind this novel concept will be introduced, an overview of the catalytic chemistry available to date will be given and the role of DNA in catalysis will be discussed. Finally, an overview of new developments of potential interest for DNA-based asymmetric catalysis will be provided.
    • Organic Co-Solvents in Aqueous DNA-Based Asymmetric Catalysis
      R.P. Megens and G. Roelfes

      Org. Biomol. Chem, 2010, 8, 1387-1393

  • 2009
    • A Kinetic and Structural Investigation of DNA-Based Asymmetric Catalysis Using First-Generation Ligands
      F. Rosati, A.J. Boersma, J.E. Klijn, A. Meetsma, B.L. Feringa, G. Roelfes

      Chem. Eur. J., 2009, 15, 9596-9605

      • 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 DielsAlder 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.
    • Enantioselective artificial metalloenzymes based on a bPP scaffold.
      D. Coquiere, J. Bos, J. Beld and G. Roelfes

      Angew. Chem. Int. Ed., 2009, 48, 5159-5162.

      • Site creation: Enantioselective artificial metalloenzymes have been created by grafting a new active site onto bovine pancreatic polypeptide through the introduction of an amino acid capable of coordinating a copper(II) ion. This hybrid catalyst gave good enantioselectivities in the DielsAlder and Michael addition reactions in water (see scheme) and displayed a very high substrate selectivity.
    • Enantioselective Friedel-Crafts Reactions in Water Using a DNA-Based Catalyst.
      A.J. Boersma, B.L. Feringa and G. Roelfes

      Angew. Chem. Int. Ed., 2009, 48, 3346-3348

      • Taking the plunge: The first example of a Lewis acid catalyzed asymmetric FriedelCrafts alkylation with olefins in water is described. By using loadings of a DNA-based copper catalyst as low as 0.15 mol %, good yields and excellent enantioselectivities were obtained in the reaction of α,β-unsaturated 2-acyl imidazoles with heteroaromatic π nucleophiles. dmbpy=4,4′-dimethyl-2,2′-bipyridine.
    • Multinuclear Non-Heme Iron Complexes for Double Strand DNA Cleavage.
      R.P. Megens, T.A. van den Berg, A.D. de Bruijn, B.L. Feringa and G. Roelfes

      Chem. Eur. J., 2009, 15, 1723-1733

      • Efficient oxidative double-strand DNA cleavage has been achieved with multinuclear non-heme iron complexes (see scheme). These complexes therefore represent model compounds that mimic the mode of action of the anti-tumor drug bleomycin. The cytotoxicity of the anti-tumor drug BLM is believed to be related to the ability of the corresponding iron complex (Fe-BLM) to engage in oxidative double-strand DNA cleavage. The iron complex of the ligand N4Py (Fe-N4Py; N4Py=N,N-bis(2-pyridyl)-N-bis(2-pyridyl)methylamine) has proven to be a particularly valuable spectroscopic and functional model for Fe-BLM. It is also a very active oxidative DNA-cleaving agent. However, like all other synthetic Fe-BLM mimics, it gives only single-strand DNA cleavage. Since double-strand DNA cleavage requires the delivery of two oxidizing equivalents to the DNA, it was envisaged that multinuclear iron complexes might be capable of effecting double-strand cleavage. For this purpose, a series of ditopic and tritopic N4Py-derived ligands has been synthesized and the corresponding iron complexes have been evaluated for their efficacy in the oxidative cleavage of supercoiled pUC18 plasmid DNA. The dinuclear iron complexes showed significantly enhanced double-strand cleavage activity compared to mononuclear Fe-N4Py, which was relatively independent of the structure of the linking moiety. Covalent attachment of a 9-aminoacridine intercalator to a dinuclear complex did not give rise to improved double-strand DNA cleavage. The most efficient oxidative double-strand cleavage agents proved to be the trinuclear iron complexes. This is presumably the result of increased probability of the simultaneous delivery of two oxidizing equivalents to the DNA.
  • 2008
    • Selenoglutaredoxin as a glutathione peroxidase mimic
      G. Casi, G. Roelfes and D. Hilvert

      ChemBioChem, 2008, 9, 1623-1631

      • Glutaredoxin (Grx1) from Escherichia coli is a monomeric, 85-amino-acid-long, disulfide-containing redox protein. A Grx1 variant in which the redox-active disulfide was replaced with a selenocysteine (C11U/C14S) was prepared by native chemical ligation from three fragments as a potential mimic of the natural selenoenzyme glutathione peroxidase (Gpx). Selenoglutaredoxin, like the analogous C14S Grx1 variant, shows weak peroxidase activity. The selenol provides a 30-fold advantage over the thiol, but its activity is four orders of magnitude lower than that of bovine Gpx. In contrast, selenoglutaredoxin is an excellent catalyst for thioldisulfide exchange reactions; it promotes the reduction of β-hydroxyethyldisulfide by glutathione with a specific activity of 130 units mg−1. This value is 1.8 times greater than that of C14S Grx1 under identical conditions, and >104 greater than the peroxidase activity of either enzyme. Given the facile reduction of the glutathionyl-selenoglutaredoxin adduct by glutathione, oxidation of the selenol by the alkyl hydroperoxide substrate likely limits catalytic turnover and will have to be optimized to create more effective Gpx mimics. These results highlight the challenge of generating Gpx activity in a small, generic protein scaffold, despite the presence of a well-defined glutathione binding site and the intrinsic advantage of selenium over sulfur derivatives.
    • DNA-Based Asymmetric Catalysis: Sequence-Dependent Rate Acceleration and Enantioselectivity
      A.J. Boersma, J.E. Klijn, B.L. Feringa and G. Roelfes

      J. Am. Chem. Soc., 2008, 130, 11783–11790

      • This study shows that the role of DNA in the DNA-based enantioselective Diels−Alder reaction of azachalcone with cyclopentadiene is not limited to that of a chiral scaffold. DNA in combination with the copper complex of 4,4′-dimethyl-2,2′-bipyridine (Cu−L1) gives rise to a rate acceleration of up to 2 orders of magnitude compared to Cu−L1 catalysis alone. Furthermore, both the enantioselectivity and the rate enhancement prove to be dependent on the DNA-sequence. These features are the main reasons for the efficient and enantioselective catalysis observed with salmon testes DNA/Cu−L1 in the Diels−Alder reaction. The fact that absolute levels of stereocontrol can be achieved with a simple and weak DNA-binding complex like Cu−L1 is a clear demonstration of the power of the supramolecular approach to hybrid catalysis.
    • DNA-based hydrolytic kinetic resolution of epoxides
      E.W. Dijk, B.L. Feringa and G. Roelfes

      Tetrahedron: Asymmetry, 2008, 19, 2374–2377

      • DNA-bound copper(II) complexes serve as catalysts for the hydrolytic kinetic resolution of 2-pyridyloxiranes in water. Selectivity factors of up to 2.7 were achieved, indicating a chirality transfer of DNA to epoxides via a coordinated metal ion.
    • Modular assembly of novel DNA-based catalysts
      N. Sancho Oltra and G. Roelfes

      Chem. Commun., 2008, 45, 6039-6041

      • A novel modular strategy towards the assembly of DNA-based catalysts containing a covalently anchored metal complex is presented.
  • 2007
    • α,β-Unsaturated 2-Acyl Imidazoles as a Practical Class of Dienophiles for the DNA-Based Catalytic Asymmetric Diels−Alder Reaction in Water
      A.J. Boersma, B.L. Feringa and G. Roelfes

      Org. Lett., 2007, 9, 3647–3650

      • α,β-Unsaturated 2-acyl imidazoles are a novel and practical class of dienophiles for the DNA-based catalytic asymmetric Diels−Alder reaction in water. The Diels−Alder products are obtained with very high diastereoselectivities and enantioselectivities in the range of 83−98%. The catalytic reaction was performed on a 1.0 mmol scale, and the imidazole auxiliary was removed readily.
    • DNA and RNA induced enantioselectivity in chemical synthesis
      G. Roelfes

      Mol. Biosyst., 2007, 3, 126-135

      • One of the hallmarks of DNA and RNA structures is their elegant chirality. Using these chiral structures to induce enantioselectivity in chemical synthesis is as enticing as it is challenging. In recent years, three general approaches have been developed to achieve this, including chirality transfer by nucleotide templated synthesis, enantioselective catalysis by RNA/DNAzymes and DNA-based asymmetric catalysis. In this article the concepts behind these strategies as well as the important achievements in this field will be discussed.
    • DNA-Based Catalytic Enantioselective Michael Reactions in Water
      D. Coquière, B.L. Feringa and G. Roelfes

      Angew. Chem. Int. Ed., 2007, 46, 9308-9311

      • High, but not dry: A highly enantioselective Michael reaction in water has been developed by using a simple DNA-based catalyst. Enantioselectivities of up to 99 % ee could be obtained by using nitromethane and dimethyl malonate as the nucleophiles and α,β-unsaturated 2-acylimidazoles as the Michael acceptors. The reactions can be performed on a preparative scale and the catalyst can be recycled.
    • Double strand DNA cleavage with a binuclear iron complex
      T.A. van den Berg , B.L. Feringa and G. Roelfes

      Chem. Commun., 2007, 180-182

      • Covalently linking two single strand DNA cleaving agents resulted in a new biomimetic binuclear iron complex capable of effecting oxidative double strand DNA cleavage.
  • 2006
    • Highly enantioselective DNA-based catalysis.
      G. Roelfes, A.J. Boersma, B.L. Feringa

      Chem. Commun., 2006, 635-637

      • A new approach to DNA-based asymmetric catalysis is presented, which gives rise to very high enantioselectivities (up to 99% ee) in the copper catalyzed DielsAlder reaction in water.
  • 2005
    • DNA-based asymmetric catalysis
      G. Roelfes and B.L. Feringa

      Angew. Chem. Int. Ed., 2005, 44, 3230-3232

      • A twist in catalysis! The chirality of DNA is transferred directly to a DielsAlder reaction by using a DNA-tethered catalyst. A catalytically active copper complex with achiral ligands self-assembles with DNA to give products with up to 90 % ee, as is the case for the exo isomer of product 1.
  • 2004
    • Non-Heme Iron Complexes for Stereoselective Oxidation: Tuning of the Selectivity in Dihydroxylation Using Different Solvents.
      M. Klopstra, G. Roelfes, R. Hage, R.M. Kellogg and B.L. Feringa

      Eur. J. Inorg. Chem., 2004, 846-856

    • Enhanced selectivity in non-heme iron catalysed oxidation of alkanes with peracids: evidence for the involvement of Fe(IV)O species.
      T.A. Van den Berg, J.W. de Boer, W.R. Browne, G. Roelfes and B.L. Feringa

      Chem. Commun., 2004, 2550-2551

      • Catalytic alkane oxidation with high selectivity using peracids and an (N4Py)Fe complex is presented and the role of [(N4Py)Fe(IV)[double bond, length as m-dash]O]2+ species, molecular oxygen and hydroxyl radicals in the catalysis is discussed.
  • 2003
    • End-on and Side-on Peroxo Derivatives of Non-Heme Iron Complexes with Pentadentate Ligands: Models for Putative Intermediates in Biological Iron/Dioxygen Chemistry.
      G. Roelfes, V. Vrajmasu, K. Chen, R.Y.N. Ho, J.-U. Rohde, C. Zondervan, R.M. la Crois, E.P. Schudde, M. Lutz, A.L. Spek, R. Hage, B.L. Feringa, E. Münck and L. Que, Jr,

      Inorg. Chem., 2003, 42, 2639-2653

      • Mononuclear iron(III) species with end-on and side-on peroxide have been proposed or identified in the catalytic cycles of the antitumor drug bleomycin and a variety of enzymes, such as cytochrome P450 and Rieske dioxygenases. Only recently have biomimetic analogues of such reactive species been generated and characterized at low temperatures. We report the synthesis and characterization of a series of iron(II) complexes with pentadentate N5 ligands that react with H2O2 to generate transient low-spin FeIII−OOH intermediates. These intermediates have low-spin iron(III) centers exhibiting hydroperoxo-to-iron(III) charge-transfer bands in the 500−600-nm region. Their resonance Raman frequencies, νO-O, near 800 cm-1 are significantly lower than those observed for high-spin counterparts. The hydroperoxo-to-iron(III) charge-transfer transition blue-shifts and the νO-O of the Fe−OOH unit decreases as the N5 ligand becomes more electron donating. Thus, increasing electron density at the low-spin Fe(III) center weakens the O−O bond, in accord with conclusions drawn from published DFT calculations. The parent [(N4Py)FeIII(η1-OOH)]2+ (1a) ion in this series (N4Py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) can be converted to its conjugate base, which is demonstrated to be a high-spin iron(III) complex with a side-on peroxo ligand, [(N4Py)FeIII(η2-O2)]+ (1b). A detailed analysis of 1a and 1b by EPR and Mssbauer spectroscopy provides insights into their electronic properties. The orientation of the observed 57Fe A-tensor of 1a can be explained with the frequently employed Griffith model provided the rhombic component of the ligand field, determined by the disposition of the hydroperoxo ligand, is 45 rotated relative to the octahedral field. EXAFS studies of 1a and 1b reveal the first metrical details of the iron−peroxo units in this family of complexes:  [(N4Py)FeIII(η1-OOH)]2+ has an Fe−O bond of 1.76 , while [(N4Py)FeIII(η2-O2)]+ has two Fe−O bonds of 1.93 , values which are in very good agreement with results obtained from DFT calculations.
    • Incorporation of selenomethionine into proteins through selenohomocysteine-mediated ligation.
      G. Roelfes and D. Hilvert

      Angew. Chem. Int. Ed., 2003, 42, 2275-2277

      • Site-selective incorporation of selenomethionine into proteins in place of methionine provides a unique spectroscopic probe of local protein structure and dynamics. This was demonstrated with seleno-bPP 1, a synthetic variant of a peptide hormone prepared by ligation of a C-terminal peptide thioester with a peptide fragment containing an N-terminal selenohomocysteine, followed by methylation of the resulting selenol (see scheme; bPP=bovine pancreatic peptide).
  • 2002
  • 2001

    • A.G.J. Ligtenbarg, P.Oosting, G. Roelfes, R.M. la Crois, M. Lutz, A.L. Spek, R. Hage, B.L. Feringa

      Chem. Commun., 2001, 385-386

  • 2000
    • Efficient DNA Cleavage with an Iron Complex Without Added Reductant
      G. Roelfes, M.E. Branum, L. Wang, L. Que, Jr., B.L. Feringa

      J. Am. Chem. Soc., 2000, 122, 11517-11518

    • Catalytic oxidation with a non-heme iron complex that generates a low spin Fe(III)OOH intermediate
      G. Roelfes, M. Lubben, R. Hage, L. Que, Jr., B.L. Feringa

      Chem. Eur. J., 2000, 6, 2152-2159

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