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Sustainable chemical processes through catalyst design.

Molecular Systems Engineering is a National Centre of Competence in Research (NCCR) funded by the Swiss National Science Foundation (SNSF), and headed by the University of Basel and the ETH Zurich. This NCCR combines expertise from chemistry, biology, physics, bioinformatics, and engineering. The overreaching aim is to develop tools and devices to monitor and manipulate off-equilibrium (bio)chemical systems. These may find applications in the synthesis of high added-value products, as innovative diagnostic tools and for the restoration of a desired cellular or organ function.

The emerging field of synthetic biology offers new opportunities to address current biological and medical challenges, by providing tools to label and influence metabolic pathways as well as approaches for the development of novel therapeutic strategies. Particularly, a catalytic approach based on Artificial Metalloenzymes (AMs) could play a fundamental role in this endeavor. This project outlines a strategy for the development of a new tumor treatment therapy. To this end, the cell surface tumor marker carbonic anhydrase IX (CAIX) is 'hijacked' as an AM by binding a CA-inhibitor, bearing a metallocofactor, to the Zn(II) ion in the active site of CA. Upon binding, the metallocofactor is activated and catalyzes the uncaging of a prodrug thus releasing a potent chemotherapeutic drug. As a complementary approach, I seek to improve the performance of CA-based AMs in cellulo by combining protein design with in vivo evolution. AMs are designed and evolved to complement existing metabolic pathways. Specifically, to form the valine precursor α-ketovaline in an auxotrophic Escherichia coli knockout strain. This project is a proof of principle and blueprint for the development of AM-based tools for studying cellular regulation to answer basic biological questions.

Tailor-Made Proteins and Peptides for Quantum Interference Experiments In recent years synthetic chemistry and quantum optics have teamed up with three major objectives in mind and are now in an encouraging position for the inclusion of molecular biology: 1 st : we face the open research challenge to test the linearity of quantum mechanics in the regime of high masses - and with it the concepts of quantum delocalization, quantum superposition and matter-wave interference. Matter-wave interferometry creates refined molecular nanopatterns that are sensitive to external fields and can be used to retrieve internal molecular properties in the presence of known forces. In the past, this has allowed us to determine molecular polarizabilities, vibrationally induced dipole moments, molecular fragmentation or to distinguish structural isomers. 3 rd : by carefully tailoring the molecular properties we were able to demonstrate the evolution of the diffraction pattern molecule by molecule and thus to unambiguously demonstrate the wave nature of the moving molecular particle. We aim to translate these achievements with tailor-made molecules into the world of biomolecular physics. Quantum experiments are expected to be compatible with neutral biomolecular beams that stay beyond a momentum of p=30.000 amu×30m/s. A key challenge is to generate a beam of slow, directed, velocity and mass-selected biomolecules in high vacuum. Peripheral chemical functionalization with perfluoroalkyl chains has proven to facilitate the promotion of intact massive molecules via thermal and laser assisted desorption methods. In this interdisciplinary approach between synthetic chemistry and bio-engineering, we propose the stepwise development of amino-acid based biomolecules for interference experiments. While the design and synthesis of these model compounds is based within the SNI at the University of Basel, the investigation of their physical properties is performed in close cooperation with the group of Markus Arndt at the University of Vienna. With the final goal of proving the wave nature of proteins, the initial challenges are mainly of chemical and biochemical nature. In particular we aim at the stepwise development of biomolecules with increasing size for interference experiments. Starting with small peptides, their volatilization (suitability for slow molecular beam formation) and ionization (detection after scattering) features shall be optimized. Profiting from these small bio-oligomers as proxy, we subsequently apply the design principles to bio-polymers of increasing size and hope to be able to push the mass limit up to suitably functionalized proteins. The sublimability of molecules is increased by reducing their intermolecular interactions and thus, peptides exposing fluorinated groups are at the focus of interest. As a starting point, lysine rich model peptides were selected for the decoration with fluorous tags. The exposed primary amino group of lysine is ideally suited for post-functionalization. Suitable electrophiles (e.g. NHS esters) enable the peripheral functionalization with groups exposing fluorous alkyl chains. The complementary conversion of carboxylic residues further permits the introduction of a photo-ionizable label to facilitate the peptide's detection by mass spectrometry. Initially we would like to investigate the number of peripheral perfluorinated alkyl groups required for the volatilization of model peptides. The modular assembly of peptides is ideally suited to study the correlation between their molecular mass and number of fluorous chains required. The features introduced by the tag can also be combined for example with a porphyrin tag exposing long perfluorinated alkyl chain. Of particular interest are photo-cleavable tags which enable future interference experiments based on optical gratings. The developed technology will subsequently be adapted to surface engineered proteins. Heterologous expression in E. coli allows control of surface residue composition by technically facile site directed mutagenesis. The outlined bio-chemical approach of protein modification also enables the global manipulation of surface charges, a particular important feature as only neutral bio-molecules are suited for interference experiments. To what extent these manipulations will interfere with the folding of the protein core will be subject of the here performed investigations. [1] S. Eibenberger, S. Gerlich, M. Arndt, M, Mayor, J. Tüxen, PhysChemPhys 2013 , 15 , 14696 [2] S. Gerlich, S. Eibenberger, M. Tomandl, S. Nimmrichter, K. Hornberger, P. Fagan, J. Tüxen, M. Mayor, M. Arndt, Nature Commun . 2011 , 2 , 263. [3] T. Juffmann, A. Milic, M. Müllneritsch, P. Asenbaum, A. Tsukernik, J. Tüxen, M. Mayor, O. Cheshnovsky, M. Arndt, Nature Nanotech. 2012 , 7 , 297. [4] P. Schmid, F. Stöhr, M. Arndt, J. Tüxen, M. Mayor, J. Am. Soc. Mass Spectr . 2013 , 24 , 602

The NCCR Molecular Systems Engineering combines competences from life sciences, chemistry, physics, biology, bioinformatics and engineering sciences. More than 100 researchers and support personnel distributed into four work packages and 31 projects work together to address systems engineering challenges by integrating novel chemical and biological modules into molecular factories and cellular systems for the production of high added-value chemicals and applications in medical diagnostics and treatment. Molecular systems engineering relies on the combination of both chemical- and biological modules. In this approach, complex dynamic phenomena emerge as the result of the integration of molecular modules designed to interact in a programmed way with their complex environment. In this manner, it should be possible to create molecular factories and cellular systems whose properties are more than the sum of the attributes of the individual modules. The commitments of the leading houses, the University of Basel and ETH Zurich, also include new (joint) professorships, and extensive training of a new generation of scientists and technologists, leading to a long-term paradigm shift in molecular sciences and a new structure of the Swiss research landscape.

Artificial metalloenzymes (ArMs) result from the incorporation of abiotic cofactors within a host protein. In collaboration with various groups of the NCCR, we aim at engineering metabolic pathways incorporating ArMs. The challenges addressed during this funding period include: i) successful compartmentalization and evolution of ArMs within the periplasm of E coli, ii) engineering enzyme cascades with ArMs and iii) cross-regulation of ArMs.

Life emerges from inanimate matter thanks to a complex network of tightly cross-regulated enzymatic reaction cascades. To mimick this, we have emplemented the cross-regulation of an artificial imine reductase which is turned on in the presence of a protease, generating a tripeptide that acts as a cross-regulator for the imine reductase.

Artificial metalloenzymes result from combining a catalytically competent organometallic moiety with a host protein. The resulting hybrid catalyst combine attractive features of both chemo- and biocatalysts. In recent years, the Ward group has exploited the biotin-streptavidin towards the creation of artificial metalloenzymes for hydrogenation, allylic alkylation, sulfoxidation, alcohol oxidation, dihydroxylation, transfer-hydrogenation and olefin metathesis. The latter two systems were shown to be particularly stable towards E. coli cellular extracts. Within this funding period, it is proposed to exploit this finding towards the implementation of directed evolution protocols for the optimization of the performance of artificial metalloenzymes. Four complementary and intedisciplinary sub-projects will be investigated: i) exploiting streptavidin expressed in the periplasm; ii) cascade reactions with artificial metalloenzymes; iii) optimization of artificial transfer-hydrogenase for the production of high-added value amines and aminoacids and iv) directed evolution of artificial metathesases. i) In order to circumvent the inhibition of the biotinylated precious metal catalyst by glutathione (present in milimolar amounts in the cytoplasm), it is proposed to target streptavidin to the periplasm. This will allow us to sidestep the lengthy purification of streptavidin prior to catalysis, eventually allowing the implementation of directed evolution protocols. ii) We have shown that artificial metalloenzymes are compatible with a variety of biocatalysts. Combining artificial metalloenyzmes with natural enzymes will lead to complex reaction cascades that can be used a) as a high-throughput colorimetric assay or b) to complement metabolic pathways. iii) The above developments will be exploited towards the preparation of a) high-added value amines via the enantioselective imine reduction and b) leucine by relying on a selection strategy based on E. coli leucine auxotrophs. iv) Thanks to the inertness of artificial metathesases based on the biotin-streptavidin technology, the performance of these will be optmized using crude E. coli cell extracts. For this purpose, we will rely screening a fluorophore-quencher substrate which, upon ring closing metathesis releases the quencher, thus becoming fluorescent. Ultimately, we aim at developing artificial metalloenzymes that outperform classical organometallic catalysts. In a biomimetic spirit and thanks to Darwinian protocols, we anticipate that the presence of an optimized second coordination sphere provided by the protein environment will allow to achieve this ambitious goal.

Overcoming Mankind's addiction to fossil fuel is perhaps the greatest scienfic challenge of the twenty-first century. Critical analysis of the key chemical reactions that may allow to address this issue, reveals that these reactions require the rigorous shuttling of multiple electrons and protons. For this purpose, Nature relies on highly evolved metalloenzymes which incorporate both metal- and organic cofactors. In a biomiemtic spirit, it is proposed to explore the synthesis, coordination chemistry and the catalytic properties of non-innocent, redox-active ligands. For this purpose, N-Heterocyclic Carbene ligands (NHC) will be derivatized with well documented Hydrogen Atom Transfer moieties (HAT, NHetCHAT for the ligand). Such HAT moieties include: sterically hindered hydroxylamines, phenols, catechols, hydrochinones, dihydropyrazines and dihydropyridines. According to the Marcus theory, fine tuning the distance between the donor-acceptor moieties as well as the thermochemical driving force of the HAT transfer may lead to improved reaction rates and contribute to lower the applied overpotential to drive the reaction. Additionally, removing some of the oxidative load from the catalytically competent metal by delocalizing it on the HAT moiety may contribute to increase the total turnover number of the catalytic systems. The following reactions will be scrutinized: alcohol oxidation, alcane oxidation as well as water oxidation. Initially, we will rely on well documented NHC-bearing organometallic catalysts precursors (typically, Ru, Pd and Ir complexes) which will be prepared and tested with NHetCHAT ligands. Having gained understanding and confidence with such systems, first-row transition metal complexes incorporating NHetCHAT ligands will be scrutinized. We believe that the approach delineated herein will yield highly relevant insight into fundamental aspects of catalytic processes relying on the transfer of multiple electrons and protons. Ultimately, it may lay the basis for novel catalytic systems for alcane or water oxidation.