NSPs: chromogenic linkers for fast, selective, and irreversible cysteine modification.

The addition of a sulfhydryl group to water-soluble N-alkyl(o-nitrostyryl)pyridinium ions (NSPs) followed by fast and irreversible cyclization and aromatization results in a stable S-C sp2-bond. The reaction sequence, termed Click & Lock, engages accessible cysteine residues under the formation of N-hydroxy indole pyridinium ions. The accompanying red shift of >70 nm to around 385 nm enables convenient monitoring of the labeling yield by UV-vis spectroscopy at extinction coefficients of ≥2 × 104 M-1 cm-1. The versatility of the linker is demonstrated in the stapling of peptides and the derivatization of proteins, including the modification of reduced trastuzumab with Val-Cit-PAB-MMAE. The high stability of the linker in human plasma, fast reaction rates (k app up to 4.4 M-1 s-1 at 20 °C), high selectivity for cysteine, favorable solubility of the electrophilic moiety and the bathochromic properties of the Click & Lock reaction provide an appealing alternative to existing methods for cysteine conjugation.

Highly sensitive single-molecule detection of macromolecule ion beams.

The analysis of proteins in the gas phase benefits from detectors that exhibit high efficiency and precise spatial resolution. Although modern secondary electron multipliers already address numerous analytical requirements, additional methods are desired for macromolecules at energies lower than currently used in post-acceleration detection. Previous studies have proven the sensitivity of superconducting detectors to high-energy particles in time-of-flight mass spectrometry. Here, we demonstrate that superconducting nanowire detectors are exceptionally well suited for quadrupole mass spectrometry and exhibit an outstanding quantum yield at low-impact energies. At energies as low as 100 eV, the sensitivity of these detectors surpasses conventional ion detectors by three orders of magnitude, and they offer the possibility to discriminate molecules by their impact energy and charge. We demonstrate three developments with these compact and sensitive devices, the recording of 2D ion beam profiles, photochemistry experiments in the gas phase, and advanced cryogenic electronics to pave the way toward highly integrated detectors.

Giving the Green Light to Photochemical Uncaging of Large Biomolecules in High Vacuum.

The isolation of biomolecules in a high vacuum enables experiments on fragile species in the absence of a perturbing environment. Since many molecular properties are influenced by local electric fields, here we seek to gain control over the number of charges on a biopolymer by photochemical uncaging. We present the design, modeling, and synthesis of photoactive molecular tags, their labeling to peptides and proteins as well as their photochemical validation in solution and in the gas phase. The tailored tags can be selectively cleaved off at a well-defined time and without the need for any external charge-transferring agents. The energy of a single or two green photons can already trigger the process, and it is soft enough to ensure the integrity of the released biomolecular cargo. We exploit differences in the cleavage pathways in solution and in vacuum and observe a surprising robustness in upscaling the approach from a model system to genuine proteins. The interaction wavelength of 532 nm is compatible with various biomolecular entities, such as oligonucleotides or oligosaccharides.

Artificial Metalloenzyme-Catalyzed Enantioselective Amidation via Nitrene Insertion in Unactivated C(sp3)-H Bonds.

Enantioselective C-H amidation offers attractive means to assemble C-N bonds to synthesize high-added value, nitrogen-containing molecules. In recent decades, complementary enzymatic and homogeneous-catalytic strategies for C-H amidation have been reported. Herein, we report on an artificial metalloenzyme (ArM) resulting from anchoring a biotinylated Ir-complex within streptavidin (Sav). The resulting ArM catalyzes the enantioselective amidation of unactivated C(sp3)-H bonds. Chemogenetic optimization of the Ir cofactor and Sav led to significant improvement in both the activity and enantioselectivity. Up to >700 TON and 92% ee for the amidation of unactivated C(sp3)-H bonds was achieved. The single crystal X-ray analysis of the artificial nitrene insertase (ANIase) combined with quantum mechanics-molecular mechanics (QM-MM) calculations sheds light on critical second coordination sphere contacts leading to improved catalytic performance.

Enantioselective Allylation of Stereogenic Nitrogen Centers.

Most tertiary amines with a stereogenic nitrogen center undergo rapid racemization at room temperature. Consequently, the quaternization of amines under dynamic kinetic resolution seems feasible. N-Methyl tetrahydroisoquinolines are converted into configurationally stable ammonium ions by Pd-catalyzed allylic alkylation. The optimization of conditions and the evaluation of the substrate scope enabled high conversions and an enantiomeric ratio of up to 10:90. We report here the first examples for the enantioselective catalytic synthesis of chiral ammonium ions.

Matter-wave interference and deflection of tripeptides decorated with fluorinated alkyl chains.

Studies of neutral biomolecules in the gas phase allow for the study of molecular properties in the absence of solvent and charge effects, thus complementing spectroscopic and analytical methods in solution or in ion traps. Some properties, such as the static electronic susceptibility, are best accessed in experiments that act on the motion of the neutral molecules in an electric field. Here, we screen seven peptides for their thermal stability and electron impact ionizability. We identify two tripeptides as sufficiently volatile and thermostable to be evaporated and interfered in the long-baseline universal matter-wave interferometer. Monitoring the deflection of the interferometric molecular nanopattern in a tailored external electric field allows us to measure the static molecular susceptibility of Ala-Trp-Ala and Ala-Ala-Trp bearing fluorinated alkyl chains at C- and N-termini. The respective values are 4 π ε 0 × 330 ± 150 Å 3 and 4 π ε 0 × 270 ± 80 Å 3 .

Neutralization of insulin by photocleavage under high vacuum.

Charge reduction and neutralization of electro-sprayed peptides are realized by selective gas-phase photocleavage of tailored covalent tags. The concept is demonstrated with four model peptides in positive and negative ion modes and tagged insulin as the largest construct.

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies.

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970's. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000's. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000's, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.

Upregulation of an Artificial Zymogen by Proteolysis.

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.

An NAD(P)H-Dependent Artificial Transfer Hydrogenase for Multienzymatic Cascades.

Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines.

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Artificial concurrent catalytic processes involving enzymes.

The concurrent operation of multiple catalysts can lead to enhanced reaction features including (i) simultaneous linear multi-step transformations in a single reaction flask (ii) the control of intermediate equilibria (iii) stereoconvergent transformations (iv) rapid processing of labile reaction products. Enzymes occupy a prominent position for the development of such processes, due to their high potential compatibility with other biocatalysts. Genes for different enzymes can be co-expressed to reconstruct natural or construct artificial pathways and applied in the form of engineered whole cell biocatalysts to carry out complex transformations or, alternatively, the enzymes can be combined in vitro after isolation. Moreover, enzyme variants provide a wider substrate scope for a given reaction and often display altered selectivities and specificities. Man-made transition metal catalysts and engineered or artificial metalloenzymes also widen the range of reactivities and catalysed reactions that are potentially employable. Cascades for simultaneous cofactor or co-substrate regeneration or co-product removal are now firmly established. Many applications of more ambitious concurrent cascade catalysis are only just beginning to appear in the literature. The current review presents some of the most recent examples, with an emphasis on the combination of transition metal with enzymatic catalysis and aims to encourage researchers to contribute to this emerging field.

Artificial metalloenzymes for the diastereoselective reduction of NAD(+) to NAD(2)H.

Stereoselectively labelled isotopomers of NAD(P)H are highly relevant for mechanistic studies of enzymes which utilize them as redox equivalents. Whereas several methods are firmly established for their generation in high diastereomeric purity by enzymatic methods, alternative methods have so far not been investigated. The article presents the stereoselective deuteration of NAD(+) at the 4-position (90% de) of the pyridinium-ring by means of an artificial metalloenzyme. The artificial metalloenzyme consists of a biotinylated iridium cofactor embedded in streptavidin isoforms and the resulting constructs have been previously shown to be compatible with natural enzymes. Alternative methods for stereoselective NAD(P)(+) reduction are expected to be of high interest for the mechanistic study of enzymes that accept NAD(P)H mimics and for the synthesis of structurally related fine chemicals.

Cytochromes P450 as useful biocatalysts: addressing the limitations.

Cytochrome P450 monooxygenases (P450s or CYPs) are a unique family of enzymes which are capable of catalysing the regio- and stereospecific oxidation of non-functionalised hydrocarbons. Despite the enormous synthetic potential of P450s, these enzymes have yet to be extensively employed for research purposes or in industry. Lack of stability, low activity, narrow substrate specificity, expensive cofactor requirements, limited solvent tolerance and electron supply are some of the main reasons why the academic and industrial implementation of these important biocatalysts remains a challenge. Considering the significance of P450s, many research groups have focused on improving their properties in an effort to make more robust catalysts with broad synthetic applications. This article focuses on some of the factors that have limited the exploitation of P450s and explores some of the significant steps that have been taken towards addressing these limitations.

Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope.

A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities.

Protein-based hybrid catalysts--design and evolution.

Artificial metalloenzymes result from the introduction of a catalytically competent non-native metal cofactor within a protein environment. In the present contribution, we summarize the recent achievements in the design and the optimization of such protein-based hybrid catalysts, with an emphasis on enantioselective transformations. The second part outlines the milestones required to achieve en masse production, screening and directed evolution of artificial metalloenzymes. In the spirit of Darwinian evolution, this will allow the full potential of such protein-based hybrid catalysts to be fully unraveled, thus complementing both homogeneous and enzymatic catalysis.