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We describe the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli in which a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer-Villiger monooxygenase is used for the synthesis of lactones. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. We estimated adapted Michaelis-Menten parameters from in vitro experiments with isolated enzymes and used these values to simulate the change in the concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated that the fastest enzyme was rate-determining because of the unexpectedly low concentration of the active form, which opens up reversible reaction channels towards byproducts. We also provide substantial experimental evidence that a low intracellular concentration of flavin and nicotinamide cofactors drastically decreased the performance of the in vivo cascade drastically. [ABSTRACT FROM AUTHOR]

Herein we highlight recent findings on the importance of water networks in proteins, and their redesign and reconfiguration as a new engineering strategy to generate enzymes with modulated binding affinity and improved catalytic versatility. Traditionally, enzyme engineering and drug design have focused on tailoring direct and favorable interactions between protein surfaces and ligands/transition states to achieve stronger binding, or an accelerated manufacturing of medicines, biofuels, fine chemicals and materials. In contrast, the opportunity to relocate water molecules in solvated binding pockets by protein design to improve overall energetics remains essentially unexplored, and fundamental understanding of the elusive processes involved is poor. Rewiring water networks in protein interiors impacts binding affinity, catalysis and the thermodynamic signature of biochemical processes through dynamic mechanisms, and thus has great potential to enhance binding specificity, accelerate catalysis and provide new reaction mechanisms and chemistry, that were not yet explored in nature. [ABSTRACT FROM AUTHOR]

This work describes the regio- and enantioselective synthesis of nitrile-containing chiral lactones from easily accessible ketone precursors using Baeyer–Villiger monooxygenases. These biocatalysts controlled the distribution of regioisomers much more tightly than commonly used stoichiometric reagents, additionally with good to excellent optical purity of products. A surprising case of strong stereoelectronic control was also observed. We tested a library of 14 catalysts using five-to eight-membered cyclic ketones with two different tether lengths to the nitrile group. In all but the largest series we found suitable wild-type enzymes for preparative scale synthesis of the target compounds. The diverse possibilities to further functionalize lactones and nitriles make this method interesting for the generation of chiral building blocks. [ABSTRACT FROM AUTHOR]

Baeyer–Villiger monooxygenases catalyze the energetically challenging oxidation of levulinates (4-oxopentanoates) to 3-hydroxypropionic acid (3-HPA) derivates under ambient conditions, replacing propellant-grade H2O2 with aerial oxygen as the oxidant. This reaction enables a new pathway to a platform for chemical 3-HPA, an important intermediate in the non-petrol based production of a variety of bulk chemicals (acrylates, malonates, 1,3-propanediol). [ABSTRACT FROM AUTHOR]

Baeyer–Villiger monooxygenases catalyze the energetically challenging oxidation of levulinates (4-oxopentanoates) to 3-hydroxypropionic acid (3-HPA) derivates under ambient conditions, replacing propellant-grade H2O2 with aerial oxygen as the oxidant. This reaction enables a new pathway to a platform for chemical 3-HPA, an important intermediate in the non-petrol based production of a variety of bulk chemicals (acrylates, malonates, 1,3-propanediol). [ABSTRACT FROM AUTHOR]

Within a newly identified set of flavin-containing monooxygenases (FMOs) from Rhodococcus jostii RHA1, we have identified three monooxygenases (FMO-E, FMO-F, and FMO-G) that are effective in catalyzing Baeyer-Villiger oxidations. These type II FMOs display relaxed coenzyme specificity by accepting both NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and NADH (reduced form of nicotinamide adenine dinucleotide), as a coenzyme, which is a novel and attractive feature among biocatalysts capable of conducting Baeyer-Villiger oxidations. We purified FMO-E and determined that the Michaelis constants for both coenzymes were in the micromolar range, whereas the activity was highest for NADH. By using the stopped-flow technique, formation of a peroxyflavin-enzyme intermediate was observed, which indicated that type II FMOs follow a catalytic mechanism similar to that of other class B flavoprotein monooxygenases. A set of cyclobutanones and cyclohexanones were used to probe the regio- and enantioselectivity of all three recombinant monooxygenases. The biocatalysts readily accepted small cyclic ketones, which enabled the conversion of previously poorly accepted substrates by other monooxygenases (especially norcamphor), and exhibited excellent and unique regio- and enantioselectivities. Sequence analysis revealed that type II FMOs that act as Baeyer-Villiger monooxygenases contain a unique N-terminal domain. Sequence conservation in this protein domain can be used to identify new NADH-dependent Baeyer-Villiger monooxygenases, which would facilitate future biocatalyst discovery efforts. [ABSTRACT FROM AUTHOR]

Smart catalysis cuts it short: Natural (S,S)‐Aerangis lactone and its epimer are synthesized with excellent optical purity by using in‐line hyphenation of continuous flow reactors and batch biotransformations. Optimization of the reaction steps and engineering of the reactor interfaces leads to a simple and efficient setup, which demonstrates how multistep syntheses can be streamlined into a single‐operation protocol. [ABSTRACT FROM AUTHOR]

Within this work a generic tool for chiral catalyst evaluation is established based on the application-oriented properties activity and selectivity; the concept aims at quantitatively comparing catalyst performance in general on a multitude of substrates. It is designed and intended to serve as decision guidance for challenges in catalysis and comprehensible information extraction from already recorded but unrefined data sets. The underlying algorithm assigns function points to catalytic entities via a statistically solid model possessing high flexibility and generates a relative ranking. This is coupled to an automated iterative refinement process towards maximum information content of results employing Shannon entropy optimization. Consequently, the developed work-flow facilitates high distinguishability between catalysts even in low-scattering data sets. The numerical ranking is complemented by a clearly arranged graphic representation permitting facile and reliable visual interpretation of generality or niche capabilities of catalysts. Usefulness of the title concept is demonstrated by the performance evaluation of cyclododecanone monooxygenase, a highly versatile Baeyer-Villiger enzyme. To retain broad applicability, an open-source MATLAB® script is provided in electronic form. [ABSTRACT FROM AUTHOR]

Abstract: Cyclopentadecanone monooxygenase (CPDMO) is one of the latest additions to the established library of Baeyer–Villiger monooxygenases. Desymmetrizations of substituted cyclobutanones and -hexanones as well as kinetic resolutions of racemic cycloketones are efficiently catalyzed by CPDMO. Moreover the enzyme shows unprecedented preference in regiodivergent oxidations of terpenones and the bicyclic Geissman–Waiss lactone precursor giving access to the optical antipode of retronecine and other pyrrolizidine alkaloids. [Copyright &y& Elsevier]

Abstract: Baeyer–Villiger monooxygenases (BVMOs) are presented as highly selective and efficient biocatalysts for the synthesis of aroma lactones via kinetic resolution of 2-substituted cycloketones, exemplified with two δ-valerolactones, the jasmine lactones and their ε-caprolactone homologs. Analytical scale screens of our BVMO library ensued by preparative whole-cell biotransformations led to the identification of two enzymes (cyclohexanone monooxygenase from Arthrobacter BP2 and cyclododecanone monooxygenase from Rhodococcus SC1) perfectly suited for the task at hand: easily accessible racemic starting materials were bio-oxidized to almost enantiopure ketones and lactones in good yields (48–74%) and optical purities (ee 93% to >99%, E >100). [Copyright &y& Elsevier]

The article presents an overview of the re-examination process within the U.S. Patent and Trademark Office (USPTO) in the U.S. It says that re-examination proceedings within USPTO are complex regulatory procedures. It observes that in certain circumstances, re-examination proceedings could save a generic manufacturer both time and money. The article further discusses how the re-examination process might be used by generic pharmaceutical companies.

The article presents an overview of recent cases involving preliminary injunctions in pharmaceutical cases in the U.S. The author said that the U.S. district courts have the power to grant preliminary and permanent injunctions. A preliminary injunction is regarded a drastic and extraordinary remedy that is not to be routinely granted. The pharmaceutical cases where a preliminary injunction was sought include Sanofi-Synthelabo versus Apotex Inc. and Abbott Laboratories versus Apotex Inc.

Magneto‐Archimedes levitation (MagLev) enables the separation of powdered mixtures of illicit drugs (cocaine, methamphetamine, heroin, fentanyl, and its analogues), adulterants, and diluents based on density, and allows the presumptive identification of individual components. Small samples (mass <50 mg), with low concentrations of illicit drugs, present a particular challenge to analysis for forensic chemists. The MagLev device, a cuvette containing a solution of paramagnetic gadolinium(III) chelate in a non‐polar solvent, placed between two like‐poles‐facing NdFeB magnets, allowed separation of seven relevant compounds simultaneously. In particular, initial separation with MagLev, followed by characterization by FTIR‐ATR, enabled identification of fentanyl in a sample of fentanyl‐laced heroin (1.3 wt % fentanyl, 2.6 wt % heroin, and 96.1 wt % lactose). MagLev allows identification of unknown powders in mixtures and enables confirmatory identification based on structure‐specific techniques. [ABSTRACT FROM AUTHOR]

Magneto‐Archimedes levitation (MagLev) enables the separation of powdered mixtures of illicit drugs (cocaine, methamphetamine, heroin, fentanyl, and its analogues), adulterants, and diluents based on density, and allows the presumptive identification of individual components. Small samples (mass <50 mg), with low concentrations of illicit drugs, present a particular challenge to analysis for forensic chemists. The MagLev device, a cuvette containing a solution of paramagnetic gadolinium(III) chelate in a non‐polar solvent, placed between two like‐poles‐facing NdFeB magnets, allowed separation of seven relevant compounds simultaneously. In particular, initial separation with MagLev, followed by characterization by FTIR‐ATR, enabled identification of fentanyl in a sample of fentanyl‐laced heroin (1.3 wt % fentanyl, 2.6 wt % heroin, and 96.1 wt % lactose). MagLev allows identification of unknown powders in mixtures and enables confirmatory identification based on structure‐specific techniques. [ABSTRACT FROM AUTHOR]

Biomolecular recognition can be stubborn; changes in the structures of associating molecules, or the environments in which they associate, often yield compensating changes in enthalpies and entropies of binding and no net change in affinities. This phenomenon-termed enthalpy/entropy (H/S) compensation-hinders efforts in biomolecular design, and its incidence-often a surprise to experimentalists-makes interactions between biomolecules difficult to predict. Although characterizing H/S compensation requires experimental care, it is unquestionably a real phenomenon that has, from an engineering perspective, useful physical origins. Studying H/S compensation can help illuminate the still-murky roles of water and dynamics in biomolecular recognition and self-assembly. This review summarizes known sources of H/ S compensation (real and perceived) and lays out a conceptual framework for understanding and dissecting-and, perhaps, avoiding or exploiting-this phenomenon in biophysical systems. [ABSTRACT FROM AUTHOR]

We report herein the first total synthesis of piperenol B, a polyoxygenated cyclohexene derivative with reported pharmacological activity isolated from Piper cubeb. The chiral building block for this synthetic approach is derived from microbial cis-ipso, ortho-dihydroxylation of sodium benzoate, which was optimized to the multi-ten-gram scale by reaction medium engineering. Final-stage deprotection of an acetonide was investigated in detail, leading to an efficient eight-step protocol for the synthesis of piperenol B with a total yield of 10 %. Most importantly, the previously assigned absolute configuration of piperenol B was revised and unequivocally established by 2D NMR analysis and the Mosher's ester method. [ABSTRACT FROM AUTHOR]

Abstract: A cyclohexanone monooxygenase (CHMO) from the xenobiotic-degrading Polaromonas sp. strain JS666 was heterologously expressed in Escherichia coli, and its ability to catalyze enantio- and regiodivergent oxidations of prochiral and racemic ketones was investigated. The expression system was also used to evaluate this enzyme''s potential role in the oxidation of cis-1,2-dichloroethene (cDCE), a groundwater pollutant for which strain JS666 is the only known assimilator. The substrate enantiopreference and -selectivity of the strain JS666 CHMO is similar to that of other CHMO-type enzymes; of note is this enzyme''s excellent stereodiscrimination of 2-substituted cyclic ketones. The expression system exhibits no activity with ethene or cDCE as substrates under the tested conditions. Phylogenetic analysis shows that sequence variability among cyclohexanone monooxygenases could be a rich source of new enzyme activities and attributes. [Copyright &y& Elsevier]

Rücktitelbild: Analysis of Powders Containing Illicit Drugs Using Magnetic Levitation (Angew. Wie J. S. Bozenko, G. M. Whitesides et al. in ihrem Forschungsartikel auf S. 884 zeigen, trennt das Verfahren die Verbindungen basierend auf ihren unterschiedlichen Dichten in dem linearen Dichtegradienten, der in der Lösung gebildet wird. Die schwebenden Verbindungen (siehe Bild, aufgenommen mit gekreuzten Polarisatoren) sind gängige polare Verfälschungs- und Verdünnungsmittel, die zum Verschneiden illegaler Drogen verwendet werden. [Extracted from the article]

This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103- to 104-fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. [ABSTRACT FROM AUTHOR]