Enantiodivergent α-Amino C–H Fluoroalkylation Catalyzed by Engineered Cytochrome P450s

The introduction of fluoroalkyl groups into organic compounds can significantly alter pharmacological characteristics. One enabling but underexplored approach for the installation of fluoroalkyl groups is selective C(sp^3)–H functionalization due to the ubiquity of C–H bonds in organic molecules. We have engineered heme enzymes that can insert fluoroalkyl carbene intermediates into α-amino C(sp3)–H bonds and enable enantiodivergent synthesis of fluoroalkyl-containing molecules. Using directed evolution, we engineered cytochrome P450 enzymes to catalyze this abiological reaction under mild conditions with total turnovers (TTN) up to 4070 and enantiomeric excess (ee) up to 99%. The iron-heme catalyst is fully genetically encoded and configurable by directed evolution so that just a few mutations to the enzyme completely inverted product enantioselectivity. These catalysts provide a powerful method for synthesis of chiral organofluorine molecules that is currently not possible with small-molecule catalysts

Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and Enhanced by Non-Native Small-Molecule Ligands

Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that Pseudomonas savastanoi ethylene-forming enzyme, a non-heme iron enzyme, can catalyze olefin aziridination and nitrene C–H insertion, and that these activities can be improved by directed evolution. The non-heme iron center allows for facile modification of the primary coordination sphere by addition of metal-coordinating molecules, enabling control over enzyme activity and selectivity using small molecules

Enantioselective Enzyme-Catalyzed Aziridination Enabled by Active-Site Evolution of a Cytochrome P450

One of the greatest challenges in protein design is creating new enzymes, something evolution does all the time, starting from existing ones. Borrowing from nature’s evolutionary strategy, we have engineered a bacterial cytochrome P450 to catalyze highly enantioselective intermolecular aziridination, a synthetically useful reaction that has no natural biological counterpart. The new enzyme is fully genetically encoded, functions in vitro or in whole cells, and can be optimized rapidly to exhibit high enantioselectivity (up to 99% ee) and productivity (up to 1,000 catalytic turnovers) for intermolecular aziridination, demonstrated here with tosyl azide and substituted styrenes. This new aziridination activity highlights the remarkable ability of a natural enzyme to adapt and take on new functions. Once discovered in an evolvable enzyme, this non-natural activity was improved and its selectivity tuned through an evolutionary process of accumulating beneficial mutations

Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein

Chiral 1,2‐amino alcohols are widely represented in biologically active compounds from neurotransmitters to antivirals. While many synthetic methods have been developed for accessing amino alcohols, the direct aminohydroxylation of alkenes to unprotected, enantioenriched amino alcohols remains a challenge. Using directed evolution, we have engineered a hemoprotein biocatalyst based on a thermostable cytochrome c that directly transforms alkenes to amino alcohols with high enantioselectivity (up to 2500 TTN and 90 % ee) under anaerobic conditions with O‐pivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive iron‐nitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering

Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein

Chiral 1,2‐amino alcohols are widely represented in biologically active compounds from neurotransmitters to antivirals. While many synthetic methods have been developed for accessing amino alcohols, the direct aminohydroxylation of alkenes to unprotected, enantioenriched amino alcohols remains a challenge. Using directed evolution, we have engineered a hemoprotein biocatalyst based on a thermostable cytochrome c that directly transforms alkenes to amino alcohols with high enantioselectivity (up to 2500 TTN and 90 % ee) under anaerobic conditions with O‐pivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive iron‐nitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering

In-Orbit Instrument Performance Study and Calibration for POLAR Polarization Measurements

POLAR is a compact space-borne detector designed to perform reliable measurements of the polarization for transient sources like Gamma-Ray Bursts in the energy range 50-500keV. The instrument works based on the Compton Scattering principle with the plastic scintillators as the main detection material along with the multi-anode photomultiplier tube. POLAR has been launched successfully onboard the Chinese space laboratory TG-2 on 15th September, 2016. In order to reliably reconstruct the polarization information a highly detailed understanding of the instrument is required for both data analysis and Monte Carlo studies. For this purpose a full study of the in-orbit performance was performed in order to obtain the instrument calibration parameters such as noise, pedestal, gain nonlinearity of the electronics, threshold, crosstalk and gain, as well as the effect of temperature on the above parameters. Furthermore the relationship between gain and high voltage of the multi-anode photomultiplier tube has been studied and the errors on all measurement values are presented. Finally the typical systematic error on polarization measurements of Gamma-Ray Bursts due to the measurement error of the calibration parameters are estimated using Monte Carlo simulations.Comment: 43 pages, 30 figures, 1 table; Preprint accepted by NIM

Enzymatic construction of highly strained carbocycles

Small carbocycles are structurally rigid and possess high intrinsic energy due to their ring strain. These features lead to broad applications but also create challenges for their construction. We report the engineering of hemeproteins that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addition to unsaturated carbon-carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed at preparative scale, and the resulting strained carbocycles can be derivatized, opening myriad potential applications

Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp^3 C–H functionalization

Although abundant in organic molecules, carbon–hydrogen (C–H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C–H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp^3 carbon. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp^3 C–H bonds through carbene C–H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C–H functionalization. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C–H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp^3 C–H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C–H functionalization reactions for applications in chemistry and synthetic biology