23 research outputs found
Genetically Tunable Enzymatic CâH Amidation for Lactam Synthesis
A major challenge in carbonâhydrogen (CâH) bond functionalization is to have the catalyst control precisely where a reaction takes place. Here we report engineered cytochrome P450 enzymes that perform unprecedented enantioselective CâH amidation reactions and control the site selectivity to divergently construct β-, Îł- and δ-lactams, completely overruling the inherent reactivities of the CâH bonds. The enzymes, expressed in Escherichia coli cells, accomplish this abiological carbonânitrogen (CâN) bond formation via reactive iron-bound carbonyl nitrenes generated from nature-inspired acyl-protected hydroxamate precursors. This transformation is exceptionally efficient (up to 1,020,000 total turnovers) and selective (up to 25:1 regioselectivity and 96% enantiomeric excess), and can be performed easily on preparative scale
An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(spÂł)âH bonds
The ability to selectively functionalize ubiquitous CâH bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(spÂł)âH bonds. These fully genetically encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad spectrum of enantioselective C(spÂł)âH amination reactions. These catalysts can aminate a variety of benzylic, allylic and aliphatic CâH bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of primary C(spÂł)âH bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quaternary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess a tertiary C(spÂł)âH bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule catalysts. Further engineering allowed for the enantioselective construction of methylâethyl stereocentres, which is notoriously challenging in asymmetric catalysis
Genetically Tunable Enzymatic CâH Amidation for Lactam Synthesis
A major challenge in carbonâhydrogen (CâH) bond functionalization is to have the catalyst control precisely where a reaction takes place. Here we report engineered cytochrome P450 enzymes that perform unprecedented enantioselective CâH amidation reactions and control the site selectivity to divergently construct β-, Îł- and δ-lactams, completely overruling the inherent reactivities of the CâH bonds. The enzymes, expressed in Escherichia coli cells, accomplish this abiological carbonânitrogen (CâN) bond formation via reactive iron-bound carbonyl nitrenes generated from nature-inspired acyl-protected hydroxamate precursors. This transformation is exceptionally efficient (up to 1,020,000 total turnovers) and selective (up to 25:1 regioselectivity and 96% enantiomeric excess), and can be performed easily on preparative scale
Site-selective enzymatic CâH amidation for synthesis of diverse lactams
A paramount challenge in carbonâhydrogen (CâH) functionalization is to control the site selectivity of the reaction. Current methods use directing groups and/or substrate control to pick out a particular CâH bond, which limits the breadth of potential substrates. Recently, an enzymatic strategy to address this challenge was reported by Professor Frances H. Arnold and coworkers Inha Cho (PhD student) and Dr. Zhi-Jun Jia (postdoctoral fellow) from the California Institute of Technology (USA). The authors used directed evolution to tune the site selectivity of CâH amidation catalyzed by heme enzymes. Professor Arnold explained: âEnzymes offer unparalleled selectivity in an array of transformations devised first by chemists and now established in natural metalloproteins. Itâs a splendid opportunity
to merge human chemical ingenuity with the power of evolution to make new, synthetically useful catalysts.
Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein
Chiral 1,2âamino 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 90â% ee) under anaerobic conditions with Oâpivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive ironânitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering
An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(spÂł)âH bonds
The ability to selectively functionalize ubiquitous CâH bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(spÂł)âH bonds. These fully genetically encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad spectrum of enantioselective C(spÂł)âH amination reactions. These catalysts can aminate a variety of benzylic, allylic and aliphatic CâH bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of primary C(spÂł)âH bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quaternary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess a tertiary C(spÂł)âH bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule catalysts. Further engineering allowed for the enantioselective construction of methylâethyl stereocentres, which is notoriously challenging in asymmetric catalysis
Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein
Chiral 1,2âamino 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 90â% ee) under anaerobic conditions with Oâpivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive ironânitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering
Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore
By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems
An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)âH bonds
The ability to selectively functionalize ubiquitous C-H bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(sp3)-H bonds. These fully genetically encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad spectrum of enantioselective C(sp3)-H amination reactions. These catalysts can aminate a variety of benzylic, allylic and aliphatic C-H bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of primary C(sp3)-H bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quaternary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess a tertiary C(sp3)-H bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule catalysts. Further engineering allowed for the enantioselective construction of methyl-ethyl stereocentres, which is notoriously challenging in asymmetric catalysis
Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA
The precise control of microRNA (miRNA) biogenesis is critical for embryonic development and normal cellular functions, and its dysregulation is often associated with human diseases. Though the birth and maturation pathway of miRNA has been established, the regulation and death pathway remains largely unknown. Here, we report the RNA-binding proteins, Lin28a and Lin28b, as posttranscriptional repressors of let-7 miRNA biogenesis. We observe that the Lin28 proteins act mainly in the cytoplasm by inducing uridylation of precursor let-7 (pre-let-7) at its 3' end. The uridylated pre-let-7 (up-let-7) fails Dicer processing and undergoes degradation. We provide a mechanism for the posttranscriptional regulation of miRNA biogenesis by Lin28 which is highly expressed in undifferentiated cells and certain cancer cells. The Lin28-mediated downregulation of let-7 may play a key role in development, stem cell programming, and tumorigenesis