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.

[1]  F. Arnold,et al.  Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE , 2014, Chembiochem : a European journal of chemical biology.

[2]  N. Scrutton,et al.  Electron transfer in flavocytochrome P450 BM3: kinetics of flavin reduction and oxidation, the role of cysteine 999, and relationships with mammalian cytochrome P450 reductase. , 2003, Biochemistry.

[3]  B. Nestl,et al.  Synthesis of Heterocyclic Terpenoids by Promiscuous Squalene–Hopene Cyclases , 2013, Chembiochem : a European journal of chemical biology.

[4]  U. Bornscheuer,et al.  Catalytic promiscuity in biocatalysis: using old enzymes to form new bonds and follow new pathways. , 2004, Angewandte Chemie.

[5]  A. Yudin.  Aziridines and Epoxides in Organic Synthesis: Yudin: Aziridines and Epoxides in Organic Synthesis O-BK , 2006 .

[6]  Frances H Arnold,et al.  Expanding P450 catalytic reaction space through evolution and engineering. , 2014, Current opinion in chemical biology.

[7]  Joseph T. Meyerowitz,et al.  Non‐natural Olefin Cyclopropanation Catalyzed by Diverse Cytochrome P450s and Other Hemoproteins , 2014, Chembiochem : a European journal of chemical biology.

[8]  C. Che,et al.  [Fe(III)(F(20)-tpp)Cl] is an effective catalyst for nitrene transfer reactions and amination of saturated hydrocarbons with sulfonyl and aryl azides as nitrogen source under thermal and microwave-assisted conditions. , 2010, Chemistry.

[9]  R. Vyas,et al.  Cobalt-catalyzed aziridination with diphenylphosphoryl azide (DPPA): direct synthesis of N-phosphorus-substituted aziridines from alkenes. , 2006, The Journal of organic chemistry.

[10]  Chelsea A. Huff,et al.  A highly effective cobalt catalyst for olefin aziridination with azides: hydrogen bonding guided catalyst design. , 2008, Organic letters.

[11]  T. Ost,et al.  Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. , 2001, Biochemistry.

[12]  T. Poulos,et al.  Structure of a cytochrome P450-redox partner electron-transfer complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Huisman,et al.  Engineering the third wave of biocatalysis , 2012, Nature.

[14]  Donald Hilvert,et al.  Minimalist active-site redesign: teaching old enzymes new tricks. , 2007, Angewandte Chemie.

[15]  Ritesh Singh,et al.  P450-Catalyzed Intramolecular sp3 C–H Amination with Arylsulfonyl Azide Substrates , 2014, ACS catalysis.

[16]  B. Nestl,et al.  Stereoselective Friedel–Crafts alkylation catalyzed by squalene hopene cyclases , 2012 .

[17]  Frances H. Arnold,et al.  Enantioselective Imidation of Sulfides via Enzyme-Catalyzed Intermolecular Nitrogen-Atom Transfer , 2014, Journal of the American Chemical Society.

[18]  D. Mansuy,et al.  Iron- and manganese-porphyrin catalysed aziridination of alkenes by tosyl- and acyl-iminoiodobenzene , 1984 .

[19]  Frances H. Arnold,et al.  Enantioselective Intramolecular C—H Amination Catalyzed by Engineered Cytochrome P450 Enzymes in vitro and in vivo. , 2014 .

[20]  I. Ryu,et al.  Iodine-catalyzed aziridination of alkenes using Chloramine-T as a nitrogen source , 1998 .

[21]  L. Wojtas,et al.  Effective synthesis of chiral N-fluoroaryl aziridines through enantioselective aziridination of alkenes with fluoroaryl azides. , 2013, Angewandte Chemie.

[22]  J. Dawson,et al.  Sulfur donor ligand binding to ferric cytochrome P-450-CAM and myoglobin. Ultraviolet-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic investigation of the complexes. , 1982, The Journal of biological chemistry.

[23]  C. Thibodeaux,et al.  Enzymatic chemistry of cyclopropane, epoxide, and aziridine biosynthesis. , 2012, Chemical reviews.

[24]  Frances H Arnold,et al.  Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran. , 2014, Angewandte Chemie.

[25]  Frances H Arnold,et al.  Enantioselective intramolecular C-H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. , 2013, Angewandte Chemie.

[26]  Michael T. Green,et al.  Iron(IV)hydroxide pKa and the Role of Thiolate Ligation in C–H Bond Activation by Cytochrome P450 , 2013, Science.

[27]  S. Minakata,et al.  Metal-free aziridination of styrene derivatives with iminoiodinane catalyzed by a combination of iodine and ammonium iodide. , 2013, Organic letters.

[28]  Frances H. Arnold,et al.  Enzyme-Controlled Nitrogen-Atom Transfer Enables Regiodivergent C–H Amination , 2014, Journal of the American Chemical Society.

[29]  L. Degennaro,et al.  Recent Advances in the Stereoselective Synthesis of Aziridines , 2014 .

[30]  L. Degennaro,et al.  Recent advances in the stereoselective synthesis of aziridines. , 2014, Chemical reviews.

[31]  Paul N. Devine,et al.  Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture , 2010, Science.

[32]  C. Che,et al.  Selective functionalisation of saturated C-H bonds with metalloporphyrin catalysts. , 2011, Chemical Society reviews.

[33]  J. Dawson,et al.  Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant. , 2011, Archives of biochemistry and biophysics.

[34]  Johan A. Kers,et al.  Cytochrome P450-catalysed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis , 2012, Nature chemical biology.

[35]  Frances H Arnold,et al.  Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. , 2015, Angewandte Chemie.

[36]  R. Fasan,et al.  Enzymatic C(sp3)-H Amination: P450-Catalyzed Conversion of Carbonazidates into Oxazolidinones , 2015, ACS catalysis.

[37]  Frances H. Arnold,et al.  A Serine-Substituted P450 Catalyzes Highly Efficient Carbene Transfer to Olefins In Vivo , 2013, Nature chemical biology.