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Olefin Cyclopropanation and Carbon-Hydrogen Amination via Carbene and Nitrene Transfers Catalyzed by Engineered Cytochrome P450 Enzymes


Coelho, Pedro de Souza Leão (2013) Olefin Cyclopropanation and Carbon-Hydrogen Amination via Carbene and Nitrene Transfers Catalyzed by Engineered Cytochrome P450 Enzymes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/HSPH-AF89.


Synthetic biology promises to transform organic synthesis by enabling artificial catalysis in living cells. I start by reviewing the state of the art in this young field and recognizing that new approaches are required for designing enzymes that catalyze nonnatural reactions, in order to expand the scope of biocatalytic transformations. Carbene and nitrene transfers to C=C and C-H bonds are reactions of tremendous synthetic utility that lack biological counterparts. I show that various heme proteins, including cytochrome P450BM3, will catalyze promiscuous levels of olefin cyclopropanation when provided with the appropriate synthetic reagents (e.g., diazoesters and styrene). Only a few amino acid substitutions are required to install synthetically useful levels of stereoselective cyclopropanation activity in P450BM3. Understanding that the ferrous-heme is the active species for catalysis and that the artificial reagents are unable to induce a spin-shift-dependent increase in the redox potential of the ferric P450, I design a high-potential serine-heme ligated P450 (P411) that can efficiently catalyze cyclopropanation using NAD(P)H. Intact E. coli whole-cells expressing P411 are highly efficient asymmetric catalysts for olefin cyclopropanation. I also show that engineered P450s can catalyze intramolecular amination of benzylic C-H bonds from arylsulfonyl azides. Finally, I review other examples of where synthetic reagents have been used to drive the evolution of novel enzymatic activity in the environment and in the laboratory. I invoke preadaptation to explain these observations and propose that other man-invented reactions may also be transferrable to natural enzymes by using a mechanism-based approach for choosing the enzymes and the reagents. Overall, this work shows that existing enzymes can be readily adapted for catalysis of synthetically important reactions not previously observed in nature.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biocatalysis, Directed Evolution, Synthetic Biology.
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Awards:The Herbert Newby McCoy Award, 2013. Demetriades-Tsafka-Kokkalis Prize in Biotechnology or Related Fields, 2013.
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Agapie, Theodor (chair)
  • Arnold, Frances Hamilton
  • Dougherty, Dennis A.
  • Peters, Jonas C.
Defense Date:27 February 2013
Non-Caltech Author Email:pc (AT)
Record Number:CaltechTHESIS:04072013-230318757
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7583
Deposited By: Pedro Coelho
Deposited On:27 Feb 2014 19:05
Last Modified:08 Nov 2023 00:11

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