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New-to-Nature Selective C-H Alkylation Using Engineered Carbene Transferases

Citation

Zhang, Juner (2022) New-to-Nature Selective C-H Alkylation Using Engineered Carbene Transferases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/4kv8-f830. https://resolver.caltech.edu/CaltechTHESIS:04252022-204451583

Abstract

Synthetic methods to selectively convert C–H bonds, a prevalent motif in organic molecules, into functionalities can significantly accelerate the syntheses and derivatization of molecules. In the past decade, many enzymatic catalysts have emerged as greener, more selective, and more versatile alternatives to small-molecule catalysts for selective C–H functionalization reactions. In nature, enzymes only catalyze a limited set of C–H functionalization reactions that are useful for chemical synthesis, as the overwhelming majority of known C–H functionalization enzymes in nature are hydroxylases. The diversity of the enzymatic reaction scope needs to be substantially expanded to make them broadly useful to synthetic chemists. This thesis will describe new enzymes, which we repurposed from one of the most prevalent C–H hydroxylases in nature, cytochromes P450, which are now able to catalyze new-to-nature C–H alkylation reactions via selective carbene transfer. Given the central role of C–C bond forming reactions in building and elaborating the carbon skeleton of organic molecules, these transformations are of high interest in many fields of research, such as medicinal chemistry and material chemistry. In Chapter 1, I review a recent surge in newly identified enzymes, repurposed enzymes, and artificial metalloenzymes which can catalyze selective C–H functionalization reactions. Chapter 2 details the development of a panel of enantiodivergent α-amino C(sp³)−H fluoroalkylases. Using directed evolution, the carbene transferases can install fluoroalkyl groups onto these C–H bonds with high activity (4,070 total turnovers, TTN) and selectivity (>99% ee). Notably, complementary regioselectivity can be achieved using an alternative enzyme, P411-PFA-(S). In Chapter 3, I report the first carbene transferase, P411-ACHF, which can transfer an α-cyanocarbene to arene C–H bonds of N-substituted benzenes. Chemodivergent C(sp²)–H and C(sp³)–H functionalization can be achieved using P411-ACHF and P411-PFA. Additionally, structural studies revealed an unprecedented backbone carbonyl flip within the long I-helix of P411-PFA, which may suggest how these enzymes have evolved to bind and activate diazo compounds for carbene transfer reactions. In Chapter 4, I discuss the efforts I took toward stabilizing an interesting but unstable P450, CYP3A4. This enzyme exhibits large active site volume and high substrate promiscuity and therefore can be a great candidate to develop late-stage carbene and nitrene transferases. I adopted consensus sequence mutagenesis and predicted five mutations which have the potential to have the strongest beneficial effects on improving CYP3A4's thermostability. In summary, this thesis work addresses the urgent need for expansion of the current enzymatic C–H functionalization reaction scope and the development of more sustainable and selective C–H functionalization catalysts which can be synthetically useful.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:directed evolution; protein engineering; biocatalysis; cytochromes P450; C–H functionalization; carbene; C-C bond formation
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Reisman, Sarah E. (chair)
  • Agapie, Theodor
  • Wei, Lu
  • Arnold, Frances Hamilton
Defense Date:9 May 2022
Record Number:CaltechTHESIS:04252022-204451583
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:04252022-204451583
DOI:10.7907/4kv8-f830
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.9b04344DOIArticle adapted for Chapter II
ORCID:
AuthorORCID
Zhang, Juner0000-0001-8181-1187
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14558
Collection:CaltechTHESIS
Deposited By: Juner Zhang
Deposited On:02 Jun 2022 19:52
Last Modified:13 Jul 2022 22:56

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