Directed Evolution of a Fe(II)- and α-Ketoglutarate-Dependent Dioxygenase for Site-Selective Azidation of Unactivated Aliphatic C-H Bonds

11 August 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Fe(II)- and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of C-H bonds via a variety of C-X bond forming reactions. Achieving high chemoselectivity for functionalization using non-native functional groups remains rare, however, particularly for non-native substrates. The current study shows that directed evolution can be used to engineer variants of an engineered dioxygenase, SadX, that address this challenge. Site-selective azidation of succinylated amino acids and a succinylated amine was achieved using variants with improved azidation yield and selectivity on a probe substrate as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C-H functionalization with other non-native functional groups.

Keywords

directed evolution
C-H Functionalization
azidation
non-heme iron
α-ketoglutarate

Supplementary materials

Title
Description
Actions
Title
Supporting Information for: Directed Evolution of Site Selective Fe(II)- and α-Ketoglutarate-Dependent Azidases
Description
Completely description of materials, methods, and compound characterization.
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.