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Leveson-Gower et al - ChemRxiv.pdf (9.3 MB)

Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase

submitted on 18.02.2021, 15:57 and posted on 19.02.2021, 13:01 by Reuben B. Leveson-Gower, Zhi Zhou, Ivana Drienovská, Gerard Roelfes
We show that the incorporation of the non-canonical amino acid para-aminophenylalanine (pAF) into the non-enzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-crafts alkylation between alpha, beta-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates towards conjugate addition via the formation of intermediate iminium ion species, whilst the protein scaffold provides rate acceleration and enantio-induction. Improved LmrR_pAF varants were identified by direted evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of enals and indoles. Analys of Michaelis-Menten kinetics of LmrR-pAF and tevolved mutants reveals that new activities emerge via evolutionary pathways that diverge from one another and specialise catalytic reactivity.


Nederlandse Organisatie voor Wetenschappelijk Onderzoek, no.724.013.003

European Research Council, Starting Grant 280010

European Research Council, Advanced Grant 885396

Ministerie van Onderwijs, Cultuur en Wetenschap, no. 024.001.035


Email Address of Submitting Author


University of Groningen


the Netherlands

ORCID For Submitting Author


Declaration of Conflict of Interest

no conflict of interest

Version Notes

version 1.0