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

Abstract

The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, we show that incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel–Crafts alkylation between α,β-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates toward conjugate addition via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis–Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many more biocatalytic transformations inspired by organocatalysis.

Tandem Friedel‐Crafts‐Alkylation‐Enantioselective‐Protonation by Artificial Enzyme Iminium Catalysis

“…Likewise, incorporation of the pAF aniline sidechain into the protein backbone was also essential, since the combination of aniline and LmrR in ratios typically used for supramolecular catalysis with LmrR [36] failed to (a) our previous work on the Friedel-Crafts alkylation of indoles with β-substituted enals using LmrR_pAF as catalyst, which takes place via a prochiral iminium-ion intermediate. [22] (b) An organocatalyst for this transformation demonstrated by Austin and MacMillan [23] . (c) This work -tandem-Friedelcrafts-alkylation-enantioselective-protonation of indoles employing α-substituted acroleins as substrates via protonation of a prochiral enamine intermediate.…”

“…The FC-EP reaction is another promiscuous activity of LmrR_pAF building on the hydrazone formation, Friedel-Crafts and synergistically catalysed processes that we have already demonstrated. [11,22,31,39] We anticipate that LmrR_pAF will find application in yet further useful and challenging reactions operating via organocatalytic processes, realizing the benefits of biocatalytic processes for a broad array of transformations. [1,2]…”

“…Similarly to our previous work, the 2-methyl substituent has a significant effect on the yields obtained, and thus products 3 e-h required increased catalyst loadings and reaction times to accumulate good yields. [22] Erosion of enantiomeric excess was also observed for these products, with shorter reaction times giving higher selectivities but lower yields, and longer reaction times improving yields at the expense of selectivity. Electron donating LmrR (no pAF) which is six repeated experiments (data from both libraries combined) and LmrR_pAF which is five repeated experiments (data from both libraries combined, one sample was calculated to be an outlier by the interquartile method).…”

“…[21] Recently we demonstrated that LmrR, with the ncAA para-aminophenyl alanine (pAF) incorporated at position 15, makes a competent and enantioselective catalyst for the Friedel-Crafts alkylation of indoles with aliphatic enal substrates, which are activated for nucleophilic attack at the β-position by iminium ion formation at the catalytic pAF residue (henceforth referred to as FC-reaction, Figure 1(a)). [22] This transformation, which was first demonstrated with organocatalysis by Austin and MacMillan in 2002 (Figure 1(b)), creates the chiral centre during the CÀ C bond forming step, and thus stereoselective formation of the iminium ion and controlled approach of indole are important for good enantioselectivity. [23,24] In this study, we turned our attention to the tandem Friedel-Crafts-alkylation-enantioselective-protonation (henceforth FC-EP reaction) of indoles with α-substituted acroleins (Figure 1(c)).…”

“…Figure1. (a) our previous work on the Friedel-Crafts alkylation of indoles with β-substituted enals using LmrR_pAF as catalyst, which takes place via a prochiral iminium-ion intermediate [22]. (b) An organocatalyst for this transformation demonstrated by Austin and MacMillan[23] .…”