Stereodivergent Evolution of Artificial Enzymes for the Michael Reaction

Abstract

Enzymes are valuable biocatalysts for asymmetric synthesis due to their exacting stereocontrol. Changing the selectivity of an existing catalyst for new applications is, however, challenging. Here we show that, in contrast, the stereoselectivity of an artificial enzyme created by design and directed evolution is readily tunable. We engineered a promiscuous artificial retro-aldolase into four stereocomplementary catalysts for the Michael addition of a tertiary carbanion to an unsaturated ketone. Notably, this selectivity is also preserved with alternative Michael nucleophiles. Complete stereodiversification of other designer enzymes should similarly be possible by extension of these approaches.

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

“…1,17,18 The iminium ion species is a highly versatile reaction intermediate 19 which has previously been exploited in designer enzymes featuring catalytic lysine or N-terminal proline residues, and in artificial enzymes with secondary-amine-containing unnatural cofactors. [20][21][22][23][24][25] However, Friedel-Crafts alkylation via an iminium ion species has not been demonstrated in these contexts, and is a distinct reaction pathway from those employed by natural enzymes for this reaction. We saw great potential in unlocking iminium catalysis to create an artificial Friedel-Crafts alkylase by using expanded genetic code technology to provide an appropriate non-canonical catalytic residue.…”

Enabling protein-hosted organocatalytic transformations

“…Iminium catalysis mediated by RA95.5-8 was used as a means to mediate carbon-carbon bond formation, including conjugate additions ( Fig. 5a and b), 40,42,44 Knoevenagel (Fig. 5c) 43 and Henry condensations (Fig.…”

“…[63][64][65][66][67][68] Nevertheless, it should be noted that, during the optimisation process, stereoselectivity may be weak because the active site undergoes reconstruction (e.g. relocating the catalytic residue); 40,41,44 eventually, stereoselectivity resumes and variants with kinetic parameters and selectivity similar to those of natural enzymes can be achieved. Furthermore, rened articial enzymes oen possess properties similar to those of natural enzymes.…”

“…Thus, laboratory evolution is used to enhance both catalytic activity and reaction profile. This pathway led to the formation of a highly competent and promiscuous de novo Kemp eliminase, 99 , 100 retro-aldolases (RA) 40 , 43 , 44 , 63 , 65 – 68 and Diels–Alderases. 52 , 101 Here, we will focus on retro-aldolases which bear a catalytically active lysine for iminium and enamine catalysis.…”

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

“…1,17,18 The iminium ion species is a highly versatile reaction intermediate 19 which has previously been exploited in designer enzymes featuring catalytic lysine or N-terminal proline residues, and in artificial enzymes with secondary-amine-containing unnatural cofactors. [20][21][22][23][24][25] However, Friedel-Crafts alkylation via an iminium ion species has not been demonstrated in these contexts, and is a distinct reaction pathway from those employed by natural enzymes for this reaction. We saw great potential in unlocking iminium catalysis to create an artificial Friedel-Crafts alkylase by using expanded genetic code technology to provide an appropriate non-canonical catalytic residue.…”