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

Abstract

The incorporation of organocatalysts into protein scaffolds holds the promise of overcoming some of the limitations of this powerful catalytic approach. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymatic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centres not only during C-C bond formation, but also by enantioselective protonation, delivering a proton to one face of a prochiral enamine intermediate. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis.

Keywords: Artificial Enzymes; Enantioselective Protonation; Friedel-Crafts Alkylations; Iminium Catalysis; Non-canonical Amino Acids.

Figure 1

(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. (b) An organocatalyst for this transformation demonstrated by Austin and MacMillan^[23]. (c) This work – tandem‐Friedel‐crafts‐alkylation‐enantioselective‐protonation of indoles employing α‐substituted acroleins as substrates via protonation of a prochiral enamine intermediate. (d) Organocatalysts for this transformation require primary‐amine moieties for iminium activation and tertiary‐amine moieties for enantioselective proton delivery^[25], steric constraints make α‐substituted acroleins challenging substrates for conventional secondary‐amine‐containing organocatalysts^[34,35].

Scheme 1

The reaction between 2‐methyl‐indole (1 a) and methacrolein (2 a) produces 3 a after reduction via a tandem enantioselective protonation process (FC‐EP reaction), whilst substitution of methacrolein with crotonaldehyde (2 b) produces 4 after reduction (FC reaction).

Figure 2

(a) Effect of reaction pH on analytical yields and enantiomeric excesses from the formation of 4 (left, blue) and 3 a (right, orange) by LmrR_pAF. Reaction conditions [LmrR_pAF]=10 μM (dimer concentration); [1 a]=1 mM; [2 a]=6 mM or [2 b]=5 mM; 300 μL volume reaction in phosphate buffer (50 mM) containing NaCl (150 mM) and DMF (8 vol %). Reactions conducted for 6 hours at 4 °C, followed by reduction to form 3 a or 4 for analysis by normal‐phase HPLC. (b) LmrR_pAF catalysed production of 3 a monitored over 48 hours, revealing product racemisation. Reaction conditions as in (a), pH=6. In both (a) and (b), errors given represent the standard deviation from two experiments with independently produced batches of protein, each conducted in duplicate.

Figure 3

(a) representative chiral normal‐phases HPLC traces obtained in competition experiments employing both substrates 2 a and 2 b together with indole 1 a to produce mixtures of products 3 a (orange) and 4 (blue) (Table 2). (b) Positions in LmrR_pAF subject to mutagenesis (PDB: 6I8N). Effect of various mutants on reaction outcomes producing product 4 (c) in blue and 3 a (d) in orange. ΔΔG^ǂ (the difference in the Gibbs’ free energy of activation for the production of the two product enantiomers) was calculated from the enantiomeric ratio (e.r.) according to the equation ΔΔG^ǂ=RTln(e.r.). In (c) and (d), errors given represent the standard deviation from two experiments with independently produced batches of protein, each conducted in duplicate.

Figure 4

(a) Catalysis results for the FC‐EP reaction producing 3 a using cell‐free extract libraries with every mutant at the N19 (top) and M89 (bottom) positions. Results are an average of a triplicate experiment, and error bars shown reflect the standard deviation of those experiments, except for the results for 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). Analytical yields and enantiomeric excesses were determined by SFC with the use of an internal standard, and are given relative to the mean of the LmrR_pAF samples. (b) Results for each library: N19 shows a weak correlation between yield and ee for the pAF containing samples, whilst M89 shows a strong correlation. LmrR (no pAF) samples shown in orange and LmrR_pAF samples shown in blue. ^[a] Value obtained by performing a linear fit of the pAF containing members of the library, i. e., LmrR without pAF was not included in the fit.

Figure 5

Substrate scope of FC‐EP reaction catalysed by LmrR_pAF. Analytical yields were determined by HPLC or SFC with the use of a calibration curve. ^[a] The absolute configuration was assigned by comparison of the order of elution of the enantiomers on chiral HPLC with that reported in the literature where the stereocentre was assigned by analogy to another product. ^[b] Small unidentified impurities also observed in the chromatogram.^[c] nal concentration of 1.5 mM was used due to low solubility of this substrate under reaction conditions. ^[b] No product was detected by HPLC. Errors given represent the standard deviation from two experiments with independently produced batches of protein, each conducted in duplicate, to give four total measurements. The errors are quoted to one decimal where they are below 1 %. Errors on ee measurements are approximately 1 % or lower in most cases. N.D.=not determined.