Engineered Biocatalytic Synthesis of β-N-Substituted-α-Amino Acids

Abstract

Non-canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,β-diamino acids. In this study, we used directed evolution to engineer the β-subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re-entry into the catalytic cycle and subsequent decomposition. Additional equivalents of l-serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β-substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

Keywords: Biocatalysis; Directed Evolution; Multiplexed Screening; Non-Canonical Amino Acids; Pyridoxal Phosphate.

Figure 1.

Non-canonical amino acids (ncAAs) and their synthesis by the pyridoxal phosphate-dependent enzyme TrpB. (A) Bioactive compounds featuring β-N-substituted amino acids. (B) TrpB has been extensively engineered for synthesis of various ncAAs. C-nucleophiles undergo a thermodynamically favorable attack into an electrophilic amino acrylate intermediate, E(A-A). The reaction does not reverse on laboratory timescales (C) Reactions with N-nucleophiles are underexplored. The reversibility of the transformation enables degradation of product through a promiscuous β-lyase reaction, challenging synthetic efforts.

Figure 2.

Pf2B9 DIT β-lyase activity. (A) DIT re-enters the catalytic cycle. Indoline is eliminated and the resultant amino acrylate intermediate breaks down to pyruvate and ammonia. (B) Progress curve monitoring degradation of DIT. β-lyase activity of Pf2B9 on DIT is shown with orange diamonds. DIT degradation in the presence of Ser is shown in red circles. DIT decay in the presence of Thr is shown in blue squares. Pf2B9 (3.2 μM), DIT (4 mM), Ser/Thr (10 mM), 3% MeOH in KPi buffer (200 mM), pH = 8.0, and 75 °C. Reactions conducted in triplicate. (C) Structure of the quinoidal species E(Q_DIT).

Figure 3.

Engineering PfTrpB for N-alkylation. (A) Substrate mixture and corresponding PfTrpB generated ncAAs. (B) PfTrpB engineering results. The left vertical axis gives the total product formed in fold-activity relative to Pf2B9 (diamond). The right vertical axis corresponds to the relative abundance of each product. PfTrpB (3.2 μM), indoline and indazole (1mM), aniline (10 mM), N-Et aniline (10 mM), morpholine (10 mM), Ser (100 mM), 10% DMSO, KPi buffer (200 mM), pH = 8.0, 75 °C, and 30 minutes. Reactions conducted in triplicate. (C) Crystal structure of Pf2B9 with active site residues in grey (PBD:6AM8) superimposed with the DIT quinonoid (E(Q_DIT)) from StTrpS (PBD:3CEP) in cyan. Atoms are colored so that nitrogen is blue, oxygen is red, and phosphorus is orange.

Figure 4.

Characterization of PfTrpB variants. (A) Formation of DIT from 1a and Ser. (B) DIT Progress curve analysis of Pf2B9 and PfA04. The vertical axis gives DIT concentration in mM over time in minutes in triplicate. PfTrpB variant (13 μM), indoline (20 mM), Ser (150 mM), 10% DMSO in KP_i buffer (200 mM), pH = 8.0, and 75 °C. (C) Kinetic characterization of β-N-substitution and β-lyase activity of Pf2B9 and PfA04. See SI for experimental design. (D) PfA04 progress curve analysis. The vertical axis gives the concentration of DIT in mM over time (minutes) in triplicate. PfA04(20 μM), indoline (4 mM), Ser (20 mM), 2% MeOH in KP_i buffer (200 mM), pH = 8.0, 64 °C, and 15 minutes.

Figure 5.

Substrate scope for the engineered TrpB catalyst, PfA04. (A) Reaction conditions are provided, and catalyst loading was varied according to the relative efficiencies of the reactions. (B) Oxidation of 1b leads to N-isotryptophan, 1c. (C) Cascade reaction of PfA04 with the H120N variant of the Trp decarboxylase from Ruminococcus gnavus, RgnTDC_H120N. The decarboxylase intercepts the amino acid 8b and produces high yields of the corresponding amino acid, out-competing depletion of 8b through the β-lyase reaction.