Engineering a norcoclaurine synthase for one-step synthesis of (S)-1-aryl-tetrahydroisoquinolines

Abstract

Tetrahydroisoquinoline alkaloids (THIQAs) are ubiquitous compounds with important pharmaceutical and biological activity. Their key N-heterocyclic structural motifs are synthesised via Pictet-Spengler (P-S) reaction by norcoclaurine synthases (NCS) in plants. The synthesis of 1-aryl-tetrahydroisoquinoline alkaloids has attracted increasing attention due to their antitumor and antivirus activities. Herein, the L68T/M97V mutant of NCS from Thalictrum flavum with improved activity was developed by semi-rational design. This mutant not only showed higher catalytic performance (> 96% conversion) toward benzaldehyde and dopamine over the wild-type enzyme, but also catalysed the P-S reaction of the bulky substrate 4-biphenylaldehyde and dopamine with high conversion (> 99%) for the effective synthesis of 1-aryl-THIQA. In terms of stereoselectivity, all products synthesised by the L68T/M97V mutant showed high optical purity (92-99% enantiomeric excess).

Keywords: Biocatalysis; Norcoclaurine synthase; Pictet–Spengler reaction; Protein engineering; Tetrahydroisoquinoline alkaloids.

Fig. 1

Some representative tetrahydroisoquinoline alkaloids (THIQAs) with pharmacological activities

Fig. 2

One-step synthesis of 1-aryl-tetrahydroisoquinolines using norcoclaurine synthase (NCS)

Fig. 3

The substrate-binding pocket of TfNCS (PDB ID: 5NON). Catalytic residues and the product mimic are coloured cyan and blue, respectively. Residues around the aldehyde substrate and the hydroxyl group of the amine substrate are coloured white and yellow, respectively

Fig. 4

Schematic diagram of the mutagenesis strategy

Fig. 5

Biotransformation of benzaldehyde and dopamine catalysed by TfNCS and its mutants. The reaction mixture was composed of 10 mM amine, 1 mM aldehyde, 20% (v/v) DMSO, 5 mM ascorbic acid, HEPES buffer (100 mM, pH 7.5) and 0.5 mg mL⁻¹ purified enzyme. Reactions were performed at 30 °C for 3 h

Fig. 6

Biotransformation of 4-bipenzaldehyde and dopamine catalysed by TfNCS and its mutants. The reaction mixture was composed of 10 mM amine, 1 mM aldehyde, 20% (v/v) DMSO, 5 mM ascorbic acid, HEPES buffer (100 mM, pH 7.5) and 4 mg mL⁻¹ purified enzyme. Reactions were performed at 30 °C for 24 h

Fig. 7

Asymmetric synthesis of bulky THIQAs using 4-bipenzaldehyde and dopamine as substrates. The reaction mixture was composed of 2 eq. amine, 1 eq. aldehyde, 20% (v/v) DMSO, 5 mM ascorbic acid, HEPES buffer (100 mM, pH 7.5) and 4 mg mL⁻¹ purified enzyme. Reactions were performed at 40 °C for 24 h

Fig. 8

Structural analysis of the substrate-binding pocket of TfNCS and its mutant L68T/M97V using POCASA 1.1. A Sectional view of the substrate pocket in TfNCS. B Sectional view of the substrate pocket in mutant L68T/M97V modelled by AlphaFold2. C Volume of the substrate-binding pocket in the WT enzyme. D Volume of the substrate-binding pocket in mutant L68T/M97V

Fig. 9

Analysis of interactions around the substrate pocket of WT TfNCS and its mutant L68T/M97V using Ligplus. A Hydrogen bonds and hydrophobic interactions between substrates and residues in the binding pocket of WT TfNCS. B Hydrogen bonds and hydrophobic interactions between substrates and residues in the binding pocket of mutant L68T/M97V