Single step syntheses of (1S)-aryl-tetrahydroisoquinolines by norcoclaurine synthases

Abstract

The 1-aryl-tetrahydroisoquinoline (1-aryl-THIQ) moiety is found in many biologically active molecules. Single enantiomer chemical syntheses are challenging and although some biocatalytic routes have been reported, the substrate scope is limited to certain structural motifs. The enzyme norcoclaurine synthase (NCS), involved in plant alkaloid biosynthesis, has been shown to perform stereoselective Pictet-Spengler reactions between dopamine and several carbonyl substrates. Here, benzaldehydes are explored as substrates and found to be accepted by both wild-type and mutant constructs of NCS. In particular, the variant M97V gives a range of (1 S)-aryl-THIQs in high yields (48-99%) and e.e.s (79-95%). A co-crystallised structure of the M97V variant with an active site reaction intermediate analogue is also obtained with the ligand in a pre-cyclisation conformation, consistent with (1 S)-THIQs formation. Selected THIQs are then used with catechol O-methyltransferases with exceptional regioselectivity. This work demonstrates valuable biocatalytic approaches to a range of (1 S)-THIQs.

Fig. 1. Pharmaceutically relevant 1-aryl-THIQs, naturally occurring 1-aryl-THIQs and a proposed route to these compounds using TfNCS (NCS isolated from Thalictrum flavum).

A range of a pharmaceutically relevant 1-aryl-THIQs and b the Cryptostylines, a variety of naturally occurring enantiopure 1-aryl-THIQs isolated from C. fulva. The analogous levorotatory compounds were isolated from C. erythroglossa^,. c The generation of a variety of (1 S)-aryl THIQ products in a single, regioselective and enantioselective step, using TfNCS as a reaction catalyst (WT or single-point variant).

Fig. 2. HPLC conversions (determined against product standards) of reactions between dopamine and 2-methyl-2-pentenal or 2-ethylbutanal.

Reaction between dopamine (1) and a 2-methyl-2-pentenal (2a) to give (3a) or b 2-ethylbutanal (2b) to give 3b. TfNCSs were used as reaction catalysts at 0.2 mg mL⁻¹ concentration. Reactions were performed in triplicate and standard deviations reported.

Fig. 3. Initial TfNCS-catalysed reactions between dopamine and aldehydes 4a–o.

a Reactions performed between dopamine (1) and aldehydes (4a–k). Reactions with aldehydes 4l–o were performed using 0.5 mg mL⁻¹ final concentration of Δ29TfNCS-M97V. b Conversions of reactions between dopamine (1) and benzaldehyde derivatives (4a–k). WT-TfNCS or active site mutants of TfNCS were used as the reaction catalyst with 0.2 mg mL⁻¹ enzyme. Samples were prepared by workup method 1, conversions were determined by monitoring product formation against standards (Supplementary Figs. 24–26 and Supplementary Methods) by analytical achiral HPLC (method 1). Product enantiomeric purities are given by indicating the amounts of R- and S-product generated and were determined by chiral HPLC analysis. Reactions were performed in triplicate and standard deviations reported.

Fig. 4. Conversions (determined against product standards) and enantiomeric excesses of products generated by a M97V-Δ29TfNCS-catalysed reaction between dopamine and a variety of aldehyde derivatives.

Reactions were performed using 0.5 mg mL⁻¹ final concentration of enzyme for 24 h. a Reactions between dopamine (1) and 4a-e at pH 7.5. b Reactions between dopamine (1) and 4f–k at pH 7.5. c Reactions between dopamine (1) and 4f–k at pH 6. Reactions were performed in triplicate and the error bars given are the standard deviations.

Fig. 5. Crystallographic investigations of M97V-TfNCS.

a Rationale of (6) design, a non-productive analogue of the iminium ion intermediate of the NCS-mediated reaction between 1 and 4b, to give 5b. b–d Location of 6 bound in the active site of M97V-Δ33TfNCS (PDB: 6Z82), with the mutation M97V given in blue.

Fig. 6. Examples of some patented meta-methoxylated, N-acylated 1,2,3,4-THIQs.

The compounds shown have been patented for the treatment of a range of neurodegenerative diseases.

Fig. 7. Achiral HPLC traces of the regioselective meta-O-methylation of two THIQ products (S)-5a and (S)-5b.

a Methylation of 5a to give 7a; b methylation of 5b to give 7b. In all cases, the methylation system generating SAM is used to avoid the addition of stoichiometric quantities of SAM and is used with the O-methyltransferases RnCOMT and MxSafC. The methyl donor, SAM is generated in situ by the enzyme EcMAT and the reaction byproduct, SAH is broken down by another enzyme, EcMTAN.