The Discovery of Imine Reductases and their Utilisation for the Synthesis of Tetrahydroisoquinolines.

Imine reductases (IREDs) are NADPH-dependent enzymes with significant biocatalytic potential for the synthesis of primary, secondary, and tertiary chiral amines. Their applications include the reduction of cyclic imines and the reductive amination of prochiral ketones. In this study, twenty-nine novel IREDs were revealed through genome mining. Imine reductase activities were screened at pH 7 and 9 and in presence of either NADPH or NADH; some IREDs showed good activities at both pHs and were able to accept both cofactors. IREDs with Asn and Glu at the key 187 residue showed preference for NADH. IREDs were also screened against a series of dihydroisoquinolines to synthesise tetrahydroisoquinolines (THIQs), bioactive alkaloids with a wide range of therapeutic properties. Selected IREDs showed high stereoselectivity, as well high THIQ yields (>90 %) when coupled to a glucose-6-phosphate dehydrogenase for NADPH cofactor recycling.

Chemoenzymatic approaches to plant natural product inspired compounds.

Covering: 2003 up to the end of 2021Complex molecules produced by plants have provided us with a range of medicines, flavour and fragrance compounds and pesticides. However, there are challenges associated with accessing these in an economically viable manner, including low natural abundance and the requirement for complex multi-step synthetic strategies. Chemoenzymatic approaches provide a valuable alternative strategy by combining traditional synthetic methods with biocatalysis. This review highlights recent chemoenzymatic syntheses towards plant natural products and analogues, focusing on the advantages of incorporating biocatalysts into a synthetic strategy.

Chemoenzymatic Cascades toward Methylated Tetrahydroprotoberberine and Protoberberine Alkaloids.

Tetrahydroprotoberberine and protoberberine alkaloids are a group of biologically active natural products with complex molecular scaffolds. Isolation from plants is challenging and stereoselective synthetic routes, particularly of methylated compounds are limited, reducing the potential use of these compounds. In this work, we describe chemoenzymatic cascades toward various 13-methyl-tetrahydroprotoberberbine scaffolds using a stereoselective Pictet-Spenglerase, regioselective catechol O-methyltransferases and selective chemical Pictet-Spengler reactions. All reactions could be performed sequentially, without the workup or purification of any synthetic intermediates. Moreover, the naturally occurring alkaloids have the (+)-configuration and importantly here, a strategy to the (-)-isomers was developed. A methyl group at C-8 was also introduced with some stereocontrol, influenced by the stereochemistry at C-13. Furthermore, a single step reaction was found to convert tetrahydroprotoberberine alkaloids into the analogous protoberberine scaffold, avoiding the use of harsh oxidizing conditions or a selective oxidase. This work provides facile, selective routes toward novel analogues of bioactive alkaloids.

Multienzyme One-Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids.

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S-Adenosylmethionine (SAM)-dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi-enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non-natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

Multienzyme One-Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids.

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S-Adenosylmethionine (SAM)-dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi-enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non-natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

Pictet-Spenglerases in alkaloid biosynthesis: Future applications in biocatalysis.

Pictet-Spenglerases provide a key role in the biosynthesis of many biologically active alkaloids. There is increasing use of these biocatalysts as an alternative to traditional organic synthetic methods as they provide stereoselective and regioselective control under mild conditions. Products from these enzymes also contain privileged drug scaffolds (such as tetrahydroisoquinoline or ß-carboline moieties), so there is interest in the characterization and use of these enzymes as versatile biocatalysts to synthesize analogs of the corresponding natural products for drug discovery. This review discusses all known Pictet-Spenglerase enzymes and their applications as biocatalysts.

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

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.