Norcoclaurine synthase: structural studies, enzyme engineering and the biocatalytic synthesis of novel alkaloids

Norcoclaurine synthase (NCS) is a biocatalyst, involved in plant alkaloid biosynthesis. In plants, NCS catalyses a stereoselective Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA), to give (S)-norcoclaurine, the first committed intermediate in benzylisoquinoline alkaloid (BIA) biosynthesis. Previous work has shown that a range of aldehydes and ketones are accepted by NCS, in place of 4-HPAA, to generate single isomer tetrahydroisoquinolines (THIQs). The THIQ moiety is found in many biologically active molecules and NCS provides a facile route towards novel analogues. The aim of this project was to rationalise and extend the substrate scope of NCS, in particular towards a-substituted aldehydes. Despite previous reports, a range of a-methyl substituted aldehydes were shown to be accepted by NCS. A kinetic resolution of the aldehyde was also observed, with the (R)-enantiomer preferentially accepted, leading to THIQ products with two well-defined stereocentres. Single point variants of NCS were shown to improve activities compared with the wild-type enzyme. One variant, M97V was particularly promising and lead to the acceptance of the benzaldehydes as substrates and so the single step syntheses of various (1S)-aryl-THIQs was achieved. To expand the amine substrate scope of NCS, N-methyl-phenylamines were also explored as substrates. Chemoenzymatic cascades were developed to increase the molecular complexity of the NCS-generated THIQs and crystallographic investigations, involving the co-crystallisation of reaction intermediate mimics helped to rationalise NCS activities. Routes towards (R)-THIQs were also explored, involving screening novel variants, NCSs from Nelumbo nucifera and another Pictet-Spenglerase, salsolinol synthase

Chemoenzymatic approaches to plant natural product inspired compounds

Complex 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

C-1 Substituted isoquinolines potentiate the antimycobacterial activity of rifampicin and ethambutol

IntroductionThe emergence of extensively drug-resistant strains of Mycobacterium tuberculosis threatens decades of progress in the treatment of a disease which remains one of the leading infectious causes of death worldwide. The development of novel antimycobacterial compounds is therefore essential to reinforce the existing antitubercular drug discovery pipeline. There is also interest in new compounds which can synergize with existing antitubercular drugs and can be deployed as part of a combination therapy. This strategy could serve to delay the emergence of resistance to first-line anti-tuberculosis drugs and increase their efficacy against resistant strains of tuberculosis. Previous research has established that several C-1 substituted tetrahydroisoquinolines have antimycobacterial activity. Here we sought to expand our understanding of their antimycobacterial structure activity relationships and their potential to act as adjunct therapies alongside existing antitubercular drugs.MethodsThree chemical series were synthesised and assayed for their antimycobacterial potency, mammalian cell toxicity, inhibition of whole-cell efflux and synergism with isoniazid, rifampicin, and ethambutol.ResultsSeveral compounds were found to inhibit the growth of mycobacteria. Potent inhibitors of whole-cell efflux were also identified, as well as compounds which exhibited synergism with rifampicin and ethambutol.ConclusionsStructure-activity relationships were identified for antimycobacterial potency, improved selectivity, whole cell efflux inhibition and synergism. Potent whole-cell efflux inhibitors and synergistic compounds were identified, suggesting potential development as adjuncts to existing anti-tuberculosis chemotherapy

C-1 Substituted isoquinolines potentiate the antimycobacterial activity of rifampicin and ethambutol

Introduction: The emergence of extensively drug-resistant strains of Mycobacterium tuberculosis threatens decades of progress in the treatment of a disease which remains one of the leading infectious causes of death worldwide. The development of novel antimycobacterial compounds is therefore essential to reinforce the existing antitubercular drug discovery pipeline. There is also interest in new compounds which can synergize with existing antitubercular drugs and can be deployed as part of a combination therapy. This strategy could serve to delay the emergence of resistance to first-line anti-tuberculosis drugs and increase their efficacy against resistant strains of tuberculosis. Previous research has established that several C-1 substituted tetrahydroisoquinolines have antimycobacterial activity. Here we sought to expand our understanding of their antimycobacterial structure activity relationships and their potential to act as adjunct therapies alongside existing antitubercular drugs./ Methods: Three chemical series were synthesised and assayed for their antimycobacterial potency, mammalian cell toxicity, inhibition of whole-cell efflux and synergism with isoniazid, rifampicin, and ethambutol. Results: Several compounds were found to inhibit the growth of mycobacteria. Potent inhibitors of whole-cell efflux were also identified, as well as compounds which exhibited synergism with rifampicin and ethambutol./ Conclusions: Structure-activity relationships were identified for antimycobacterial potency, improved selectivity, whole cell efflux inhibition and synergism. Potent whole-cell efflux inhibitors and synergistic compounds were identified, suggesting potential development as adjuncts to existing anti-tuberculosis chemotherapy.

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 NAD

The acceptance and kinetic resolution of alpha-Methyl Substituted Aldehydes by Norcoclaurine Synthase

Norcoclaurine synthase (NCS) catalyzes a stereoselective Pictet-Spengler reaction to give the key intermediate, (S)-norcoclaurine in benzylisoquinoline alkaloid (BIA) biosynthesis. This family of alkaloids contains many bioactive molecules including morphine and berberine. Recently, NCS has been demonstrated to accept a variety of aldehydes and some ketones as substrates, leading to a range of chiral tetrahydroisoquinoline (THIQ) products. Here, we report the unusual acceptance of a-substituted aldehydes, in particular a-methyl substituted aldehydes, by wild-type Thalictrum flavum NCS (Δ33TfNCS) to give THIQ products. Moreover, the kinetic resolution of several a-substituted aldehydes to give THIQs with two defined chiral centers in a single step with high conversions was achieved. Several dopamine analogues were also accepted as substrates and reactions were amenable to scale-up. Active site mutants of TfNCS were then used which demonstrated the potential to enhance the stereoselectivities in the reaction and improve yields. Rationale for the acceptance of these substrates and improved activity with different mutants has been gained from a co-crystallized structure of Δ33TfNCS with a non-productive mimic of a reaction intermediate bound in the active site. Finally, molecular dynamics simulations were performed to study the binding of dopamine and an a-substituted aldehyde and provided further insight into the reaction with these substrates

Single step syntheses of (1 S)-aryltetrahydroisoquinolines 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 cocrystallised 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)-THIQ

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 offers 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

The acceptance and kinetic resolution of alpha-Methyl Substituted Aldehydes by Norcoclaurine Synthase

Norcoclaurine synthase (NCS) catalyzes a stereoselective Pictet-Spengler reaction to give the key intermediate, (S)-norcoclaurine in benzylisoquinoline alkaloid (BIA) biosynthesis. This family of alkaloids contains many bioactive molecules including morphine and berberine. Recently, NCS has been demonstrated to accept a variety of aldehydes and some ketones as substrates, leading to a range of chiral tetrahydroisoquinoline (THIQ) products. Here, we report the unusual acceptance of a-substituted aldehydes, in particular a-methyl substituted aldehydes, by wild-type Thalictrum flavum NCS (Δ33TfNCS) to give THIQ products. Moreover, the kinetic resolution of several a-substituted aldehydes to give THIQs with two defined chiral centers in a single step with high conversions was achieved. Several dopamine analogues were also accepted as substrates and reactions were amenable to scale-up. Active site mutants of TfNCS were then used which demonstrated the potential to enhance the stereoselectivities in the reaction and improve yields. Rationale for the acceptance of these substrates and improved activity with different mutants has been gained from a co-crystallized structure of Δ33TfNCS with a non-productive mimic of a reaction intermediate bound in the active site. Finally, molecular dynamics simulations were performed to study the binding of dopamine and an a-substituted aldehyde and provided further insight into the reaction with these substrates