One-Pot Multienzyme Cascades for Stereodivergent Synthesis of Tetrahydroquinolines.

The tetrahydroquinoline (THQ) framework is commonly found in natural products and pharmaceutically relevant molecules. Apart from the use of transition metal catalysts and chiral phosphoric acids, the chiral 2-substituted 1,2,3,4-THQs are synthesized using amine oxidase biocatalysts. However, the use of imine reductases (IREDs) in their asymmetric synthesis remained unexplored. In the current work, IREDs are employed in telescopic multienzyme cascades to catalyze the intramolecular reductive amination leading to chiral 2-alkyl and 2-aryl substituted-1,2,3,4-tetrahydroquinolines starting from inexpensive nitroalkenones. The cascades containing NtDBR (an ene reductase), NfsB (a nitro reductase) with either Na2S2O4 or V2O5, various IREDs, and glucose dehydrogenase (for NADPH regeneration) are used to synthesize a broad range of (R)/(S)-2-alkyl-substituted (THQs) (26 examples) with high yield (up to 93%) and excellent ee (up to 99%) in one-pot. The method further facilitates the one-pot biocatalytic synthesis of chiral 2-aryl substituted THQs (26 examples) from amino chalcones. Lastly, the asymmetric synthesis of several (R)- and (S)-THQ based intermediates of Hancock alkaloids showed the practical application of the newly developed biocatalytic cascades.

Anthrol reductases: discovery, role in biosynthesis and applications in natural product syntheses.

Covering: up to 2023Short-chain dehydrogenase/reductases (SDR) are known to catalyze the regio- and stereoselective reduction of a variety of substrate types. Investigations of the deoxygenation of emodin to chrysophanol has led to the discovery of the anthrol reductase activity of an SDR, MdpC involved in monodictyphenone biosynthesis of Aspergillus nidulans and provided access to (R)-dihydroanthracenone, a putative biosynthetic intermediate. This facilitated the identification of several MdpC-related enzymes involved in the biosynthesis of aflatoxins B1, cladofulvin, neosartorin, agnestins and bisanthraquinones. Because of their ability to catalyze the reduction of hydroanthraquinone (anthrols) using NADPH, they were named anthrol reductases. This review provides a comprehensive summary of all the anthrol reductases that have been identified and characterized in the last decade along with their role in the biosynthesis of natural products. In addition, the applications of these enzymes towards the chemoenzymatic synthesis of flavoskyrins, modified bisanthraquinones, 3-deoxy anthraquinones, chiral cycloketones and ß-halohydrins have been discussed.

Asymmetric synthesis of (+)-teratosphaerone B, its non-natural analogue and (+)-xylarenone using an ene- and naphthol reductase cascade.

Herein, a one-pot bienzymatic cascade containing an ene and a naphthol reductase is developed. It is applied for the synthesis of (+)-(3R,4R)-teratosphaerone B, its non-natural regioisomer in both cis- and trans-forms and (+)-xylarenone by the reduction of chemically synthesized naphthoquinone precursors in high yields (76-92%) and excellent ee (>99%). This work implies similar biosynthetic steps in the formation of the synthesized natural products.

Synthesis of rhein and diacerein: a chemoenzymatic approach using anthrol reductase of Talaromyces islandicus.

Herein, we report two methods for the synthesis of the osteoarthritis drug rhein and its prodrug diacerein using a chemoenzymatic approach. The strategy relies on the use of an NADPH-dependent anthrol reductase of Talaromyces islandicus (ARti-2), which mediates the regioselective and reductive deoxygenation of anthraquinones. The work further implies similar biosynthesis of rhein in fungi.

Chemoenzymatic total synthesis of nodulones C and D using a naphthol reductase of Magnaporthe grisea.

Herein, the asymmetric and chemoenzymatic synthesis of (R)-nodulone C, cis-nodulone D and related (R)-dihydronaphthalenone is reported. It involves multistep chemical synthesis of putative biosynthetic substrates followed by regio- and stereoselective reduction using a NADPH-dependent tetrahydroxynaphthalene reductase of Magnaporthe grisea to obtain chiral nodulones in a biomimetic fashion.

Insights into the Role of Ketoreductases in the Biosynthesis of Partially Reduced Bacterial Aromatic Polyketides.

Partially reduced aromatic polyketides are bioactive secondary metabolites or intermediates in the biosynthesis of deoxygenated aromatics. For the antibiotic GTRI-02 (mensalone) in different Streptomyces spp., biosynthesis involving the reduction of a fully aromatized acetyltrihydroxynaphthalene by a naphthol reductase has been proposed and shown in vitro with a fungal enzyme. However, more recently, GTRI-02 has been identified as a product of the ActIII biosynthetic gene cluster from Streptomyces coelicolor A3(2), for which the reduction of a linear polyketide precursor by ActIII ketoreductase, prior to cyclization and aromatization, has been suggested. We have examined three different ketoreductases from bacterial producer strains of GTRI-02 for their ability to reduce mono-, bi-, and tricyclic aromatic substrates. The enzymes reduced 1- and 2-tetralone but not other aromatic substrates. This strongly suggests a reduction of a cyclized but not yet aromatic polyketide intermediate in the biosynthesis of GTRI-02. Implications of the results for the biosynthesis of other secondary polyketidic metabolites are discussed.

Chemoenzymatic reduction of citreorosein and its implications on aloe-emodin and rugulosin C (bio)synthesis.

A chemoenzymatic reduction of citreorosein by the NADPH-dependent polyhydroxyanthracene reductase from Cochliobolus lunatus or MdpC from Aspergillus nidulans in the presence of Na2S2O4 gave access to putative biosynthetic intermediates, (R)-3,8,9,10-tetrahydroxy-6-(hydroxymethyl)-3,4-dihydroanthracene-1(2H)-one and its oxidized form, (R)-3,4-dihydrocitreorosein. Herein, we discuss the implications of these results towards the (bio)synthesis of aloe-emodin and (+)-rugulosin C in fungi.

A Redox-Based Superoxide Generation System Using Quinone/Quinone Reductase.

Superoxide (O2.- ) generation in biological systems is achieved through some of the most complex enzymatic systems. Of these, only xanthine/xanthine oxidase has been used for in vitro biochemical studies. However, it suffers from limitations such as a lack of suitable heterologous expression system for xanthine oxidase and the irreversible consumption and low solubility of xanthine under physiological conditions. Herein, we report a redox-based, enzyme-catalyzed system, in which autoxidation of hydroquinone to quinone via semiquinone results in superoxide generation. Quinone is reduced back to hydroquinone by using the NfsB (oxygen-insensitive nitroreductase) enzyme of Escherichia coli strain K-12 and nicotinamide adenine dinucleotide phosphate hydride (NADPH; which is regenerated by using the glucose/glucose dehydrogenase system). This new system relies on quinones that can be recycled and have superior water solubility, as well as enzymes that are heterologously expressed. By using a variety of quinones and reaction conditions, along with a comparison of real-time fluorescence, menadione has been identified as the optimal substrate for superoxide generation. The new redox-based system presents a viable alternative for studying the biochemistry of superoxide under different physiological and pathological conditions.

Monomeric Dihydroanthraquinones: A Chemoenzymatic Approach and its (Bio)synthetic Implications for Bisanthraquinones.

Modified bisanthraquinones are complex dimeric natural products containing a cage-like structural motif. For their biosynthesis, monomeric dihydroanthraquinones have been proposed as key intermediates despite not being isolated from natural sources or synthesized as of yet. Here, isolation and characterization of dihydroemodin, as well as dihydrolunatin, synthesized by a biomimetic and chemoenzymatic approach using NADPH-dependent polyhydroxyanthracence reductase (PHAR) from Cochliobolus lunatus followed by Pb(OAc)4 oxidation is reported. Subsequent dimerization through a hetero-Diels-Alder reaction of the dihydroemodin and dihydrolunatin resulted in (-)-flavoskyrin (68 %) and (-)-lunaskyrin (62 %), respectively. Pyridine treatment of (-)-flavoskyrin and (-)-lunaskyrin gave (-)-rugulosin and (-)-2,2'-epi-cytoskyrin A in 64 % and 60 % yield, respectively, through a cascade that involves two dimeric intermediates. Implications of the described synthesis for the biosynthesis of bisanthraquinones by a combination of enzymatic and spontaneous steps are discussed.

Phylogenetic Studies, Gene Cluster Analysis, and Enzymatic Reaction Support Anthrahydroquinone Reduction as the Physiological Function of Fungal 17ß-Hydroxysteroid Dehydrogenase.

17ß-Hydroxysteroid dehydrogenase (17ß-HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)-dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17ß-HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17ß-HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17ß-HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17ß-HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17ß-HSDcl in the biosynthesis of other anthrahydroquinone-derived natural products is proposed; hence, 17ß-HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR).

Unprecedented role of hydronaphthoquinone tautomers in biosynthesis.

Quinones and hydroquinones are among the most common cellular cofactors, redox mediators, and natural products. Here, we report on the reduction of 2-hydroxynaphthoquinones to the stable 1,4-diketo tautomeric form of hydronaphthoquinones and their further reduction by fungal tetrahydroxynaphthalene reductase. The very high diastereomeric and enantiomeric excess, together with the high yield of cis-3,4-dihydroxy-1-tetralone, exclude an intermediary hydronaphthoquinone. Labeling experiments with NADPH and NADPD corroborated the formation of an unexpected 1,4-diketo tautomeric form of 2-hydroxyhydronaphthoquinone as a stable intermediate. Similar 1,4-diketo tautomers of hydronaphthoquinones were established as products of the NADPH-dependent enzymatic reduction of other 1,4-naphthoquinones, and as substrates for different members of the superfamily of short-chain dehydrogenases. We propose an essential role of hydroquinone diketo tautomers in biosynthesis and detoxification processes.

Tautomers of anthrahydroquinones: enzymatic reduction and implications for chrysophanol, monodictyphenone, and related xanthone biosyntheses.

Reduction of emodin by sodium dithionite resulted in the formation of two tautomeric forms of emodin hydroquinone. Subsequent conversion by the short-chain dehydrogenase/reductase (SDR) MdpC into the corresponding 3-hydroxy-3,4-dihydroanthracen-1(2H)-one implies that deoxygenation is the first step in monodictyphenone biosynthesis. Implications for chrysophanol formation as well as reaction sequences in the related xanthone, ergochrome, and bianthraquinone biosyntheses are discussed.

Tetrahydroxynaphthalene reductase: catalytic properties of an enzyme involved in reductive asymmetric naphthol dearomatization.

In reduced circumstances: tetrahydroxynaphthalene reductase shows a broad substrate range including alternate phenolic compounds and cyclic ketones. Structural modeling reveals major enzyme-substrate interactions; C-terminal truncation of the enzyme causes an altered substrate preference, in accordance with stabilization of the substrate by the C-terminal carboxylate. This effect allows the identification of a homologous enzyme.

Synthesis of (-)-Flavoskyrins by Catalyst-Free Oxidation of (R)-Configured Dihydroanthracenones in Aqueous Media and Its (Bio)synthetic Implications.

A catalyst-free method for the synthesis of dimeric (-)-flavoskyrins has been developed. It involves the autoxidation of chemoenzymatically synthesized (R)-configured dihydroanthracenones in the presence of molecular oxygen in buffer of pH 6.0 followed by spontaneous [4 + 2] cycloaddition in stereocontrolled exo-anti fashion to form (-)-flavoskyrins. The method is applied to obtain several homo- as well as heterodimerized flavoskyrins (nine examples) in 27-72% yield and implies the involvement of a similar pathway in the (bio)synthesis of modified bisanthraquinones and their analogues.

Chemoenzymatic, biomimetic total synthesis of (-)-rugulosin B, C and rugulin analogues and their biosynthetic implications.

Herein, we report the chemoenzymatic synthesis of a heterodimeric (-)-rugulosin B, homodimeric (-)-rugulosin C, and several rugulin analogues in three to four steps starting from anthraquinones. This work supports dimerization between variously substituted putative monomeric intermediates during the biosynthesis of naturally occurring (+)-rugulosin B and C.

Asymmetric synthesis of enantiomerically pure 2-substituted tetrahydro-3-benzazepines and their affinity to sigma1 receptors.

A very short asymmetric synthesis of enantiomerically pure 2-substituted tetrahydro-3-benzazepines is described. First, 3-phenyl-2,3,11,11a-tetrahydro[1,3]oxazolo[2,3-b][3]benzazepin-5(6H)-ones 3a-d and 4a-d were synthesized by condensation of 2-(2-oxoalkyl)phenylacetic acids 1a-d with (R)-phenylglycinol (2). With the exception of the 11a-phenyl derivatives 3d/4d (ratio 91:9), the ratio of the diastereomeric 11a-alkyl derivatives 3a-c/4a-c was almost 50:50. The configuration of the newly formed chiral center in position 11a was proved by NOE experiments as well as X-ray crystal structure analysis. The reduction of the oxazolo[2,3-b][3]benzazepin-5(6H)-ones trans-3 and cis-4 with AlCl(3)/LiAlH(4) (1:3) took place with retention of configuration and yielded 2,3-disubsituted tetrahydro-3-benzazepines 10 and11. In the final step, removal of the N-(2-hydroxy-1-phenylethyl) residue from 10 and 11 by hydrogenolysis provided four pairs of enantiomerically pure 2-substituted tetrahydro-3-benzazepines 12a-d and ent-12a-d, which were tested for their sigma(1), sigma(2), and NMDA receptor affinities. The 2-butyl and the 2-phenyl derivatives 12c and 12d show very high sigma(1) affinity with K(i) values of 16 and 50 nM, respectively. The eudismic ratios are greater than 50, reflecting highly stereoselective interaction with the sigma(1) receptor. Both sigma(1) ligands are very selective against the sigma(2) subtype and the PCP binding site of the NMDA receptor.

Asymmetric Synthesis of Natural cis-Dihydroarenediols Using Tetrahydroxynaphthalene Reductase and Its Biosynthetic Implications.

Asymmetric reduction of hydroxynaphthoquinones to secondary metabolites, (3 S,4 R)-3,4,8- and (2 S,4 R)-2,4,8-trihydroxy-1-tetralone, a putative biosynthetic diketo intermediate and a probable natural analogue, (3 S,4 R)-7-acetyl-3,4,8-trihydroxy-6-methyl-3,4-dihydronaphthalene-1(2 H)-one, using NADPH-dependent tetrahydroxynaphthalene reductase (T4HNR) of Magnaporthe grisea is described. This work implies the involvement of T4HNR or related enzymes during the (bio)synthesis of other dihydroarenediols by reduction of the hydroxynaphthoquinone scaffold containing substrates.

Biomimetic asymmetric synthesis of (R)-GTRI-02 and (3S,4R)-3,4-dihydroxy-3,4-dihydronaphthalen-1(2H)-ones.

The NADPH-dependent tetrahydroxynaphthalene reductase (T4HNR) from Magnaporthe grisea was used for the biomimetic synthesis of (R)-GTRI-02 by stereoselective reduction of 1-(3,6,8-trihydroxy-1-methylnaphthalen-2-yl)ethanone. This also led to the isolation of a (3S,4R)-cis-ketodiol formed by T4HNR-catalyzed reduction of the corresponding hydroxynaphthoquinone. Flaviolin and lawsone also reduced to corresponding cis-ketodiols in good yields.