An NmrA-like enzyme-catalysed redox-mediated Diels–Alder cycloaddition with anti-selectivity

Peroxidase-induced C–N bond formation via nitroso ene and Diels–Alder reactions

“…After the successful development of an enzyme-driven methodology that works both in intra- and intermolecular nitroso ene-type reactions, we turned our attention to the second reactivity pattern of the NO derivatives, that is, their ability to act as dienophiles in hetero Diels–Alder reactions. Diels–Alder cycloadditions have for a long time been a topic of interest in enzyme catalysis development and over the years, both native Diels–Alderases 20 and artificial biocatalytic tools for [4 + 2]-cycloadditions have been discovered. 21 We used 1,3-cyclohexadiene ( 10 ) as benchmark reaction partner and assumed, considering the similarities to cyclohexene, that the emulsified medium would also offer advantages in the [4 + 2]-cycloaddition.…”

Fungal P450 Deconstructs the 2,5-Diazabicyclo[2.2.2]octane Ring En Route to the Complete Biosynthesis of 21R-Citrinadin A

“…21 R -Citrinadin A ( 10 ) is one of the main metabolites discovered inP. citrinumATCC 9849, and its putative BGC ctd contains 21 genes based on previous studies and bioinformatic analysis (Figure A). , To pinpoint genes related to the deconstruction of 2,5-diazabicyclo[2.2.2]­octane, we individually knocked out uncharacterized genes in the ctd gene cluster that might have a putative redox function, including four P450-encoding genes ( ctdT , ctdF , ctdG , and ctdY ), two α -ketoglutarate ( α -KG)-dependent oxygenase-encoding genes ( ctdV and ctdJ ), and one NmrA-like enzyme-encoding gene ( ctdR ) by the split marker recombination approach (Figure S4). As revealed by liquid chromatography–mass spectrometry (LC–MS), Δ ctdT and Δ ctdF mutants still produce 10 (Figure B­(vii,viii)), indicating that CtdT and CtdF are not involved in the biosynthesis of 10 , while Δ ctdV , Δ ctdJ , Δ ctdG , Δ ctdR , and Δ ctdY mutants completely abolished the production of 10 (Figures B and S5A,B).…”

“…Furthermore, the ΔctdR mutant generated an iminium intermediate 19 identified in previous studies (Figure 2B-B(v),C). 15 All intermediates purified from the above mutants still have the 6/5/5/6/6 pentacyclic scaffold, indicating there is another enzyme responsible for the deconstruction of the S6A), indicating the loss of one carbon atom and one oxygen atom compared with 20, CtdY might remove a carbonyl group to produce a putative iminium cation intermediate 21 (Figure 3B). However, the structure of 21 is difficult to be characterized by NMR data due to its instability.…”

“…Followed by the reverse prenylation of the indole moiety in the cyclodipeptide precursor, the azadiene moiety and the dienophile isoprene undergo an intramolecular [4 + 2] hetero-Diels–Alder (DA) reaction to construct the 2,5-diazabicyclo[2.2.2]­octane system . Recently, several different classes of Diels-Alderases (DAases) were reported to facilitate the DA reaction and strictly control the specific stereochemistry of bridged bicyclic adducts. − In addition to the PIAs containing the 2,5-diazabicyclo[2.2.2]­octane ring structure, another distinct class of fungal PIAs including cyclopiamine A ( 7 ), citrinalin A ( 8 ), PF1270 A ( 9 ), and citrinadin A ( 10 ) possess unique 6/6/5/5/6/5 hexacyclic or 6/5/5/6/6 pentacyclic spiroindolinone scaffolds (Figure A). − …”

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“…The enzyme CtdP, a bifunctional oxidoreductase-Diels Alderase, responsible for the α- anti -selective cyclisation to generate the bicyclo[2.2.2]diazaoctane scaffold of (+)-precitrinadin A 30 , a prenylated indole alkaloid from Penicillium citrinum ATCC 9849, has been characterised. 28 Computational studies revealed that CtdP promotes an oxidation to an electron-deficient diene that then undergoes an inverse electron demand Diels–Alder reaction. Directed evolution of the iron( ii )- and α-ketoglutarate-dependent dioxygenase SadX has generated a biocatalyst capable of selective C–H functionalisation of amino acids.…”