Variation of terpene alkaloids in Daphniphyllum macropodum across plants and tissues.

Daphniphyllum macropodum produces alkaloids that are structurally complex with polycyclic, stereochemically rich carbon skeletons. Understanding how these compounds are formed by the plant may enable exploration of their biological function and bioactivities. We employed multiple metabolomics techniques, including a workflow to annotate compounds in the absence of standards, to compare alkaloid content across plants and tissues. Different alkaloid structural types were found to have distinct distributions between genotypes, between tissues and within tissues. Alkaloid structural types also showed different isotope labelling enrichments that matched their biosynthetic relationships. The work suggests that mevalonate derived 30-carbon alkaloids are formed in the phloem region before their conversion to 22-carbon alkaloids which accumulate in the epidermis. This sets the stage for further investigation into the biosynthetic pathway.

Unspecific Peroxygenase (UPO) can be Tuned for Oxygenation or Halogenation Activity by Controlling the Reaction pH.

Unspecific Peroxygenases (UPOs) are increasingly significant enzymes for selective oxygenations as they are stable, highly active and catalyze their reactions at the expense of only hydrogen peroxide as the oxidant. Their structural similarity to chloroperoxidase (CPO) means that UPOs can also catalyze halogenation reactions based upon the generation of hypohalous acids from halide and H2O2. Here we show that the halogenation and oxygenation modes of a UPO can be stimulated at different pH values. Using simple aromatic compounds such as thymol, we show that, at a pH of 3.0 and 6.0, either brominated or oxygenated products respectively are produced. Preparative 100 mg scale transformations of substrates were performed with 60-72 % isolated yields of brominated products obtained. A one-pot bromination-oxygenation cascade reaction on 4-ethylanisole, in which the pH was adjusted from 3.0 to 6.0 at the halfway stage, yielded sequentially brominated and oxygenated products 1-(3-bromo-4-methoxyphenyl)ethyl alcohol and 3-bromo-4-methoxy acetophenone with 82 % combined conversion. These results identify UPOs as an unusual example of a biocatalyst that is tunable for entirely different chemical reactions, dependent upon the reaction conditions.

Stereoselective synthesis of an advanced trans-decalin intermediate towards the total synthesis of anthracimycin.

Progress towards the total synthesis of the macrolide natural product anthracimycin is described. This new approach utilises an intermolecular Diels-Alder strategy followed by epimeirsation to form the key trans-decalin framework. The route culminates in the stereoselective synthesis of an advanced tricyclic lactone intermediate, containing five contiguous sterogenic centres with the correct relative and absolute stereochemistry required for the anthracimycin core motif.

Cascade ring expansion reactions for the synthesis of medium-sized rings and macrocycles.

This Feature Article discusses recent advances in the development of cascade ring expansion reactions for the synthesis of medium-sized rings and macrocycles. Cascade ring expansion reactions have much potential for use in the synthesis of biologically important medium-sized rings and macrocycles, most notably as they don't require high dilution conditions, which are commonly used in established end-to-end macrocyclisation methods. Operation by cascade ring expansion method can allow large ring products to be accessed via rearrangements that proceed exclusively by normal-sized ring cyclisation steps. Ensuring that there is adequate thermodynamic driving force for ring expansion is a key challenge when designing such methods, especially for the expansion of normal-sized rings into medium-sized rings. This Article is predominantly focused on methods developed in our own laboratory, with selected works by other groups also discussed. Thermodynamic considerations, mechanism, reaction design, route planning and future perspective for this field are all covered.

Expanding the scope of the successive ring expansion strategy for macrocycle and medium-sized ring synthesis: unreactive and reactive lactams.

New methods are described that expand the scope of the Successive Ring Expansion (SuRE) with respect to synthetically challenging lactams. A protocol has been developed for use with 'unreactive' lactams, enabling SuRE reactions to be performed on subsrates that fail under previously established conditions. Ring expansion is also demonstarted on 'reactive' lactams derived from iminosugars for the first time. The new SuRE methods were used to prepare a diverse array of medium-sized and macrocyclic lactams and lactones, which were evaluted in an anti-bacterial assay against E. coli BW25113WT.

The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

A Modular Strategy for the Synthesis of Macrocycles and Medium-Sized Rings via Cyclization/Ring Expansion Cascade Reactions.

Macrocycles and medium-sized rings are important in many scientific fields and technologies but are hard to make using current methods, especially on a large scale. Outlined herein is a strategy by which functionalized macrocycles and medium-sized rings can be prepared using cyclization/ring expansion (CRE) cascade reactions, without resorting to high dilution conditions. CRE cascade reactions are designed to operate exclusively via kinetically favorable 5-7-membered ring cyclization steps; this means that the problems typically associated with classical end-to-end macrocyclization reactions are avoided. A modular synthetic approach has been developed to facilitate the simple assembly of the requisite linear precursors, which can then be converted into an extremely broad range of functionalized macrocycles and medium-sized rings using one of nine CRE protocols.

Divergent Cascade Ring-Expansion Reactions of Acryloyl Imides.

Macrocyclic and medium-sized ring ketones, lactones and lactams can all be made from common acryloyl imide starting materials through divergent, one-pot cascade ring-expansion reactions. Following either conjugate addition with an amine or nitromethane, or osmium(VIII)-catalysed dihydoxylation, rearrangement through a four-atom ring expansion takes place spontaneously to form the ring expanded products. A second ring expansion can also be performed following a second iteration of imide formation and alkene functionalisation/ring expansion. In the dihydroxylation series, three- or four-atom ring expansion can be performed selectively, depending on whether the reaction is under kinetic or thermodynamic control.

Ring expansion reactions of PO-containing molecules.

A series of ring expansion reactions of PO-containing molecules have been developed for the synthesis of medium-sized ring cyclic phosphonate esters and phosphonamidates. The reactivity trends initially appear to be counter-intuitive, compared with more well established ring expansion reactions of lactam derivatives, but are explained by considering the differences in heteroatom bonding to P and C respectively.

Ring Expansion Strategies for the Synthesis of Medium Sized Ring and Macrocyclic Sulfonamides.

Two new ring expansion strategies are reported for the synthesis of medium sized ring and macrocyclic sulfonamides. Both methods can be performed without using classical protecting groups, with the key ring expansion step initiated by nitro reduction and amine conjugate addition respectively. Each method can be used to make diversely functionalised cyclic sulfonamides in good to excellent yields, in a range of ring sizes. The ring size dependency of the synthetic reactions is in good agreement with the outcomes modelled by Density Functional Theory calculations.

Preparative-Scale Biocatalytic Oxygenation of N-Heterocycles with a Lyophilized Peroxygenase Catalyst.

A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is a highly effective catalyst for the oxygenation of a diverse range of N-heterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ca. 100 mg scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic N-heterocycles and indoles. H2 O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co-substrate. Good synthetic yields (up to 84 %), regioselectivity and enantioselectivity (up to 99 % ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts.

Comparing the Catalytic and Structural Characteristics of a 'Short' Unspecific Peroxygenase (UPO) Expressed in Pichia†pastoris and Escherichia†coli.

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an 'artificial' peroxygenase (artUPO), close in sequence to the 'short' UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the 'long' UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short' UPOs.

Visible-Light-Mediated Energy Transfer Enables the Synthesis of ß-Lactams via Intramolecular Hydrogen Atom Transfer.

The synthesis of 2-azetidinones (ß-lactams) from simple acrylamide starting materials by visible-light-mediated energy transfer catalysis is reported. The reaction features a C(sp3 )-H functionalization via a variation of the Norrish-Yang photocyclization involving a carbon-to-carbon 1,5-hydrogen atom transfer (supported by deuterium labelling and DFT calculations) and can be used for the construction of a diverse range of ß-lactam products.

Selectivity, Speciation, and Substrate Control in the Gold-Catalyzed Coupling of Indoles and Alkynes.

A convenient and mild protocol for the gold-catalyzed intermolecular coupling of substituted indoles with carbonyl-functionalized alkynes to give vinyl indoles is reported. This reaction affords 3-substituted indoles in high yield, and in contrast to the analogous reactions with simple alkynes which give bisindolemethanes, only a single indole is added to the alkyne. The protocol is robust and tolerates substitution at a range of positions of the indole and the use of ester-, amide-, and ketone-substituted alkynes. The use of 3-substituted indoles as substrates results in the introduction of the vinyl substituent at the 2-position of the ring. A combined experimental and computational mechanistic study has revealed that the gold catalyst has a greater affinity to the indole than the alkyne, despite the carbon-carbon bond formation step proceeding through an η2(π)-alkyne complex, which helps to explain the stark differences between the intra- and intermolecular variants of the reaction. This study also demonstrated that the addition of a second indole to the carbonyl-containing vinyl indole products is both kinetically and thermodynamically less favored than in the case of more simple alkynes, providing an explanation for the observed selectivity. Finally, a highly unusual gold-promoted alkyne dimerization reaction to form a substituted gold pyrylium salt has been identified and studied in detail.

Synthesis of medium-ring lactams and macrocyclic peptide mimetics via conjugate addition/ring expansion cascade reactions.

A novel conjugate addition/ring expansion (CARE) cascade reaction sequence is reported that enables medium-sized ring and macrocyclic bis-lactams to be prepared from primary amines and cyclic imides. The reactions are simple to perform, generally high yielding, and very broad in scope, especially with respect to the primary amine component. CARE reactions can also be performed iteratively, enabling ß-peptoid-based macrocyclic peptide mimetics to be 'grown' via well controlled, sequential 4-atom ring expansion reactions, with the incorporation of varied functionalised amines during each iteration.

Indole-ynones as Privileged Substrates for Radical Dearomatizing Spirocyclization Cascades.

Indole-ynones have been established as general substrates for radical dearomatizing spirocyclization cascade reactions. Five distinct and varied synthetic protocols have been developed─cyanomethylation, sulfonylation, trifluoromethylation, stannylation and borylation─using a variety of radical generation modes, ranging from photoredox catalysis to traditional AIBN methods. The simple and easily prepared indole-ynones can be used to rapidly generate diverse, densely functionalized spirocycles and have the potential to become routinely used to explore radical reactivity. Experimental and computational investigations support the proposed radical cascade mechanism and suggest that other new methods are now primed for development.

A Thiol-Mediated Three-Step Ring Expansion Cascade for the Conversion of Indoles into Functionalized Quinolines.

An operationally simple, high yielding three-step cascade process is described for the direct conversion of indole-tethered ynones into functionalized quinolines. A single "multitasking" thiol reagent is used to promote a three-step dearomatizing spirocyclization, nucleophilic substitution, and one-atom ring expansion reaction cascade under remarkably mild conditions. In addition, a novel route to thio-oxindoles is described, which was discovered by serendipity.

Synthesis of macrocyclic and medium-sized ring thiolactones via the ring expansion of lactams.

A side chain insertion method for the ring expansion of lactams into macrocyclic thiolactones is reported, that can also be incorporated into Successive Ring Expansion (SuRE) sequences. The reactions are less thermodynamically favourable than the analogous lactam- and lactone-forming ring expansion processes (with this notion supported by DFT data), but nonetheless, three complementary protecting group strategies have been developed to enable this challenging transformation to be achieved.

Phosphoranyl Radical Fragmentation Reactions Driven by Photoredox Catalysis.

Photocatalytic generation of phosphoranyl radicals is fast emerging as an essential method for the generation of diverse and valuable radicals, typically via deoxygenation or desulfurization processes. This Perspective is a comprehensive evaluation of all studies using phosphoranyl radicals as tunable mediators in photoredox catalysis, highlighting how two distinct methods for phosphoranyl radical formation (radical addition and nucleophilic addition) can be used to generate versatile radical intermediates with diverse reactivity profiles.