What can a chemist learn from nature’s macrocycles? – A brief, conceptual view

Macrocyclic natural products often display remarkable biological activities, and many of these compounds (or their derivatives) are used as drugs. The chemical diversity of these compounds is immense and may provide inspiration for innovative drug design. Therefore, a database of naturally occurring macrocycles was analyzed for ring size, molecular weight distribution, and the frequency of some common substructural motifs. The underlying principles of the chemical diversity are reviewed in terms of biosynthetic origin and natures strategies for diversity and complexity generation in relation to the structural diversity and similarities found in the macrocycle database. Finally, it is suggested that synthetic chemists should use not only natures molecules, but also natures strategies as a source of inspiration. To illustrate this, the biosynthesis of macrocycles by non-ribosomal peptide synthetases and terpene and polyketide cyclases, as well as recent advances of these strategies in an integrated synthesis/biotechnology approach are briefly reviewed.     

Enzymology of Pyran Ring A Formation in Salinomycin Biosynthesis

Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X‐ray crystal structure of SalBIII, and structure‐guided mutagenesis of putative active‐site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+β barrel fold proteins.     

Insights into the Dual Activity of a Bifunctional Dehydratase‐Cyclase Domain

Oxygen‐containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran‐forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran‐polyketides by combinatorial biosynthesis.     

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

Di‐ and sesterterpene synthases produce C₂₀ and C₂₅ isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene ( 1 ), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.     

A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran‐Forming Cyclase

Hydropyran rings are a common structural motif in reduced polyketides. Information on their biosynthetic formation and particularly the biochemical characterization of the responsible enzymes has only been reported in few cases. The dehydratase domain AmbDH3 from the ambruticin polyketide synthase was investigated. Through in vitro assay of the recombinant domain with synthetically‐derived substrate surrogates, it was shown that it has a second catalytic activity as a cyclase that performs oxa‐conjugate addition. Probing AmbDH3 with synthetic substrate analogues revealed stereoselectivity and substrate tolerance in both substeps. This is the first characterization of a pyran‐forming cyclase from a cis‐AT PKS system and the first report of a polyketide synthase domain with this kind of dual activity. Finally, it was revealed that this domain shows potential for application in chemoenzymatic synthesis.     

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor‐Induced Combinatorial Pairing of Stig Cyclases

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three‐step cascade involving Cope rearrangement, 6‐exo‐trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca²⁺, to cooperatively control the stereochemistry of hapalindole natural products.     

Exploiting the Synthetic Potential of Sesquiterpene Cyclases for Generating Unnatural Terpenoids

The substrate flexibility of eight purified sesquiterpene cyclases was evaluated using six new heteroatom‐modified farnesyl pyrophosphates, and the formation of six new heteroatom‐modified macrocyclic and tricyclic sesquiterpenoids is described. GC‐O analysis revealed that tricyclic tetrahydrofuran exhibits an ethereal, peppery, and camphor‐like olfactoric scent.     

Trichodiene Synthase: Synthesis and Inhibition Kinetics of 12‐Fluoro‐farnesylphosphonophosphate for Sesquiterpene Cyclases

trans, trans‐Farnesyl diphosphate (FPP) serves as a universal substrate for a large family of sesquiterpene cyclases that are responsible for biosynthesis of more than 300 structurally diverse sesquiterpenes in nature. A new FPP substrate analogue, 12‐fluoro‐farnesylphosphonophosphate (12‐F‐F‐CH₂PP), was synthesized in this paper for applications on kinetic and mechanistic studies of the enzyme family. Trichodiene synthase (TS), a sesquiterpene cyclase, catalyzes the conversion of trans, trans‐farnesyl diphosphate (FPP) to trichodiene. 12‐F‐F‐CH₂PP was tested as a potential inhibitor of TS. Inactivation and inhibition kinetic experiments showed that 12‐F‐F‐CH₂PP was not a mechanism‐based inactivator for TS; instead, a mixed‐type reversible inhibition was observed with inhibition constants K_i1 = 2.33 ± 0.50 μM and K_i2 = 25.80 ± 7.70 μM, values close to those previously determined for farnesylphosphonophosphate, K_i1 = 3.25 μM and K_i2 = 9.10 μM. Although 12‐F‐F‐CH₂PP did not irreversibly inactivate TS, this new analogue serves as a potential active‐site directed inactivator and mechanistic probe of other sesquiterpene cyclases and FPP‐utilizing enzymes, which utilize FPP as a common acyclic substrate.