Biomimetic polyene cyclizations.

Vinigrol (1) is a fungal diterpenoid consisting of a decahydro-1,5-butanonaphthalene ring system with no analogs in nature. Despite immense efforts in synthetic studies, the vinigrol biosynthesis pathway remains largely unknown. Herein, we identified a biosynthetic gene cluster for 1 and fully elucidated the biosynthetic pathway. By employing an AlphaFold-generated model structure, we identified the possible catalytic residues of the noncanonical terpene cyclase and analyzed their function by site-directed mutagenesis. We found that the G340A mutation opened a cryptic pathway for an unprecedented tetracyclic diterpene, defined here as virgarene. Retro-biosynthetic theoretical analysis provided a solid foundation for the complex cyclization pathway for the vinigrol scaffold, its chemical transformation to a structurally distinct bonnadiene, and redirection of the enzymatic cyclization cascade to virgarene. Close inspection of the terpene cyclization pathway via integrated experimental and theoretical approaches would allow efficient exploration of novel terpenoid chemistries.

Cyclization↗

PubMed2025

Enzymatic Anti-Baldwin Ring-Closure Cascade for Fused Bicyclic Ether Formation.

Pyrenulic acids are cytotoxic polyketides isolated from the ascomycete Pyrenula sp. derived from Vietnamese lichen that are characterized by complex fused cyclic core structures. Genome sequencing, in silico sequence analysis, and RT-PCR studies identified the pyrenulic acid (pya) biosynthetic gene cluster. Based on a functional analysis of the enzymes by expression of each gene in a heterologous host using Aspergillus nidulans, we discovered two cytochrome P450s PyaJ and PyaG that effect epoxidation and hydroxylation of the alkyl chain terminal, respectively, and an α/β hydrolase PyaF that constructs a 6- and 7-membered fused bicyclic diether skeleton by catalyzing successive epoxide ring-opening 6-endo and 7-endo cyclization reactions. To elucidate the detailed mechanism of pyrenulic acid formation, we obtained PyaF as a recombinant enzyme and performed an in vitro experiment, which confirmed catalysis by PyaF of the cyclization reaction. In addition, we performed alignment analysis of PyaF with α/β hydrolases with known functions, as well as an in-depth computational study. In-depth computational analyses of the cyclization reaction pathways with density functional theory quantum mechanics and detailed characterization of PyaF by Chai-1-based protein structure modeling with molecular dynamics simulations and site-specific mutagenesis predicted the active amino acid residues of this serine α/β hydrolase to be an unusual catalytic serine tetrad involving Ser170, Asn342, Asp314, and His169, with Tyr255 and His284 acting as general bases to facilitate opening of the epoxides. Our study provides insight into how regioselectivity of enzymatic anti-Baldwin epoxide ring-opening cascades for the formation of a fused cyclic ether structure is controlled.

Cyclization↗

PubMed2007

Spin-polarized conceptual density functional theory study of the regioselectivity in ring closures of radicals.

The regioselectivity of ring-forming radical reactions is investigated within the framework of the so-called spin-polarized conceptual density functional theory. Two different types of cyclizations were studied. First, a series of model reactions of alkyl- and acyl-substituted radicals were investigated. Next, attention was focused on the radical cascade cyclizations of N-alkenyl-2-aziridinylmethyl radicals (a three-step mechanism). In both of these reactions, the approaching radical (carbon or nitrogen centered) adds to a carbon-carbon double bond within the same molecule to form a radical ring compound. In this process, the number of electrons is changing from a local point of view (a charge transfer occurs from one part of the molecule to another one) at constant global spin number Ns (both the reactant and the product ring compound are in the doublet state). It is shown that the experimentally observed regioselectivities for these ring-closure steps can be predicted using the spin-polarized Fukui functions for radical attack, f0NN(r).

Cyclization↗