The naphterpins and marinones are naphthoquinone meroterpenoids with an unusual aromatic oxidation pattern that is biosynthesized from 1,3,6,8-tetrahydroxynaphthalene (THN). We propose that cryptic halogenation of THN derivatives by vanadium-dependent chloroperoxidase (VCPO) enzymes is key to this biosynthetic pathway, despite the absence of chlorine in these natural products. This speculation inspired a total synthesis to mimic the naphterpin/marinone biosynthetic pathway. In validation of this biogenetic hypothesis, two VCPOs were discovered that interconvert several of the proposed biosynthetic intermediates.
The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon−carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.
The atomic coordinates and structure factors for the Aspergillus fumigatus sliding clamp can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 5TUP.
A concise and divergent strategy for the synthesis of the naphterpin
and marinone meroterpenoid families has been developed. The approach
features a succession of pericyclic reactionsan aromatic Claisen
rearrangement, a retro-6π-electrocyclization, and two Diels–Alder
reactionswhich facilitated the first total synthesis of naphterpin
itself in five steps from 2,5-dimethoxyphenol, alongside similar syntheses
of 7-demethylnaphterpin and debromomarinone. Late-stage oxidation
and bromination reactions were also investigated, leading to the first
total syntheses of naphterpins B and C and isomarinone.
Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in
fungi, macroalgae, and
other bacteria by catalyzing organohalogenating reactions with strict
regiochemical and stereochemical control. While this group of enzymes
collectively uses hydrogen peroxide to oxidize halides for incorporation
into electron-rich organic molecules, the mechanism for the controlled
transfer of highly reactive chloronium ions in the biosynthesis of
napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution
crystal structures of two homologous VHPO family members associated
with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive
chemical reactions in the construction of meroterpenoid natural products.
The structures, combined with site-directed mutagenesis and intact
protein mass spectrometry studies, afforded a mechanistic model for
the asymmetric alkene and arene chlorination reactions catalyzed by
NapH1 and the isomerase activity catalyzed by NapH3. A key lysine
residue in NapH1 situated between the coordinated vanadate and the
putative substrate binding pocket was shown to be essential for catalysis.
This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating
species much as proposed in structurally distinct flavin-dependent
halogenases. Unexpectedly, NapH3 is modified post-translationally
by phosphorylation of an active site His (τ-pHis) consistent
with its repurposed halogenation-independent, α-hydroxyketone
isomerase activity. These structural studies deepen our understanding
of the mechanistic underpinnings of VHPO enzymes and their evolution
as enantioselective biocatalysts.
Motivated by the biosynthesis of azamerone, we report the first example of a diazo-Hooker reaction, which involves the formation of a phthalazine ring system by the oxidative rearrangement of a diazoketone. Computational studies indicate that the diazo-Hooker reaction proceeds via an 8π-electrocyclization followed by ring contraction and aromatization. The biosynthetic origin of the diazoketone functional group was also chemically mimicked using a related natural product, naphterpin, as a model system.
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