Enzyme-catalyzed cationic epoxide rearrangements in quinolone alkaloid biosynthesis

Abstract

Epoxides are highly useful synthons and biosynthons for the construction of complex natural products during total synthesis and biosynthesis, respectively. Among enzyme-catalyzed epoxide transformations, a reaction that is notably missing, in regard to the synthetic toolbox, is cationic rearrangement that takes place under strong acid. This is a challenging transformation for enzyme catalysis, as stabilization of the carbocation intermediate upon epoxide cleavage is required. Here, we discovered two Brønsted acid enzymes that can catalyze two unprecedented epoxide transformations in biology. PenF from the penigequinolone pathway catalyzes a cationic epoxide rearrangement under physiological conditions to generate a quaternary carbon center, while AsqO from the aspoquinolone pathway catalyzes a 3-exo-tet cyclization to forge a cyclopropane-tetrahydrofuran ring system. The discovery of these new epoxide-modifying enzymes further highlights the versatility of epoxides in complexity generation during natural product biosynthesis.

Figure 1. Versatility of epoxide as a (bio)synthon and the quinolone alkaloid biosynthetic pathways

(a) Biosynthetic reactions using epoxide as a biosynthon. Examples shown are for lasalocid and lanosterol formation catalyzed by epoxide hydrolase and oxidosqualene cyclase, respectively. (b) The cationic rearrangement of epoxide under strongly acidic conditions in organic synthesis. The semi-pinacol, Meinward and Jung rearrangements all involve the formation of a carbocation intermediate followed by C-C bond migration to yield aldehydes or ketones. A comparable reaction has not been reported in Nature. (c) Knowledge of the quinolone alkaloid biosynthetic pathways at the onset of this study. Penigequinolone A (1a) and B (1b) are from the pen pathway in Penicillium sp., while aspoquinolone A (2a) and B (2b) are from the asq pathway in Aspergillus nidulans. The alcohol 4 is oxidized into the epoxide 5 by the FMO in the pathways, which can spontaneously form the 5-exo-tet product 6. Previously, 1 was proposed to derive from 6, while 2 was proposed to derive from the 6-endo-tet product 7. The common uncharacterized genes in the two pathways are shown, and the two CrtC enzymes were investigated in this work to possibly catalyze the formation of 1 and 2 from either 6, 7, or 5.

Figure 2. In vitro reconstitution of the conversion of 5 to 1

Since 5 cannot be stably isolated given its rapid conversion to 6, a coupled assay using PenE and 4 were performed. The assays demonstrate PenF can convert 5 to hemiacetal 8, which is dehydrated and reduced to 1 by the SDR-like reductase PenD.

Figure 3. Proposed reactions catalyzed by PenF and AsqO on the epoxide substrates

AsqC catalyzes the dehydration of 5 to yield the stable 10, as well as that of 4 to yield the shunt product 9. The 5-exo-product 6 is proven to be an off-pathway product in the absence of PenF. PenF catalyzes the cationic rearrangement of epoxides 5 and 10 to yield the corresponding aldehydes. Both the aldehyde 11 and ketone 12 can be formed from 10 in the presence of BF₃·Et₂O. In sharp contrast to PenF, AsqO catalyzes the formation of 2 from 10 via a 3-exo-tet cyclization. We propose revision of the stereochemistry of 2 to be either pair of diastereomers shown in parentheses.

Figure 4. Computational studies on model epoxides

(a) General acid and general acid/general base catalyzed 5-exo and 6-endo cyclization and C8-O cleavage of a model for epoxide 5. (b) General acid catalyzed 3-exo and C8-O cleavage of a model for epoxide 10. Lowest-energy transition state (TS) structures calculated at M06-2X/6-311++G(d,p)-IEFPCM(water)//M06-2X/6-31G(d)-IEFPCM(water) level, activation free energies (kcal mol⁻¹), and forming/breaking bond distances (in Ångstroms) are given.

Figure 5. In vitro reconstitution of activities of asq enzymes and enzymatic synthesis of 2

(a) Confirming the activity of AsqC as a dehydratase in the asq pathway. Comparison of substrate promiscuity of AsqC and AsqG showed AsqG functions upstream of AsqC, and 10 is the on-pathway product. (b) AsqO, a second CrtC enzyme in the asq cluster, catalyzes the 3-exo-tet reaction of 10 to yield 2. In contrast, PenF converts 10 to 11 via a cationic rearrangement, further confirming its unique activity.