Two New Phomaligols from the Marine-Derived Fungus Aspergillus flocculosus and Their Anti-Neuroinflammatory Activity in BV-2 Microglial Cells

Abstract

Two new phomaligols, deketo-phomaligol A (1) and phomaligol E (2), together with six known compounds (3–8) were isolated from the culture broth of the marine-derived fungus Aspergillus flocculosus. Compound 1 was first isolated as a phomaligol derivative possessing a five-membered ring. The structures and absolute configurations of the new phomaligols were determined by detailed analyses of mass spectrometry (MS), nuclear magnetic resonance (NMR) data, optical rotation values and electronic circular dichroism (ECD). In addition, the absolute configurations of the known compounds 3 and 4 were confirmed by chemical oxidation and comparison of optical rotation values. Isolated compounds at a concentration of 100 μM were screened for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Among the compounds, 4 showed moderate anti-neuroinflammatory effects with an IC50 value of 56.6 μM by suppressing the production of pro-inflammatory mediators in activated microglial cells without cytotoxicity.

Reisolation and Structure Revision of Asperspiropene A

“…The revised structure of ring B is closely similar to that of sarcosone derivatives, which are common fungal polyketides containing a 3-methoxycyclohexenone ring as a substructure, − and we have isolated several 3-methoxycyclohexenone analogues from the same strain. , Therefore, we compared the chemical shifts of the revised structure of ring B and 3-methoxycyclohexenone derivatives and found that they are well matched to each other (Figure S19). Moreover, the UV spectrum of 1 (Figure S27) displayed a strong absorption peak at 256 nm, which was similar to that of other 3-methoxycyclohexenones, and the IR spectrum (Figure S28) showed an absorption band at 1643 cm –1 corresponding to an α,β-conjugated carbonyl as in the revised structure (unconjugated carbonyl as in the proposed structure absorbs at >1700 cm –1 ). , Additionally, an extensive literature review revealed that the chemical shift of the methyl group in methyl esters of glucuronic acid (similar to the proposed structure) is smaller than 53 ppm (δ C 51–53) (Table S2).…”

“…During the course of our ongoing research for novel marine natural products, we have isolated a number of oxygenated cyclohexenone derivatives from two strains of Aspergillus flocculosus , 01NT-1.1.5 and 168ST-16.1. − Among them, compound 1 was isolated as a pure compound with identical 1D and 2D NMR data to those of asperspiropene A. However, the HRESIMS data of 1 were different from the previously published data for asperspiropene A. Additionally, we found that some 2D NMR data of asperspiropene A (COSY and HMBC) were incorrectly interpreted .…”

“…The theoretical ECD spectra were calculated by Gaussian 16 software using the time-dependent DFT (TD-DFT) method at the CAM-B3LYP/6-311+G (d,p) level in MeOH with the Polarizable Continuum Model (PCM) using the integral equation formalism variant (IEFPCM) model. The Boltzmann distribution of each conformer was calculated based on their Hartree energy, and the calculated ECD spectra were averaged according to the Boltzmann distributions. , …”

“…It is noteworthy that trichothosporon A, trichothecrotocins D–G, and the proposed structure of asperspiropene A are the only six representatives of a rare meroterpenoid class containing an oxygenated 7-hydroxydihydro-2 H -pyran-6­(4 H )-one substructure (Figure S20). − In contrast, the revised structure of asperspiropene A and sarcosone derivatives belongs to a wide group of fungal polyketides containing a 3-methoxycyclohexenone substructure. ,,, Interestingly, the chemical shifts of the oxygenated 7-hydroxydihydro-2 H -pyran-6­(4 H )-one (ring B in the proposed structure and in trichothecrotocins and trichothosporon A) and the 3-methoxycyclohexenone ring (ring B in the revised structure and in sarcosone derivatives) are very similar (Figures S19 and S20), suggesting that the ring structures in trichothecrotocins and trichothosporon A could be wrongly determined. So we carefully re-examined 2D NMR data of these analogues of the proposed structure of asperspirone A. Consequently, we found that the same strong HMBC correlation of H-7/C-8 was observed for trichothecrotocins D–G (Figures S21–S24), which is an uncommon four-bond correlation and also was not taken into consideration when Yang and co-workers elucidated the structures of trichothecrotocins D–G.…”

Brief overview of recently reported misassigned natural products and their in silico revisions enabled by DU8ML, a machine learning-augmented DFT computational NMR method

“…Culture broth from marine-derived fungus, Aspergillus flocculosus , produced two new compounds, phomaligol E ( 15 ), and deketo-phomaligol A ( 17 ). 9 Our analysis showed that phomaligol E ( 15 ), needs revision to 4-epimer ( 16 ), while deketo-phomaligol A ( 17 ) required a more extensive revision into α,β-unsaturated cyclopentanone 18 .…”