The chemical structure of ergostane-type steroids is typically determined by nuclear magnetic resonance (NMR) spectral analysis. The ergostane-type steroids from the Pleurotus genus mushrooms typically show two singlets of tertiary methyl groups at the C-18 and C-19 positions. Further, they exhibit four doublets of secondary methyl groups at C-21, C-26, C-27, and C-28, and two doublets of doublets of the double bond between C-22 and C-23. Except for a hydroxy or epoxy group at the C-4 position, the ergostane-type steroids of the Pleurotus genus mushrooms have a hydroxy group at the C-3 position of the A ring, and the hydroxymethine at the C-3 position appears as a triplet of triplets in 1H NMR spectra [6, 15,16,17]. This splitting pattern is also observed in steroids derived from sources other than Pleurotus genus mushrooms [27,28,29].

A tertiary hydroxyl group is sometimes present at the C-5 position. This configuration can be determined by confirming the pyridine-induced deshielding effect on surrounding protons such as H-1α, H-3, and H-9. That is, near one hydroxyl group, the δH shifts approximately 0.3 ppm downfield in C5D5N measurements compared with that in CDCl3 or CD3OD measurements (Fig. 5a) [30,31,32,33,34,35]. Further, this can be applied to the determination of 14-OH orientation such as 21 (Fig. 5b).

The pyridine-inducing deshielding effects in compounds 50 (a) and 21 (b)

In case an epoxy group exists at C-5, the δC of C-5 under CDCl3 measurement is typically observed at approximately 63–68 ppm [6, 15, 18], whereas when it is a hydroxyl group, signals above δC 70 ppm are observed [6, 8, 17, 18, 20]. Further, several compounds with chemical modifications such as olefination or epoxidation between C-8 and C-14 or C-9 and C-11 and hydroxylation or carbonylation at positions C-14 and C-15 of the C and D rings, have been isolated. The configuration at C-20 in ergostanes isolated from the Pleurotus genus mushrooms has been consistently identified as S configuration. This can be determined using NOESY, showing NOE correlations such as H-16α/H-22, H3-18/H-20, and H3-28/H-14 based on the calculated stable conformations of (20R) and (20S) [6]. However, the configuration at C-24 in ergost-22,23-ene structures can be determined from 13C NMR signals, as there are differences in the chemical shift values of δC, particularly in the CDCl3 of 24R [δC 42.9 (C-24) and 17.7 (C-28)] and 24S [δC 43.2 (C-24) and 18.1 (C-28)] methylcholestane-type steroids [36].

For 8,14-seco and 9,11-secoergostanes, such as (3β,5α,22E)-3,5-dihydroxy-8,14-secoergosta-6,22-diene-8,14-dione (55), (3β,5α,22E)-3,5,11-trihydroxy-9,11-secoergosta-7,22-diene-6,9-dione (56) [6], (3β,5α,6β,22E)-3,5,6-trihydroxy-9,11-secoergosta-7,22-dien-9-one (57) [16], and (3β,5α,6α,22E)-3,5,6,11-tetrahydroxy-9,11-secoergosta-7,22-dien-9-one (58) [17], correlations from the methyl groups of H3-18 or H3-19 to the keto carbonyl group are observed in the HMBC spectrum. This facilitates the prediction of C–C bond cleavage with the keto carbonyl group (Fig. 6). In addition, for 9,11-secoergostanes, it is noteworthy that the CH2 signals at C-11 are replaced by oxymethylene or primary methyl group signals. For the configuration of side chains, the method of determining those of the normal ergost-22-ene side chain can be applied. In NMR spectrum analysis, when overlapping signals hinder analysis, changing the NMR measurement solvent can be an effective means to deduce the correct structure. For the structure determination of (3β,5α,22E)-3,5,11-trihydroxy-9,11-secoergosta-7,22-diene-6,9-dione (56), the signals of H-22 and H-23 on the side chain overlap in CDCl3 measurement; however, in C5D5N measurement, it shows J = 15.6 Hz, and it has been determined to have an E configuration [6].

Key HMBC and 1H–.1H COSY correlations of secoergostanes (55–59)

Conversely, in 5,6-seco and 13,14-secoergostanes, the C–C bonds between C-5 and C-6, and C-13 and C-14 are cleaved, with ether bridges existing between them. Eringiacetal A (59), a 5,6-secoergostane, possesses a cage-like structure with three acetal carbons forming three ether bridges, which is unique among other steroids [13]. In the structural analysis of this compound, the HMBCs from of H-6 to the acetal carbons of C-5 and C-7, from 7-OH to the acetal carbons of C-6 and C-7, and the olefinic carbon of C-8 are observed. These HMBCs were crucial in elucidating the cage-like structure of the B ring of the steroid skeleton (Fig. 6).

In addition to HMBC, NOESY has proven to be an important tool in the structure determination of 13,14-secoergostane, eringiacetal B (60) [14]. Correlations were observed from H3-18 to a tertiary oxycarbon (C-13) and an acetal carbon (C-14), suggesting the presence of C–C bond cleavage and ether bond formation. Regarding the conformation at the C-13 and C-14 positions, the configuration of the C/D ring in this compound cannot be (13R,14S) or (13S,14R) because of the strain, which means that the hydroxy group at the C-14 position is too hindered to be stable [37, 38]. It is proposed that the cis configuration, i.e., CH3-18–C-13–O–C-14–OH, takes a W-shaped conformation. The relative configuration was determined by combining NOE correlations (Fig. 7).

Key NOE correlations of eringiacetal B (60)

In the structural elucidation of the abeo-steroids isolated from the Pleurotus genus mushrooms, the HMBCs from H3-18 or H3-19 methyl to carbonyl groups are observed as in the seco type, suggesting the cleavage of the ergostane skeleton. However, 1H–1H COSY and HMBCs confirm the formation of new C–C bonds. In the 1H and 13C NMR spectra of pleurocin A (61), an 11(9 → 7)abeo-ergostane steroid, four tertiary methyl groups and two secondary methyl groups are observed, like in normal ergostane-type steroids [14]. Conversely, the HMBC from H3-19 to the carbonyl group at C-9 and the 1H–1H COSYs of H-6–H-7–H2-11 have been observed. These correlations indicate the inversion of the A/B rings and the presence of a C–C bond between C-7 and C-11. Simultaneously, pleurocin B (62), which is the 22,23-dihydro derivative, has been isolated and structurally determined [14].

In addition, the 15(14 → 22)abeo-ergostanes, strophasterols E (63) and F (64) [16], have been isolated from P. eryngii mushrooms. In the HMBC experiments of the 15(14 → 22)abeo-ergostanes, correlations from H3-18 to the carbonyl group at C-14 are observed. Furthermore, 1H–1H COSYs between H-15–H-22(–H-20)–H-23 have been observed, revealing the cleavage of the C–C bond between C-14 and C-15 and the formation of the cyclopentane ring by the bonding of C-15 and C-22. Strophasterols E (63) and F (64) are the 23R and 23S epimers.

Regarding pleurocorol A (65) having a six-membered B ring and a five-membered C ring connected by a spiro carbon at the C-8 position, the HMBCs from H2-7, H2-11, H-12α, and H-14 to quaternary carbon of C-8 have been observed. This indicated that the C–C bond exists between C-11 and C-8 instead of C-11 and C-9 [7]. Conversely, in pleurocorol B (66) with a five-membered B ring and a six-membered C ring, HMBCs from the proton of the formyl group, H-6, to C-7 and C-8, and from H-8 and H-11β to the formyl carbon, C-6, were observed. This indicated the presence of a formyl group at C-6 and the cleavage of the C–C bond between C-5 and C-6 [7].

X-ray crystallography, along with mass spectrometry and NMR, is an effective means for determining detailed structures, and also enables the determination of absolute configuration, although it requires suitable crystals. If the crystallization of abeo-ergostane steroids is difficult, the preparation of its p-bromobenzoate derivative is an effective method for crystallization. The absolute configurations of pleurocin A (61), strophasterols E (63) and F (64), and pleurocorols A (65) and B (66) have been confirmed through the X-ray crystallographic analysis of their p-bromobenzoate derivative (61a, 63a, 64a, 65a, and 66a) (Fig. 8) [7, 14, 16].

Oak-Ridge thermal ellipsoid plots of compounds 61a and 63a–66a