New Tricholomalides D–G from the Mushroom Tricholoma ustaloides Grown in an Italian Beech Wood

Four novel seconeodolastane diterpenoids, named tricholomalides D–G, were isolated, together with the known tricholomalide C, from the fruiting bodies of Tricholoma ustaloides Romagn., a species belonging to the large Tricholoma genus of higher mushrooms (Basidiomycota, family Tricholomataceae). They were isolated through multiple chromatographic separations, and the structures, including the absolute configuration, were established through a detailed analysis of MS, NMR, and CD spectral data and comparison with related compounds reported in the literature, which has been thoroughly revised.


Introduction
More than 250 species are included in the genus Tricholoma (Fr.) Staude [1], which is the largest in the family Tricholomataceae of the order Agaricales (Basidiomycota).The species have a worldwide distribution and are mainly found in temperate and subtropical zones in both the southern and northern hemispheres [2], from Australia and China to North America and Europe [3][4][5][6][7][8].
The use of molecular methods based on nuclear rDNA internal transcribed spacer ITS1-5.8S-ITS2(ITS) sequences is becoming more and more important in phylogenetic studies of higher mushrooms [7][8][9], accompanying or even substituting the traditional studies of fungal morphological characters.Thus, it has often been demonstrated that mushrooms growing in different habitats/countries but classified as the same species based on morphological characters, must instead be placed in distinct clades/subclades or even different taxa [8,10,11].In this context, the distinct chemical contents determined through phytochemical analysis have often confirmed the possible existence of different varieties or taxa [12].
Our interest in the chemical analysis of Tricholoma species grown in Italy was thus motivated by taxonomical reasons, as the morphological characters alone make it difficult to differentiate some species, e.g., those included in the section Genuina [7].However, not less important was the fact that the fruiting bodies of most Tricholoma produce a wide variety of secondary metabolites, exhibiting unusual/rare structures and important biological activities, including cytotoxic and antimicrobial properties, neurite outgrowth stimulation effects, acetylcholinesterase inhibitory activity, etc. [13].
Continuing our studies on Tricholoma species grown in Italy, our attention was recently drawn to the phytochemical study of Tricholoma ustaloides Romagn.(Figure 1A), which has been included by Heilmann-Clausen and Christensen in the critical section Genuina [7].This mushroom, which is considered inedible in Europe, is generally rare in Italy, where it typically grows from late summer to late autumn in association with oak, chestnut, hornbeam, and beech trees [4,5] (Figure 1B).
Molecules 2023, 28, x FOR PEER REVIEW 2 of 13 Continuing our studies on Tricholoma species grown in Italy, our attention was recently drawn to the phytochemical study of Tricholoma ustaloides Romagn.(Figure 1A), which has been included by Heilmann-Clausen and Christensen in the critical section Genuina [7].This mushroom, which is considered inedible in Europe, is generally rare in Italy, where it typically grows from late summer to late autumn in association with oak, chestnut, hornbeam, and beech trees [4,5] (Figure 1B).In our initial phytochemical investigation of an aqueous methanol sub-extract of an EtOAc extract of T. ustaloides fruiting bodies, we isolated two rare C-30 terpenoids, saponaceolides J (1) and F (2), three cyclic lactone-containing lanostane triterpenoids, tricholidic acid (3), the new tricholidic acids B (4) and C (5), and the rare tricholomenyn C (6) (Figure 2).In addition, mixtures of undetermined triglycerides and free fatty acids, together with five unidentified diterpenoids (A1-A5), were isolated [14].In this paper we report the structures of compounds A1-A5, which were established through interpretation of IR, MS, 1D, and 2D NMR spectral data.The assignment of the absolute configuration (AC) to these compounds was based on the interpretation of electronic circular dichroism (ECD) spectra.In general, determination of the absolute configuration of natural products using ECD compares the spectrum of a new compound having an unknown AC to those of analogous compounds of known ACs [15].However, AC determination by predicting the sign of one or more bands in the ECD spectrum using empirical, semiempirical, or nonempirical rules is often an option [15,16].Another widely used option is to compare calculated and experimental ECD spectra [15].
The assignment of stereostructures to the compounds A1-A5 heavily depended on those of related diterpenoids previously isolated from other Tricholoma species.Since there are several discrepancies in the literature about the structures of the latter compounds [13], especially about their ACs, the discussion of the structures of A1-A5 is preceded by a short critical review of the existing literature.

Diterpenoids Isolated from Tricholoma Fruiting Bodies: A Short Critical Review
Diterpenoids are a class of terpenes formally composed of four isoprene units, that are biosynthesized by plants, animals, and fungi, including higher mushrooms (Basidiomycota) [17], via the HMG-CoA reductase pathway, with geranylgeranyl pyrophosphate being a primary intermediate [18].Only a few examples of diterpenoids have been isolated so far from fruiting bodies of Tricholoma [13].

Trichoaurantianolides and Tricholomalides
All the diterpenoids isolated from fruiting bodies of Tricholoma species have a rare rearranged terpenoid skeleton, which has been named seconeodolastane in accordance with the proposed biosynthetic pathway (vide infra, Scheme 1).They belong to two diastereomeric families, the trichoaurantianolides and the tricholomalides, respectively, depending on the stereochemistry of the OH group at the C-8 position of the seconeodolastane skeleton (vide infra).
Molecules 2023, 28, x FOR PEER REVIEW 5 of 13 interpretation of the CD data applying the empirical rule of twisted cyclopentanones [16], instead of the octant rule used in the paper reporting the isolation of tricholomalides [23], have strongly suggested that the configuration is identical to that of trichoaurantianolides, thus inverting the original assignment [23].In fact, like trichoaurantianolide C (9) [20], tricholomalide A (15) exhibited a positive Cotton effect at 302 nm (Δε + 0.31) [23], indicating a similar "positively twisted" conformation of the cyclopentanone ring (see the discussion above).In addition, tricholomalide B ( 16) was converted to 15 during storage at 4 °C in DMSO [23], and the CD spectra of the cisoid α,β-unsaturated ketones 16 and 17 revealed CEs with the same signs for the corresponding peaks at 250 (Δε − 0.57) and 336 nm (Δε + 0.53) for 16 and at 234 (Δε − 0.22) and 344 nm (Δε + 0.16) for 17 [23].
Structures 7-11 were established using 2D NMR data and using single-crystal Xray analysis in the case of alcohol 8 [20,21].Moreover, the absolute configurations of trichoaurantianolides B (8), C (9), and D (10) (Figure 3) were firmly established using anomalous X-ray diffraction and chemical interconversions [20,21], and enantioselective total synthesis [22].Thus, the absolute stereochemistry of trichoaurantianolide C (9) was identical to the one initially assigned to this compound on the basis of the weak Cotton effect (CE) observed at 300 nm (∆ε = +0.14)[20].In fact, based on an empirical rule [16], the positive sign of the CE of compound 9, for which the molecular modeling indicated a "twisted" conformation of the cyclopentanone ring, corresponded to the signs of the octants occupied by the carbons of the ring (see structure A in Figure 4).Instead, misapplication of the octant rule [16] to the positive CE of trichoaurantianolide C resulted in the erroneous assignment of the enantiomeric configuration ent-9 (see structure ent-A in Figure 4) [23].Tricholomalides A-C were isolated from the methanol extract of fresh fruiting bodies of an undetermined species of Tricholoma collected in Japan [23].Noteworthily, these diterpenoids significantly induced neurite outgrowth in rat pheochromocytoma cells at concentrations of 100 µM [23].The stereostructures of tricholomalides A-C, which were similar to those of trichoaurantianolides 7-11, were definitely established as 12-14 (Figure 5) using total synthesis and single-crystal X-ray diffraction.Thus, the stereochemistry at C-2 in tricholomalides A ( 12) and B (13) [24] was established to be opposite to the one originally assigned to these compounds based on spectral data only [23].Moreover, H-8 was cis to H3-19, while it was trans in compounds 7-11.Tricholomalides A-C were isolated from the methanol extract of fresh fruiting bodies of an undetermined species of Tricholoma collected in Japan [23].Noteworthily, these diterpenoids significantly induced neurite outgrowth in rat pheochromocytoma cells at concentrations of 100 µM [23].The stereostructures of tricholomalides A-C, which were similar to those of trichoaurantianolides 7-11, were definitely established as 12-14 (Figure 5) using total synthesis and single-crystal X-ray diffraction.Thus, the stereochemistry at C-2 in tricholomalides A ( 12) and B (13) [24] was established to be opposite to the one originally assigned to these compounds based on spectral data only [23].Moreover, H-8 was cis to H3-19, while it was trans in compounds 7-11.Tricholomalides A-C were isolated from the methanol extract of fresh fruiting bodies of an undetermined species of Tricholoma collected in Japan [23].Noteworthily, these diterpenoids significantly induced neurite outgrowth in rat pheochromocytoma cells at concentrations of 100 µM [23].The stereostructures of tricholomalides A-C, which were similar to those of trichoaurantianolides 7-11, were definitely established as 12-14 (Figure 5) Molecules 2023, 28, 7446 5 of 13 using total synthesis and single-crystal X-ray diffraction.Thus, the stereochemistry at C-2 in tricholomalides A (12) and B (13) [24] was established to be opposite to the one originally assigned to these compounds based on spectral data only [23].Moreover, H-8 was cis to H 3 -19, while it was trans in compounds 7-11.Tricholomalides A-C were isolated from the methanol extract of fresh fruiting bodies of an undetermined species of Tricholoma collected in Japan [23].Noteworthily, these diterpenoids significantly induced neurite outgrowth in rat pheochromocytoma cells at concentrations of 100 µM [23].The stereostructures of tricholomalides A-C, which were similar to those of trichoaurantianolides 7-11, were definitely established as 12-14 (Figure 5) using total synthesis and single-crystal X-ray diffraction.Thus, the stereochemistry at C-2 in tricholomalides A (12) and B (13) [24] was established to be opposite to the one originally assigned to these compounds based on spectral data only [23].Moreover, H-8 was cis to H3-19, while it was trans in compounds 7-11.The absolute configuration of tricholomalides A-C has not yet been determined unambiguously.However, biosynthetic considerations (vide infra, Scheme 1) and interpretation of the CD data applying the empirical rule of twisted cyclopentanones [16], instead of the octant rule used in the paper reporting the isolation of tricholomalides [23], have strongly suggested that the configuration is identical to that of trichoaurantianolides, thus inverting the original assignment [23].In fact, like trichoaurantianolide C (9) [20], tricholomalide A (15) exhibited a positive Cotton effect at 302 nm (∆ε + 0.31) [23], indicating a similar "positively twisted" conformation of the cyclopentanone ring (see the discussion above).In addition, tricholomalide B ( 16) was converted to 15 during storage at 4 • C in DMSO [23], and the CD spectra of the cisoid α,β-unsaturated ketones 16 and 17 revealed CEs with the same signs for the corresponding peaks at 250 (∆ε − 0.57) and 336 nm (∆ε + 0.53) for 16 and at 234 (∆ε − 0.22) and 344 nm (∆ε + 0.16) for 17 [23].
Molecules 2023, 28, x FOR PEER REVIEW 5 of 13 interpretation of the CD data applying the empirical rule of twisted cyclopentanones [16], instead of the octant rule used in the paper reporting the isolation of tricholomalides [23], have strongly suggested that the configuration is identical to that of trichoaurantianolides, thus inverting the original assignment [23].In fact, like trichoaurantianolide C (9) [20], tricholomalide A (15) exhibited a positive Cotton effect at 302 nm (Δε + 0.31) [23], indicating a similar "positively twisted" conformation of the cyclopentanone ring (see the discussion above).In addition, tricholomalide B ( 16) was converted to 15 during storage at 4 °C in DMSO [23], and the CD spectra of the cisoid α,β-unsaturated ketones 16 and 17 revealed CEs with the same signs for the corresponding peaks at 250 (Δε − 0.57) and 336 nm (Δε + 0.53) for 16 and at 234 (Δε − 0.22) and 344 nm (Δε + 0.16) for 17 [23].In summary, the absolute stereochemistry depicted in Figure 6 is proposed for tricholomalides A-C (15-17).Biosynthetically, trichoaurantianolides and tricholomalides should originate from an enzymatically controlled stereoselective cyclization of geranylgeranyl pyrophosphate (18) producing nonracemic dolabellane cation 19.This intermediate would be the precursor of the dolastane (20), neodolabellane (21), and neodolastane (guanacastane) (22) skeletons through a series of stereospecific transannular cyclizations and Wagner-Meerwein migrations (Scheme 1).The seconeodolastane skeleton (23) of trichoaurantianolides and tricholomalides would finally result from oxidation and cleavage of the six-membered ring in 22 (Scheme 1), while oxidative decoration of the structures at C-2, C-8, and C-12 would likely occur in the latest steps of the biosynthetic path to trichoaurantianolides and tricholomalides.It is interesting to note that diterpenoids originating from a common biogenetic pathway in algae, fungi, liverworts, and higher plants are enantiomeric to the corresponding metabolites isolated from species of marine invertebrates [13,25].

Tricholomalides from Tricholoma Ustaloides
In the following paragraphs, we will describe the determination of the structures of compounds A1-A5, whose isolation from T. ustaloides fruiting bodies was reported in a recent paper [14].A1-A4 were new diterpenoids, named tricholomalides D-G (24-27 in Figure 7), respectively, while A5 was identified as the known tricholomalide C (17) [23].At first, we will discuss the structures of tricholomalides D ( 24), E (25), and G (27), which showed a γ-lactone ring cis-fused at C-2 and C-7 with the central cycloheptene ring of the seconeodolastane skeleton.Subsequently, we will describe the structure of tricholomalide F (26), in which the γ-lactone ring is cis-fused with the cycloheptene ring at C-7 and C-8.
likely occur in the latest steps of the biosynthetic path to trichoaurantianolides and tricholomalides.It is interesting to note that diterpenoids originating from a common biogenetic pathway in algae, fungi, liverworts, and higher plants are enantiomeric to the corresponding metabolites isolated from species of marine invertebrates [13,25].
linked C-13 to C-16 and included the H-15 (δH 1.75-1.85,1H, m) to H3-17 linkage.The spin system of the unit II was an isolated AB quartet (δH 2.29 and 2.88, 2H, JAB = 18.5 Hz), which was assigned to an isolated methylene (H2-6) attached to the γ-lactone carbonyl group (δC 175.2, s, C-5) by HMBC correlations (Figure 8A).The moiety III gave rise to a distorted AMX system which includes a methylene (H2-9; δH 1.79 (1H, dd, 15.0, 11.5) and δH 2.15 (1H, dd, 15.0, 2.0)) and a methine hydrogen (H-8; δH 4.72 (1H, ddd, 11.5, 2.0, 1.0)).This proton was geminal to the acetoxy carbonyl carbon (δC 169.4,s, C-21) via an HMBC cross peak with C-21 (Figure 8A).Assembling these partial structures using the two-and threebond C-H connectivity data from HMBC correlations (Figure 8A), we unambiguously assigned structure 24 to tricholomalide D. The structure of the ring system was determined by (i) the bonds around the angular methyl (H3-19) attached to C-7 (δC 49.8 s), which linked C-6 (δC 35.3 t) and C-9 (δC 37.  8B), suggested that tricholomalide D ( 24) was the C-8 epimer of trichoaurantianolide A ( 7) [19].Thus, the 8-acetoxy group was trans to H3-19 in 24, while it was cis in 7 [19].This finding was confirmed by the significantly different chemical shift and coupling constants of H-8 in 24 (δH 4.72; J8β-9β = 2.0 and J8β-9α = 11.5 Hz) and in 7 (δH 5.15; J8α-9β = 9.5 and J8α-9α = 1.5 Hz) [19].S1 in the Supplementary Materials) clearly indicated that it was the deacetyl derivative of tricholomalide D (24).In fact, the signals of an acetyl group were missing in the 1 H and 13 C NMR spectra of 25 (Figures S11 and S13 in the Supplementary Materials), while the signals of H-8 (δH 3.63, br d, 11.5) and C-8 (δC 71.6 d) in the 1 H and 13 C NMR spectra, respectively, of alcohol 25 moved upfield by about 1 and 3 ppm, respectively, in comparison with the corresponding signals of the acetate 24 (vide supra).Finally, the structure of 25 was confirmed with standard acetylation with Ac2O/Py, which afforded a product that was identical with acetate 24.S1 in the Supplementary Materials) clearly indicated that it was the deacetyl derivative of tricholomalide D (24).In fact, the signals of an acetyl group were missing in the 1  Tricholomalide G (27), with the molecular formula C 20 H 26 O 5 from the EIMS (Figure S27 in the Supplementary Materials) and the 1 H and 13 C NMR spectra (Figures S23-S26 in the Supplementary Materials), was identified as the dihydroderivative of aldehyde 24 through the following significant differences between the NMR spectra of the two compounds (Figures S23-S26 and Table S1 in the Supplementary Materials): (i) the presence of two diastereotopic protons (H 2 -4) at δ H 4.22 (1H, dd, 13.0, 1.1) and 4.27 (1H, br d, 13) in the 1 H NMR spectrum of 27, assignable to an allylic CH 2 OH group, which replaced the signal H-4 (δ H 9.70 (1H, s)) of aldehyde 24; (ii) the upfield shift of the H 2 -20 protons (δ H 5.32 (1H, s) and δ H 5.67 (1H, t, 1.1)) and the C-20 carbon (δ C 118.3, t), respectively, in the 1 H and 13 C NMR spectra of 27, compared with the corresponding signals of the βCH 2 group of the acrolein moiety in 24.Finally, the structure of compound 27 was confirmed through the oxidation of the allylic alcohol with PDC (pyridinium dichromate), which afforded a product indistinguishable from aldehyde 24.
The NMR spectral data (Figures S1-S3 in the Supplementary Materials) and the signs of the CD maxima of compound A5 (Figure S4 in the Supplementary Materials) were identical with the data reported in the literature for tricholomalide C (17) [23].Moreover, selective oxidation of A5 (≡17) with PDC gave a product indistinguishable from aldehyde 25.

Fungal Material
The fruiting bodies of Tricholoma ustaloides Romagn.were collected in a beech wood at the end of September-beginning of October 2021 and identified by Alfredo Gatti, as reported in the inaugural study of this mushroom [14].A sample specimen (accession code: TU001) was deposited at the Department of Chemistry, University of Pavia, Italy.

Conclusions
The isolation of four novel seconeodolastane diterpenoids, tricholomalides D-G, from T. ustaloides has confirmed that the fruiting bodies of Tricholoma mushrooms are rich sources of new compounds, most of which have unique chemical structures.The rare seconeodolastane skeleton of tricholomalides seems to be a characteristic feature of the diterpenoids present in the fruiting bodies of Tricholoma; however, epimeric derivatives, e.g., compounds 7 and 24, are produced by different species, and this finding may be chemotaxonomically significant.So far, besides being isolated from fruiting bodies of the genus Tricholoma, seconeodolastane diterpenoids have only been isolated from a mycelial culture of Lepista sordida [27].Interestingly, the genera Tricholoma and Lepista both belong to the family Tricholomataceae (Agaricales).
In the future, we will extend our investigations to the chemical contents of other Tricholoma species grown in the Italian woods, and, regarding tricholomalides C-G, we intend to study their biological activities using in vitro tests.

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules28217446/s1: 1 H NMR graphical spectra of tricholomalides C-G; COSY graphical spectra of tricholomalides D-G; 13 C NMR graphical spectra of tricholomalides C-G; DEPT 13 C NMR graphical spectra of tricholomalides C-G; EIMS graphical spectra of tricholomalides D-G; CD graphical spectra of tricholomalides C-G; tabulated NMR spectral data of tricholomalides D-G.

Figure 1 .
Figure 1.Specimens of Tricholoma ustaloides (A) collected in a beech wood (B) in Northern Apen near Pavia, Italy (photos provided by Teresio Restelli and Alfredo Gatti).

Figure 1 .
Figure 1.Specimens of Tricholoma ustaloides (A) collected in a beech wood (B) in Northern Apennines near Pavia, Italy (photos provided by Teresio Restelli and Alfredo Gatti).

Figure 1 .
Figure 1.Specimens of Tricholoma ustaloides (A) collected in a beech wood (B) in Northern Apennines near Pavia, Italy (photos provided by Teresio Restelli and Alfredo Gatti).

Figure 6 .
Figure 6.Absolute configurations proposed for tricholomalides A-C.Figure 6. Absolute configurations proposed for tricholomalides A-C.

Figure 6 .
Figure 6.Absolute configurations proposed for tricholomalides A-C.Figure 6. Absolute configurations proposed for tricholomalides A-C.

3. 1 . 1 .
Tricholomalides C, D, E, and G The molecular formula of tricholomalide D (24) was established as C 22 H 28 O 6 by EIMS (Figure S9 in the Supplementary Materials), elemental analysis, and 1 H and 13 C NMR spectra (Table S1 in the Supplementary Materials).It indicated nine degrees of unsaturation.The oxygen-containing functionalities in the molecule were established as one cyclopentanone (C-12), one formyl (C-4), one γ-lactone (C-5), and one acetoxy (C-21) group from the IR bands (1783, 1736, 1699 cm −1 ) and the characteristic chemical shifts (δ C 205.0, 191.8, 175.2, and 169.4 ppm, respectively) of the corresponding carbonyl carbons in the 13 C NMR spectrum (Figures S7 and S8 in the Supplementary Materials).

Figure 8 .
Figure 8. HMBC (H→C) correlations (A) and 1D NOE correlations (H→H) (B) observed for tricholomalide D (24).The molecular formula of tricholomalide E (25), C20H26O5, was established from the EIMS (Figure S15 in the Supplementary Materials) and the 1 H and 13 C NMR spectra (Figures S11-S14 in the Supplementary Materials).The NMR spectral data (TableS1in the Supplementary Materials) clearly indicated that it was the deacetyl derivative of tricholomalide D(24).In fact, the signals of an acetyl group were missing in the 1 H and13 C NMR spectra of 25 (FiguresS11 and S13in the Supplementary Materials), while the signals of H-8 (δH 3.63, br d, 11.5) and C-8 (δC 71.6 d) in the 1 H and 13 C NMR spectra, respectively, of alcohol 25 moved upfield by about 1 and 3 ppm, respectively, in comparison with the corresponding signals of the acetate 24 (vide supra).Finally, the structure of 25 was confirmed with standard acetylation with Ac2O/Py, which afforded a product that was identical with acetate 24.

Figure 8 .
Figure 8. HMBC (H → C) correlations (A) and 1D NOE correlations (H → H) (B) observed for tricholomalide D (24).The molecular formula of tricholomalide E (25), C 20 H 26 O 5 , was established from the EIMS (Figure S15 in the Supplementary Materials) and the 1 H and 13 C NMR spectra (Figures S11-S14 in the Supplementary Materials).The NMR spectral data (TableS1in the Supplementary Materials) clearly indicated that it was the deacetyl derivative of tricholomalide D(24).In fact, the signals of an acetyl group were missing in the1 H and 13 C NMR spectra of 25 (Figures S11 and S13 in the Supplementary Materials), while the signals of H-8 (δ H 3.63, br d, 11.5) and C-8 (δ C 71.6 d) in the 1 H and 13 C NMR spectra, respectively, of alcohol 25 moved upfield by about 1 and 3 ppm, respectively, in comparison with the corresponding signals of the acetate 24 (vide supra).Finally, the structure of 25 was confirmed with standard acetylation with Ac 2 O/Py, which afforded a product that was identical with acetate 24.Tricholomalide G (27), with the molecular formula C 20 H 26 O 5 from the EIMS (Figure S27 in the Supplementary Materials) and the 1 H and 13 C NMR spectra (Figures S23-S26 in the Supplementary Materials), was identified as the dihydroderivative of aldehyde 24 through the following significant differences between the NMR spectra of the two compounds (Figures S23-S26 and Table S1 in the Supplementary Materials): (i) the presence of two diastereotopic protons (H 2 -4) at δ H 4.22 (1H, dd, 13.0, 1.1) and 4.27 (1H, br d, 13) in the 1 H H and 13 C NMR spectra of 25 (Figures S11 and S13 in the Supplementary Materials), while the signals of H-8 (δ H 3.63, br d, 11.5) and C-8 (δ C 71.6 d) in the 1 H and 13 C NMR spectra, respectively, of alcohol 25 moved upfield by about 1 and 3 ppm, respectively, in comparison with the corresponding signals of the acetate 24 (vide supra).Finally, the structure of 25 was confirmed with standard acetylation with Ac 2 O/Py, which afforded a product that was identical with acetate 24.