Cyclohumulanoid Sesquiterpenes from the Culture Broth of the Basidiomycetous Fungus Daedaleopsis tricolor

A series of cyclohumulanoids, i.e., tricocerapicanols A–C (1a–1c), tricoprotoilludenes A (2a) and B (3), tricosterpurol (4), and tricoilludins A–C (5–7) were isolated along with known violascensol (2b) and omphadiol (8) from the culture broth of Daedaleopsis tricolor, an inedible but not toxic mushroom. The structures were fully elucidated on the basis of NMR spectroscopic analysis, and the suggested relative structures were confirmed via density functional theory (DFT)-based chemical shift calculations involving a DP4 probability analysis. In the present study, the 1H chemical shifts were more informative than the 13C chemical shifts to distinguish the diastereomers at C-11. The absolute configurations of 1–5 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. For 6 and 7, the same chirality was assigned according to their biosynthetic similarities with the other compounds. The successful assignment of some Cotton effects was achieved by utilizing DFT calculations using simple model compounds. The plausible biosynthesis of 1–7 was also discussed on the basis of the structural commonality and general cyclohumulanoid biosynthesis. Compounds 2a and 5 were found to simultaneously induce hyphal swelling and branching at 5.0 μg/mL against a test fungus Cochliobolus miyabeanus.


Results
D. tricolor sampled in 2019 at the Shirakami Natural Science Park of Hirosaki Univ sity located in the Shirakami Mountains, Japan, was cultured in potato dextrose medi for 2 weeks under shaking conditions. The ethyl acetate extracts from both the cult broth and the fungus body were subjected to a series of chromatographic separations obtain tricocerapicanols A-C (1a-1c), tricoprotoilludenes A (2a) and B (3), tricosterpu (4), tricoilludins A-C (5-7), and known violascensol (2b) [30] and omphadiol (8) as sho in Figure 1 [31]. Table 1 summarizes the 1 H and 13 C NMR data of the new compoun The spectral data of known 2b [30] and 8 [31] agreed with those reported in the literatu Although the molecular ion of 8 was not observed in the electron spray ionization-ti of flight-mass spectrometry (ESI-TOFMS) analysis, its formula was confirmed after c version into its 10-O-benzoate (8-OBz, Supplementary Materials SI-93). Despite being dressed in various studies [30,32], the absolute configuration of 2b was not fully est lished yet; this was successfully elucidated in the present study. The absolute configu tion of 8 is discussed on the basis of our own investigations, although it was previou established synthetically [33].    OR PEER REVIEW 5 of 20 The molecular ion of tricocerapicanol A (1a) was observed at m/z 237.1869 in the ESI-TOFMS spectrum (SI-6), suggesting that its molecular formula is C15H24O2 ([M + H] + : 237.1849). This was confirmed by the presence of 15 resonances in the 13 C NMR spectrum. A 2D spectral analysis involving heteronuclear single quantum coherence (HSQC, SI-10) and heteronuclear multiple bond coherence (HMBC, SI-11) spectra suggested a cerapicane framework for 1a [34,35]. Its 1 H NMR spectral profile (SI-7) resembled that of repraesentin B, which was isolated by Makabe et al. in 2003 from the fungus Lactarius repraesentaneus [36]; however, there were some considerable differences, despite fact that CDCl3 was used as the solvent in both cases. For example, the H-2 of 1a was observed at 2.27 ppm, whereas it appears at 2.37 ppm for repraesentin B. This chemical shift difference suggests that they are different compounds. The irradiation of the H-2 resonance of 1a afforded nuclear Overhauser effects (NOEs) with Hβ-4, Hβ-5, H-9, and H2-12 as shown in Figure 2 (SI-12), revealing that 1a is a C-11 epimer of repraesentin B. Other correlations observed in the 2D nuclear Overhauser effect spectroscopy (NOESY) spectrum not only confirmed the relative structure but also allowed assigning the prochiral methylene 1 H signals, except for the conformationally flexible H2-12 (SI-13). Considering other novel compounds isolated in the present study, this compound was named tricocerapicanol A. The ESI-TOFMS spectrum of tricocerapicanol B (1b) exhibited the molecular ion at m/z 253.1802 (SI-15). The mass difference from 1a (Δm/z 15.9933) indicated that 1b contains one more oxygen than 1a. The similar 2D spectral analysis as above allowed establishing that 1b is a 5-hydroxylated derivative of 1a (SI-18, 19, and 20). A NOESY correlation between H-5 and H-9 revealed the α-configuration for 5-OH (SI-21). Although the H-2 and H-9 signals appeared too close (2.28 and 2.35 ppm, respectively) to observe the NOESY The ESI-TOFMS spectrum of tricocerapicanol B (1b) exhibited the molecular ion at m/z 253.1802 (SI-15). The mass difference from 1a (∆m/z 15.9933) indicated that 1b contains one more oxygen than 1a. The similar 2D spectral analysis as above allowed establishing that 1b is a 5-hydroxylated derivative of 1a 19,and 20). A NOESY correlation between H-5 and H-9 revealed the α-configuration for 5-OH (SI-21). Although the H-2 and H-9 signals appeared too close (2.28 and 2.35 ppm, respectively) to observe the NOESY correlation between them, these were assigned to take a cis-relationship because Hβ-4 afforded NOESY correlations with both H-2 and H-9. Another NOESY correlation between H-2 and H 2 -12 defined the relative configuration at C-11.
Tricocerapicanol C (1c) was found in the polar fraction eluted by silica gel column chromatography using 80% EtOAc/hexane. In the 1 H NMR spectrum (SI-24), this molecule afforded two sets of broad AB doublets at the medium frequency region (3.14 and 3.20 ppm, J = 10.3 Hz (H 2 -12); 3.38 and 3.60 ppm, J = 11.0 Hz (H 2 -14)), suggesting the existence of two hydroxymethyl groups in the molecule. A 2D NMR spectral analysis (SI-26, 27, and 28) allowed us to conclude that 1c is a C-14 hydroxylated congener of 1a, which is in accordance with the observed molecular ion at m/z 253.1802 (C 15 H 25 O 3 + , [M + H] + : 253.1798) in the ESI-TOFMS spectrum (SI-23). The NOESY signals between H-2 and H-12, H-2 and Hβ-4, and Hβ-5 and H-9 were diagnostic of the relative structure depicted in Figure 1. Although the C-5 resonance (29.7 ppm) was overlapped with the solvent signal (acetone-d 6 , 29.9 ppm), this signal was assigned on the basis of the HSQC and HMBC spectra (SI-27 and 28, respectively).
The 1 H NMR spectral data of tricoprotoilludene A (2a, SI-32) resembled those of known protoilludane violascensol 2b (SI-39), except for the absence of the H 2 -12 signal in 2a [30]. The C-12 resonance was observed at 182.0 ppm, suggesting that 2a is a carboxylic acid derivative of 2b. This was confirmed by ESI-TOFMS spectrum (m/z 249.1482, C 15  Since the extracts also afforded 2b, 2a can be assumed to possess the same stereochemical relationship as 2b by considering their biosynthesis. Hence, the prochiral methylene protons were tentatively assigned according to those of 2b. The proposed relative configuration was supported by the theoretical 13 C and 1 H chemical shifts obtained by density functional theory (DFT) calculations, as will be described later.
The Tricosterpurol (4) gave the largest ion at m/z 219.1760 indicating C 15 H 23 O + (calcd. 219.1743) in the ESI-TOFMS spectrum (SI-51). This ion was tentatively assigned as the dehydrated ion because two oxygenated carbon signals were observed at 73.4 and 70.5 ppm (C-4 and C-12, respectively) in the 13 C NMR spectrum (SI-53). Thus, C 15 H 24 O 2 can be assigned as the molecular formula of 4. The methylene protons H 2 -1 appeared at 2.07 and 2.25 ppm as broad AB doublets (SI-52), which showed HMBC correlations with the C-2 (140.1 ppm) and C-3 (126.6 ppm) sp 2 carbons. The COSY spectrum indicated a long-range spin coupling between H 2 -1 and H 3 -14 (SI-54), although the signal splittings were not observed in the regular one-dimensional 1 H NMR spectrum. These observations suggest a tetrasubstituted C-2/C-3 double bond. In the HMBC spectrum, the quaternary C-4 signal showed correlations with H 2 -5 and H 3 -14, and the other quaternary C-7 resonance (43.8 ppm) correlated with H 2 -6, H 2 -8, and H 3 -15, revealing the presence of a C-4/C-5/C-6/C-7 cyclobutane ring. This allowed us to assign a sterpurane framework for 4 [37]. The irradiation of H-9 (2.48 ppm) resulted in NOEs with Hβ-6 and H 2 -12, which unequivocally defined the relative configuration of 4 (SI-57). An analysis of the NOESY spectrum enabled the full assignment of the 1 H and 13 C NMR signals, except for the conformationally flexible prochiral H 2 -12 (SI-58).
Tricoilludin A (5) produced the largest ion at m/z 219.1738 in the ESI-TOFMS spectrum (SI-60), which corresponds to C 15 H 23 O + (calcd. 219.1743). Similarly to 4, the presence of two oxygenated carbon signals at 70.4 and 70.6 ppm in the 13 C NMR spectrum indicates that this signal can be assigned to the dehydrated molecular ion (SI-62). Accordingly, the molecular formula of 4 was determined to be C 15 H 24 O 2 . The 1 H NMR spectrum showed two characteristic sets of methylene proton signals at a low-frequency region (at 0.69 and 0.75 ppm (H 2 -4) and at 0.48 and 0.81 ppm (H 2 -5), SI-61) which showed the HMBC correlations with a quaternary carbon at 30.2 ppm (SI-65). The carbon atoms bonded to these protons resonated also at low frequency (5.7 and 7.7 ppm). These results reveal the presence of a spiro-cyclopropane ring (C-4/C-5/C-6) in the molecule. Since these cyclopropane methylene protons further correlated with quaternary carbons at 70.4 (C-7) and 125.2 ppm (C-3) in the HMBC spectrum, the cyclopropane ring can be assumed to be sandwiched between them. Further analysis of the 2D NMR spectra allowed establishing an illudane framework for 5, [38] as illustrated in Figure  . Furthermore, the 1 H NMR spectra of both compounds were similar (SI-83). Nevertheless, a notable difference was found; one of the oxymethylene signals at 3.57 ppm (Hα-15) of 7 showed a long-range coupling with Hα-8 (1.70 ppm, 4 J Hα-8/Hα-15 = 1.8 Hz), whereas a similar long-range coupling but between H-2 and Hβ-14 was observed for 6. Accordingly, 7 contains a cyclic ether between C-3 and C-15 instead of the ether linkage between C-7 and C-14 in 6. The HMBC spectrum supported the proposed relative structure (SI-87). The NOESY correlations observed between H-2 and H-9, H-2 and H 2 -12, and H-9 and Hβ-15 allowed establishing the relative configuration of 7 shown in Figure 2 (SI-88). Moreover, the NOESY signals between H R -4 and H 3 -14 and between H S -5 and Hα-1 enabled us to differentiate the prochiral H 2 -1, H 2 -4, and H 2 -5 methylene protons.
The relative structures of 1a-7 and known 2b and 8 were further investigated by DFT-based NMR chemical shift calculations because the variety of the framework of 1a-8 constituted interesting examples to evaluate the reliability of this methodology [39][40][41][42] and to clearly establish the relative structure of 2a. Calculations were performed with the NMR chemical shift calculation protocol equipped on Spartan'18 without changing the default settings (Hehre's protocol) [43]. The protocol features the chemical shift calculations using ωB97X-D/6-31G*, the evaluation of the energies of individual conformers with ωB97X-V/6-311+G(2df,2p)[6-311G*]//ωB97X-D/6-31G*, and the empirical correction based on the type of carbons, attached atoms, and bond lengths. Some molecules (1a, 1b, 4, and 5) were calculated using the ent-forms ((11S)-enantiomers) of the natural products because the chirality investigation was performed later. However, in this article, their NMR and electronic circular dichroism (ECD) properties are discussed after interpretation that the natural products are all (11R)-enantiomers for convenience. In the statistical analysis of 1 H NMR signals, the methylene protons of the proposed diastereomers were arranged according to the observed NOEs except for unassigned protons such as H 2 -12 of 1a-7 and H 2 -15 of 3, which were set to reduce the chemical shift difference with the calculated values. Similarly, all prochiral nuclei in other diastereomers were arranged so that the chemical shift difference with the calculated values was smaller. Note that the signals were arranged so that the incorrect structures became rather advantageous in the analysis. The calculated 1 H and 13 C chemical shifts were directly subjected to statistical and DP4 analysis without empirical correction [44], since Hehre's protocol involved more sophisticated corrections [43]. Although this protocol has been proved to be more accurate than Goodman's method [39], Goodman's standard deviation (σ: 2.306 and 0.185 ppm for δ 13 C and δ 1 H, respectively) and freedom (ν: 11.38 and 14.18 for δ 13 C and δ 1 H, respectively) were applied in the DP4 analysis [44] because these parameters have not been published for Hehre's protocol. Accordingly, the DP4 analysis in the present study tended to be slightly less sensitive toward structural differences than that using the appropriate parameters (SI-98-122). Table 2 summarizes the results. The proposed structures of 1a-8 afforded satisfying small root-mean-square of the deviation (RMSD) values for δ 13 C and δ 1 H from the experimental data. The δ 13 C DP4 probability for 6 was not the highest (47.9%), and the 11-epimer gave the best score (52.1%). However, the δ 13 C RMSD values for these isomers were small enough (1.1 and 1.0 ppm, respectively) when considering the average error of this protocol (2.0 ppm) [43]. In other words, the small difference of the calculated δ 13 C values between 6 and its 11-epimer hindered their differentiation. In contrast, the δ 1 H DP4 probability for 6 was quite high (98.5%), which compensated for the unsatisfactory results of the δ 13 C DP4 analysis. Although the relative configuration of 2a based on the spectroscopic analysis remained unclear, both δ 1 H and δ 13 C DP4 scores strongly supported the proposed structure (δ 13 C: 87.3%, δ 1 H: 99.9%, δ 13 C + δ 1 H: 100%). The 13 C + 1 H DP4 values were above 99% probability in all cases. It is worth noting that the δ 13 C DP4 probability of 8 was the highest and exclusive (99.9%) among the 32 possible diastereomers. As described above, these chemical shift calculations not only supported the relative structures of 1a-8 obtained by the spectral analysis but also demonstrated the efficiency of this methodology.
The calculation results must be discussed more holistically. The δ 1 H DP4 values generally gave higher scores than the δ 13 C DP4 values for the correct diastereomers of 1a-7. The 13 C chemical shift is known to depend on parameters such as the electronegativity of neighboring functional groups, bond angles, and bond lengths. Since the substituents methyl and hydroxymethyl groups at C-11 are located at the sterically less hindered end of the molecules, the effect of its geometrical difference on the conformations of the other moieties in 1a-7 and on the δ 13 C values can be considered insignificant. In contrast, the anisotropic effect of 12-OH significantly affects the magnetic shielding of the nearby hydrogen atoms. This effect is small for carbon nuclei probably because carbons are located inside the molecules and are usually shielded by hydrogen atoms. This would explain why only the 1 H chemical shifts are sensitive to the geometry of 12-OH. Despite the high δ 13 C DP4 score obtained for 8, its δ 1 H DP4 is less conclusive (30.7%). This result is usual in calculations of this type and demonstrates the difficulty of the stereostructural elucidation only based on δ 1 H DP4 values.  Then, the absolute configurations of the natural products were investigated. Tricocerapicanol A (1a) provided a characteristic negative Cotton effect (∆ε −5.7) at 297 nm attributed to the R-band (n→π * transition) of the C-5 carbonyl group in the ECD spectrum ( Figure 3, spectra A). This was reproduced by a B3LYP/def2-TZVP model [45,46] when the (11R)-enantiomer of 1 was applied ( Figure 3, spectra C, SI-124). However, the calculated and experimental spectra were considerably different in the 200-230 nm region. The Cotton effect in this region depended on the individual stable conformers (Figure 3, spectra D), whereas the negative Cotton effects at approximately 300 nm were constant regardless of the conformers, suggesting that the Cotton effect at approximately 300 nm is more reliable for the chirality assessment than that at approximately 200 nm. It is known that the octant rule can be applied for Cotton effects at the R-band of the ketone group [47]. The bicyclo[1-3]-8-octanone framework in 1a is highly symmetric; thus, this moiety is not responsible for the Cotton effect. Similarly, the Cotton effects of the 14-and 15-methyl groups should cancel each other's the Cotton effects. Meanwhile, Hα-1 (highlighted with a green sphere in the 3D and 2D models in Figure 3), which is located at the front right lower octant, could be expected to contribute to the negative Cotton effect at approximately 300 nm. This assumption was verified with the simplified virtual models I (the model without 12-CH 2 OH and 13-CH 3 ), II (the model in which the Hα-1 of model I was replaced with a chlorine atom), and III (the ∆ 1,12 model); the calculations of model I afforded a negative Cotton effect at approximately 300 nm similar to that of the experimental spectrum of 1a, and the intensity of the negative Cotton effect increased in model II because of the presence of a polarized C-Cl bond there, whereas that of model III was inversed because of the absence of Hα-1 (see SI-145), according to the calculations (SI-145, 146, and 147).
replaced with a chlorine atom), and III (the Δ 1,12 model); the calculations of model I afforded a negative Cotton effect at approximately 300 nm similar to that of the experimental spectrum of 1a, and the intensity of the negative Cotton effect increased in model II because of the presence of a polarized C-Cl bond there, whereas that of model III was inversed because of the absence of Hα-1 (see SI-145), according to the calculations (SI-145, 146, and 147).  1a, 1b, and 1c (spectra A and B), calculated ECD spectra (spectra C), calculated ECD spectra of the individual conformers of 1a (spectra D), three-dimensional structure of 1a, and structures of models I-III.
In contrast, 1b and 1c showed only faint, negative Cotton effects at approximately 300 nm (Figure 3, spectra A), which were moderately well reproduced by the calculations (Figure 3, spectra C, SI-126-131). Consequently, the 5-OH group of 1b, located at the rear left upper octant, contributes to the positive Cotton effect and cancels the negative Cotton effect observed in 1a. Although the reason for this observation remains unclear, the 14-  1a, 1b, and 1c (spectra A and B), calculated ECD spectra (spectra C), calculated ECD spectra of the individual conformers of 1a (spectra D), three-dimensional structure of 1a, and structures of models I-III.
In contrast, 1b and 1c showed only faint, negative Cotton effects at approximately 300 nm (Figure 3, spectra A), which were moderately well reproduced by the calculations (Figure 3, spectra C, SI-126-131). Consequently, the 5-OH group of 1b, located at the rear left upper octant, contributes to the positive Cotton effect and cancels the negative Cotton effect observed in 1a. Although the reason for this observation remains unclear, the 14-OH group of 1c likely weakens the negative Cotton effect observed in 1a. Note that the DFT calculations can describe these effects more quantitatively. On the basis of these results, it can be concluded that 1a, 1b, and 1c possess the (11R)-configuration.
Violascensol (2b) was first isolated by Vidari's group in 1998 and was reported to show a negative Cotton effect at 332 nm [30]. However, its absolute configuration could not be determined on the basis of this observation and was tentatively assigned as 11R for 2b according to its structural and biosynthetic resemblance to related natural products. In 2002, Ferlek mentioned that the general helicity rule for cisoidal enones is not applicable to 2b in their review discussing the chirotopical properties of cisoidal enones [32]. Both 2a and 2b exhibited negative Cotton effects at approximately 335 nm, which were in accordance with Vidari's report (Figure 4, spectra A and B), along with positive and negative Cotton effects at around 260 and 220 nm, respectively. All these Cotton effects were nicely reproduced for the (11R)-enantiomers of both 2a and 2b using a B3LYP/def2-TZVP model (SI-134-137). Interestingly, the simplified virtual model IV consisting of the substructure highlighted in red in 2a and 2b (Figure 4) also well reproduced the experimental ECD spectrum when the dihedral angle ∠O/C-5/C-6/C-7 was set to 15 • (SI-148 and 149), which was nearly identical to that of the stable conformer of 2a (the numbering followed that used for 2a). Consequently, the three Cotton effects observed in 2a and 2b are most likely caused by the chiral torsion of their cisoidal α,β-unsaturated ketone moiety.
Compounds 3, 4, and 5 weakly absorbed UV light at approximately 200 nm, which can be attributed to the K-band (π→π* transition) of their isolated double bonds ( Figure 5,  spectra A-C). Coincidentally, all these compounds showed positive Cotton effects at this wavelength region in their ECD spectra, which were well reproduced when the (11R)-enantiomers of 3-5 were subjected to ECD calculations (SI-136-142). Although these compounds involved more than 30 stable conformers within 10 kJ/mol from the global minimum, the 10 most stable conformers showed positive Cotton effects at around 210 nm in all cases (spectra D-F), which confirms the reliability of the above argument. In the UV/ECD spectral reproductions for 3, and the BHLYP functional provided a better match than B3LYP (SI-152), whereas the latter functional afforded satisfying UV/ECD spectra for other compounds. This highlights the necessity of using various functionals for a reliable elucidation of the chirality. The suggested chirality of 3 was consistent with that of 2a and 2b, which was expected from the viewpoint of their biosynthesis. For 3-5, attempts at applying Scott's empirical octant rule for olefins [48] were not conclusive.
2002, Ferlek mentioned that the general helicity rule for cisoidal enones is not applicable to 2b in their review discussing the chirotopical properties of cisoidal enones [32]. Both 2a and 2b exhibited negative Cotton effects at approximately 335 nm, which were in accordance with Vidari's report (Figure 4, spectra A and B), along with positive and negative Cotton effects at around 260 and 220 nm, respectively. All these Cotton effects were nicely reproduced for the (11R)-enantiomers of both 2a and 2b using a B3LYP/def2-TZVP model (SI-134-137). Interestingly, the simplified virtual model IV consisting of the substructure highlighted in red in 2a and 2b (Figure 4) also well reproduced the experimental ECD spectrum when the dihedral angle ∠O/C-5/C-6/C-7 was set to 15° (SI-148 and 149), which was nearly identical to that of the stable conformer of 2a (the numbering followed that used for 2a). Consequently, the three Cotton effects observed in 2a and 2b are most likely caused by the chiral torsion of their cisoidal α,β-unsaturated ketone moiety. Compounds 3, 4, and 5 weakly absorbed UV light at approximately 200 nm, which can be attributed to the K-band (π→π* transition) of their isolated double bonds ( Figure  5, spectra A-C). Coincidentally, all these compounds showed positive Cotton effects at this wavelength region in their ECD spectra, which were well reproduced when the (11R)enantiomers of 3-5 were subjected to ECD calculations (SI-136-142). Although these compounds involved more than 30 stable conformers within 10 kJ/mol from the global minimum, the 10 most stable conformers showed positive Cotton effects at around 210 nm in all cases (spectra D-F), which confirms the reliability of the above argument. In the UV/ECD spectral reproductions for 3, and the BHLYP functional provided a better match than B3LYP (SI-152), whereas the latter functional afforded satisfying UV/ECD spectra for other compounds. This highlights the necessity of using various functionals for a reliable elucidation of the chirality. The suggested chirality of 3 was consistent with that of 2a and 2b, which was expected from the viewpoint of their biosynthesis. For 3-5, attempts at applying Scott's empirical octant rule for olefins [48] were not conclusive. The absolute configuration of omphadiol (8) was established to be the (10R)-enantiomer in the enantioselective total synthesis reported by Romo [33]. Nevertheless, its 10-O-benzoate 8-OBz was independently investigated in the present study. Natural 8 The absolute configuration of omphadiol (8) was established to be the (10R)-enantiomer in the enantioselective total synthesis reported by Romo [33]. Nevertheless, its 10-Obenzoate 8-OBz was independently investigated in the present study. Natural 8 showed no remarkable Cotton effects at the UV/vis region, whereas 8-OBz showed a characteristic ECD spectrum, as shown in Figure 6. Such complexed Cotton effects most likely stem from the interaction of the π character of the cyclopropane ring [49] with the benzoyl chromophore. The experimental ECD spectrum was successfully reproduced by the DFT B3LYP/def2-TZVP functional (SI-143 and 144), confirming the (10R)-configuration. Interestingly, the experimental ECD spectrum of 8-OBz could be roughly reproduced with the simplified virtual model V, which only contains the substructure highlighted in red in 8-OBz (Figure 6), when the dihedral angle ∠C-7/C-9/C-10/O was set to 170 • , which is the angle of the corresponding atoms in the stable conformation of 8 (SI-150 and 151). These demonstrated that the DFT-based modeling calculation is an efficient tool not only for chirality determinations but also for revealing the major factors causing the Cotton effects. Although tricoilludins B (6) and C (7) do not possess either appropriate functio or chromophores for chirality elucidation, the (11R)-configuration could be assigned considering that they were isolated from the same culture broth as 5.
These molecules belong to the cyclohumulanoid family, and except for 8, conta C-1/C-2/C-9/C-10/C-11 cyclopentane ring sharing methyl (C-13) and hydroxymethyl 12) groups at C-11 with (R)-configuration, although the absolute configurations of 6 7 were not unequivocally established. A protoilludane derivative 10 can be envisage the key biosynthetic intermediate for the compounds obtained in the present study. toilludane 10 is likely derived from farnesyl pyrophosphate via humulenyl cation shown in Scheme 1. According to the established configurations, the oxidation of C occurs stereoselectively right after cyclization because all compounds except for 8 con an (11R)-hydroxymethyl group. Humulenyl cation 9 also produces 8 in another tand cyclization at C-7/C-9 and C-2/C-6; however, a different type of enzyme may be respo ble for this process [8,10]. Protoilludanes 2a, 2b, and 3 are obtained by oxidation(s) o Epoxide 11 can be proposed as a versatile intermediate yielding cerapicanes 1a-c, ster rane 4, and illudanes 5-7. An epoxide ring opening reaction at C-6 induces a ring cont tion of cyclobutene into a cyclopropane, affording illudanes 5-7. Another epoxide opening at C-7 promotes a ring expansion into cyclopentane, generating cerapican cation 12. Deprotonation at 5-OH affords ceapicanes 1a-1c, whereas another deproto tion at H-2 produces a concerted olefin formation and ring contraction into cyclobu to give tricosterpurol (4).
Finally, an antifungal assay was performed using Cochliobolus miyabeanus as the fungus to reveal that 2a and 5 simultaneously produced swelling and branching at a c centration of 5 μg/mL (SI-153), whereas other compounds did not show obvious inh tory effect even at higher concentrations. Although tricoilludins B (6) and C (7) do not possess either appropriate functionals or chromophores for chirality elucidation, the (11R)-configuration could be assigned by considering that they were isolated from the same culture broth as 5.
These molecules belong to the cyclohumulanoid family, and except for 8, contain a C-1/C-2/C-9/C-10/C-11 cyclopentane ring sharing methyl (C-13) and hydroxymethyl (C-12) groups at C-11 with (R)-configuration, although the absolute configurations of 6 and 7 were not unequivocally established. A protoilludane derivative 10 can be envisaged as the key biosynthetic intermediate for the compounds obtained in the present study. Protoilludane 10 is likely derived from farnesyl pyrophosphate via humulenyl cation 9 as shown in Scheme 1. According to the established configurations, the oxidation of C-12 occurs stereoselectively right after cyclization because all compounds except for 8 contain an (11R)-hydroxymethyl group. Humulenyl cation 9 also produces 8 in another tandem cyclization at C-7/C-9 and C-2/C-6; however, a different type of enzyme may be responsible for this process [8,10]. Protoilludanes 2a, 2b, and 3 are obtained by oxidation(s) of 10. Epoxide 11 can be proposed as a versatile intermediate yielding cerapicanes 1a-c, sterpurane 4, and illudanes 5-7. An epoxide ring opening reaction at C-6 induces a ring contraction of cyclobutene into a cyclopropane, affording illudanes 5-7. Another epoxide ring opening at C-7 promotes a ring expansion into cyclopentane, generating cerapicanium cation 12. Deprotonation at 5-OH affords ceapicanes 1a-1c, whereas another deprotonation at H-2 produces a concerted olefin formation and ring contraction into cyclobutane to give tricosterpurol (4).

Experimental
General Experimental Procedures. UV spectra were obtained on a HITACHI U-2010 spectrometer (Hitachi High-Tech, Tokyo, Japan) using a 10 mm length cell. ECD spectra were recorded on a JASCO J-1100 spectropolarimeter (JASCO Cooperation, Tokyo, Japan) with a 10 mm length cell. Fourier transform infrared spectroscopy was conducted using a HORIBA FT-720 spectrometer (Horiba Ltd, Kyoto, Japan) and a KBr cell. 1 H and 13 C NMR spectra were recorded on a JEOL JNM-ECX500 spectrometer ( 1 H: 500 MHz, 13 C: 125 MHz, (JEOL Ltd., Tokyo, Japan). Tetramethylsilane (0 ppm) was used as the internal standard for both types of spectra when CDCl3 was used as the solvent. When acetone-d6 was used, the signals of CHD2COCD3 (2.05 ppm) and 13 CD3COCD3 (29.92 ppm) were used as the internal standard for the 1 H and 13 C NMR spectra, respectively. ESI-TOFMS spectra were obtained using a HITACHI NanoFrontier LD spectrometer equipped with a HITACHI 2100 high-performance liquid chromatography (HPLC) pump, a HITACHI L-2420 UV detector (Hitachi High-Tech, Tokyo, Japan), and a HITACHI L-2300 column oven. Calibration was performed with a mixture of tetrabutylammonium ion (m/z 242. Finally, an antifungal assay was performed using Cochliobolus miyabeanus as the test fungus to reveal that 2a and 5 simultaneously produced swelling and branching at a concentration of 5 µg/mL (SI-153), whereas other compounds did not show obvious inhibitory effect even at higher concentrations.

Experimental
General Experimental Procedures. UV spectra were obtained on a HITACHI U-2010 spectrometer (Hitachi High-Tech, Tokyo, Japan) using a 10 mm length cell. ECD spectra were recorded on a JASCO J-1100 spectropolarimeter (JASCO Cooperation, Tokyo, Japan) with a 10 mm length cell. Fourier transform infrared spectroscopy was conducted using a HORIBA FT-720 spectrometer (Horiba Ltd, Kyoto, Japan) and a KBr cell. 1 H and 13 C NMR spectra were recorded on a JEOL JNM-ECX500 spectrometer ( 1 H: 500 MHz, 13 C: 125 MHz, (JEOL Ltd., Tokyo, Japan). Tetramethylsilane (0 ppm) was used as the internal standard for both types of spectra when CDCl 3 was used as the solvent. When acetone-d 6 was used, the signals of CHD 2 COCD 3 (2.05 ppm) and 13 CD 3 COCD 3 (29.92 ppm) were used as the internal standard for the 1 H and 13 C NMR spectra, respectively. ESI-TOFMS spectra were obtained using a HITACHI NanoFrontier LD spectrometer equipped with a HITACHI 2100 high-performance liquid chromatography (HPLC) pump, a HITACHI L-2420 UV detector (Hitachi High-Tech, Tokyo, Japan), and a HITACHI L-2300 column oven. Isolation. D. tricolor was cultured in a potato dextrose medium (200 mL in a 500 mL baffled Erlenmeyer flask × 45) on a rotary shaker (110 rpm) at 26 • C for 14 days. MeOH (150 mL) was then added to each flask to precipitate the fungus body. After filtration through cotton gauze, the MeOH of the combined filtrate was removed with a rotary evaporator. The resulting aqueous suspension was extracted with ethyl acetate (3 L × 3), and the organic layer was combined and concentrated under vacuum conditions to obtain the crude extracts (1.7 g). These operations were repeated to obtain the second crude extracts (1.6 g). The first crude extracts were diluted with EtOAc (100 mL), and then, silica gel (~6.0 g) was added. The resulting suspension was carefully concentrated under reduced pressure. The obtained residual powder was loaded on a silica gel column (300 g) and eluted with 0→100% EtOAc/hexane to give fraction A to fraction G. Fraction C eluted with 20-30% EtOAc/hexane in the first column chromatography (113 mg) was further subjected to ODS medium-pressure column chromatography (YAMAZENE ODS universal column type S, 50-100% MeOH/H 2 O for 20 min, flow rate 5 mL/min) to give 1a (11.6 mg). Fraction D eluted with 50-60% EtOAc/hexane in the first column chromatography (205 mg) was similarly subjected to the ODS medium-pressure column chromatography (YAMAZENE ODS universal column type S, 50-100% MeOH/H 2 O for 30 min, flow rate 5 mL/min) to give fractions D-1 to D-4. Fraction D-2 (35.0 mg) was subjected to a preparative silica gel TLC (30% EtOAc/hexane, two developments) to give 8 (1.5 mg, R f = 0.3), 2b (1.8 mg, R f = 0.45), and 2a (1.0 mg, R f = 0.2). Fraction E eluted with 70-80% EtOAc/hexane in the first column chromatography (186 mg) was further subjected to ODS medium-pressure column chromatography (YAMAZENE ODS universal column type S, 50-100% MeOH/H 2 O for 20 min, flow rate 5 mL/min) to give crude 1b (40 mg). Analytical 1b (10 mg) was obtained by preparative silica gel chromatography (70% EtOAc/hexane). The second crude extracts were also dispersed in silica gel (6.0 g) and subjected to silica gel column chromatography as described above to obtain fraction A to fraction J . Similar separations afforded 1a (13.4 mg) and 2a (1.4 mg). Fraction A eluted with 10% EtOAc/hexane (8.0 mg) was subjected to preparative silica gel chromatography (30% EtOAc/hexane) to obtain 5 (5.5 mg, R f = 0.3). Fraction J eluted with 80% EtOAc/hexane (434 mg) was dispersed on diatomaceous earth powder (1.0 g) in MeOH (20 mL), and the solvent was then carefully evaporated under reduced pressure. The obtained dried powder was placed in a small column and connected to a YAMAZENE ODS universal column type L, which was developed using gradient conditions (5→100% MeOH/H 2 O (containing 1% AcOH) for 250 min, flow rate 3 mL/min). The eluents were fractionated into 85 test tubes. Each fraction was checked with silica gel TLC and integrated into eight fractions (J -1 to J -8). Fraction J -2 eluted with 10-15% MeOH/H 2 O (22.7 mg) was further subjected to silica gel column chromatography (~5 g, 50% EtOAc/hexane) to yield 6 (1.2 mg).
Fraction   Physical data of 6: This compound showed no considerable absorption in the UV region. IR (film) 3400, 2923, 2869, 1025 cm −1 ; the 1 H and 13 C NMR data in CDCl 3 are shown in Table 1; ESI-TOFMS (rel. int (%), assignment) m/z 275.1623 (45, [M + Na] + : 275.1623), D/6-31G* model, followed by conformer narrowing by setting the threshold at 15 kJ/mol from the global minimum conformer; structural optimization with the ωB97X-D/6-31G* model, followed by conformer narrowing by setting the threshold at 10 kJ/mol from the global minimum conformer; energy estimation applying the ωB97X-V/6-311+G(2df,2p)[6-311G*] model, followed by conformer narrowing by setting the threshold at 10 kJ/mol from the global minimum conformer; chemical shift calculations using ωB97X-D/6-31G*, followed by the empirical correction [39]. The obtained chemical shifts were directly compared with the experimental data, and the result data (RMSD, maximum deviation, and DP4) were calculated using Microsoft Excel for Microsoft 365. The methylene protons of the proposed diastereomers except for H-12 were arranged according to the assignments on the basis of the NOE analysis, and H 2 -12 and all the methylene signals in other isomers were set to reduce the chemical shift difference with the calculated values. The obtained 1 H and 13 C chemical shifts were directly subjected to statistical analysis without corrections on the basis of the slopes and intercepts. In the DP4 analysis, the original parameters provided by Goodman's method (σ: 2.306 ppm for 13 C and 0.185 ppm for 1 H; ν: 11.38 for 13 C and 14.18 for 1 H) were used [44].
ECD spectral calculations. The stable conformer sets obtained in the chemical shift calculations were further optimized with B3LYP/def2-TZVP on TmoleX 2021. Only the most 10 stable conformers were subjected to the ECD calculations when more than 10 stable conformers within 10 kJ/mol from the global minimum conformers were obtained with ωB97X-V/6-311+G(2df,2p)[6-311G*//ωB97X-D/6-31G* (1b, 1c, 3, 4, 5, 6, and 7). After vibrational analysis, UV/ECD calculations were performed with B3LYP/def2-TZVP. The UV and ECD spectra of the individual conformers were constructed according to the frequencies, oscillator strength, and rotatory strengths using the NORM.DIST function in Microsoft Excel for Microsoft 365. The widths of the UV and ECD bands were set to reproduce the spectra appropriately. The UV/ECD spectra of the individual conformers were constructed by Boltzmann averaging on the basis of the free energy. The wavelengths of the UV spectra were corrected according to the experimental spectra, and those of the ECD spectra were corrected using the identical number. The ECD spectra of 1a, 1b, 4, and 5 were expressed as the values obtained after multiplying by −1.
Model I was constructed on Spartan'18 by removing nonessential atoms from the most stable conformer of 1a. For model II, Hα-1 of model I was replaced with chlorine. Model III was prepared from model I by introducing a double bond between C-1 and C-11. The resulting structures were then optimized with B3LYP/def2-TZVP and subjected to ECD calculations as described above. The most stable conformation of 8-OBz was obtained by performing a conformational search with MMFF, and the 6 stable conformers were subjected to the ECD calculations in a similar manner to that above. Model IV and model V were prepared by removing nonessential atoms from the most stable conformations of 2a and 8-OBz, respectively. The structures obtained by setting the dihedral angles ∠O/C-5/C-6/C-7 in model IV and ∠C-7/C-9/C-10/O in model V to 15 • and 170 • , respectively, were optimized with B3LYP/def2-TZVP by fixing the set torsions and then subjected to UV/ECD calculations. The calculated UV/ECD spectra were constructed similarly to those above.
Antifungal assay. A series of suspensions of spores of Cochliobolus miyabeanus (1.0 mL) containing 1, 5, 25, and 100 µg/mL of samples in Petri dishes were prepared and incubated at 25 • C for 24 h. Then, microscopic observation was performed using an Olympus CKX-41 binocular inverted microscope equipped with a ×10 objective lens and ×10 eyepieces.

Conclusions
In summary, nine cyclohumulanoids were successfully identified from D. tricolor. The present study proves the usefulness of DFT-based modeling calculations for the determination of the relative and absolute configurations of polycyclic terpenoids and the suitability of calculations using simplified models for elucidating the reasons behind the Cotton effects.