Koninginins X-Z, Three New Polyketides from Trichoderma koningiopsis SC-5

Koninginins X-Z (1–3), three novel polyketides, were isolated from the solid fermentation of the endophytic fungus Trichoderma koningiopsis SC-5. Their structures, including the absolute configurations, were comprehensively characterized by a combination of NMR spectroscopic methods, HRESIMS, 13C NMR, DFT GIAO 13C NMR, and electronic circular dichroism calculations as well as single crystal X-ray diffraction. In addition, all the compounds were evaluated for antifungal activity against Candida albicans.


Introduction
Plant pathogens are ubiquitous microorganisms that have profound impacts on the agricultural, industrial, pharmaceutical, and medical domains [1].Beyond causing crop diseases, they play a dual role by also serving as biological control agents in agriculture, safeguarding crops [2,3].Their unique characteristics enable applications in biofuel and industrial chemical production, while their secondary metabolites, rich in biological activities, hold promise for new drug developments in medicine [4,5].The genus Trichoderma, a widely distributed fungal group in nature, mainly exists in the ecological environment of plant inter-root soil, foliage, seeds, and bulbs.It stands as the most extensively researched and utilized phytopathogenic antagonist fungus among biopreventive fungi employed for plant disease control [6,7].Notably, Trichoderma is one of the best-represented plant antagonistic fungi, contributing to biological control and the discovery of new natural products, such as polyketides, alkaloids, and terpenoids [8][9][10][11][12].Koninginin derivatives, a kind of typical polyketides produced by the species of the Trichoderma genus, most of which share an intriguing bicyclic pyran skeleton with a characteristic hemiketal or ketal moiety [13][14][15][16][17][18][19][20][21][22], have successfully aroused our keen interest in medicinal discoveries.
During our continuing search for novel microbial natural products [23][24][25][26][27], we have previously reported two novel koninginin derivatives sharing unprecedented nitrogenous fused polycyclic polyketide skeletons from Trichoderma koningiopsis SC-5 [28].With the examination of other similar structurally unique and biologically meaningful koninginins, the further chemical investigation of Trichoderma koningiopsis SC-5 successfully led to the the examination of other similar structurally unique and biologically meaningful koninginins, the further chemical investigation of Trichoderma koningiopsis SC-5 successfully led to the isolation of three novel koninginin derivatives, koninginins X-Z (1-3) (Figure 1).Herein, the isolation, structural elucidation, and antifungal activity evaluation of these three isolates are described in detail.
Molecules 2023, 28, 7848 2 of 12 the examination of other similar structurally unique and biologically meaningful koninginins, the further chemical investigation of Trichoderma koningiopsis SC-5 successfully led to the isolation of three novel koninginin derivatives, koninginins X-Z (1-3) (Figure 1).Herein, the isolation, structural elucidation, and antifungal activity evaluation of these three isolates are described in detail.
The planar structure of 1 was established by a thorough analysis of its 1 H-1 H COSY and HMBC spectra (Figure 2).Firstly, two spin-spin coupling segments of H2-2/H2-3/H-4 and H-7/H-8/H-9/H-10/H2-11/H2-12/H2-13/H2-14/H2-15/H3-16 were observed in the 1  Therefore, the planar structure of 1 was completely illustrated, and it was recommended to share an identical framework with that of trichodermaketone B [9].Similarly, an obvious negative Cotton effect at 286 nm (∆ε −3.26) was observed in the CD spectrum of 1, which informatively suggested the absolute configuration to be 4R [9].In addition, compound 1 possessed the same relative configuration as that of trichodermaketone B, referring to the key NOE correlations (Figure 2), while the absolute configuration has not been completely identified.The chemical structure of 1 was further affirmed by 13 C NMR calculation, and the 13 C NMR calculated data for the 4R*,7R*,8R*,9R*,10S*-1 conformer showed a correlation coefficient (R 2 ) of 0.9991 (Figure 3) and a small mean absolute error (MAE) of 1.15.Moreover, a minute root mean square (RMS) of 1.60 and the critical Prel value of 100% were also obtained for the 4R*,7R*,8R*,9R*,10S*-1 conformer, which thus further evidenced the probability of the calculated result (Table 2).Subsequently, we determined the absolute configuration of 1 by theoretical ECD calculation, and its absolute configuration was finally revealed to be 4R,7R,8R,9R,10S, as depicted in Figure 4. Consequently, the absolute configuration of 1 was absolutely elucidated and given a trivial name, (4R,7R,8R,9R,10S)-koninginin X.
Compound 2 was gathered as a stereoisomer of 1 and had the same molecular formula, C 16 H 24 O 5 , determined by HRESIMS-(+) analysis (m/z 297.1710 [M + H] + and 319.1530 [M + Na] + ).The NMR data (Table 1) of 2 and key 1 H-1 H COSY and HMBC correlations (Figure 2) also demonstrated that both compounds should share the same planar structure.However, according to the 13 C NMR spectrum, the chemical shift of C-2 at δ C 35.6 in trichodermaketone B was upshifted to δ C 22.5 in 2, while the chemical shift of C-4 at δ C 63.6 in trichodermaketone B was downshifted to δ C 71.7 in 2; the aforementioned informative results tentatively indicated the relative configuration of C-4 of 2 might differ from that of the known trichodermaketone B [9,29,30].Interestingly, the CD experiment of 2 showed a clear positive Cotton effect for the n → π* transition at 302 nm (∆ε + 3.25), which further suggested the absolute configuration for 2 to be 4S and consolidated the aforementioned deduction.Notably, the 4S configuration of 2 was absolutely opposite to 4R for the known compounds, trichodermaketones A and B [9].Furthermore, in the NOE difference experiments (Figure 2), the irradiation of H-8 (δ H 5.05) led to the enhancement of H-7 (δ H 5.65) and H-9 (δ H 4.16), the irradiation of H-7 (δ H 5.65) led to the enhancement of H-8 (δ H 5.05) and H-9 (δ H 4.16), and the irradiation of H-10 (δ H 3.43) resulted in the enhancement of H-9 (δ H 4.16).Based on these NOE signals, the chiral protons of H-7, H-8, and H-9 were then suggested to be on the same side, while H-10 should be on the other orientation.Therefore, the relative configuration of 2 was completely established.
Subsequently, with the aim to verify the relative configuration and determine the absolute configuration of 2, the time-dependent density functional (TDDFT)-ECD calculations in Gaussian 16 were applied.In Figure 4, the calculated ECD curve of 4S,7S,8S,9S,10S-2 was consistent with that of the experimental one by a close comparison, thus ambiguously suggesting that the absolute configuration of 2 was 4S,7S,8S,9S,10S.As a result, the structure of 2 was fully determined and given the trivial name, (4S,7S,8S,9S,10S)-koninginin Y.
Compound 3 was obtained as a yellow oil; its molecular formula was confirmed as C 18   Then, a detailed comparison of the NMR data of 3 with those of 7-O-methylkoninginin D [9], a koninginin derivative isolated from the marine fungus Trichoderma koningii, implied that 3 should also be a closely similar derivative of koninginin D. The NMR data for both compounds are very similar, excepting that the methoxy group [δ C 59.6 (C-17)] in 7-Omethylkoninginin D was changed to be an ethoxy group [δ C 65.4 (C-17) and δ C 16.0 (C-18)] in 3.Moreover, the cross peak between H 2 -17 and H 3 -18 in the 1 H-1 H COSY spectrum, together with the critical HMBC correlations from H 3 -18 to C-17 and H 2 -17 to C-7, further strengthened the presence of the ethoxy functional group and suggested its location at the C-7 position.Moreover, other observed HMBC correlations and 1 H-1 H COSY cross peaks (Figure 2) further established the planar structure of 3.
Nevertheless, the relative configuration of compound 3 was unable to be established by the NOESY experiment, because there were no diagnosable correlative signals obviously observed, and the configuration of 7-O-methylkoninginin D was previously determined from an inference of biogenetic perspective [9].Then, the J values of 1 H NMR were used to determine the relative configuration of 3 [29][30][31].In the 1 H NMR spectrum, the coupling constants for H-7/H-8α with J = 2.4 Hz, H-8α/H-8β with J = 14.4 Hz, and H-9/H-8β with J = 2.4 Hz were essentially located H-7 and H-9 in a trans-diequatorial relationship.Additionally, the coupling constant (J = 12.6 Hz) for H-10/H-9 also indicated H-9 and H-10 in a trans-diequatorial relationship.Moreover, a negative Cotton effect at 286 nm (∆ε − 2.05) for the n → π* transition in the CD spectrum of 3 also suggested the absolute configuration of 4R [9,32].
Subsequently, the absolute configuration of 3 was further confirmed by comparison of the experimental and the simulated circular dichroism (CD) spectra.As shown in Figure 4, the calculated ECD curve of 4R,8R,9S,10S-3 was perfectly matched with the experimental one, thus strongly illustrating that the absolute configuration of 3 was 4R,8R,9S,10S.Satisfactorily, the absolute configuration of 3 with 4R,8R,9S,10S was further ambiguously verified by single crystal X-ray diffraction with a Flack parameter of 0.15 (11) (Figure 5), Therefore, the absolute configuration of compound 3 was completely determined and given the trivial name, (4R,8R,9S,10S)-koninginin Z.
inginin D [9], a koninginin derivative isolated from the marine fungus Trichoderma koningii, implied that 3 should also be a closely similar derivative of koninginin D. The NMR data for both compounds are very similar, excepting that the methoxy group [δC 59.6 (C-17)] in 7-O-methylkoninginin D was changed to be an ethoxy group [δC 65.4 (C-17) and δC 16.0 (C-18)] in 3.Moreover, the cross peak between H2-17 and H3-18 in the 1 H-1 H COSY spectrum, together with the critical HMBC correlations from H3-18 to C-17 and H2-17 to C-7, further strengthened the presence of the ethoxy functional group and suggested its location at the C-7 position.Moreover, other observed HMBC correlations and 1 H-1 H COSY cross peaks (Figure 2) further established the planar structure of 3.
Nevertheless, the relative configuration of compound 3 was unable to be established by the NOESY experiment, because there were no diagnosable correlative signals obviously observed, and the configuration of 7-O-methylkoninginin D was previously determined from an inference of biogenetic perspective [9].Then, the J values of 1 H NMR were used to determine the relative configuration of 3 [29][30][31].In the 1 H NMR spectrum, the coupling constants for H-7/H-8α with J = 2.4 Hz, H-8α/H-8β with J = 14.4 Hz, and H-9/H-8β with J = 2.4 Hz were essentially located H-7 and H-9 in a trans-diequatorial relationship.Additionally, the coupling constant (J = 12.6 Hz) for H-10/H-9 also indicated H-9 and H-10 in a trans-diequatorial relationship.Moreover, a negative Cotton effect at 286 nm (∆ε − 2.05) for the n → π* transition in the CD spectrum of 3 also suggested the absolute configuration of 4R [9,32].
Subsequently, the absolute configuration of 3 was further confirmed by comparison of the experimental and the simulated circular dichroism (CD) spectra.As shown in Figure 4, the calculated ECD curve of 4R,8R,9S,10S-3 was perfectly matched with the experimental one, thus strongly illustrating that the absolute configuration of 3 was 4R,8R,9S,10S.Satisfactorily, the absolute configuration of 3 with 4R,8R,9S,10S was further ambiguously verified by single crystal X-ray diffraction with a Flack parameter of 0.15 (11) (Figure 5), Therefore, the absolute configuration of compound 3 was completely determined and given the trivial name, (4R,8R,9S,10S)-koninginin Z.  Conclusively, the type of chemical structure for the koninginin family was first reported in 1989 [33]; nowadays, it has been increasingly arousing the great interest of many pharmacists with an aim to discover novel lead drugs with structurally fascinating skeletons and biologically significant activities [10,17,21], which thus boosts the reporting of novel compounds in the koninginin family [9][10][11][12]28].However, the intractable challenge and imperfect issue clouding the koninginin family is that the configurations for many chemical structures had not been thoroughly solved [9,18,21].In this research, the chemical structures, including the absolute configuration of these novel isolates, were comprehensively characterized with the help of the combination of NMR spectroscopic methods, HRESIMS, electronic circular dichroism calculations, and single crystal X-ray diffraction.We believe that these reliable methodologies towards efficient structure resolution in this study could act as a promisingly applicable strategy to resolve the configuration determination of other koninginin derivatives.
Notably, the genus Trichoderma has been revealed in recent years to be a high producer of natural products [34].Polyketides are the critical characteristic chemical constituents for the genus Trichoderma and reported extensively.They usually share a variety of intriguing structures and novel carbon-chain skeletons [34].As exemplified by the sorbicillinoid-based compounds, saturnispols A-H, all of them share a novel cyclic hexaketide nucleus and a typical sorbyl sidechain in their chemical scaffold [35].Moreover, trichorenins A-C with a unique tetracyclic carbon skeleton were also discovered from the genus Trichoderma [36], and tandyukisins G-I possess an attractive chromone core with a natural rarely occurring 4-oxo-4H-1-benzopyran scaffold representing a class of unprecedented polyketides that are widely distributed in Trichoderma sp.[37].Although the genus Trichoderma has been widely studied due to the diversity of natural environmental systems, the genus Trichoderma parasitized by different biological groups is likely to produce more natural products with novel structures and significant biological activities under the influence of an environmental host and their own evolution [35,38,39].Therefore, the genus Trichoderma is a significantly promising strategic bio-resource for the excavation of structurally novel lead natural products, and further extensive research efforts on their chemical constituents are still called upon for their great research value and spacious application prospect.
In the previously reported literature [18,19,40], the natural products derived from the genus Trichoderma usually exhibit considerable antifungal activity against a variety of different pathogenic fungi.For example, trichodermatide B from Emericella nidulans showed significant antifungal activity against Cryptococcus neoformans, with an IC 50 value of 4.9 µg/mL [37].Therefore, compounds 1-3 were further evaluated for antifungal activity against Candida albicans, where they did not exhibit any notable antifungal effect at the concentration of 100 µg/mL.Notably, we evaluated the antibacterial and cytotoxic activities of the isolated koninginin compounds from Trichoderma koningiopsis SC-5 in our previous experiments [28], and none of them showed any significant activity.In the future, more pathogenic fungi should be considered to evaluate the antifungal effects of these koninginin compounds, and many other biological activities such as antiviral and anti-inflammatory activities also worthy of testing.

General Experimental Procedures
The HRESIMS analyses were carried out on an Agilent 6500 series Q-TOF mass spectrometer (Agilent, Singapore) in the positive ion mode.The 1D and 2D NMR data were measured on a Bruker AVIII-600 spectrometer (Bruker, Karlsruhe, Germany) using TMS as an internal standard.Optical rotations were recorded on an Anton Paar MCP-500 spectropolarimeter (Anton Paar, Graz, Austria).Experimental ECD spectra were acquired from Applied PhotoPhysics' Chiascan circular dichroism spectrometer (APL, Surrey, UK).The single crystal data were collected on an Agilent Xcalibur Novasingle crystal diffractometer equipped with CuKα radiation.The reversed-phase preparative HPLC was conducted using an Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) instrument connected to an Innoval ODS-2 column (5 µm, 10 mm × 250 mm) with a VWD detector.The UV spectra were recorded with the use of a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan).Silica gel (Qingdao Marine Chemical Factory, Qingdao, China), macroporous resin (Hebei, China), Sephadex LH-20 (Toyopearl Tosoh, Tokyo, Japan), and octadecylsilane reversed-phase silica gel (50 µm, Fuji, Kasugai, Japan) were utilized for column chromatography.All the solvents were of analytical grade.

Fungal Material
The strain of Trichoderma koningiopsis SC-5 was isolated from Pedicularis integrifolia in Li County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province.It was collected in July 2021.This endophytic strain was subsequently identified by the sequence analysis of the rDNA ITS region, and the sequence of its rDNA ITS region was submitted to GenBank (Accession: OP646773).This endophytic fungus is preserved in the Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South University.

Fermentation, Extraction, and Isolation
The fungus was cultured in autoclaved rice solid medium (120 × 500 mL Erlenmeyer flasks, each containing 100 g of grain and 120 mL of ultrapure water).The fermentation was performed under static conditions at a constant temperature of 28 • C for 4 weeks.The fungal culture (5 kg) was extracted with EtOAc (10 L) three times, and the extract was subsequently decompression-concentrated to provide 110.0 g crude products.

Quantum Chemistry Calculations
A conformational search was performed by Crest [41], followed by optimization on a GFN2-xTB [42] level with a 4 kcal/mol energy window to remove high-energy conformers.The optimization and frequency calculation of each conformer was implemented on a B3LYP-D3(BJ)/TZVP [43,44] level of theory by using a Gaussian 16 software package with the keyword: g09defaults [45].The DFT GIAO 13 C NMR calculation was performed on the ωB97x-D/6-31G* level [46], and the data processing followed the reported STS protocol [47].The ECD calculations were by TDDFT on the ωB97x-D/TZVP level of theory and were Boltzmann-averaged according to the Gibbs free energy.SpecDis v1.71 [48] was used to simulate the ECD curves of compounds 1-3, with sigma/gamma values of 0.30 eV, 0.30 eV, and 0.40 eV, respectively.The averaged calculated ECD curves of compounds 1-2 were adjusted by blue shifting for 15 nm and 10 nm, respectively.

Figure 3 .
Figure 3. Regression analyses of experimental and calculated 13 C NMR chemical shifts of 1.

Figure 5 .
Figure 5. ORTEP drawing of the X-ray structure of 3.

Figure 5 .
Figure 5. ORTEP drawing of the X-ray structure of 3.
a Record in CDCl 3 , 600 MHz for 1 H, and 150 MHz for13C, δ in ppm.b Record in CD 3 OD, 600 MHz for 1 H, and 150 MHz for13C, δ in ppm.

Table 2 .
Calculated13C chemical shifts (CDCl 3 ) of structure 1 fitting to the experimental data of compound 1.
a Mean absolute error.b Root mean square.c Absolute deviation of calcd.δ C .