Combining the Elicitor Up-Regulated Production of Unusual Linear Diterpene-Derived Variants for an In-Depth Assessment of the Application Value and Risk of the Medicinal and Edible Basidiomycete Schizophyllum commune

To better assess the practical value and avoid potential risks of the traditionally medicinal and edible basidiomycete Schizophyllum commune, which may arise from undescribed metabolites, a combination of elicitors was introduced for the first time to discover products from cryptic and low-expressed gene clusters under laboratory cultivation. Treating S. commune NJFU21 with the combination of five elicitors led to the upregulated production of a class of unusual linear diterpene-derived variants, including eleven new ones (1–11), along with three known ones (12–14). The structures and stereochemistry were determined by 1D and 2D NMR, HRESIMS, ECD, OR and VCD calculations. Notably, the elongation terminus of all the diterpenes was decorated by an unusual butenedioic acid moiety. Compound 1 was a rare monocyclic diterpene, while 2–6 possessed a tetrahydrofuran moiety. The truncated metabolites 4, 5 and 13 belong to the trinorditerpenes. All the diterpenes displayed approximately 70% scavenging of hydroxyl radicals at 50 μM and null cytotoxic activity at 10 μM. In addition, compound 1 exhibited potent antifungal activity against the plant pathogenic fungi Colletotrichum camelliae, with MIC values of 8 μg/mL. Our findings indicated that this class of diterpenes could provide valuable protectants for cosmetic ingredients and the lead compounds for agricultural fungicide development.


Introduction
The basidiomycete Schizophyllum commune has generated considerable attention as a kind of valuable medicinal and edible mushroom [1][2][3].The species S. commune is wellknown for being rich in highly nutritious supplements, such as a wealth of amino acids and essential trace elements for the human body, high-quality protein, etc., and thus it is unusually regarded as a delicious dish [4].S. commune has also shown great potential for drug development, particularly in the area of macromolecular exopolysaccharide substances [1].
A notable example is schizophyllan, an exopolysaccharide used as a biological-response modifier in combination with chemo and radiation therapy [4].
In 2023, the crude extract of S. commune was approved as a cosmetic ingredient for use as a skin moisturizer and protectant [5], wherein the exopolysaccharide plays a crucial role, by the China National Regulatory Commission.Unlike macromolecules in higher fungi, the potential applications of small molecules remain a mystery [6][7][8].Currently, according to available bioinformatics data, this extremophilic fungus is known to encode at least 19 biosynthetic gene clusters, including those related to terpenes, non-ribosomal peptide synthetases (NRPSs), etc. [9].To date, only four classes of small molecules, including terpenoids [10], alkaloids [4,11], aromatics [12] and polyketides [13], have been identified.It is evident that most chemotypes of small molecules, encoded by gene clusters that are cryptic and low-expressed, have not yet been characterized.Given the multiple values in drug development, and the uses of this organism as an edible mushroom and a raw material for cosmetics, it is necessary to explore the yet-undiscovered small molecules.It is also important to comprehensively assess the in-depth potentials and risks associated with the safety and new applications of higher fungi.
A variety of methods have been applied to activate natural products, including One Strain Many Compounds (OSMACs) [14,15], co-culture [16], chemical epigenetic regulation [17,18] and heterologous expression [19] methods, and so on.The High-Throughput Elicitors Screening (HITES) strategy is another highly effective approach for activating silent gene clusters.It offers wide applicability and represents a cutting-edge method for activating microbial secondary metabolites.Initially applied primarily to actinomycetes and bacteria, the use of HITES in fungi was first reported by Professor Mohammod in 2022 [20].During the elicitor screening process, single-factor experiments are typically employed, which tend to be time-consuming and not conducive to high-throughput screening.The utilization of combined elicitors might offer a higher probability of activating new substances.This approach also has the potential to enhance efficiency when screening a large number of microorganisms.However, the application of a combinatorial elicitor strategy in microbial screening has not yet been reported.
Herein, we developed a combination of elicitors to significantly enhance the likelihood of discovering previously undescribed small molecules in fungi.This approach was applied to activate and up-regulate the production of small molecules in S. commune NJFU21.As a result, a series of linear diterpene-derived variants 1-14 (Figure 1), were successfully produced in higher quantities.These small molecules enable us to more thoroughly investigate the practical value and assess the potential risks associated with the small molecules from S. commune, as well as their biological functions.

Selection and Screening
Previous studies have provided sufficient evidence that a single elicitor could activate the expression of metabolites of a cryptic or extremely low-expressed gene cluster.Thus, it is logically hypothesized that elicitors regulating gene expression in various manners could synergistically affect each other; the combination strategy of elicitors was able to activate and up-regulate the metabolite production as much as possible, once.
To verify the hypothesis, five function-verified elicitors-Sodium Laurate, Aniline, p-Trifluoromethylaniline, Vorinostat (SAHA) and 5-Azacytidine (5-Aza) were selected and eight concentration gradients were set to acquire the suitable work concentration of each

Selection and Screening
Previous studies have provided sufficient evidence that a single elicitor could activate the expression of metabolites of a cryptic or extremely low-expressed gene cluster.Thus, it is logically hypothesized that elicitors regulating gene expression in various manners could synergistically affect each other; the combination strategy of elicitors was able to activate and up-regulate the metabolite production as much as possible, once.
To verify the hypothesis, five function-verified elicitors-Sodium Laurate, Aniline, p-Trifluoromethylaniline, Vorinostat (SAHA) and 5-Azacytidine (5-Aza) were selected and eight concentration gradients were set to acquire the suitable work concentration of each elicitor and the universal applicability for most fungi.The mycelium growth and changes in secondary metabolite profiles were considered as indication signs.The change in secondary metabolite profile was analyzed by HPLC.As results, 0.1 mM of sodium laurate, aniline, and p-trifluoromethylaniline, respectively, and 0.075 mM of SAHA and 0.05 mM of 5-Azacytidine were determined as the suitable work concentration at which each elicitor does not affect the growth of fungi.When the combination of the five elicitors with individual concentrations was added into the culture of S. commune NJFU21, a series of new peaks with similar UV absorption appeared by HPLC analysis, indicating a class of secondary metabolites which was unregulated (Figure 2).
Schizostatin F (5) was obtained as yellow oil.The molecular formula was determined as C 17 H 26 O 6 based on the HR-ESI-MS ions at m/z 327.1800 ([M + H] + , calcd for C 17 H 27 O 6 , 327.1802).Detailed analyses of its 1 H and 13 C NMR (Tables 1 and 2) and 2D NMR spectra revealed the structure of 5 was almost identical to that of 4. The only difference between 5 and 4 is that the double-bond group of C-9/C-10 in 4 disappeared and was replaced by the OH-10 group at C-10 in 5.The total E-geometry of the double bond of C-6/C-7 was confirmed by key NOESY correlations of H 3 -19/H 2 -5 and H-6/H 2 -8 (Figure 4).Thus, 5 was also a truncated and tetrahydrofuran-containing monocyclic diterpene.However, the Mosher reaction was not carried out, so that the absolute configuration of OH-10 was not determined.Considering the common biosynthesis between 5 and 2, the absolute configuration of C-11 in 5 was proposed to be S.
cm −1 showed that the configuration S is more consistent with the experimental v [21,22] (Figure 6).Thus, the absolute configuration of 4 was determined as 2E, 6E 11S.Schizostatin F (5) was obtained as yellow oil.The molecular formula was determ as C17H26O6 based on the HR-ESI-MS ions at m/z 327.1800 ([M + H] + , calcd for C17H 327.1802).Detailed analyses of its 1 H and 13 C NMR (Tables 1 and 2) and 2D NMR sp revealed the structure of 5 was almost identical to that of 4. The only difference bet 5 and 4 is that the double-bond group of C-9/C-10 in 4 disappeared and was replac the OH-10 group at C-10 in 5.The total E-geometry of the double bond of C-6/Cconfirmed by key NOESY correlations of H3-19/H2-5 and H-6/H2-8 (Figure 4).Thus, also a truncated and tetrahydrofuran-containing monocyclic diterpene.Howeve Mosher reaction was not carried out, so that the absolute configuration of OH-10 wa determined.Considering the common biosynthesis between 5 and 2, the absolute co uration of C-11 in 5 was proposed to be S.  1 and 2) revealed the structure of 6 was similar to that of 14.The main difference between NMR data 6 and 14 is that the methyl group of 14 is replaced with oxygenated methylene carbon at the C-18 position, and the sp 3 methylene group at the C-13 position is replaced by oxygenated methine carbon.The HMBC correlations (Figure 3) from All the compounds 7-11 were observed to contain twenty carbons and are noncyclic, as deduced from their HRESIMS and 1D NMR spectra (Tables 1 and 2), indicative of the fact that they possessed an unabridged linear diterpene-type skeleton.The detailed interpretation of 2D NMR spectra of 7-11 established the identical fragments from C-1 to C-9, including the existence of the butenedioic acid moiety, and the E-configuration of the double bond of C-6/C-7 (Figure 4).The structures of 7-11 should be oxidized variants of schizostatin A ( 14), a potent squalene synthase inhibitor which was also co-isolated from the fungus.
Schizostatin H (7) and schizostatin I (8) were obtained as yellow oil and were found to be  1 and 2), except for the replacement of the double bond of C-14/C-15 in 15 with the pinacol group in 7, evidenced by the key HMBC correlations from H 3 -17 (δ C/H 25.0/1.12)/H 3 -16 (δ C/H 25.6/1.15) to C-15 (δ C 73.8) and C-14 (δ C 79.1), as well as the COSY correlations of H 2 -12/H 2 -13/H-14 (Figure 3).The structure of schizostatin I (8) was determined to be very similar to that of 7, except for the loss of the signal corresponding to the methylene carbon at C-12, and replacement with an oxygenated methine carbon in 8.It was confirmed by correlations of H-12/H 2 -13/H-14 in the COSY spectrum and of Me-18 (δ C/H 11.0/1.61)with C-10 (δ C 128.2), C-11 (δ C 137.0) and C-12 (δ C 78.0) in the HMBC spectrum.The absolute configuration of C-14 in both 7 and 8 was proposed as S, which was deduced by the biosynthetically related product 2.
The molecular formula of C 20 H 32 O 6 of schizostatin J (9) was same as that of Schizostatin H (7). Comparison of NMR data between 9 and 7 indicated that the structure of 9 is similar to that of 7. The difference is that the pinacol group was positioned at C-10 and C-11 and the double bond at C-14/C-15 remained, supported by the related COSY and HMBC correlations.Due to the limited amount, the absolute configuration of C-10 and C-11 was unresolved.
The molecular formula of C 20 H 30 O 5 of schizostatin L (11) was the same as that of 12.Comparison of NMR data between 11 and 12 indicated that the structure of 11 was almost identical to that of 12, with the difference in chemical shifts of C-16 (δ C/H 61.4/4.05 for 11 vs. δ C/H 69.0/3.9 for 12) and C-17 (δ C/H 21.5/1.75 for 11 vs. δ C/H 13.7/1.6 for 12).The strong NOESY correlations of H 3 -17/H-14 and H 2 -16/H 2 -13 allowed us to assign the Z-configuration of C-14/C-15.
Compounds 1-14 were tested for cytotoxic, antimicrobial and OH scavenging activities.None of them displayed any cytotoxic activity at 10 µM.Compound 1 showed potent antifungal activity against plant pathogenic fungi Colletotrichum camelliae with MIC values of 8 µg/mL, while others did not exhibit any antifungal activity at a concentration of 64 µg/mL.
At a concentration of 50 µM, the inhibition rates of compounds 1-14 ranged from 42.3% to 76.0%.For comparison, the inhibition rate of the positive control, V C, was 81.69%, which is shown in Table 4.The hydroxyl radical scavenging data suggested that compounds 1-14 are beneficial for the interest in preservation of foodstuffs, drug products and cosmetics.

Fungi Materials
The strain NJFU21 was isolated from the larval intestines of the cutworm, which was collected from the Zi Jin Mountain in Nanjing, Jiangsu province, China, in 2020.The voucher specimen has been deposited in our laboratory collection.The fungus was identified as Schizophyllum commune through sequence-based rDNA ITS region analysis, as evidence by its GenBank accession number OP761877 (Supporting Information).

The Screening of Elicitors
Five elicitors with various regulatory mechanisms were selected in the study, including sodium laurate, SAHA, 5-Aza, p-(trifluoromethyl)aniline and aniline.Each elicitor was screened individually to determine a suitable concentration for S. commune NJFU21, acting as a control.Following this, the selected elicitors were combined and added into the fermentation of S. commune NJFU21 in the suitable concentration, individually.HPLC analysis was conducted to analyze and compare the changes in the metabolites of S. commune NJFU21 among themselves.

Fermentation and Extraction
S. commune NJFU21 was cultured on potato dextrose agar at 28 • C for 7 days.Fresh mycelium blocks (3 mm × 3 mm, 1 block per flask) were then added to seventy 1000 mL Erlenmeyer flasks, each containing 80.0 g of rice, 120 mL of H 2 O, and a combination of five selected elicitors: 0.1 mM (Sodium Laurate, Aniline and p-Trifluoromethylaniline), 0.075 mM (SAHA), and 0.05 mM (5-Aza).After 30 days of cultivation, the secondary metabolites were extracted with an equal volume of ethyl acetate, three times.The ethyl acetate extract was then concentrated under reduced pressure to yield 60.4 g of crude extract.

Electric Circular Dichroism (ECD) Calculation Assay
Conformational searches were run employing the 'systematic' procedure implemented in Spartan'14, based on the MMFF (Merck Molecular Force Field).All conformers were further optimized with Density functional theory (DFT) calculations at the B3LYP/6-31+G(d) level by using the Gaussian 09 program [25].

Regarding the Optical Rotation (OR) Calculation Assay
For OR computation, all conformers of compound 6 were first optimized at the B3LYP/6-31+G(d) in MeOH (PCM).DFT at the B3LYP/6-31+G(d) level in Gaussian 09 was used to theoretically calculate the OR in MeOH for each conformer of compound 6 [26].

Vibrational Circular Dichroism (VCD) Calculation Assay
The conformational search for the molecule R-compound-4 and S-compound-4 was carried out using the MMFF94 force field by the MOE 2019.01 software (Chemical Computing Group ULC).A total of 129 stable conformers for R-compound-4 and 140 stable conformers for S-compound-4 were recorded with relative energy within a 5 kcal/mol energy window.DFT calculations were used to optimize the conformers at the B3LYP/6-31G(d) and B3LYP/6-311+G(d) levels, respectively.The VCD calculations for the stable conformers were performed by Gaussian 09 (Gaussian Inc., Wallingford, CT, USA) software.VCD calculations for R-compound-4 and S-compound-4 were carried out at the B3LYP/6-311+G(d) level in the gas phase.Boltzmann statistics were used for final simulations of the VCD for these molecules.The VCD spectrum obtained from R-compound-4 and S-compound-4 both have a half-peak width of 0.16 and a wavelength range of 1100 to 1800 [27].

Assay of Antimicrobial Activity
The major plant pathogenic fungi C. camelliae was used to evaluate the antifungal activity of each compound, with carbendazim serving as the benchmark control at a concentration of 64 µg/mL in DMSO.The compounds were dissolved in DMSO to generate 128 mg/mL stock solutions.A total of 1 × 10 5 cells/mL of C. camelliae were inoculated into each well of a 96-well plate [28].Subsequently, the stock solutions were then serially diluted with PDB liquid medium to afford working concentrations of 128 to 2 µg/mL.Following a 48 h incubation at 28 • C, we determined the minimum inhibitory concentration (MIC) based on the growth outcomes in the 96-well plates.Carbendazim was used as a positive control.

Hydroxyl Radical Scavenging Assay
The salicylate technique was utilized to assess the hydroxyl radical scavenging capacity [29,30].The hydroxyl radical scavenging test was performed in 96-well microplates.Twenty-five microliters of samples (200 µM) was mixed with 25 µL of FeSO 4 •7H 2 O (9 mM) and 25 µL of ethanolic salicylic acid (9 mM), which were thoroughly mixed using a vortex mixer.Subsequently, 25 µL of H 2 O 2 (8.8 mM) was added to the reaction mixture and then incubated at 37 • C for 30 min.Vitamin C was prepared as positive control and the absorbance was measured at 510 nm.

Conclusions
In conclusion, with the aim of assessing the practical value and potential risk of the traditionally medicinal and edible higher fungus Schizophyllum commune, induced by natural undescribed small molecules, we implemented a combination strategy of elicitors to characterize products of cryptic and extremely low-expressed gene clusters.The resulting combination of five elicitors with different concentrations and induction mechanism was selected and added into fermentation medium.As a result, we were able to significantly upregulate a class of linear diterpene-derived metabolites.In total, fifteen linear diterpenederived variants were isolated and identified, including eleven new ones and three known ones.All the isolated terpenes contain an unusual butenedioic acid moiety in the elongation terminus.Compound 1 was a rare monocyclic diterpene, while 2-6 possessed a tetrahydrofuran moiety.The compounds 4, 5 and 13 are considered as trinorditerpene-type metabolites.In contrast to polycyclic terpene products, the linear terpene-derived products are unusual in nature.Recently, the Zou group [31] had been characterized as a globinlike enzyme, TutaA, in the Schizophyllum commune, which is responsible for truncating schizostain A (14) to form trinorditerpene product 2-butenedioic acid (13), indicating that the production of the new truncated compounds 4 and 5 is probably involved in a similar formation mechanism.
All the diterpene compounds displayed the ability for the scavenging of hydroxyl radicals and showed null cytotoxic activity at 10 µM.Considering that the crude extract of Schizophyllum commune has been approved as a cosmetic ingredient in China, the diterpenes would be beneficial protectants for a cosmetic ingredient.In addition, compound 1 showed potent antifungal activity, which highlights the potential of 1 as a lead compound for a novel agricultural fungicide development.

Molecules 2024 ,
29,  x FOR PEERREVIEW  4 o    elicitor and the universal applicability for most fungi.The mycelium growth and chang in secondary metabolite profiles were considered as indication signs.The change in s ondary metabolite profile was analyzed by HPLC.As results, 0.1 mM of sodium laura aniline, and p-trifluoromethylaniline, respectively, and 0.075 mM of SAHA and 0.05 m of 5-Azacytidine were determined as the suitable work concentration at which each el tor does not affect the growth of fungi.When the combination of the five elicitors w individual concentrations was added into the culture of S. commune NJFU21, a series new peaks with similar UV absorption appeared by HPLC analysis, indicating a class secondary metabolites which was unregulated (Figure2).

Figure 2 .
Figure 2. Changes in secondary metabolite profiles of S. commune NJFU21 analyzed by HPLC Five elicitors were concurrently introduced into S. commune NJFU21 strain.b-g: Representing individual S. commune NJFU21 and the elicitors added independently, respectively.

Figure 2 .
Figure 2. Changes in secondary metabolite profiles of S. commune NJFU21 analyzed by HPLC.a: Five elicitors were concurrently introduced into S. commune NJFU21 strain.b-g: Representing the individual S. commune NJFU21 and the elicitors added independently, respectively.

Figure 5 .
Figure 5. Experimental and calculated ECD spectra of 1 and 2 (in MeOH).Schizostatin K (3) was isolated as yellow oil and the molecular formula was determined as C 20 H 32 O 7 by the HR-ESI-MS ions at m/z 385.2220 [M + H] + (calcd for C 20 H 33 O 7 , 385.2219), indicating five degrees of unsaturation.Comparison of NMR data of 3 and those of 2 revealed that the skeleton outline of 3 is very similar to that of 2. The only difference was that the double bond at C-9/C-10 which disappeared in 2 was replaced by the OH-10 group at C-10 in 3, supported by COSY correlations of H 2 -8/H 2 -9/H-10 and HMBC correlations of H 2 -9 (δ C/H 77.1/1.34,1.71) with C-11 (δ C 86.8) and H 3 -18 (δ C/H 23.0/1.13)with C-10 (δ C 77.1).The relative configuration of tetrahydrofuran moiety was deduced by the NOESY correlation of H 3 -18/H 3 -16.Considering the common biosynthetic origin, the absolute configuration of both C-14 and C-11 in 3 was proposed as S, which is consistent with those of 2. Schizostatin E (4) was obtained as white powder, and was found to be C 17 H 24 O 5 , based on positive HR-ESI-MS at m/z 309.1691 [M + H] + , indicating it was a truncated diterpene, like compound 13.The unsaturation degree of compound 4 is 6.Analysis of

Figure 6 .
Figure 6.The VCD (left) and IR (right) spectra of compound 4 (measures in DMSO) with those calculated for 11R−4, 11S−4.Schizostatin G (6) was obtained as yellow oil.The molecular formula of C 20 H 28 O 5 was determined according to the HR-ESI-MS ions at m/z 349.2006 [M + H] + (calcd for C 20 H 29 O 5 , 349.2009).Detailed inspection of 1D NMR (Tables1 and 2) revealed the structure of 6 was similar to that of 14.The main difference between NMR data 6 and 14 is that the methyl group of 14 is replaced with oxygenated methylene carbon at the C-18 position, and the sp 3 methylene group at the C-13 position is replaced by oxygenated methine carbon.The HMBC correlations (Figure3) from H 2 -18 (δ C/H 69.0/4.38,4.22) to C-12 (δ C 40.2), C-11 (δ C 139.7), C-10 (δ C 121.3), C-13 (δ C 77.5) and from H-13 (δ C/H 77.5/4.57) to C-15 (δ C 138.0), H 2 -12 (δ C/H 40.2/2.59,2.20) to C-10 (δ C 121.3), along with the COSY correlations between H 2 -12, H-13 and H-14 allowed us to determine the presence of a tetrahydrofuran in compound 6.The NOESY correlations of H 2 -18/H 2 -9 and H-10/H 2 -12 provided the evidence for Z-configuration of C-10/C-11.The optical rotation calculations were carried out to ascertain the exact configuration of C-13 at the B3LYP/6-31+G(d) level.Comparing the calculated optical rotation of R-6 (+234.90)with the experimental data of 6-(+4.21), the absolute configuration of C-13 was assigned as R. Thus, the absolute configuration of 6 was determined as 2E, 10Z, 13R.All the compounds 7-11 were observed to contain twenty carbons and are noncyclic, as deduced from their HRESIMS and 1D NMR spectra (Tables1 and 2), indicative of the fact that they possessed an unabridged linear diterpene-type skeleton.The detailed interpretation of 2D NMR spectra of 7-11 established the identical fragments from C-1 to C-9, including the existence of the butenedioic acid moiety, and the E-configuration of the double bond of C-6/C-7 (Figure4).The structures of 7-11 should be oxidized variants of schizostatin A (14), a potent squalene synthase inhibitor which was also co-isolated from the fungus.Schizostatin H (7) and schizostatin I (8) were obtained as yellow oil and were found to be C 20 H 32 O 6 and C 20 H 32 O 7 , based on positive HR-ESI-MS at m/z 351.2173 [M-H 2 O + H] + and 385.2219 [M + H] + (calcd for C 20 H 31 O 5 , 351.2166 for 7; 385.2221 for 8), respectively.A careful comparison of 1D NMR data between 7 and 15 showed an almost identical planar structure (Tables1 and 2), except for the replacement of the double bond of C-14/C-15 in 15 with the pinacol group in 7, evidenced by the key HMBC correlations from H 3 -17 (δ C/H 25.0/1.12)/H 3 -16 (δ C/H 25.6/1.15) to C-15 (δ C 73.8) and C-14 (δ C 79.1), as well as the COSY correlations of H 2 -12/H 2 -13/H-14 (Figure3).The structure of schizostatin I (8) was determined to be very similar to that of 7, except for the loss of the signal corresponding to the methylene carbon at C-12, and replacement with an oxygenated methine carbon H 2 -18 (δ C/H 69.0/4.38,4.22) to C-12 (δ C 40.2), C-11 (δ C 139.7), C-10 (δ C 121.3), C-13 (δ C 77.5) and from H-13 (δ C/H 77.5/4.57) to C-15 (δ C 138.0), H 2 -12 (δ C/H 40.2/2.59,2.20) to C-10 (δ C 121.3), along with the COSY correlations between H 2 -12, H-13 and H-14 allowed us to determine the presence of a tetrahydrofuran in compound 6.The NOESY correlations of H 2 -18/H 2 -9 and H-10/H 2 -12 provided the evidence for Z-configuration of C-10/C-11.The optical rotation calculations were carried out to ascertain the exact configuration of C-13 at the B3LYP/6-31+G(d) level.Comparing the calculated optical rotation of R-6 (+234.90)with the experimental data of 6-(+4.21), the absolute configuration of C-13 was assigned as R. Thus, the absolute configuration of 6 was determined as 2E, 10Z, 13R.
C 20 H 32 O 6 and C 20 H 32 O 7 , based on positive HR-ESI-MS at m/z 351.2173 [M-H 2 O + H] + and 385.2219 [M + H] + (calcd for C 20 H 31 O 5 , 351.2166 for 7; 385.2221 for 8), respectively.A careful comparison of 1D NMR data between 7 and 15 showed an almost identical planar structure (Tables

Funding:
We are grateful for the financial support provided by the Start-up Research Fund from Nanjing Forestry University, China (Grant No. 163030196), the Student Practice Innovation and Training Program of Nanjing Forestry University (Grant Nos.2021NFUSPITP0035, 2022NFUSPITP0061, and 2023NFUSPITP0063), and the National Natural Science Foundation of China (Grant No. 22207030).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.
a Signals were overlapped.
a Positive control.