Antifungal Nafuredin and Epithiodiketopiperazine Derivatives From the Mangrove-Derived Fungus Trichoderma harzianum D13

A new polyketide derivative, nafuredin C (1), a novel heterocyclic dipeptide, trichodermamide G (3), together with four known biogenetically related compounds nafuredin A (2), trichodermamide A (4), aspergillazin A (5), and peniisocoumarin H (6), were isolated from the mangrove-derived fungus Trichoderma harzianum D13. Their structures, including their absolute configurations, were determined by spectroscopic analysis and time-dependent density functional theory-electronic circular dichroism (ECD) calculations. Trichodermamide G was found to be a novel epithiodiketopiperazine derivative with an unprecedented cyclic system containing a sulfur bridge, and nafuredin C represented the third nafuredin derivative of these homologous compounds. The new compound nafuredin C exhibited obvious antifungal activity against Magnaporthe oryzae with a minimum inhibitory concentration (MIC) of 8.63 μM, which is on the same order of magnitude as the positive control carbendazim (MIC = 3.27 μM).


INTRODUCTION
Rice blast disease is the most serious disease affecting cultivated rice, a staple food for nearly 50% of the world's population, and seriously threatens global food security. Each year, rice blast disease is responsible for the loss of 10-30% of the rice harvest, which is enough to feed more than 60 million people (Martin-Urdiroz et al., 2016;Zhou, 2016). Rice blast is caused by Magnaporthe oryzae, an ascomycete fungus that causes diseases in a wide range of economically important crops including barley, oats, rye grass, and millets. It was first reported in Brazil in 1985 and then rapidly spread to other South American countries, causing significant yield losses. The disease captured public attention in 2016 when it appeared in Bangladesh, resulting in thousands of rice fields being burned to prevent further spread of the disease (Ma and Xu, 2019). In China, rice blast occurs everywhere rice is cultivated, particularly in hilly areas. In epidemic years of the disease, yield loss can reach 40-50% and in severe cases can result in complete losses in major rice production areas . Therefore, searching for new biological pesticides to control rice blast is necessary for sustainable development of the rice industry and food security worldwide.
Beneficial microbes can function as biocontrol organisms that help plants defend themselves from attack by pathogens. Fungi in the genus Trichoderma are excellent mycoparasites of plant pathogens and directly protect plants against them. In addition, Trichoderma spp. can enhance the plant defense system, which enables the plant to respond in a fast and strong manner to pathogen attack (Guzman-Guzman et al., 2019). These fungi are also prolific producers of numerous secondary metabolites with pharmaceutical and biotechnological importance, including polyketides, non-ribosomal peptides, siderophores, peptaibols, and volatile and non-volatile terpenes (Contreras-Cornejo et al., 2016). Several studies have focused on the application of Trichoderma spp. for controlling rice blast disease with apparent effects; however, marine-derived Trichoderma spp. have not been evaluated for use in plant protection, and few studies have reported their antifungal effects against M. oryzae.
During our ongoing search for new anti-phytopathogenic fungal secondary metabolites from marine-derived fungi in the Yellow Sea and South China Sea (Huang et al., 2018;Zhao et al., 2018;Zhao D. L. et al., 2019), we found that the extract of mangrove-derived fungus Trichoderma harzianum D13, collected from Hainan province, China, displayed a strong activity against fungal plant pathogens. Further chemical investigation of the ethyl acetate (EtOAc) extracts led to the isolation of six compounds (Figure 1), including two nafuredin derivatives, nafuredins C and A (1 and 2); three epithiodiketopiperazine derivatives, trichodermamide G (3), trichodermamide A (4), and aspergillazin A (5); and one isocoumarin, peniisocoumarin H (6). Among them, nafuredin C and trichodermamide G are new compounds. Herein, we report the isolation, structural elucidation, and antifungal activities of these compounds.

Fungal Material
The fungus was isolated from the internal tissues of the root of mangrove plant Excoecaria agallocha Linn. collected from Hainan province, China, in 2016, and was identified as T. harzianum (GenBank accession number MG827165) by sequence analysis of the internal transcribed spacer region of the rDNA. A voucher strain of this fungus was deposited in the Marine Agriculture Research Center, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, China.

Structural Elucidation of the Isolated Compounds
Nafuredin C (1) was isolated as a white, amorphous powder. Its molecular formula, C 22 H 34 O 4 , was established from highresolution ESIMS (HRESIMS), 1 H and 13 C nuclear magnetic resonance (NMR) data (Supplementary Figures S1, S2, S7), indicating six indexes of hydrogen deficiency. The 1 H NMR ( Table 1)  2), two quaternary carbons (one oxygenated, one olefinic), three methylene groups, 11 methines (seven olefinic, two oxygenated), and five methyl groups. These spectroscopic data are similar to those of nafuredin B, isolated from a mixed culture of the deep-sea-derived fungus Talaromyces aculeatus and mangrove-derived fungus Penicillium variabile (Zhang et al., 2017). The major differences were replacement of the sp 2 double bond at C-2 and C-3 in nafuredin B by a hydroxyl group at C-2 in 1, which were consistent with the downfield shifts of C-1/C-4 (δ C 163.3/68.0 in nafuredin B vs 177.2/84.8 in 1) and upfield shifts of C-2/C-3 (δ C 118.0/156.3 in nafuredin B vs 68.4/37.9 in 1), supported by the COSY correlations of H-2/H-3, and key HMBC from H-2 to C-1/C-4, from H-3 to C-1/C-5, from H-5 to C-3/C-4, from 2-OH to C-1/C-2/C-3, and from H 3 -19 to C-3/C-4/C-5 (Figure 2 and Supplementary Figures S4, S5). The relative configuration of the δ-lactone ring was established based on the NOESY spectrum (Figure 2 and Supplementary Figure S6). The correlations of 4-OH with 2-OH and H-5 and of H-6 with H 3 -19 indicated a cofacial relationship among 4-OH, 2-OH, and H-5. The geometry of double bonds in the olefinic chain was E elucidated from the J values and NOESY correlations (Table 1 and Figure 2). Considering the biosynthesis and coisolation of nafuredin A (2), whose absolute configuration was determined based on asymmetric synthesis  Zhang et al., 2017), the chirality of C-10 and C-16 was determined to be 10R and 16S, which was supported by the identical NMR data. Comparing the computed ECD spectra with experimental results is a valid method of assigning the absolute configurations of natural products (Cao et al., 2020). To determine the absolute configuration of the δ-lactone ring in 1, ECD computations for all B3LYP/6-311+G(d)-optimized conformers (50 structures) were carried out at the B3LYP/6-311++G(2d,p) level, and six structures with relative energy less than 2.5 kcal/mol were obtained. Boltzmann statistics were performed for ECD simulations with a standard deviation of σ 0.25 eV. The experimental and calculated ECD spectra for the (2R,4S,5R,10R,16S)-1 showed good agreement (Figure 3 and Supplementary Table S1), thus suggesting the absolute configuration. Trichodermamide C (3) was obtained as a yellow, amorphous powder, with the molecular formula C 20 H 20 N 2 O 8 S based on its HRESIMS, 1 H and 13 C NMR data, indicating 12 degrees of unsaturation (Supplementary Figures S8, S9, S15). The 13 C NMR and DEPT spectroscopic data ( Table 2) revealed 20 carbon signals containing two carbonyl groups, two methoxyl groups, 10 methines (with five olefinic or aromatic, two thiogenated, one nitrogenated, two oxygenated), and six quaternary carbons  (with five sp 2 and one sp 3 ). In examining the 1 H NMR and HSQC data (Supplementary Figures S8, S10), an amide proton at δ H 10.09, two ortho-aromatic protons attributed to a 1,2,3,4tetrasubstituted phenyl unit at δ H 7.04 (1H, d, J = 9.0 Hz) and 6.61 (1H, d, J = 9.0 Hz), and three olefinic protons including two derived from a cis-coupled double bond at δ H 6.50 (1H, t, J = 8.0 Hz) and 6.04 (1H, d, J = 8.0 Hz) were observed in the deshielded region of the spectrum, whereas shielded signals for five methines at δ H 5.18 (1H, s), 4.02 (1H, s), 3.63 (1H, s), 3.42 (1H, d, J = 9.5 Hz), and 3.29 (1H, s) were also present. Additionally, two O-methyl groups (δ H 3.81 and 3.69) were observed in the 1 H NMR spectrum of 3. Inspection of the above NMR data indicated that the isocoumarin portion of 3 was similar to that of aspergillazine A, a modified dipeptide isolated from the marine-derived fungus Spicaria elegans (Liu et al., 2005), whereas the signals of oxazine were obviously changed, possibly because of the different location of the sulfur bridge. The characteristic NMR data of C-3 (δ H 3.29, δ C 44.6) and C-7 (δ H 3.42, δ C 37.8) indicated they were linked by a sulfur atom, which was confirmed from the COSY correlation of H5/H6/H7/H8/H9 and HMBC correlations from H-3 to C-5/C-7/C-9, from H-5 to C-7/C-9, from H-6 to C-4/C-8, and from H-7 to C-9 (Figure 2 and Supplementary Figures S11, S12). In the NOE difference spectra (Figure 2 and Supplementary Figures S13, S14), irradiation of H-2 and H-3 enhanced the resonance of H-9. This information, in addition to the similar coupling constant of H8/H-9 (brs) to trichodermamide A and aspergillazine A (Capon et al., 2005;Liu et al., 2005), indicates a syn relationship among H-2, H-3, H-9, and 8-OH. 4-OH was assigned on the opposite face of the sulfur bridge, as the cis relationship did not lead to a reasonable model according to 3D simulations.
To determine the absolute configuration of 3, ECD calculations were carried out. Monte Carlo conformational searches were applied using Spartan's 10 software with the Merck molecular force field. Conformers with a Boltzmann-population of over 10% were selected for ECD calculations and were initially optimized at the B3LYP/6-31g (d,p) level in MeOH using the conductor-like polarizable continuum calculation model. Theoretical calculation of the ECD was conducted in MeOH using time-dependent density functional theory at the B3LYP/6-31+g (d,p) level for all conformers of compound (2R,3S,4S,7S,8S,9S)-3. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the programs SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (GraphPad, Inc., La Jolla, CA, United States) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV. The predicted ECD spectrum agreed well with the experimental result (Figure 4 and Supplementary Tables S2, S3), indicating that the absolute configuration of 3 was 2R,3S,4S,7S,8S,9S.
Epipolythiodiketopiperazine alkaloids are fungal metabolites with a highly complex molecular architecture comprising a densely functionalized core structure with many stereogenic centers. In the past decade, an increasing number of studies have discovered powerful new biological processes involving these molecules, including cytotoxic, antileukemic, antiviral, antibiotic, and antinematodal activities (Kim and Movassaghi, 2015); however, epithiodiketopiperazines have not been widely examined. Notably, trichodermamide G is a novel epithiodiketopiperazine derivative with an unprecedented cyclic system containing a sulfur bridge.
Compounds 2 and 4-6 were identified as nafuredin A , trichodermamide A (Liu et al., 2005), aspergillazine A (Liu et al., 2005), and peniisocoumarin H (Cai et al., 2018) by comparison of their spectroscopic data with those in the literature.

Antifungal Activity
In the present study, all isolated compounds were evaluated to determine their anti-phytopathogenic fungal activities against B. cinerea, M. oryzae, P. theae, P. parasitica, and V. mali. The nafuredin derivatives 1 and 2 exhibited obvious antifungal activities against M. oryzae, with minimum inhibitory concentrations (MICs) of 8.63 and 17.4 µM, respectively. These data indicate that the antifungal activity toward M. oryzae of new compound 1 was the same magnitude as that of the positive control carbendazim (MIC = 3.27 µM). Compounds 1 and 2 also displayed weak antifungal activity against V. mali and P. theae compared to carbendazim ( Table 3).

CONCLUSION
In summary, we report three polyketide derivatives, nafuredin C (1), nafuredin A (2), and peniisocoumarin H (6), and three epithiodiketopiperazine derivatives, trichodermamide G (3), trichodermamide A (4), and aspergillazin A (5), isolated from the mangrove-derived fungus T. harzianum D13. Among them, nafuredin C (1) and trichodermamide G (3) are new compounds. Their structures were assigned based on extensive NMR spectroscopic data, time-dependent density functional theory ECD calculations together with comparison of their ECD spectra. Trichodermamide G contains a unique sulfur bridge compared to the homologous compounds. The polyketide derivatives nafuredin C (1) and nafuredin A (2) exhibited distinct antifungal activity against M. oryzae. T. harzianum have been widely used as biocontrol agents and commercially marketed as biopesticides. Our study expands the source of Trichoderma which used for biocontrol, and provides basic material for the discovery of new antifungal pesticides.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.