Sulfur-Containing Compounds from Endophytic Fungi: Sources, Structures and Bioactivities

Endophytic fungi have attracted increasing attention as an under-explored source for the discovery and development of structurally and functionally diverse secondary metabolites. These microorganisms colonize their hosts, primarily plants, and demonstrate diverse ecological distribution. Among endophytic fungal natural products, sulfur-containing compounds feature one or more sulfur atoms and possess a range of bioactivities, e.g., cytotoxicity and antimicrobial activities. These natural products mainly belong to the classes of polyketides, nonribosomal peptides, terpenoids, and hybrids. Here, we reviewed the fungal producers, plant sources, chemical structures, and bioactivities of 143 new sulfur-containing compounds that were reported from 1985 to March 2022.


Introduction
Sulfur is one of the prime elements on Earth and the eighth most abundant element in the human body. It is a group 6A (or VIA) member of the periodic table, with a larger atomic size and a weaker electronegativity than oxygen. Sulfur has unique characteristics, such as five different oxidation states, and sulfur-containing molecules often participate in biological redox reactions and electron transfer processes. Notably, two essential amino acids, L-methionine and L-cysteine, both contain a sulfur atom, further highlighting the importance and indispensability of sulfur in biology [1]. Indeed, one fifth (20%) of the FDA-approved drugs contain at least one sulfur atom. These sulfur-containing drugs have different structure skeletons such as sulfonamides, β-lactams, thioethers, thiazoles, thiophenes, phenothiazines, sulfoxides, S=C and S=P structures, thionucleotides, sulfones, sulfates and macrocyclic disulfides. Of note, many sulfur-containing drugs are natural products or their derivatives (i.e., rosuvastatin, ecteinascidin 743 and ixabepilone) [2].
Fungi are a major group of microorganisms that produce a broad array of compounds with novel structures and unique bioactivities. One type of fungi colonizes the intercellular and/or intracellular regions of healthy plant tissues at a particular time and has no interference with and causes no pathogenic symptoms to the host [3]. These endophytic microorganisms are an important but less-explored source for the discovery of structurally novel natural products in drug research. This paper reviews new sulfur-containing compounds isolated from endophytic fungi since 1985 (Table 1). Based on their major chemical structurally novel natural products in drug research. This paper reviews new sulfur-containing compounds isolated from endophytic fungi since 1985 (Table 1). Based on their major chemical features, these compounds will be categorized into peptides, disulfides, polyketides, hybrids and terpenoids. The fungal strains that producing sulfur-containing compounds, host plants, structure uniqueness and biological activities of these compounds will be discussed (Table 1). structurally novel natural products in drug research. This paper reviews new sulfur-containing compounds isolated from endophytic fungi since 1985 (Table 1). Based on their major chemical features, these compounds will be categorized into peptides, disulfides, polyketides, hybrids and terpenoids. The fungal strains that producing sulfur-containing compounds, host plants, structure uniqueness and biological activities of these compounds will be discussed (Table 1). structurally novel natural products in drug research. This paper reviews new sulfur-containing compounds isolated from endophytic fungi since 1985 (Table 1). Based on their major chemical features, these compounds will be categorized into peptides, disulfides, polyketides, hybrids and terpenoids. The fungal strains that producing sulfur-containing compounds, host plants, structure uniqueness and biological activities of these compounds will be discussed (Table 1). structurally novel natural products in drug research. This paper reviews new sulfur-containing compounds isolated from endophytic fungi since 1985 (Table 1). Based on their major chemical features, these compounds will be categorized into peptides, disulfides, polyketides, hybrids and terpenoids. The fungal strains that producing sulfur-containing compounds, host plants, structure uniqueness and biological activities of these compounds will be discussed (Table 1). structurally novel natural products in drug research. This paper reviews new sulfur-containing compounds isolated from endophytic fungi since 1985 (Table 1). Based on their major chemical features, these compounds will be categorized into peptides, disulfides, polyketides, hybrids and terpenoids. The fungal strains that producing sulfur-containing compounds, host plants, structure uniqueness and biological activities of these compounds will be discussed (Table 1). Penicillium raciborskii (TRT59) Rhododendron tomentosum [7] Epicoccum nigrum

Sulfide (R-S-R′)
A rare diketopiperazine bionectin D (1) (Figure 1) was obtained from a fungal strain Bionectria sp. Y1085 that was isolated from the plant Huperzia serrata. Bionectin D (1) consists of a tryptophan and a threonine moiety, and the α-carbon of its tryptophan moiety

Sulfide (R-S-R′)
A rare diketopiperazine bionectin D (1) (Figure 1) was obtained from a fungal strain Bionectria sp. Y1085 that was isolated from the plant Huperzia serrata. Bionectin D (1) consists of a tryptophan and a threonine moiety, and the α-carbon of its tryptophan moiety OSO

Sulfide (R-S-R′)
A rare diketopiperazine bionectin D (1) (Figure 1) was obtained from a fungal strain Bionectria sp. Y1085 that was isolated from the plant Huperzia serrata. Bionectin D (1) consists of a tryptophan and a threonine moiety, and the α-carbon of its tryptophan moiety OSO

Sulfide (R-S-R′)
A rare diketopiperazine bionectin D (1) (Figure 1) was obtained from a fungal strain Bionectria sp. Y1085 that was isolated from the plant Huperzia serrata. Bionectin D (1) consists of a tryptophan and a threonine moiety, and the α-carbon of its tryptophan moiety

Sulfide (R-S-R )
A rare diketopiperazine bionectin D (1) (Figure 1) was obtained from a fungal strain Bionectria sp. Y1085 that was isolated from the plant Huperzia serrata. Bionectin D (1) consists of a tryptophan and a threonine moiety, and the α-carbon of its tryptophan moiety carries a single methylthio substitution. Compound 1 exhibited antibacterial activity against Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium ATCC 6539 with the same minimal inhibitory concentration (MIC) of 25 µg/mL [4]. Lasiodiplines A-C (2-4) and E-F (5-6) are new sulfureous diketopiperazines that were produced by Lasiodiplodia pseudotheobromae F2 isolated from the apparently normal flower of Illigera rhodantha. The structure elucidation of these compounds was accomplished using a combination of spectroscopic and computational approaches, and the structure of 2 was further confirmed in conjunction with low-temperature (100 K) single-crystal X-ray diffraction. Lasiodiplines E (5) displayed antibacterial activity against Veillonella parvula, Actinmyces israelili, Streptococcus sp., Bacteroides vulgates and Peptostreptococcus sp. with the MIC values of 0.25, 32.0, 0.12, 0.12 and 0.12 µg/mL, respectively [5].
Two new compounds, Sch 54794 (34) and Sch 54796 (35) (Figure 2), were separated from the fermentation culture of ToJypocJadium sp. The microorganism ToJypocJadium sp. was isolated from dead twigs from a Quercus virginiana Miller, an old live oak tree in the state of Tamalupas, Mexico. The structures of Sch 54794 (34) and Sch 54796 (35) were determined as cis and trans isomers in the spectroscopic analysis. The trans isomer, which was similar to other diketopiperazines reported as platelet-activating factor (PAF) inhibitors in the literature, displayed weak inhibitory activity in PAF assay with an IC 50 of 50 µM. However, the cis isomer appeared inactive (IC 50 > 100 µM) [16].
The fungal strain was isolated from the fresh leaf tissues of the medicinal plant C. speciosus collected from Colombo, Sri Lanka. Rostratazine B (57) inhibited porcine pancreatic alpha-amylase activity with an IC 50 of 578 µM [25]. A pentacyclic diketopiperazine with a 4,5-dihydrooxepine moiety versicolor A (59) was isolated from Aspergillus versicolor 0312. The fungal strain was isolated from the stems of Paris polyphylla var. yunnanensis collected in Kunming, Yunnan Province, P. R. China. Compound 59 displayed cytotoxicity against the contraction of the MOLT-4 cell line with an IC 50 of 29.6 µM [26].
An amide of a coumarin moiety and L-phenylalanine-derived 1,2-oxazadecaline moiety, trichodermamide G (112), was isolated from Trichoderma harzianum D13. The fungal strain was isolated from the internal tissues of the root of Excoecaria agallocha, distributed in the mangrove regions of various parts of India [43].

Disulfide
A new natural compound, a symmetrical disulfide dimer dodecyl 3,3"-dithiodipropionate (117) (Figure 5), was isolated from the ethyl acetate extract of fermentation broth of an endophytic fungus, Sphaceloma sp. LN-15. The fungal strain was isolated from the leaves of Melia azedarach L., commonly known as the chinaberry tree, pride of India, Persian lilac, and some other names [46]. The structure of 117 was determined by NMR and MS and was further confirmed by chemical synthesis.

Sulfoxide
LC-UV/MS-based metabolomics analysis of the Hawaiian endophytic fungus Paraphaeosphaeria neglecta FT462 led to the identification of unique mercaptolactated γ-pyranolγ-lactams, paraphaeosphaerides G (118). The fungal strain was isolated on potato dextrose agar (PDA) medium from a healthy leaf of the Hawaiian indigenous plant Lycopodiella cernua (L.) Pic. Serm, which was collected in the Mokuleia Forest Reserve in 2014 [36].

Sulfates and Sulfonates
Two new alkyl sulfate-containing aromatic compounds, penixylarins B (121) and D (122), were isolated from a mixed culture of the Antarctic deep-sea-derived fungus Penicillium crustosum PRB-2 and the fungus Xylaria sp. HDN13-249 [48]. The extracts of cultures grown in liquid or on solid rice media of the fungal endophyte Ampelomyces sp. isolated from the medicinal plant Urospermum picroides exhibited considerable cytotoxic activity against L5178Y cells. The extract obtained from liquid cultures afforded two sulfated anthraquinones, macrosporin-7-O-sulfate (125) and 3-Omethylalaternin-7-O-sulfate (126) [50]. However, neither compound showed any cytotoxic or antimicrobial activities.

Sulfides
A fungal strain Pestalotiopsis sp. HS30 was isolated from the fresh stems of Isodon xerophilus collected at Kunming Botanical Garden, Yunnan Province, P. R. China [54]. Pestaloamides A (131) and B (132), two novel alkaloids featuring an unprecedented spiro[imidazothiazoledione-alkylidenecyclopentenone] scaffold, were obtained from the cultures of Pestalotiopsis sp. HS30. Compounds 131 and 132 were derived from a polyketide and a Phe-Cys dipeptide together with C 2 and C 5 moieties. Both compounds could enhance the cell surface engagement of NKG2D ligands in HCT116 cells at 40 µM [54].

Discussion and Conclusions
From 1985 to March 2022, 143 new sulfur-containing compounds were obtained from endophytic fungi. This review summarized the fungal producers, host plants, chemical structures and biological activities of these fungal metabolites ( Table 1). The majority of these compounds (109 out of 143) were reported in 2010, 2014, 2015, 2017, 2019 and 2020 ( Figure 6). There was a trend that more sulfur-containing compounds were reported in recent years except 2021. Only one sulfur-containing compound was reported in 2021, most likely due to the outbreak of COVID-19 in 2020. A total of 24 journals reported these compounds (Figure 7). The J. Nat. Prod. has published the highest number of papers (16) that reported sulfur-containing compounds, followed by Phytochemistry (8) (Figure 7). This is not unexpected because both J. Nat. Prod. and Phytochemistry are prominent natural product journals.  These sulfur-containing compounds demonstrate functional and structural diversity and exhibited many bioactivities. Among the reported biological activities, 42% of these compounds were antimicrobial, while 37% were cytotoxic (Figure 8), which is not surprising because the majority of the FDA-approved antimicrobial and anticancer drugs are either natural products or derived from natural products. For example, Secoemestrin D (69), a diketopiperazine, was very active against a panel of seven cancer cell lines with IC50 values ranging from 0.06 to 0.24 μM [27], while PM181110 (133) [55] and FE399 (134) [56], hybrids of polyketides and peptides, exhibited potent anticancer activity with IC50 values at the nM level. These compounds also possess other bioactivities. For instance, oreganic acid (128), a fatty acid derivative, inhibited FPTase with an IC50 of 14 nM [49]. The majority  These sulfur-containing compounds demonstrate functional and structural diversity and exhibited many bioactivities. Among the reported biological activities, 42% of these compounds were antimicrobial, while 37% were cytotoxic (Figure 8), which is not surprising because the majority of the FDA-approved antimicrobial and anticancer drugs are either natural products or derived from natural products. For example, Secoemestrin D (69), a diketopiperazine, was very active against a panel of seven cancer cell lines with IC50 values ranging from 0.06 to 0.24 μM [27], while PM181110 (133) [55] and FE399 (134) [56], hybrids of polyketides and peptides, exhibited potent anticancer activity with IC50 values at the nM level. These compounds also possess other bioactivities. For instance, oreganic acid (128), a fatty acid derivative, inhibited FPTase with an IC50 of 14 nM [49]. The majority The numbers of papers These sulfur-containing compounds demonstrate functional and structural diversity and exhibited many bioactivities. Among the reported biological activities, 42% of these compounds were antimicrobial, while 37% were cytotoxic (Figure 8), which is not surprising because the majority of the FDA-approved antimicrobial and anticancer drugs are either natural products or derived from natural products. For example, Secoemestrin D (69), a diketopiperazine, was very active against a panel of seven cancer cell lines with IC 50 values ranging from 0.06 to 0.24 µM [27], while PM181110 (133) [55] and FE399 (134) [56], hybrids of polyketides and peptides, exhibited potent anticancer activity with IC 50 values at the nM level. These compounds also possess other bioactivities. For instance, oreganic acid (128), a fatty acid derivative, inhibited FPTase with an IC 50 of 14 nM [49]. The majority of sulfur-containing compounds (92) were peptides, followed by polyketides (38), hybrids (6), terpenoids (5) and others (2) (Figure 9). All 92 of these peptides are diketopiperazines, and the sulfur atoms in these molecules are mainly derived from L-cysteine that contains a reactive sulph-hydryl group. of sulfur-containing compounds (92) were peptides, followed by polyketides (38), hybrids (6), terpenoids (5) and others (2) (Figure 9). All 92 of these peptides are diketopiperazines, and the sulfur atoms in these molecules are mainly derived from L-cysteine that contains a reactive sulph-hydryl group.

Prospects
Some plants are rich in sulfur, for example, allium vegetables, legumes and cruciferous plants. These plants should be great sources of endophytic fungi that produce sulfurcontaining compounds. Large amounts of sulfur are released during volcanic eruptions. Hence, plants in volcanic areas and hot springs might also be excellent sources for endophytic fungi producing sulfur-containing compounds.  of sulfur-containing compounds (92) were peptides, followed by polyketides (38), hybrids (6), terpenoids (5) and others (2) (Figure 9). All 92 of these peptides are diketopiperazines, and the sulfur atoms in these molecules are mainly derived from L-cysteine that contains a reactive sulph-hydryl group.

Prospects
Some plants are rich in sulfur, for example, allium vegetables, legumes and cruciferous plants. These plants should be great sources of endophytic fungi that produce sulfurcontaining compounds. Large amounts of sulfur are released during volcanic eruptions. Hence, plants in volcanic areas and hot springs might also be excellent sources for endophytic fungi producing sulfur-containing compounds.

Prospects
Some plants are rich in sulfur, for example, allium vegetables, legumes and cruciferous plants. These plants should be great sources of endophytic fungi that produce sulfur-containing compounds. Large amounts of sulfur are released during volcanic erup-tions. Hence, plants in volcanic areas and hot springs might also be excellent sources for endophytic fungi producing sulfur-containing compounds.
Most of the compounds reviewed in this article were tested for their antimicrobial and antiproliferative or anticancer activities. We believe that other biological properties could be identified if fungal metabolites were evaluated in a broader range of biological settings. For example, sinuxylamides A and B were obtained from Xylaria sp. FM1005, an endophytic fungus isolated from Sinularia densa (leather coral) collected in the offshore region of the Big Island, Hawaii [64]. Sinuxylamides A and B showed no antibacterial activity or cytotoxicity at 40 µM, but they strongly inhibited the binding of fibrinogen to purified integrin IIIb/IIa in a dose-dependent manner with IC 50 values of 0.89 and 0.61 µM, respectively.
Diketopiperazines are expected to be biosynthetically assembled from two amino acid building blocks by nonribosomal peptide synthetases [65]. On the other hand, the biogenesis of many sulfur-containing compounds remains incompletely understood. For example, the structures of compounds 20 [10], 40 [18], 98 [37], 136 [58], 142 [62] and 143 [63] are unique. It would be interesting to investigate how these molecules are biogenetically synthesized. Presumably, the 4,5-dihydrooxepine ring in 20 is derived from the benzene ring of L-phenylalanine through ring expansion. On the other hand, the spiro[cyclopenta[b]pyrrole-5,2 -furan] moiety in 40 might be formed through the constriction of the benzene ring of L-phenylalanine followed by the merge of the octahydrocyclopenta[b]pyrrole ring with an isoprenyl (C 5 ) group. We previously isolated compound 98 [37]. The precursor of the side chain at the 14-position in compound 98 could be L-cysteine, which is converted to mercaptolactate. The nucleophilic addition of the mercaptolactate thiol to C-14 of paraphaeosphaeride C generates an intermediate that is oxidized to another intermediate. It is also plausible that the second intermediate is generated from mercaptopyruvate and paraphaeosphaeride C. The nitrogen atom in the second intermediate undergoes intramolecular nucleophilic addition to the ketone of the mercaptopyruvate moiety, leading to the formation of the third intermediate. The dehydration of the third intermediate yields the final product 98 [37]. However, the experimental details of the biosynthesis of compound 98 are still not available. Compound 136 is composed of five fragments, including a 2amino benzoic acid moiety, an L-alanine, a 2-amino-2-methylsuccinic acid fragment that might be derived from an isoprenyl group (C 5 ), and L-glycine and L-cysteine-derived 3-mercaptopropanoic acid moieties. Compound 142 carries a 2-hydroxyl propanoic acid ester. The thiazole ring in 142 is probably derived from acetate and L-cysteine, while the linker (-CH 2 -CH 2 -) might be derived from another acetate. It would be interesting to investigate how 142 is synthesized biogenetically. Investigating the biosynthesis of diamond-like compound 143 should be very challenging and interesting. Recent advances in genome mining and synthetic biology offer new opportunities to discover new natural products [66]. It becomes routine to sequence the (meta)genomes of fungal isolates, and capable bioinformatics tools (e.g., antiSMASH fungal version) [67] are increasingly available for identifying potential biosynthetic gene clusters (BGCs) of fungal natural products [68]. These predicted BGCs can suggest new chemotypes, enzymology and bioactivities. Subsequently, native and engineered BGCs can be expressed in multiple synthetic biology chasses, such as Aspergillus nidulans [69] and Saccharomyces cerevisiae [70]. In this regard, biosynthetic research is critical for laying the basis for the genome mining of BGCs of new fungal sulfur-containing compounds with bioactivities, particularly those whose biogenesis remains unclear.