Investigation of Diaporthe phaseolorum Chemical Compounds toward Mass Spectral Molecular Networking and in silico Substructure Annotation

Abstract

Diaporthe phaseolorum, an endophyte isolated from Combretum lanceolatum, has exhibited diverse biological activities including antimicrobial, leishmanicidal, antitumoral, and herbicidal properties. Regardless of these activities, there remains limited research on its chemical constituents. Therefore, this study aimed to investigate the metabolome of D. phaseolorum employing mass spectrometry data dereplication through molecular networking and MolNetEnhancer tools. Twenty two molecular families were annotated, including prenol lipids, benzenoids, organoheterocyclic compounds, and nucleosides. Among the 41 nodes annotated, 36 metabolites were confirmed via spectral references, including alismol, farnesol, linoleic acid, tyramine, N-acetyl-tyramine, 5-methoxy-1H-indole-3-carbaldehyde, 6-pentyl-2H-pyran-2-one, and adenosine. Further node annotation was achieved through MolNetEnhancer, resulting in the classification of an additional 68 nodes into various molecular families. Consequently, this study represents a significant contribution to the identification of the D. phaseolorum metabolome, highlighting its secondary metabolites and providing valuable chemical insights to elucidate its demonstrated biological activities.

Keywords:
alismol; dereplication; Diaporthe phaseolorum; farnesol; GNPS; molecular networking

Introduction

Natural products serve as a remarkable source of biologically active small molecules with diverse applications. For instance, phomanolide is an antiviral nordrimane-type sesquiterpenoid,¹ stemphyperylenol acts as a fungicide polyketide,² and 3-hydroxypropionic acid exhibits antibacterial properties.³ Additionally, compounds like the alkaloids antidesmone and arborinine act as photosynthesis inhibitors,⁴,⁵ while graveoline demonstrates herbicidal activity.⁶ Furthermore, pestaloficiol derivatives exhibit antitumor properties,⁷ and chenopodolin and sphaeropsidin C serve as larvicides against Aedes aegypti.⁸ Notably, (-)-mammea A/BB demonstrates anti mycobacterium tuberculosis activity.⁹ Overall, research involving natural products is a relevant area for the discovery and development of new biologically active compounds.¹⁰-¹⁵

The dereplication approach has emerged as a prominent method in metabolome studies, particularly for fast investigation of chemical profiles of large and complex spectrometric datasets.¹⁶-²⁰ In recent years, research integrating analytical techniques with computational tools for studying endophytic fungi metabolomes has significantly expanded. This expansion has broadened the scope of annotated metabolites within fungal metabolomes, thereby facilitating the identification of bioactive natural products derived from fungi.²¹-²⁴

In the current context, molecular networking (MN) dereplication emerges as a valuable strategy for identifying secondary metabolites, as this method avoids the need for isolating and determining the structure of known molecules. MN has recently been applied to evaluate fungal metabolomes, enabling the identification of various secondary bioactive metabolites. For instance, MN was used for the discovery of secondary cytotoxic metabolites in Colletotrichum strains isolated from leaves of the tropical palm species Astrocaryum sciophilum,²⁵ as well as the identification of cytosporone derivatives in Phomopsis sp. isolated from Casearia arborea leaves.²⁶ Furthermore, MN has been applied in the chemical evaluation of Aspergillus fumigatus collected from the lateral buds of Crocus sativus²⁷ and in the metabolomic study of Cophinforma mamane and Fusarium solani isolated from Bixa orellana L. and Plantago lanceolate, respectively.²⁸

Among fungal endophytes, the genus Diaporthe has exhibited numerous biological activities²⁹,³⁰ and several bioactive secondary metabolites have been isolated from it. These include alkaloids,³¹,³² terpenoids,³³ cytosporones,³⁴ dothiorelone derivatives,³⁵ bisanthroquinones,³⁶ chloroazaphilone derivatives,³⁷ cytochalasins and polyketides.³⁸ Diaporthe phaseolorum, for instance, is an endophyte isolated from Combretum lanceolatum roots,³⁹ and the extract obtained from its fermentation has demonstrated various biological activities, including antioxidant and leishmanicidal effects,⁴⁰ antimicrobial properties,⁴¹,⁴² antitumoral activity⁴³ and herbicidal potential.⁴⁴

Previous chemical profile studies have revealed that the D. phaseolorum produces various bioactive secondary metabolites, including alkaloids like 18-des-hydroxycytochalasin H,⁴⁰ anthraquinones such as 1,1-bislunatin, 5-deoxybostrycoidin and 1-hydroxy-8 methoxyanthraquinone,⁴⁵ chromones like phomopsichins A and B and diaporchromanones C and D,³² benzofuran derivatives like 7-methoxy-4,6-dimethyl-benzofuran and 3H-isobenzofuran-1-one,⁴⁶ as well as xanthone fomoxanthone A,⁴³ among others.⁴⁷,⁴⁸ Considering the biological potential exhibited by D. phaseolorum endophyte and the limited number of metabolites described for this fungus, our study aims to explore the D. phaseolorum metabolome using MN dereplication, employing in silico bioinformatics tools such as substructure annotation (MS2LDA), network annotation propagation (NAP), and MolNetEnhancer to enhance metabolite identification.

Experimental

Reagents and solvents

Acetonitrile and methanol of high-performance liquid chromatography (HPLC) grade were purchased from Mallinckrodt Baker (St. Louis, MO, USA). Ethyl acetate and formic acid were purchased from Synth (São Paulo, Brazil) and Sigma-Aldrich (St. Louis, USA), respectively. Potato dextrose agar was obtained from Merck (São Paulo, Brazil). Ultrapure deionized water was obtained from a Milli-Q water purification system (Millipore Corporation, Watford, UK).

Preparation of D. phaseolorum crude extract

The crude extract of D. phaseolorum (Access GenBank KF555229)³⁹ was prepared following a modified literature procedure.⁴⁴ Initially, the fungal strain was grown on potato dextrose agar for 7 days. Subsequently, the culture was transferred to Erlenmeyer flasks containing 0.150 kg of parboiled rice and incubated statically in the dark. After 20 days of incubation, ethyl acetate (150 mL) was added, and the mixture was stirred for 24 h at 75 rpm. Finally, the mixture was sonicated for 10 s in an ultrasonic bath (UltraSonic Cleaner, Model USC3380/Unique Ind. Brasileira, São Paulo, Brazil), filtered, and the solvent was evaporated under reduced pressure. The resulting crude extract was then stored at -20 °C until further analysis.

Data acquisition

The D. phaseolorum extract was analyzed using a Shimadzu UHPLC system (Shimadzu, Kyoto, Japan) equipped with a reverse-phase column (Waters Acquity UPLC BEH C-18, 100 mm × 2.1 mm inner diameter, particle size 1.8 μm) maintained at 40 °C. The mobile phases consisted of 0.1% (v/v) formic acid in ultrapure water (A) and acetonitrile (B). Elution condition was performed under the following conditions: 0-12 min, linear gradient from 5 to 100% B, followed by column reconditioning with 5% B. The flow rate was set at 0.30 mL min^-1, and 1 μL of D. phaseolorum extract solution in methanol at 0.5 mg mL^-1 was injected for analysis.

High-resolution mass spectrometry (HRMS) analyses were carried out using an electrospray ionization quadrupole time-of-flight (ESI-qTOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). The MS analysis employed data-dependent acquisition MS¹ and MS² modes, with the mass spectra ranging from 50 to 1100 Da. The ionization source operated in positive electrospray ionization (ESI) mode, with a capillary voltage set at 3600 V and a plate end potential of 450 V. Dry gas parameters were set to 8.0 L min^-1 at 180 °C, with a nebulization gas pressure of 4.0 bar. Subsequently, the MS/MS data were converted to mzXML format, compressed using the Core FTP LE online server,⁴⁹ and then transferred to the Global Natural Products Social (GNPS)⁵⁰ platform for MN analysis.

Molecular networking

The MN was generated following a modified literature procedure.⁵¹ The MS/MS data was uploaded to the GNPS MN server to generate the chemical map (ID = c65a5130891e4634ade323c360da3345). The MN was obtained using a parent mass tolerance of 0.02 Da and the mass variances of ion fragments within ± 0.02 Da for each group to create consensus spectrum. Network edges were produced only if the cosine score exceeded 0.7 and with a minimum correspondence of six peaks in the fragmentation spectrum. Additionally, the MN spectra were compared against the GNPS spectral libraries, and the data obtained were visualized using the Cytoscape 3.8.1 software.⁵² Fragmentation spectra showing similarities to the mass spectral libraries underwent manual verification, and the mass error was calculated with a mass error with a tolerance of less than 5 ppm.

Network annotation propagation

The MN generated by GNPS was utilized to perform in silico annotation propagation using NAP (ID = 152bb9c9f0f442f684b7c63b2cac304b). The parameters employed for this annotation propagation were as follows: considering the 10 candidates for consensus score, an accuracy of exact mass search set at 15 ppm, and utilizing structural databases including GNPS, HMDB,⁵³ SUPNAT,⁵⁴ CHEBI,⁵⁵ DRUGBANK,⁵⁶ and FooDB.⁵⁷

MS2LDA substructural model

Mass2Motifs inspection and annotation were carried out using the MS2LDA⁵⁸ web platform (ID = 2df7012ed58d4e609f55f5aff62b874e), following a modified literature procedure.⁵⁹ The MS2LDA parameters were configured as follows: a m/z tolerance of 5.0 ppm, a retention time (t_R) tolerance of 10.0 s, minimum MS1 intensity of 10.0 au, minimum MS2 intensity of 100.0 au, 1000 interactions, 300 Mass2Motifs, and no duplicated filtering.

Integration of annotation data using MolNetEnhacer

To enhance chemical structural information within the MN, the in silico annotations obtained from NAP and Mass2Motifs extracted by MS2LDA were incorporated into the network using the GNPS MolNetEnhancer workflow (ID = 35744a1aa5bc4ff8951d2a7cdfcc844b). Chemical class annotations were conducted utilizing the ClassyFire chemical ontology to identify the most prevalent chemical classes per molecular family (MF) based on GNPS structural library hits. The integration of the standard distribution of Mass2Motifs on the network was accomplished using the R package (rMolNetEnhancer).

Results and Discussion

To investigate the chemical profile of D. phaseolorum fungus, a crude extract was prepared using ethyl acetate after seven days of fungal inoculation in a culture medium. The extract was then analyzed by HPLC-HRMS/MS employing an ESI qTOF. The analyses were conducted using a data-dependent acquisition procedure, which generated fragmentation spectra for all precursor ions above a defined threshold. The obtained fragmentation data was organized into MNs using the GNPS platform. This approach facilitated the exploration of the main chemical classes and enabled the annotation of metabolites produced by D. phaseolorum.

Molecular networking

The chemical profile of D. phaseolorum was investigated using a dereplication strategy towards GNPS spectra library analysis. This approach generated a MN comprising 238 nodes, with 65.12% of the nodes organized into a total of 22 MFs, each consisting of two or more nodes with similarity scores (CScore ≥ 0.7). Additionally, 83 nodes were identified as singletons, lacking molecular relatives. Considering the limited information contained in natural product spectral libraries, automated spectral similarity led to the addition of 41 putative annotations among all registered data in the MN. Subsequently, all node annotations underwent mirror match, cosine scores, and the number of shared peaks, resulting in 36 hits with level 2 and 3 annotations according to the 2007 Metabolomics Standards Initiative.⁶⁰

These annotations were confirmed through individual analyzes, fragmentation (MS/MS), and comparison to literature spectra. To enhance metabolite annotation, the MolNetEnhancer approach, a computational tool for MS/MS based untargeted metabolomics, was employed. Most MFs were annotated for their specific chemical classes using the GNPS library matching and in silico annotation obtained from the NAP approach. Furthermore, computational class annotations were double-checked to prevent false annotations.⁶¹

Based on the MS2LDA, Mass2Motifs 79, 80, 130, and 256 were extracted from MS/MS spectra and annotated as prenol lipids. Mass2Motifs 130 and 79 indicate the presence of a fragment ion m/z 95 [C₇H₁₁]⁺ and the neutral loss of 18 Da (H₂O), respectively. Mass2Motifs 80 and 256 indicate the presence of fragment ion at m/z 109 [C₈H₁₃]⁺ and m/z 123 [C₉H₁₅]⁺, respectively. These Mass2Motifs were annotated to represent terpene and fatty acid scaffolds, which are different in their degrees of unsaturation and their functional groups.

Furthermore, Mass2Motifs 15, 97, 120, and 173 were annotated as benzenoid derivatives. Mass2Motif 15 was annotated to show a neutral loss of 59 Da (C₂H₅NO), indicating the presence of small generic losses such as C₂H₂O and NH₃. Mass2Motif 173 represents the neutral loss of 46 Da (CH₂O₂), which is caused by the loss of H₂O and CO, suggesting phenolic derivative substructures like aromatic amino acids and carboxylic acids. Metabolites annotated with Mass2Motif 120 exhibited fragment ions at m/z 95 [C₆H₇O]⁺, 107 [C₇H₇O]⁺, and 119 [C₈H₇O]⁺, indicating substructures derived from tyrosine.

The MNs associated with MolNetEnhancer facilitated the annotation of new compounds for this fungus, making the first documentation of these metabolites. The described metabolites were categorized into four main groups, underscoring the chemical diversity of this fungal species. These groups comprised a significant cluster of prenol lipids, a secondary group containing benzenoid derivatives, two smaller clusters consisting of nucleosides, and heterocyclic compounds. This delineation highlights the diverse array of chemical compounds produced by D. phaseolorum and underscores its potential significance in natural product discovery and drug development.

Prenol lipids annotation

The prenol lipid family comprises two distinct clusters designed as terpenoids and fatty acids. This categorization arises from the diverse ion adducts commonly detected, each exhibiting distinct fragmentation behaviors during collisional activation. Such variations frequently lead to the inadvertent segregation of MN into subnetworks and pose limitations on the propagation of library annotations throughout the networks.⁶² This observation underscores the challenges encountered in accurately delineating and annotating metabolites within complex MNs, necessitating further refinement and optimization of analytical approaches.

The MF of prenol lipid exhibited ten nodes associated with terpenoid compounds (Figure 1), characterized by similar daughter ions observed at m/z 95.0858 [C₇H₁₀ + H]⁺ and a common neutral fragment loss of 42 Da (C₃H₆) and 56 Da (C₄H₈). The node with m/z 203.1800 [C₁₅H₂₄O - H₂O + H]⁺ (calcd. 203.1794), alongside fragments indicating the loss of 28.0317 (C₂H₄), 42.0467 (C₃H₆), 54.0487 (C₄H₆), 56.0629 (C₄H₈), and 68.0634 (C₅H₈) Da, suggests a fragmentation pattern typical of terpenoid. The MS² data of this node match those of the sesquiterpene alismol (1).⁶³ Another node at m/z 205.1958 [C₁₅H₂₆O - H₂O + H]⁺ (calcd. 205.1951), exhibiting fragment ions observed at m/z 121.1018 [C₉H₁₂ + H]⁺ (calcd. 121.1012) and m/z 109.1017 [C₈H₁₂ + H]⁺ (calcd. 109.1012), corresponds to isolongifolol (2), another sesquiterpene, based on comparison with literature data.⁶⁴

Figure 1
MFs and chemical structures of prenol lipid compounds.

The fragment at m/z 149.1331 [C₁₁H₁₆ + H]⁺ (calcd. 149.1325) detected in the MS spectra emerged as a base peak ion, derived from the precursor m/z 223.2058 [C₁₅H₂₆O + H]⁺ (calcd. 223.2056), as observed in the node mass spectrum. Additionally, the fragment resulting from the loss of H₂O (18 Da) appeared at m/z 205.1956 [C₁₅H₂₄ + H]⁺ (calcd. 205.1951), and the fragmentation pattern indicated the loss of 14 Da (CH₂) units, suggesting an aliphatic compound. Based on this data, along with literature evidence the structure of farnesol (3), a structural isomer of isolongifolol, is proposed.⁶⁵ Another terpenoid identified from D. phaseolorum metabolism was ferutinol (4), represented by the node at m/z 221.1902 [C₁₅H₂₆O₂ - H₂O + H]⁺ (calcd. 221.1900), with a daughter ion observed at m/z 203.1807 [C₁₅H₂₂ + H]⁺ (calcd. 203.1794), corresponding to the loss of additional H₂O molecule.⁶⁶

The node at m/z 219.1734 [C₁₅H₂₄O₂ - H₂O + H]⁺ (calcd. 219.1743), identified as the base peak ion in the LC-MS spectra, represents the fragment derived from the loss of H₂O (18 Da). Additionally, fragments resulting from the loss of H₂O (18 Da) and C₄H₈ (56 Da) were observed at m/z 201.1643 [C₁₅H₂₀ + H]⁺ (calcd. 201.1638) and m/z 145.1022 [C₁₁H₁₂ + H]⁺ (calcd. 145.1012), respectively. These data indicate the presence of 3,6-epidioxybisa-bola-1,10-diene (5).⁶⁷ Another terpenoid node at m/z 237.1846 [C₁₅H₂₆O₃ - H₂O + H]⁺ (calcd. 237.1849) led to the fragment ions m/z 219.1732 [C₁₅H₂₂O + H]⁺ (calcd. 219.1743) and m/z 201.1632 [C₁₅H₂₀ + H]⁺ (calcd. 201.1638) attributed to the loss of one (18 Da) and two (36 Da) water molecules, respectively. These MS/MS data align with those of the sesquiterpene 5-hydroxyculmorin (6).⁶⁸

The node at m/z 263.1993 [C₁₇H₂₈O₃ - H₂O + H]⁺ afforded the fragment ion at m/z 203.1800 [C₁₅H₂₂ + H]⁺ (calcd. 203.1794) after losing the acetate ester (60 Da). Additionally, a fragment resulting from the loss of C₄H₈ (56 Da) was observed at m/z 147.1172 [C₁₁H₁₄ + H]⁺ (calcd. 201.1178), followed by the loss of C₃H₂ (38 Da), providing m/z 109.1017 [C₈H₁₂ + H]⁺ (calcd. 109.1012). This fragment pattern is similar to that observed for chrysanthemol terpene,⁶⁹ with a difference of 42 Da, suggesting an acetylated derivative (7). Another terpenoid node at m/z 201.1647 [C₁₅H₂₂O - H₂O + H]⁺ (calcd. 201.1038) produced fragment ions at m/z 173.1328 [C₁₃H₁₆ + H]⁺ (calcd. 173.1325) and m/z 159.1173 [C₁₂H₁₄ + H]⁺ (calcd. 159.1168) after losing C₂H₄ (28 Da) and C₃H₆ (42 Da), respectively. Based on these results, along with the literature data, the structure of the sesquiterpenoid nootkatone (8) is proposed.⁷⁰

Furthermore, the nodes at m/z 409.2966 [C₂₄H₄₀O₅ + H]⁺ (calcd. 409.2949) and m/z 345.2406 [C₂₀H₃₄O₃ + Na]⁺ (calcd. 345.2400) were annotated as hyocholic acid (9) and a labdane derivative (10), respectively. Hyocholic acid was identified based on its MS/MS data, considering the fragment ion resulting from the loss of H₂O (18 Da) and CO₂ (44 Da), providing m/z 347.2938 [C₂₀H₂₂O₂ + H]⁺ (calcd. 347.2945).⁷¹ In the mass spectrum of compound 10, fragment ions at m/z 149.1330 [C₁₁H₁₆ + H]⁺ (calcd. 149.1325) and 135.1171 [C₁₀H₁₄ + H]⁺ (calcd. 135.1178) were observed, which align the labdane fragmentation pattern.

Within the prenol lipid group, 13 nodes were identified as fatty acid derivatives (Figure 1), exhibiting similar fragment ions at m/z 95 [C₇H₁₀ + H]⁺, m/z 109 [C₈H₁₂ + H]⁺, and m/z 123 [C₉H₁₄ + H]⁺. This similarity arises from their structural characteristics, featuring a long unsaturated hydrocarbon chain terminated with a carboxyl group. Additionally, common neutral fragment losses of 18 Da (H₂O) and units of 14 Da (CH₂) were observed in the mass spectra.

Through tandem MS comparison utilizing mass spectra libraries, the fatty acid derivatives were annotated as follows: one saturated fatty acid, dodecanedioic acid (11) at m/z 213.1497 [C₁₂H₂₂O₄ - H₂O + H]⁺ (calcd. 213.1485),⁷² and four unsaturated fatty acids: elaidic acid (12) at m/z 283.2645 [C₁₈H₃₄O₂ + H]⁺ (calcd. 283.2632),⁷³ (9Z)-12,13-dihydroxy-9-octadecenoic acid (13) at m/z 332.2800 [C₁₈H₃₄O₄ + NH₄]⁺ (calcd. 332.2795),⁷⁴ and the constitutional isomers 12,13-epoxyoctadecenoic acid (14) and 9,10-epoxyoctadecenoic acid (15) at m/z 297.2409 [C₁₈H₃₂O₃ + H]⁺ (calcd. 297.2424)⁷⁵ and m/z 279.2326 [C₁₈H₃₂O₃ -H₂O + H]⁺ (calcd. 279.2319),⁷⁶ respectively.

GNPS further annotated several polyunsaturated fatty acids within the prenol lipid group, including: (9Z,11E)-13-oxo-9,11-octadecadienoic acid (16) at m/z 295.2271 [C₁₈H₃₀O₃ + H]⁺ (calcd. 295.2268),⁷⁷ (10E,12Z)-10,12-octadecadienoic acid (17) at m/z 263.2379 [C₁₈H₃₂O₂ - H₂O + H]⁺ (calcd. 263.2369), and stearidonic acid (18) at m/z 277.2168 [C₁₈H₂₈O₂ + H]⁺ (calcd. 277.2162).⁷⁸ Additionally, three fatty acid derivatives were annotated as esters: ethyl linoleate (19) at m/z 309.2786 [C₂₀H₃₆O₂ + H]⁺ (calcd. 309.2788),⁷⁹ ethyl (6Z,9Z,12Z,15Z) 6,9,12,15 octadecatetraenoate (20) at m/z 307.2639 [C₂₀H₃₄O₂ + H]⁺ (calcd. 307.2632),⁸⁰ and 2,3-dihydroxypropyl (6Z,9Z,12Z,15Z) 6,9,12,15 octadecatetraenoate (21) at m/z 353.2693 [C₂₁H₃₆O₄ + H]⁺ (calcd. 353.2686).⁸¹

Furthermore, two singletons were identified as fatty acid derivatives: (9Z,12Z)-N-(2-hydroxyethyl)-9,12 octadecadienamide (22) at m/z 324.2906 [C₂₀H₃₇NO₂ + H]⁺ (calcd. 324.2897)⁸² and ceratodictyol derivative (23) at m/z 299.2583 [C₁₉H₃₈O₄ - CH₃OH + H]⁺ (calcd. 299.2581).⁸³ The fragmentation pattern observed in prenol lipids displayed consistent neutral losses, including CH₂ (14 Da), C₂H₄ (28 Da), and C₄H₈ (56 Da), alongside characteristics fragment ions such as m/z 95.0861 [C₇H₁₀ + H]⁺, 109.1017 [C₈H₁₂ + H]⁺, and 123.1173 [C₉H₁₄ + H]⁺. Identified terpenes in this study, including seven sesquiterpenes like alismol and isolongifolol, suggest the involvement of the mevalonate pathway in their biosynthesis.⁸⁴ These metabolites have been associated with various biological activities such as hormone regulation, pigment production, electron transport in photosynthesis, ecological interaction mediation such as pollinator attraction, and roles as allelopathic and antimicrobial agents, among others.⁸⁵,⁸⁶

Benzenoid derivatives annotation

The MF assigned to benzenoid derivatives revealed nine nodes, with six related to amino acid derivatives such as tyrosine, phenylalanine, and proline (Figure 2). The node with m/z 165.0550 [C₉H₁₁NO₃ - NH₃ + H]⁺ (calcd. 165.0546) and fragments at m/z 123.0440 [C₇H₆O₂ + H]⁺ (calcd. 123.0441) and 121.0645 [C₈H₈O + H]⁺ (calcd. 121.0648), resulting from the loss of 42 Da (C₂H₂O) and 44 Da (CO₂), respectively, signifies a fragmentation pattern characteristic of the amino acid tyrosine (24).⁸⁷ Additionally, two derivatives of tyrosine were identified: tyramine (25) at m/z 121.0654 [C₈H₁₁NO - NH₃ + H]⁺ (calcd. 121.0648)⁸⁸ and N-acetyl tyramine (26) at m/z 180.1019 [C₁₀H₁₃NO₂ + H]⁺ (calcd. 180.1019).⁸⁹ The mass spectra of these metabolites exhibited a similar fragmentation pattern with fragment ions at m/z 95, 103, and 121 Da, corresponding to [C₆H₆O + H]⁺, [C₈H₆ + H]⁺, and [C₈H₈O + H]⁺, respectively.

Figure 2
MFs and chemical structures of benzenoids, organoheterocyclic derivatives, and nucleosides.

The observed fragmentation patterns for benzenoids in the MS data are indicative of their structural features and functional groups, allowing for the annotation of specific compounds within the benzenoid family. These patterns include characteristic losses of various moieties such as H₂O, NH₃, CO₂, CO, and CH₂CO, corresponding to specific mass losses of 18, 17, 44, 28, and 42 Da, respectively. For instance, the MS² fragmentation pattern of the node at m/z 208.0974 [C₁₁H₁₃NO₃ + H]⁺ (calcd. 208.0968) suggests the presence of N-acetylphenylalanine (27),⁹⁰ as evidenced by the fragments at m/z 190.0871 [C₁₁H₁₁NO₂ + H]⁺ (calcd. 190.0863) and m/z 166.0864 [C₉H₁₁NO₂ + H]⁺ (calcd. 166.0863), resulting from the loss of water (18 Da) and acetyl group (42 Da), respectively. Similarly, N-acetyl-2 phenylethylalanine (28)⁹¹ and N-prolylphenylalanine (29)⁹² were identified based on their characteristic fragmentation pattern and mass spectral data.

These phenylalanine derivatives exhibit fragment ions at m/z 95, 105, and 120 Da, corresponding to [C₇H₁₀ + H]⁺, [C₈H₈ + H]⁺, and [C₈H₉N + H]⁺, respectively. The biological activities attributed to D. phaseolorum, such as antibacterial and antifungal effects, may be attributed with the presence to these aromatic compounds identified in the extract.³,⁴⁶,⁹³ Overall, the annotation of benzenoid derivatives in D. phaseolorum extract provides insights into the chemical composition and potential bioactive properties of this fungal endophyte.

The nodes corresponding to m/z 123.0440 [C₇H₆O₂ + H]⁺ (calcd. 123.0441), m/z 139.0390 [C₇H₆O₃ +H]⁺ (calcd. 139.0390), and m/z 107.0493 [C₇H₈O₂ - H₂O + H]⁺ (calcd. 107.0491) were identified as 4-hydroxybenzaldehyde (30),⁹⁰ 4-hydroxybenzoic acid (31),⁹⁴ and 3-hydroxybenzyl alcohol (32),⁹⁵ respectively. These phenol derivatives exhibit a common fragmentation pattern characterized by the presence of the ion at m/z 95 [C₆H₆O +H]⁺ resulting from the loss of CO (28 Da), CO₂ (44 Da) and CH₂O (30 Da) for metabolites 30, 31, and 32, respectively.

The identification of these phenolic compounds in the D. phaseolorum extract is significant, as these compounds have been previously associated with phytotoxic effects on grapevine leaf disks and leaf absorption bioassays employing Diaporthe eres fungus.⁹⁶ This suggests that these phenolic derivatives may play a role in the ecological interactions of D. phaseolorum with its host plant, potentially serving as chemical defenses or signaling molecules. Furthermore, the presence of these compounds suggests potential bioactive properties associated with phenolic compounds, which are known for their antioxidant, antimicrobial, and anti-inflammatory activities.

Organoheterocyclic and nucleoside derivatives annotation

The MFs assigned to organoheterocyclic and nucleoside compounds in the D. phaseolorum extract are represented by two nodes each (Figure 2). In the organoheterocyclic group, one of the nodes corresponds to m/z 176.0710 [C₁₀H₉NO₂ + H]⁺ (calcd. 176.0706), which upon fragmentation produced the fragment ion m/z 148.0757 [C₉H₉NO + H]⁺ (calcd. 148.0757) after losing CO (28 Da), followed by the loss of HCN (27 Da), resulting in m/z 121.0653 [C₈H₈O + H]⁺ (calcd. 121.0648). Based on these fragmentation patterns and literature data, this compound was identified as 5-methoxy-1H-indole-3-carbaldehyde (33).⁹⁷ Another node at m/z 167.1071 [C₁₀H₁₄O₂ + H]⁺ (calcd. 167.1067) showed fragmentation indicative of the presence of an alkyl chain and a pyranone scaffold. Fragment ions at m/z 125.0598 [C₇H₈O₂ + H]⁺ (calcd. 125.0597), m/z 111.0444 [C₆H₆O₂ + H]⁺ (calcd. 111.0441), and 97.0289 [C₅H₄O₂ + H]⁺ (calcd. 97.0284), supported the identification of this compound as 6-pentyl-2-pyrone (34).⁹⁸

In the nucleoside cluster, two nodes were identified as adenosine (35)⁹⁹ at m/z 268.1048 [C₁₀H₁₃N₅O₄ + H]⁺ (calcd. 268.1040) and 5’-deoxyadenosine (36)⁹⁹ m/z 252.1080 [C₁₀H₁₃N₅O₃ + H]⁺ (calcd. 252.1091), based on their mass spectra showing similar MS/MS patterns with ions at m/z 136, which represent the purine scaffold of adenine.

The metabolites annotated in this study represent novel findings for the Diaporthe genus, with only a few exceptions. Elaidic acid and stearidonic acid have been previously described for Diaporthe cuppatea,¹⁰⁰ while ethyl linoleate has been identified from Diaporthe schini biomass using supercritical carbon dioxide extraction.¹⁰¹ Additionally, the amino acid derivative tyramine has been reported by various Diaporthe species, including Diaporthe cynaroidis, Diaporthe eres, Diaporthe helianthi, and D. phaseolorum.¹⁰² Phenolic derivatives such as 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid has been isolated from the fungi Diaporthe gulyae¹⁰³ and D. eres.⁹⁶ The analysis using MNs and MolNetEnhancer has elucidated secondary metabolites produced through two stablished metabolic pathways such as acetate and shikimate pathways. These discoveries not only enhance comprehension of the chemical variation within Diaporthe species but also are useful for future research on the chemistry of natural products and ecological interactions involving plants and fungi.

Conclusions

In summary, our study successfully annotated 36 metabolites present in the crude extract of the endophytic fungus D. phaseolorum using dereplication analysis with MN. Remarkably, 31 of these metabolites are reported here for the first time for both this particular endophytic fungus and the Diaporthe genus. Additionally, following the Metabolomics Standard Initiative reporting standards, MolNetEnhancer notably enhanced chemical annotations, elevating them from putative annotations to well-defined chemical classification at level 3 through GNPS library matching. These findings underscore the importance of investigating the chemical profiles of endophytic fungi, as they provide valuable insights into their metabolic potential and potential bioactivities. This knowledge not only contributes to expanding our understanding of fungal secondary metabolism but also holds promise for the discovery of novel bioactive compounds with potential applications in various fields, including pharmaceuticals, agriculture, and biotechnology.

Supplementary Information

Supplementary information, containing detailed data related to mass spectrometry and chromatographic analysis, including molecular formula, adduct, experimental and calculated ion mass, mass accuracy, CScore value, retention time, mirror plot GNPS, and fragmentation proposal of all identified metabolites, is available free of charge at http://jbcs.sbq.org.br as PDF file.PDF

Acknowledgments

We gratefully acknowledge INCT-INAU II (421733/2017-9), FAPEMAT (grant 0217830/2017 and 0250685/2017), CNPq (grant 430089/2016-3), and UFMT for financial support and fellowships. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

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Edited by

Editor handled this article: Paula Homem-de-Mello (Associate)

Publication Dates

• Publication in this collection
  31 May 2024
• Date of issue
  2025

History

• Received
  20 Dec 2023
• Accepted
  30 Apr 2024