New Cyclic Pentapeptides from the Mangrove-Derived Aspergillus fumigatus GXIMD 03099

Four new cyclic pentapeptides, avellanins D–G (1–4), together with four known compounds (5–8), were isolated from a mangrove-derived Aspergillus fumigatus GXIMD 03099 fungus from Acanthus ilicifolius L. Their structures were elucidated by analysis of HRESIMS, NMR, and ESI-MS/MS data. Their absolute configurations were determined by X-ray diffraction analysis and Marfey’s method. Compounds 1–8 were screened for insecticidal and antibacterial activities. Compound 2 showed insecticidal activity against newly hatched larvae of Culex quinquefasciatus with an LC50 value of 86.6 µM; compound 4 had weak activity against Vibrio harveyi with an MIC value of 5.85 µM.


Introduction
The secondary metabolites of marine-derived fungi contain a variety of structural types, such as polyketides, terpenoids, alkaloids, and peptides, which are recognized as potential resources for drug discovery [1].Peptides have been widely used in the clinic, and the structure of cyclic peptides is one of the most special peptides [2][3][4][5].Cyclic peptides (or cyclopeptides), which originate from natural resources, are cyclic compounds mainly composed of proteinogenic or non-proteinogenic amino acids linked together by amide bonds [6].According to the composition of amino acid residues in the core ring, the structure of fungal cyclic peptides can be divided into cyclic dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, etc. [7].Most of the cyclic peptides showed significant biological activities, such as cytotoxicity [8], antitubercular [9], antiviral [10], anti-inflammatory [11], antimicrobial [12], and pancreatic lipase (PL) inhibitory activities [13].Due to the particularly complex and interesting chemical structures and wide diversity of biological activities of cyclic peptides, they have been attracting significant attention from chemists and pharmacologists [14][15][16].

Structural Elucidation
In this study, the suitable crystals of compound 5 were obtained from methanol solutions.Analysis of the single-crystal X-ray diffraction data using Cu Kα radiation (with a Flack parameter of 0.06( 6)) confirmed the absolute configuration of compound 5, which was determined to be L-Pro-Ant-L-Ile-D-Ala-N-Me-D-Phe and named avellanin A.
Furthermore, detailed analysis of the heteronuclear single quantum correlation (HSQC), correlation spectroscopy (COSY), heteronuclear multiple bond correlation

Structural Elucidation
In this study, the suitable crystals of compound 5 were obtained from methanol solutions.Analysis of the single-crystal X-ray diffraction data using Cu Kα radiation (with a Flack parameter of 0.06( 6)) confirmed the absolute configuration of compound 5, which was determined to be L -Pro-Ant-L -Ile-D -Ala-N-Me-D -Phe and named avellanin A.

Biological Activity
Compounds 1-8 were subjected to several biological assays available in our laboratories, including insecticidal and antibacterial activity tests.The result showed that compound 2 exhibited insecticidal activity against newly hatched larvae of Culex quinquefasciatus with an LC50 value of 86.6 µM, and compound 4 had weak activity against Vibrio harveyi with an MIC value of 5.85 µM.

Fungal Material, Isolation, and Purification
The fungal strain Aspergillus fumigatus GXIMD 03099 was isolated from the mangrove Acanthus ilicifolius L., collected in the Guangxi Shankou Mangrove Nature Reserve in July, 2020, and was identified according to its morphological characteristics and a molecular biological protocol by 18S rRNA amplification and sequencing of the ITS region.The sequence data have been submitted to GenBank, with accession number ON668102.The fungal strain was cultivated in rice solid-substrate medium (100 Erlenmeyer flasks, each containing 80 g of rice and 0.5 g of sea salt in 100 mL of distilled H2O in 1 L Erlenmeyer flasks) at room temperature under static conditions and daylight for 30 days.
The solid rice medium was extracted with EtOAc.The organic extract was concentrated in vacuo to yield an oily residue (76.5 g), which was subjected to silica gel Compound 2 was obtained as yellow powder.Its HRESIMS spectrum showed the ion at m/z 598.3001 [M + Na] + (calcd for 598.3005), suggesting its molecular formula as C 32 H 41 N 5 O 5 with 15 degrees of unsaturation.The 1 H and 13 C NMR data (Table 1) of compound 2 were similar to those of compound 5, the only obvious difference being the additional presence of one methylene (δ H 1.49 and 1.30, and δ C 17.6) in compound 2. Detailed analysis of the NMR spectra indicated that the Pro residue in compound 5 was replaced by a Pip unit in compound 2. The results were confirmed by the COSY correlations of H-2 (δ H 4.86) to H-3 (δ H 1.69 and 1.34), H-4 (δ H 1.49 and 1.30), H-5 (δ H 1.62), and H-6 (δ H 3.57 and 3.40), and HMBC correlations of H-2 (δ H 4.86) to C-1 (δ C 173.7), C-4 (δ C 17.6), and Ant-CO (δ C 171.9), and H-6 (δ H 3.57 and 3.40) to C-4 (δ C 17.6) and Ant-CO (δ C 171.9) (Figure 2).Therefore, the amino acid sequence of compound 2 was assigned as Pip-Ant-Ile-Ala-N-Me-Phe by the key 2D NMR correlations (Figure 2) and further supported by ESI-MS/MS analysis (Figures 3 and S21).The absolute configurations of the amino acid residues of compound 2 were identified using Marfey's method (Figure S22) [25].In addition, basis on the absolute configuration of avellanins A and B (5 and 6) [18], and a shared biogenesis in the same fungus.Thus, the completed structure of compound 2 was elucidated as L -Pip-Ant-L -Ile-D -Ala-N-Me-D -Phe and named avellanin E.
Compound 3 was obtained as a yellow amorphous powder.It is assigned the molecular formula C 29 H 35 N 5 O 5 on the basis of HRESIMS spectrum, which revealed 15 degrees of unsaturation.Carefully analysis of the 1 H and 13 C NMR data (Table 2) of compound 3 closely resemble those of compound 1, indicating that compound 3 was an analogue of compound 1, the main difference being one amino acid constitution, corresponding to the loss of a methylene group from compound 1.The above results were confirmed by the COSY correlations of H-4 (δ H 0.88) and H-5 (δ H 0.86) to H-3 (δ H 2.31) and H-2 (δ H 5.29), and HMBC correlations of H-3 (δ H 2.31) to C-1 (δ C 168.9) and C-2 (δ C 58.7), and H-2 (δ H 5.29) to C-1 (δ C 168.9), C-3 (δ C 29.4), C-4 (δ C 19.0), C-5 (δ C 16.9), and Gly-CO (δ C 169.4) (Figure 2).Indeed, analysis of 2D NMR data established the amino acid sequence of compound 3 to be Pro-Ant-Val-Gly-N-Me-Phe.The ESI-MS/MS data also supported the sequence order of amino acid residues (Figures 3 and S32).In addition, the absolute configuration of compound 3 was identified using Marfey's method (Figure S33) [25].Thus, the completed structure of compound 3 was elucidated as L -Pro-Ant-L -Val-Gly-N-Me-D -Phe and named avellanin F.

Biological Activity
Compounds 1-8 were subjected to several biological assays available in our laboratories, including insecticidal and antibacterial activity tests.The result showed that compound 2 exhibited insecticidal activity against newly hatched larvae of Culex quinquefasciatus with an LC 50 value of 86.6 µM, and compound 4 had weak activity against Vibrio harveyi with an MIC value of 5.85 µM.

Fungal Material, Isolation, and Purification
The fungal strain Aspergillus fumigatus GXIMD 03099 was isolated from the mangrove Acanthus ilicifolius L., collected in the Guangxi Shankou Mangrove Nature Reserve in July, 2020, and was identified according to its morphological characteristics and a molecular biological protocol by 18S rRNA amplification and sequencing of the ITS region.The sequence data have been submitted to GenBank, with accession number ON668102.The fungal strain was cultivated in rice solid-substrate medium (100 Erlenmeyer flasks, each containing 80 g of rice and 0.5 g of sea salt in 100 mL of distilled H 2 O in 1 L Erlenmeyer flasks) at room temperature under static conditions and daylight for 30 days.

X-ray Crystallographic Analyses of Compound 5
Colorless crystals of compound 5 were obtained from MeOH/H 2 O. Single-crystal Xray diffraction data were collected on an Xcalibur, Atlas, Gemini ultra-diffractometer with Cu Kα radiation (λ = 1.54184Å) at 99.9 (3) K, respectively.The structure was solved by direct methods (ShelXT) and refined with the ShelXL refinement package using least squares minimization.All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined relatively isotropically with a riding model.Crystallographic data of compound 5 have been deposited in the Cambridge Crystallographic Data Centre with deposition number CCDC 2323898.Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44-(0)1223-336033, or E-mail: deposit@ccdc.cam.ac.uk).

Acid Hydrolysis and Marfey's Analysis Methods
Hydrolysis and Marfey's analysis were carried out according to Xiao Lin et al. [25].Compounds 1-4 (100 µg each) were dissolved in 100 µL of 6 M HCl and hydrolyzed in a pressure-resistant reaction flask at 110 • C for 24 h, then the HCl was removed by evaporation under a stream of N 2 gas.The hydrolysis product was dissolved in 50 µL of 1 M NaHCO 3 to adjust the pH to 7-8 and reacted with 10 µL of Marfey's reagent (1-fluoro-2, 4-dinitrophenyl-5-L -leucinamide, L -FDLA; 1% solution in acetone) at 40 • C for 1 h, and then 50 µL of 1 M HCl was used to neutralize the reactant pH to 2-3.Finally, the mixture was diluted with MeCN (800 µL) and filtered.Amino acid standards L -Pip, L -Pro, L -Val, L -Ile, and L -Abu were derivatized with L -FDLA.The L -Pro, L -Ile, D -Ala, and N-Me-D -Phe from compound 5 crystals were used as standards for comparison with the hydrolysate derivatives of compounds 1-4.Marfey's derivatives of the compounds and the L-FDAA standards described above were analyzed using HPLC-DAD-MS in positive ion mode using a WATERS Xevo G2-S Qtof Quadrupole Timeof-Flight Mass Spectrometry (Waters, Milford, MA, USA).

Insecticidal Activities
The insecticidal activity against Culex quinquefasciatus larvae was evaluated according to methods reported in the literature [21].Newly hatched larvae were housed at 25 ± 1 • C and 80% relative humidity.Two sets of replicates were set up with dimethyl sulfoxide as a

-2. 1 a 2 b Unit Position δ H , (J in Hz) δ C , Type Unit Position δ H , (J in Hz) δ C , Type
a Measured in DMSO-d 6 at 500 MHz for 1 H NMR and 125 MHz for13C NMR.b Measured in DMSO-d 6 at 400 MHz for 1 H NMR and 100 MHz for13C NMR.