Xylapeptide A, an Antibacterial Cyclopentapeptide with an Uncommon L-Pipecolinic Acid Moiety from the Associated Fungus Xylaria sp. (GDG-102)

Two new cyclopentapeptides, xylapeptide A (1) with an uncommon L-pipecolinic acid moiety, and xylapeptide B (2) having a common L-proline residue were identified from an associated fungus Xylaria sp. isolated from the Chinese medicinal plant Sophora tonkinensis. Their planar structures were elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra. The absolute configurations were determined by Marfey’s method and single-crystal X-ray diffraction (Cu Kα) analysis. Xylapeptide A (1) is the first example of cyclopentapeptide with L-Pip of terrestrial origin and showed strong antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 μg/mL.

. Chemical structures of xylapeptides A (1) and B (2). 170.2, six nitrogenated sp 3 carbon resonance at δ C 64.2, 59.0, 57.0, 48.1, 47.5 and 47.4. These above NMR features accounted for 9 of the 11 unsaturations. Further analysis of 2D NMR spectrum allowed five subunits to be established, an N-Me-phenylalanine (N-Me-Phe), a valine (Val), an alanine (Ala), a leucine (Leu), and a pipecolinic acid (Pip) (Fig. 2). Compound 1 was concluded to be a cyclopeptide on the basis of these spectral characteristics. The sequence of residues in 1 was elucidated on the basis of HMBC correlations (Fig. 2). The HMBC correlations from Pip-H-6 to Leu-CO, Leu-NH to Ala-CO, Ala-NH to Val-CO, Val-NH to N-Me-Phe-CO, and N-Me-Phe-α-H to Pip-CO allowed the definition of the residue sequence of 1 as cyclo- were also observed which confirmed the cyclic structure for 1. The configurations of the amino acid residues of 1 was determined by acid hydrolysis, derivatization with N α -(2,4-dinitro-5-fluorophenyl)-L-alalinamide (L-FDAA, the advanced Marfey's method 12 ), and UPLC-MS analysis of the derivatives with comparison to the standards ( Figure S17). Retention times (min) of the standard amino acid derivatives were as follows: L-Ala, D-Ala, L-Val D-Val, L-Leu, D-Leu, N-Me-L-Phe and N-Me-D-Phe were 5.817, 6.811, 7.692, 8.977, 8.996, 10.212, 9.281, and 9.736 min, respectively. Retention times of the derivatives of the acid hydrolysate of 1 were 6.793, 7.676, 8.975, and 9.263 min. It indicated that Leu, Val, and N-Me-Phe had the L-configuration, while Ala was D-configured. Finally, the absolute configuration of 1 was confirmed unambiguously by single-crystal X-ray analysis which indicated an L-configuration of Pip (Fig. 3).
Xylapeptide B (2) was also obtained as a colorless crystal. Its molecular formula C 29 H 43 N 5 O 5 (eleven degrees of unsaturation) was determined on the basis of HRESIMS data. The 1 H and 13 C NMR spectrum ( Table 2) indicated that 2 was very similar to 1 with the additional high-field resonances (δ H 0.79/1.57, δ C 23.2 (CH 2 )) in 1 were absent. Examination of the 2D NMR spectra revealed that 2 shared the same N-Me-Phe, Val, Ala, and Leu units with 1, the only difference is that the Pip in 1 was replaced by a proline (Pro) subunit in 2. The sequence of the  amino acids of 2 was also assigned by HMBC correlations (Fig. 4) and fully supported by the ESI MS 2 experiment results ( Fig. S16) which revealed the same linkage with 1. The configurations of the amino acid residues were also determined by Marfey's method. The retention times (min) of the standard L-Pro and D-Pro were 6.262 and 6.689 min, respectively. The retention times for the acid hydrolysate of 2 were 6.285, 6.818, 7.707, 9.004, and 9.318 min, respectively, which correspond to L-Pro, L-Leu, L-Val, N-Me-L-Phe, and D-Ala ( Figure S17). Besides, by slow crystallization from CH 3 OH, single crystals of 2 suitable for X-ray diffraction analysis using Cu Kα radiation was also obtained, allowing the complete structure to be established unambiguously (Fig. 3).    Naturally occurring cyclic peptides with nonproteinogenic amino acids mainly contained pipecolinic acid (Pip) and anthranilic acid (Ant) 13 . Up to now, nearly twenty cyclic peptides with Pip residues isolated from nature sources have been reported which occupied a very small proportion of the identified peptide. Most of the cyclic peptides with Pip residues reported so far were of a marine origin 14 27 ). Cyclic peptides with Pip from terrestrial origins are rare including cyclohexapeptides PF1171 A−G (unknown ascomycete OK-128 13 ), and cyclodecadepsipeptides, clavariopsins A and B (aquatic hyphomycetes Clavariopsis aquatic 28 ). Up to now, the Pip in all the peptides is L-configuration. Xylapeptide A (1) is the first cyclopentapeptide from a terrestrial origin with L-Pip.
The transformation of Lys to Pip in plants and fungus has been studied by radioactive labeling experiment. In the Ca-deficient wheat plants, the 14 C-L-Lys was transported rapidly in to the shoots and then degraded to Pip in many pathways 29,30 . In the Rhizoctonia leguminicola fungus, the L-[U-14 C]-Lys is used to form L-Pip 31,32 . Although Gatto and co-workers reported the first in vitro characterization of a lysine cyclodeaminase (RapL) which utilized the NAD + as a cofactor in a catalytic manner in Streptomyces hygroscopicus to catalyst lysine into Pip 33 , the genes controlling the biosynthesis of Lys to Pip have not been described yet. It is suggested that the Pip in xylapeptide A (1) may also be converted from lysine, and a radioactive labeling experiment is needed.
Xylapeptides A (1) and B (2) were evaluated for their antibacterial activity. Compound 1 showed strong and selective antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 μg/mL. Compound 2 exhibited strong and broad antibacterial spectrum against B. subtilis, B. cereus, B. megaterium, Micrococcus luteus, Staphylococcus aureus, and Shigella castellani with MIC values of 12.5, 6.25, 6.25, 12.5, 12.5, and 12.5 μg/ mL, respectively. Compound 2 also showed a strong antifungal activity against Canidia albicans with MIC value of 12.5 μg/mL. This suggested that the L-Pip residue and L-Pro residue play different roles in the antimicrobial activity. The cytotoxic, α-glucosidase inhibitory, and antiviral activities of 1 and 2 were also evaluated. However, they were all inactive.
In summary, two new cyclopentapeptides, xylapeptides A (1) and B (2), were isolated from a Xylaria sp. fungus cultured from the Chinese medicinal plant S. tonkinensis. Xylapeptide A (1) contains a non-proteinogenic amino acid, L-pipecolinic acid (L-Pip), which is the first example of cyclopentapeptide from a terrestrial origin with L-Pip. Compound 1 displayed a strong antibacterial activity against Bacillus subtilis and B. cereus. Xylapeptide B (2) has a broader antibacterial spectrum as well as antifungal activity.

Methods
General experimental procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were determined on a Shimadzu double-beam 210 A spectrometer. IR spectra were taken on a Bruker EQUINOX 55 spectrometer using KBr pellets. 1D and 2D NMR spectra were obtained on an Agilent DD2 400 MHz NMR spectrometer or a DRX-500 spectrometer, respectively, with TMS as the internal standard. ESIMS and HRESIMS spectra were obtained from a Micromass Q-TOF spectrometer and a Thermo Scientific LTQ Orbitrap XL spectrometer. Semi-preparative HPLC was performed on a Waters 1525 system using a C18 (Kromasil, 5 μm, 10 × 250 mm) column coupled with a Waters 2996 photodiode array detector. UPLC MS was performed on Waters UPLC ® system using a C18 column [ACQUITY UPLC ® BEH C18, 2.1 × 50 mm, 1.7 μm; The leaves were designed to undergo a process described as surface sterilization 34 . The surface-sterilized leaves were aseptically sectioned into small pieces and plated onto the PDA plates containing an antibiotic to suppress bacterial growth (composition of isolation medium: potatoes 200 g/L, glucose 20 g/L, agar 15 g/L and ampicillin sodium 0.2 g/L in water). After incubation at 27 °C, the fungal strain under investigation was isolated from the growing cultures by repeated reinoculation on PDA plates. The fungus was identified as Xylaria sp. (GenBank accession number KU645984) using a molecular biological protocol by DNA amplification and sequencing of the ITS region as described in ref. 34. Fermentation, extraction, and isolation. The fungal strain was cultivated in 20 L of liquid medium (composition of medium: 200 g/L cooked and sliced potatoes, 20 g/L glucose in water, in 1 L Erlenmeyer flasks each containing 500 mL of culture broth) at room temperature without shaking for 7 weeks. Then the culture was filtered to separate the culture broth from the mycelia. The culture broth was extracted with an equal volume of ethyl acetate (EtOAc) and the fungal mycelia were extracted with CH 2 Cl 2 -CH 3 OH (v:v, 1:1) for three times, respectively. The mycelia extraction were concentrated to about 0.3 L and extracted with EtOAc. The EtOAc layer was evaporated to dryness under reduced pressure to give broth extract (4.5 g) and mycelia extract (4.8 g), respectively. The broth extract was subjected to silica gel CC (petroleum ether (PE)−EtOAc, v-v, gradient) to afford five fractions (Fr.1-Fr.5). Fr.4 was repeated purified by ODS CC (CH 3 OH−H 2 O) and then recrystallized in MeOH to obtain 2 (20 mg). In search for similar compounds, the mycelia extraction was subjected to silica gel CC (PE−EtOAc, v:v, gradient) to afford six fractions (Fr.   Table 2 Biological assays. The antibacterial activity against B. subtilis, B. cereus, B. megaterium, Micrococcus luteus, Staphylococcus aureus, and Shigella castellani, and antifungal activity against C. albicans were evaluated by using 96-well microtitre plates 35 , with ampicillin as positive control. The cytotoxic activity against human colon carcinoma (HCT-116), human cervical cancer (HeLa), non-small cell lung carcinoma (A549), human breast cancer (MCF-7), human pancreatic carcinoma (BXPC-3), and chronic myelocytic leukemia (K562) cell lines was evaluated by the SRB method 36 and MTT method 37 , with adriamycin as a positive control. The antiviral activity against human cytomegalovirus (HCMV), and herpes simplex virus type 1 (HSV-1) virus was evaluated by the cytopathic effect inhibition assay 38 , with cidofovir and acyclovir as positive control. The α-glucosidase inhibitory activity was evaluated by Yilmazer-Musa's method 39 , with acarbose as a positive control.