Penicitroamide, an Antimicrobial Metabolite with High Carbonylization from the Endophytic Fungus Penicillium sp. (NO. 24)

Penicitroamide (1), a new metabolite with a new framework, was isolated from the ethyl acetate extract of the PDB (Potato Dextrose Broth) medium of Penicillium sp. (NO. 24). The endophytic fungus Penicillium sp. (NO. 24) was obtained from the healthy leaves of Tapiscia sinensis Oliv. The structure of penicitroamide (1) features a bicyclo[3.2.1]octane core unit with a high degree of carbonylization (four carbonyl groups and one enol group). The chemical structure of penicitroamide (1) was elucidated by analysis of 1D-, 2D-NMR and MS data. In bioassays, penicitroamide (1) displayed antibacterial potency against two plant pathogens, Erwinia carotovora subsp. Carotovora (Jones) Bersey, et al. and Sclerotium rolfsii Sacc. with MIC50 at 45 and 50 μg/mL. Compound 1 also showed 60% lethality against brine shrimp at 10 μg/mL. Penicitroamide (1) exhibited no significant activity against A549, Caski, HepG2 and MCF-7 cells with IC50 > 50 μg/mL. Finally, the possible biosynthetic pathway of penicitroamide (1) was discussed.


Introduction
Endophytic fungi from plants can yield an extremely large amount of natural products with unique frameworks including high biological potency and low side effects [1][2][3][4][5][6][7]. Natural products scientists focus most of their attention to the secondary metabolites derived from plant endophytic fungi rather than from the traditional medicinal plants or folk medicines. Meanwhile, genetic biologists are involved in the whole genome sequencing of the filamentous fungi genera-Aspergillus, Penicillium and Fusarium [8]. Currently we can find out which gene clusters control the biosynthesis of natural products using methods of biological information analysis. Thus, there is great potency for controllable fungi natural products manufacturing using genetic engineering technology. In our ongoing search for new bioactive secondary metabolites from plant endophytic fungi in the Shennongjia District [9][10][11][12], a chemical investigation of an endophytic Penicillium sp. (NO. 24), isolated from the healthy leaves of Tapiscia sinensis Oliv., was performed, and led to the isolation of a new alkaloid with a new bicyclo[3.2.1]octane core structural framework-penicitroamide (1). Interestingly, there are four carbonyl groups and a stable enol group in penicitroamide (1), which is rare in natural products.
Its structure was proven by the analysis of 1D-, 2D-NMR, EI-MS, HR-EI-MS and CD data. Herein, we reported the isolation, structure elucidation, and biological activities of penicitroamide (1). (1) Penicitroamide (1) was obtained as a brown oil, [α] 25 D = −19 (c, 0.01). Its molecular formula was determined as C 29 H 39 NO 6 by HR-EI-MS with m/z 497.2775 (calcd. 497.2777). From the results of mass spectra, there was (2n + 1) nitrogen atoms in compound 1 according to the Nitrogen Rule of the mass spectrum. Simultaneously, 11 degrees of unsaturation were obtained from the molecular formula of compound 1, and three carbonyl groups, one ester or amide carbonyl group and eight olefinic carbons in the carbon resonances were due to eight degrees of unsaturation; thus, the three remaining unsaturation degrees can be ascribed to the three aliphatic rings in penicitroamide (1).

Structural Elucidationof Penicitroamide
There were five methyl groups, six methylene groups, 10 methyne groups and eight quaternary carbons, including three carbonyl groups and one ester or amide carbonyl group at δ 208.5 (C-7), l97.3 (C-11), 206.8 (C-20) and 170.7 (C-18) in the 13  Both the cross peaks between 170.7 (C-18) and 4.65 (H-5), 2.75 (H-2b) in the HMBC spectrum and the chemical shifts of C-2 and C-5 at δ 47.6 and 61.2 hinted that there may be a nitrogen atom among the C-2, C-5 and C-18, which was verified by the HR-EI-MS. Additionally, from the correlations of 4.65 (H-5) to 47.6 (C-2), 29.2 (C-3) and 34.9 (C-4), from 3.71/2.75 (H-2) to 34.9 (C-4) and 61.2 (C-5), and from 1.50 (H-3) to 61.2 (C-5), the substructure of part A was inferred (see Figure 1 and Supplementary Materials). A similar structural subunit of part A was found in Perinadine A and Scalusamide A [13,14]. Comparing their 1 H-and 13 C-NMR data, the chemical shifts of C-2-C-5, C-18-C-27 and C-30 in penicitroamide (1) were similar to those in Perinadine A and Scalusamide A, considering the different chemical surroundings of C-4 and C-5 and the deuterium solvent effect between DMSO-d 6 and CDCl 3 . The E-geometry of the double bond was hinted at by the References [13,14], because the two olefinic protons on C-25 and C-26 were overlapped in penicitroamide (1).
The relative conformation of penicitroamide (1) was established by NOESY correlations. In the NOESY spectrum, the correlation from 3.35 (H-4) to 4.65 (H-5) proved the cis relationship between H-4 and H-5 (see Figure 2). Since compound 1 was a bridge-ring compound, the groups on the two bridgehead carbons must lie on the same side. The correlations from 4.65 (H-5) to 0.99 (H-28) and from 3.29 (H-9) to 1.07 (H-29) established the relative conformations of C-4, C-5, C-8 and C-9 (see Figure 2), meaning that the H-4, H-5, H-28, H-9 and H-29 were all a cis relationship in the core [5/6/5] ring systems. Unfortunately, the relative conformation of H-19 could not be determined using hints from the NOESY spectrum and the methyl group (H-30) located in the linear chain structure. The CD spectrum of penicitroamide (1) showed a positive Cotton effect at 360 (+7.3) and 220 (+1.4) nm and a negative Cotton effect at 281 (−7.8) nm (see Figure 3), and the value of specific optical rotation was −19 with the concentration of 0.01 mg/mL in the CH3OH solvent. Since the physical state is an oil, we cannot determine the absolute configuration of six chiral carbons using X-ray diffraction of penicitroamide (1), and only the relative configuration of penicitroamide (1) was reported in this paper.  1]octane core unit through C-6 and C-9, and the six-membered ring (B ring) was fused with the five-membered ring containing a nitrogen atom at C-4 and C-5. Finally, the planar structure of compound 1 was built, a new carbon skeleton named penicitroamide (1).
The relative conformation of penicitroamide (1) was established by NOESY correlations. In the NOESY spectrum, the correlation from 3.35 (H-4) to 4.65 (H-5) proved the cis relationship between H-4 and H-5 (see Figure 2). Since compound 1 was a bridge-ring compound, the groups on the two bridgehead carbons must lie on the same side. The correlations from 4.65 (H-5) to 0.99 (H-28) and from 3.29 (H-9) to 1.07 (H-29) established the relative conformations of C-4, C-5, C-8 and C-9 (see Figure 2), meaning that the H-4, H-5, H-28, H-9 and H-29 were all a cis relationship in the core [5/6/5] ring systems. Unfortunately, the relative conformation of H-19 could not be determined using hints from the NOESY spectrum and the methyl group (H-30) located in the linear chain structure. The CD spectrum of penicitroamide (1) showed a positive Cotton effect at 360 (+7.3) and 220 (+1.4) nm and a negative Cotton effect at 281 (−7.8) nm (see Figure 3), and the value of specific optical rotation was −19 with the concentration of 0.01 mg/mL in the CH 3 OH solvent. Since the physical state is an oil, we cannot determine the absolute configuration of six chiral carbons using X-ray diffraction of penicitroamide (1), and only the relative configuration of penicitroamide (1) was reported in this paper.   Figure 3), and the value of specific optical rotation was −19 with the concentration of 0.01 mg/mL in the CH3OH solvent. Since the physical state is an oil, we cannot determine the absolute configuration of six chiral carbons using X-ray diffraction of penicitroamide (1), and only the relative configuration of penicitroamide (1) was reported in this paper.   (1) Prolines play an important role in plants for responding to environmental and biotic stress [15]. When the plants underwent drought [16], saline [17], UV radiation [18], heavy metal pollution [19] and plant pathogen invasion [20,21], the amount of prolines massively accumulated. After proline accumulation, the endophytic fungi residing in the plants may take full advantage of the massive amount of prolines to synthesize secondary metabolites.   (1) Prolines play an important role in plants for responding to environmental and biotic stress [15]. When the plants underwent drought [16], saline [17], UV radiation [18], heavy metal pollution [19] and plant pathogen invasion [20,21], the amount of prolines massively accumulated. After proline accumulation, the endophytic fungi residing in the plants may take full advantage of the massive amount of prolines to synthesize secondary metabolites.  (1) Prolines play an important role in plants for responding to environmental and biotic stress [15]. When the plants underwent drought [16], saline [17], UV radiation [18], heavy metal pollution [19] and plant pathogen invasion [20,21], the amount of prolines massively accumulated. After proline accumulation, the endophytic fungi residing in the plants may take full advantage of the massive amount of prolines to synthesize secondary metabolites. Apparently, there was a proline core pyrrole ring in the structure of compound 1; the pyrrole ring was possibly derived from proline by the decarboxylation reaction by the proline decarboxylase (see Scheme 1). The other parts of 1 can be decomposed into two polyketide parts, a and b (see Scheme 1). The two polyketide parts, a and b, could originate from the two six-acetyl coenzyme units in different binding modes, respectively. Furthermore, part a and part c could possibly be formed from the intermediate d by amidation. Then, the intermediates b and d could combine together to form the nucleus structure of 1 by the Diels-Alder reaction. Finally, the intermediate e could undergo epoxidation, epoxide cleavage, oxidation and hydroxylation resulting in compound 1. (1) Compound 1 was evaluated against four cancer cell lines, A549, Caski, HepG2 and MCF-7, and the anticancer assay results showed no significant activity to the above four cancer cell lines with IC 50 > 50 µg/mL. Then the antimicrobial assay against plant-pathogenic Erwinia carotovora subsp. Carotovora (Jones) Bersey et al. and Sclerotium rolfsii Sacc. were carried out using the well diffusion method; penicitroamide (1) displayed moderate inhibiting activity on two plant pathogens with MIC 50 at 45 and 50 µg/mL. The positive control, mitomycin, was employed with MIC 50 at 0.0.5-1 µg/mL. Finally, brine shrimp lethality assay was used in the laboratory, and the results showed 60% lethality to brine shrimp at 10 µg/mL.

General Procedures
UV spectra were obtained on a SCINCO Spectrometer; IR spectra were recorded on Nicoler Auatar Spectrometer series FT360 spectrophotometer. 1D-and 2D-NMR spectra were recorded using a Bruker Ultrashield-400 MHz NMR spectrometer (Fällanden, Switzerland). Mass spectra were obtained on an EI mass spectrometer (Harrisburg, PA, USA). Silica gel GF254 (10-40 µm) was used for TLC and silica gel (200-300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Fractions were monitored by TLC, and the spots were visualized under ultraviolet lamp with 254 and in iodine cylinder. Semi-preparative HPLC was performed on Dionex Ultra-3000 (Sunnyvale, CA, USA) and 1525 Waters (USA) using a Cosmosil C-18 column (10 µm × 20 mm × 250 mm and 5 µm × 4.6 mm × 250 mm). All organic solvents were analytical grade and were redistilled before use. The deuterium solvent DMSO-d 6 was from CIL Company (Tewksbury, MA, USA). The CD spectrum was obtained on JASCO J-810 Spectrometer (Japan). The HPLC CH 3 OH and CH 3 CN were purchased from Tedia Company (Tianjin, China) and the water was purchased from WaHaHa (Wuhan, China) and YiBao Companies (Hangzhou, China).

Isolation and Identification of the Strain
The healthy leaves of Tapiscia sinensis Oliv. were collected in the Shennongjia National Forest Park and washed under running water, then immersed in the 5%-10% NaClO solution for 5 min, in 75% ethanol solution for 3 min, finally, washed by aseptic water three times, and then the aseptic water was removed by sterile filter papers. The sterilized leaves were cut into small pieces 0.5 cm × 0.5 cm 2 , and those pieces were placed on PDA plates in the incubator under 28 • C. After the microorganisms on the PDA plates were ready for purification, the growing fungi were repeatedly purified using the Streak method, until single colonies were obtained. The pure single colonies were then deposited on the slants under 4 • C. In total, 20 endophytic fungi were obtained from the leaves of Tapiscia sinensis Oliv., then those fungi were fermented in 200 mL PDB liquid medium with 500 mL Erlenmeyer flasks. The fungus (NO. 24) was selected for further chemical constituents investigation according to the results of the bioactivity assays and HPLC-DAD analysis-guiding. The fungus (NO. 24) was cultured in 50 L PDB liquid medium with 250,500 mL-Erlenmeyer flasks on the electronic oscillator under 28 • C with the speed at 120 r/min for 20 days. During the identification of the fungus NO. 24, we found that the 18S rDNA results shared 99% similarity to the fungus Penicillium citrinum, thus the specie of fungus NO. 24 was identified as Penicillium citrinum. (1) The broth and mycelium were collected by eight layers of gauze, and the 50 L broth was extracted with 20 L ethyl acetate for five times, then the extract was condensed to a crude extract of 26 g under vacuum. Then 20 g of the crude extract was then subjected to silica gel column chromatography and eluted from light petroleum ether to ethyl acetate, then to methanol. The subfractions were merged into six fractions using TLC plates. The fraction D (0.5 g) was further subjected to silica gel column chromatography with the gradient elution from 30% petroleum ether/ethyl acetate to 40% petroleum ether/ethyl acetate in volume, then the fraction D was fractionated into two subfractions. And the subfraction D-1 (80 mg) was further purified by semi-preparative reverse-phase HPLC with constant elution using 45% acetronitrile-H 2 O to afford compound 1 (t R 15.5 min, 4.5 mg).

The Isolation and Purification of Penicitroamide
Penicitroamide (1)

Cytotoxic Activity against Four Cancer Cell Lines In Vitro
A549, Caski, HepG2 and MCF-7 cells were cultured in RPMI 1640 medium (HyClone) supplemented with 10% FBS (HyClone). The cells were kept in 5% CO 2 at 37 • C. The 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, Kawasaki, Japan) colorimetric assay was used to evaluate cell proliferation in the presence of different chemicals. The cells were seeded in 96-well culture plates and treated with desired concentrations of chemicals for a further 24 h. After treatment, the cells were incubated at 37 • C with MTT (10 µL/well, 5 mg/mL) for 4 h, and the cell growth response to the chemicals was determined by measuring the absorbance at 570 nm on a plate reader. Three replicates were used for each treatment. In the anticancer activity in vitro experiment, mitomycin was employed as positive control for the anticancer assay.

Antimicrobial Assay
Antimicrobial assay against plant-pathogenic Erwinia carotovora subsp. Carotovora (Jones) Bersey et al. and Sclerotium rolfsii Sacc. were carried out using the well diffusion method [22]. Amphotericin B was used as positive control for the antimicrobial assay.

Brine Shrimp Lethality Assay
The eggs of brine shrimp (Artemia salina) were purchased from Qingdao Haina Baichuan Biological Engineering Limited Company (Qingdao, China), and were incubated in artificial sea water. The artificial sea water contained 7.5 g bay salts in every liter of water, then was boiled and filtrated for the culture mediums of brine shrimp (Artemia salina). The 15 mg of eggs of brine shrimp were suspended in the 300 mL artificial sea water medium in a 500 mL flask for 48 h pumping in air and water, bathing at 25 • C. The hatchability of brine shrimp was between 85% and 88%. The 1 mg of penicitroamide (1) was dissolved in 200 µL DMSO. 10 µL, 5 µL and 2 µL of penicitroamide (1) was dropped into three wells containing 990 µL, 995 µL and 998 µL medium on a 24 wells plate, respectively. Each concentration sample was provided with three parallel samples. There was a DMSO control group and a medium blank group in a 24-well plate. There were 20 brine shrimps in every well. The 24-well plate containing the samples was incubated for 24 h at 25 • C. The lethality of brine shrimp and LC 50 value were calculated according to Soli improved method.

Conclusions
Penicitroamide (1), a new metabolite with a new framework, was isolated from the endophytic fungus Penicillium sp. (NO. 24), isolated from the healthy leaves of Tapiscia sinensis Oliv. Penicitroamide (1) features a bicyclo[3.2.1]octane core unit with a high degree of carbonylization (four carbonyl groups and one enol group). The chemical structure of 1 was elucidated by analysis of 1D, 2D-NMR and MS data. In the bioassay, penicitroamide (1) displayed antibacterial potency to plant-pathogenic Erwinia carotovora subsp. Carotovora (Jones) Bersey et al. and Sclerotium rolfsii Sacc. with MIC 50 at 45 and 50 µg/mL, and compound 1 also showed 60% lethality to brine shrimp at 10 µg/mL, but exhibited no significant activity toward A549, Caski, HepG2 and MCF-7 cells with IC 50 > 50 µg/mL. Finally, the possible biosynthetic pathway of penicitroamide (1) was discussed; 1 possibly was evolved through the hybridization of prolines and the polyketide biosynthesis pathways by a key Diels-Alder reaction in Penicillium sp. (NO. 24).