Two new linear peptides from the marine-derived fungus Penicillium sp. SCSIO 41512

Abstract Two new linear peptides, penicamides A and B (1 and 2), together with four known analogous were isolated from the extracts of the marine-derived fungus Penicillium sp. SCSIO 41512. Their structures were elucidated by analysis of 1D/2D NMR data and HRESI-MS. Their absolute configurations were established by Marfey’s methods and quantum chemical calculations. Graphical Abstract


Introduction
Marine derived fungi are valuable sources of structurally diverse bioactive lead drugs [1]. Most bioactive peptides isolated from marine environments are obtained from symbiont microorganisms [2]. Peptides are specific protein fragments, and display diverse pharmacological and biological activities, such as cytotoxic, antimicrobial, antiviral, lipid lowering, and anti-inflammatory activities [3][4][5][6].

Results and discussion
Compound 1 was obtained as pale yellow oil. Its molecular formula was established as C 16 11.5). Parts of these NMR data were similar to those of maremycins A-B [13] and asperochramides C-D [14], which suggested that 1 was also an indole alkaloid with a different peptide substituent. Detailed analysis of the HSQC, HMBC, and COSY data of 1 (Figure 2) proved the existence of a 3-hydroxyl-2-indolone skeleton in 1, the same as asperochramides C-D [14], and also suggested the presence of an isoleucine  (Ile) residue in 1. Furthermore, the HMBC correlations from NH (d H 7.81) to C-9 (d C 168.5)/C-11 (d C 56.2)/C-16 (d C 172.6) suggested the Ile amino acid residue connected with 3-hydroxyl-2-indolone skeleton by an amido group. Hydrolysis of 1 in 6 N HCl followed by Marfey's method and HPLC analysis with a chiral chromatographic column established the configuration of Ile in 1 as L-Ile ( Figure S17).
The absolute configuration of C-2 in 1 was determined as 2 R by comparing its experimental ECD spectrum with that of asperochramide D ( Figure S19 in the supplementary material), which was further supported by electronic circular dichroism (ECD) calculations (Tables S1). The calculated weighted ECD spectrum of model-(2R)-1a agreed well with the experimental ECD spectrum of 1 ( Figure 3). Thus, the structure of 1 was established as shown in Figure 1.
The molecular formula of compound 2 was determined to be C 16 H 23 N 3 O 4 on the basis of HRESIMS (m/z 322.1765 [M þ H] þ ) and NMR data ( Table 1). Analysis of 1 H and 13 C NMR spectroscopic data of 2 (Table 1) revealed that the structural feature of 2 closely resembled that of (S)-methyl 2-acetamido-4-(2 0 -(methylamino)phenyl)-4oxobutanoate [15], and the obvious difference between them was the additional presence of two methines in 2. Detailed analysis of the HSQC, HMBC, and COSY data (  probability DP4þ method [16]. The results showed that (9S,13S)-2b with high DP4þ probability score of 93.24% (Tables S2-S4 and Figure S20) was the correct structure for 2. Thus, the structure of 2 was established as shown.
The antifungal activities of all of the compounds were estimated against four phytopathogenic fungi, but none of the compounds showed antifungal activities at 100 lg/disc.

General experimental procedures
The procedures were the same as previously reported [7,8].

Fungal material
The procedures were the same as previously reported [7]. Briefly, the fungus Penicillium sp. SCSIO 41512 (GenBank: MT436778) was isolated from a soft coral of the South China Sea. The ITS sequence of this strain showed 99% similarity to that of Penicillium sp. (GenBank: GU985208).

Fermentation and extraction
The procedures were almost the same as previously reported [7]. Briefly, the spores of the fungus Penicillium sp. SCSIO 41512 were added to 150 ml potato dextrose (PD) medium in 500 ml Erlenmeyer flasks, and fermented for 3 days at 28 C. Then 3 ml of spore suspension was transferred into 300 ml PD medium, and a total of 100 L was fermented. Static fermentation was performed for 25 days at 26 C. Then, two crude extracts (39.0 and 36.6 g) were obtained from the mycelia and broth parts by the methods as previously described [7], respectively, and combined for further isolation.

Isolation and purification
The crude extracts (75 g) were fractionated by a medium-pressure column using a stepped gradient elution with CH 2

Determination of amino acid residues' absolute configurations of 1-2 (Marfey's method)
The procedures were similar to the previous report [17]. Briefly, compounds 1-2 (0.5 mg, each) were hydrolyzed in 1 ml of 6 N HCl in a sealed glass bottle at 115 C for 21 h, respectively. The hydrolyzate was dried and dissolved in 100 ll H 2 O. Then, 20 ll of 1 M NaHCO 3 solution and 100 ll FDAA solution (Marfey's reagent, 1 mg dissolved in 100 ll acetone) were added to the hydrolyzates, and the mixture was incubated at 40 C for 1 h. The reaction was quenched by adding 20 ll of 2 M HCl. Finally, the dried mixture was dissolved in MeOH and analyzed by HPLC on an Agilent Technologies 1260 Infinity system [column: YMC-Pack ODS-A column, 250 Â 4.6 mml.D., S-5 lm, 12 nm; mobile phase: CH 3 CN/H 2 O (0.03% TFA in H 2 O), linear gradients started with 15% CH 3 CN and finished with 55% CH 3 CN in 55 min; flow rate was 1 ml/min, with UV detection at an absorbance of 340 nm]. The standard amino acids D/L-Val, D/L-Ile and L-allo-Ile were treated using the above process.
The retention times for the standard amino acid derivatives were as follows: L-Val 26.1 min, D-Val 32.8 min, L-Ile 31.1 min, D-Ile 38.1 min, L-allo-Ile 31.1 min. FDAA derivatives of 1-2 and standard amino acids were compared with retention times. The L-Ile-FDAA and L-allo-Ile-FDAA and compound 1's FDAA derivative was dissolved in EtOH and analyzed by chiral column [column: CHIRALPAK IA 4.6 Â 250 mm, mobile phase: isocratic elution with 12% isopropanol (contain 0.1& TFA)-n-hexane (contain 0.2& TFA), flow rate was 1 ml/min, with UV detection at an absorbance of 340 nm], FDAA derivate of 1 and standard amino acids were compared with respect to their retention times (see Figure S17-18 in the Supporting Information).

Antifungal assay
The methods were the same as previously reported [18]. Briefly, the antifungal activities of compounds 1-6 were preliminarily tested by disc diffusion method. The phytopathogenic fungi including Fusarium oxysporum f. sp. cubense, Pyricularia oryzae, Altemaria solani, and Fusarium oxysporum f. sp. momordicae were used as indicators. Paper disc (d ¼ 5 mm) loaded with 100 lg samples were placed on the inoculated plate. Carbendazim (12.5 lg/disc) was used as positive control.

Computational methods
The methods were almost the same as previously reported [19]. Briefly, molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan'14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program package (Gaussian, Inc., Pittsburgh PA), respectively, using default grids and convergence criteria. The conformers of 1 and 2 (Tables S1 and S2) were initially optimized at the B3LYP/6-31G(d,p) level in MeOH using the polarizable continuum model (PCM). The theoretical ECD calculation was carried out using Time-dependent Density functional theory (TDDFT) at the B3LYP/6-311G(d,p) level for all conformers of 1. The ECD spectra were generated using the program SpecDis-1701 and Excel from dipole-length rotational strengths by applying Gaussian band shapes with sigma 0.20 to 0.30 eV. The B3LYP/6-31G(d) optimized geometries in the ECD calculation of 2 were adopted for further NMR computation. Gauge-Independent Atomic Orbital (GIAO) calculations of the 13 C NMR chemical shifts were accomplished by DFT at the mPW1PW91/6-311G(d, p) level in DMSO with PCM. The calculated 13 C NMR spectroscopic data were averaged according to the foregoing Boltzmann distribution.