Bioactive Phenylalanine Derivatives and Cytochalasins from the Soft Coral-Derived Fungus, Aspergillus elegans

One new phenylalanine derivative 4′-OMe-asperphenamate (1), along with one known phenylalanine derivative (2) and two new cytochalasins, aspochalasin A1 (3) and cytochalasin Z24 (4), as well as eight known cytochalasin analogues (5–12) were isolated from the fermentation broth of Aspergillus elegans ZJ-2008010, a fungus obtained from a soft coral Sarcophyton sp. collected from the South China Sea. Their structures and the relative configurations were elucidated using comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical synthesis and Marfey’s method. All isolated metabolites (1–12) were evaluated for their antifouling and antibacterial activities. Cytochalasins 5, 6, 8 and 9 showed strong antifouling activity against the larval settlement of the barnacle Balanus amphitrite, with the EC50 values ranging from 6.2 to 37 μM. This is the first report of antifouling activity for this class of metabolites. Additionally, 8 exhibited a broad spectrum of antibacterial activity, especially against four pathogenic bacteria Staphylococcus albus, S. aureus, Escherichia coli and Bacillus cereus.


Results and Discussion
4′-OMe-asperphenamate (1) was obtained as a white powder. Its molecular formula was established as C 33 [13,14]. The only difference in the 1 H NMR spectrum was the presence of a singlet methyl signal at δ H 3.74 (3H, s) in 1 instead of an aromatic proton signal at δ H 7.30 (1H, m) in 2. Furthermore, in the 13 C NMR spectrum, the C-4′ signal moved downfield significantly [δ C 158.9 (C) in 1 vs. 126.8 (CH) in 2], indicating a methoxy group was located at C-4′. The location of the methoxy group at C-4′ was confirmed by the heteronuclear multiple bond correlation (HMBC) correlation from 4′-OCH 3 at δ H 3.74 (3H, s) to C-4′ at δ C 158.9 (C). The planar structure of 1 was further confirmed by the 1 H-1 H COSY and HMBC experiments ( Figure 2).  The absolute configurations of C-8 and C-8′ in 1 were determined by chemical synthesis and Marfey's method [22]. The major acid hydrolysis products of 1 with 6 N HCl at 105 °C for 19 h were Phe and 2-amino-3-(4-methoxyphenyl)-1-propanol. The hydrolysates of 1, as well as the standard L-Phe and DL-Phe, were derivatized with Marfey's reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA). Analysis of the FDAA derivatives of the hydrolysates from 1 by HPLC, compared with the FDAA derivatives from L-Phe and DL-Phe, revealed the presence of an L-Phe moiety in 1 ( Figure S32). Therefore, the absolute configuration at C-8 was assigned as S.
To determine the absolute configuration at C-8′, (R/S)-2-amino-3-(4-methoxyphenyl)-1-propanol (14a) and (S)-2-amino-3-(4-methoxyphenyl)-1-propanol (14b) were synthesized. Compounds 14a and 14b were prepared from DL-Tyr (13a) and L-Tyr (13b) (Figure 3), respectively, according to the literature method [23]. Also, 14a and 14b were derivatized with Marfey's reagent (FDAA). Analysis of the FDAA derivatives of the hydrolysates from 1 by HPLC, compared with the FDAA derivatives from 14a and 14b, revealed the presence of a 14b moiety in 1 ( Figure S32). Accordingly, the absolute configuration at C-8′ was assigned as S. The 8S8′S configurations in 1 were consistent with those in asperphenamate (2), confirming that these two compounds share the same biogenetic path way. Aspochalasin A1 (3) was isolated as a white powder. Its molecular formula was established as C 24 H 35 NO 5 (eight degrees of unsaturation) by HRESIMS, combined with 1 H and 13 C NMR spectroscopic data ( Table 2). The 1 H NMR spectrum recorded in CDCl 3 intuitively revealed five methyl groups, two olefinic protons [δ H 6.00, 1H, d, J = 11.4 Hz, H-13; δ H 5.37, 1H, br s, H-7], an amide group (δ H 5.93, 1H, br s, NH), six methine protons, five methylene units, along with one hydroxy group (Table 2). These features characteristically revealed the structure of 3 as possessing a (2-methylpropyl) isoindolone moiety, consistent with a cytochalasin skeleton [16]. These structural features were also confirmed by the 13 C NMR and DEPT spectra ( Table 2). In the 13 C NMR spectrum, except for the carbon signals of the (2-methylpropyl) isoindolone moiety, there were also ten resonance signals revealed that 3 possessed a 12-membered lactone macrocycle, containing a methyl group (δ C 15.9, CH 3 , C-25), a double bond [δ C 125.6 (CH), C-13 and δ C 136.2 (C), C-14] and two carbonyl groups [δ C 212.7 (C), C-17 and δ C 171.8 (C), C-21]. The 1 H and 13 C NMR spectra of 3 were closely related to those of aspochalasin M [24]. The obvious difference was the absence of the macrocyclic ketone moiety as in aspochalasin M at δ C 214.5 (C-21) and the presence of a lactone at δ C 171.8 (C-21) in 3. The differences were also observed for the chemical shifts of H-18/C-18 [δ H 5.00 (1H, ddd, J = 10.8, 3.6, 3.6 Hz) and δ C 74.0 (CH) in 3 vs. δ  The relative stereochemistry of 3 was determined by NOESY experiments and comparison of NMR data with those of aspochalasin M [24]. In the NOESY experiments (Figure 4), the signal of H-3 showed correlations with H-10 and H-11 and H-4 with H-5 and H-8, indicating that the relative configurations of the perhydroisoindol-1-one moiety in 3 were in accord with those of reported cytochalasins. The modified Mosher's method [25] was tried to determine the absolute configuration of C-18 in 3; unfortunately, the reactions failed. A literature search revealed that the stereochemistry of the cyclohexane and isoindole moieties in all isolated cytochalasins, so far, are the same [26]. Therefore, based on the above data and the biogenesis consideration, 3 was determined as aspochalasin A1. Cytochalasin Z24 (4) was also obtained as a white power. Its molecular formula was established as C 28 H 35 NO 5 by HRESIMS. The general features of its NMR spectroscopic data ( Table 2) were markedly similar to those of cytochalasin Z22 [27]. Detailed comparison of NMR data of these two compounds suggested that they had the same 10-phenyl-substituted 6,7-epoxyperhydroisoindol-1-one skeleton. The only significant difference in the 1  The structures of known compounds (2, 5-12) were identified by comparison of their spectroscopic data with those in the literature [13][14][15][16][17][18][19][20][21]. Among them, asperphenamate (2), an uncommon phenylalanine derivative, has already been isolated from some bioactive natural sources, such as fungi, Aspergillus flavipes [29], Penicillium megasporum [30], P. brevicompactum [31] and P. canadense [32], and plants, Anaphalis subumbellata [33], Artemisia anomala [34] and Croton hieronymi [13]. In the present paper, this is the first report of isolated asperphenamate from marine-derived fungus.
Cytochalasins are a large group of fungal alkaloids with a wide range of biological activities targeting cytoskeletal processes [35]. Cytochalasin biosynthesis has been revealed by the formation of an acetate-and methionine-derived octa-or nonaketide chain and the attachment of an amino acid. The type of cytochalasins depends on the incorporated amino acids as structural subunits [36,37]. Compounds 3-9, 11 and 12 are a class of cytochalasins with 12-membered or 11-membered carbocyclic (or oxygen-containing) rings connecting the C-8 and C-9 positions of a perhydroisoindol-1-one moiety. The substituents at C-3 in compounds 3 and 5-9 is a 2-methylpropyl group; and in compounds 4, 11 and 12 is a phenyl group, belonging to the class of 10-phenyl- [11]-cytochalasin. Compound 10 belongs to an unusual type of aspochalasins with a pentacyclic system, of which only three examples have been reported.
All the isolated compounds were evaluated for antifouling activity against the larval settlement of the barnacle, Balanus amphitrite. Compounds 5, 6, 8 and 9 showed strong antifouling activity with the EC 50 values of 34, 14, 6.2 and 37 μM, respectively. Despite the slight structural differences in their macrocycles, 8, bearing an α,β-unsaturated ketone moiety, was found to be considerably more active than 6, with an α,β-unsaturated lactone moiety, suggesting the importance of an electrophilic α,β-unsaturated carbonyl moiety for the antifouling activity of these cytochalasins. Compound 8 displayed more activity than 9, indicating that the double-bond at C-19 and C-20 might be essential for the antifouling activity of cytochalasins. This is the first report of antifouling activity for this class of metabolites.
The antibacterial activity of all isolated compounds were also assessed against six terrestrial pathogenic bacteria and two marine pathogenic bacteria. Compounds 1, 2, 5, 8 and 10 exhibited selective antibacterial activity (Table 3)

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer. 1 H and 13 C NMR spectra were recorded on a JEOL Eclips-600 spectrometer at 600 MHz for 1 H and 150 MHz for 13 C in DMSO-d 6 or CDCl 3 . Chemical shifts δ are reported in ppm, using TMS as internal standard, and coupling constants (J) are in Hz. ESIMS and HRESIMS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. HPLC separation was performed using a Hitachi L-2000 prep-HPLC system coupled with a Hitachi L-2455 photodiode array detector. A Kromasil C18 preparative HPLC column (10 × 250 mm, 5 μm and 4.6 × 250 mm, 5 μm) was used. Analysis of FDAA derivatives by HPLC was performed using Waters 2695 prep-HPLC system coupled with a Waters 2489 UV detector. A Waters

Identification of Fungus
The Fungus was identified according to its morphological characteristics and a molecular biological protocol by 16S rRNA amplification and sequencing of the ITS region. The sequence data have been submitted to GenBank, with an accession number JF694928, and the fungal strain was identified as Aspergillus elegans.

Biological Assays
Antifouling activity against the larval-attachment was determined using cyprids of the barnacle, B. amphitrite Darwin, according to literature procedures [38]. Adults of B. amphitrite exposed to air for more than 6 h were collected from the intertidal zone in Hong Kong and then were placed in a container filled with 0.22 μm of filtered seawater (FSW) to release nauplii. The collected nauplii were reared to the cyprid stage according to the method described by Thiyagarajan et al. [38]. When kept at 26-28 °C and fed with Chaetoceros gracilis, larvae developed to cyprids on the fourth day. Fresh cyprids were used in the tests. Larval settlement assays were performed using 24-well polystyrene plates (Becton Dickinson 353047 [39]).
Antibacterial activity was determined against six terrestrial pathogenic bacteria, including Staphylococcus epidermidis, S. aureus, Escherichia coli, Bacillus subtilis, B. cereus and Micrococcus luteus, and two marine pathogenic bacteria, Vibrio parahaemolyticus and Listonella anguillarum, by the microplate assay method [40].

Conclusions
Twelve secondary metabolites, including two phenylalanine derivatives (1,2) and ten cytochalasins (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), have been isolated from the fermentation broth of a soft coral-derived fungus, Aspergillus elegans ZJ-2008010. Compound 1 is a new phenylalanine derivative, and 3 and 4 are new cytochalasin analogues. Their structures and the relative configurations were elucidated using comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical synthesis and Marfey's method. Asperphenamate (2) is the first report of isolated asperphenamate from marine-derived fungus. Compound 10 belongs to an unusual type of aspochalasins with a pentacyclic system, of which only three examples have been reported. Cytochalasins showed strong antifouling activity against the larval settlement of the barnacle, B. amphitrite. This is the first report of antifouling activity for this class of metabolites.