Psychrophilin E, a new cyclotripeptide, from co-fermentation of two marine alga-derived fungi of the genus Aspergillus

Chemical investigation of the mycelial extract of a mixed culture of two marine alga-derived fungal strains of the genus Aspergillus has yielded one new cyclotripeptide, psychrophilin E (1), the recently reported oxepin-containing alkaloids, protuboxepin A (2) and oxepinamide E (3), together with three other polyketide derivatives (4–6). The chemical structure and relative and absolute configurations of psychrophilin E (1) were unambiguously established based on HRMS, 1D, 2D NMR and chiral-phase HPLC analysis of its hydrolysate. All the isolated compounds were assessed for their anti-proliferative activity against four different human cancer cell lines and some of them revealed selective activities.


Introduction
Marine-derived fungi continue to be a prolific source providing a plethora of hitherto unprecedented chemical scaffolds and/or new metabolites with potent pharmacologically significant activities (Rateb & Ebel 2011). This notion was proven by the increasing research efforts directed towards marine-derived fungi during the last few decades, which yielded a sixfold increase in the number of identified marine fungal metabolites in 2007 compared with those reported from 1965 to 2005 (Imhoff et al. 2011).
Recently, co-culturing of two or more marine-derived fungal strains in order to imitate their natural environment and/or to induce other biosynthetic interactions has gained a considerable attention aiming at identifying novel chemical skeletons produced through different biosynthetic pathways such as aspergicin, a potent anti-microbial alkaloid recently reported from a mixed culture of two marine-derived mangrove epiphytic fungal strains of the genus Aspergillus .
Psychrophilins (Psp's) are a class of cyclic nitrotripeptides comprising four metabolites that have been reported from psychrotolerant fungal species of the genus Penicillium (Dalsgaard et al. 2004a(Dalsgaard et al. , 2004b2005). The core structural elements of these peptides are one anthranilic acid (2-aminobenzoic acid) and one tryptophan residue together with variable amino acid residues including L-proline (Psp A) (Dalsgaard et al. 2004a), L-valine (Psp B) (Dalsgaard et al. 2004b), L-alanine (Psp C) (Dalsgaard et al. 2004b) and L-leucine (Psp D) (Dalsgaard et al. 2005). During our ongoing search for bioactive secondary metabolites from marine-derived fungi through cocultures, we have studied an EtOAc extract of a mixed culture of two marine alga-derived fungal strains belonging to the genus Aspergillus which were derived from the brown algae Sargassum sp. collected off the North Sea shores in Germany. The two fungi were cultivated as a co-culture under static conditions at 288C for four weeks till the dark brown pigment of conidia was noticed and then EtOAc was added to the culture mixture to end the fermentation process and to extract the whole biomass.

Results and discussion
Chemical investigation of EtOAc extract yielded one new psychrophilin congener (Psp E, 1), two recently reported oxepin-containing pyrimidine alkaloids, protuboxepin A (2)  and oxepinamide E (3) (Lu et al. 2011), together with three mycotoxins, namely sterigmatocystin (4) (Seto et al. 1974;Shao et al. 2007), 5-methoxysterigmatocystin (5) (Seto et al. 1974;Shao et al. 2007;Cai et al. 2011) and aversin (6) (Shao et al. 2007 In addition, UV spectrum of 1 in MeOH indicated the presence of aromatic and indole moieties in the compound as revealed by psychrophilins A -D (Dalsgaard et al. 2004a(Dalsgaard et al. , 2004b2005). Based on the literature search, a match was found in psychrophilins, a class of cyclic nitrotripeptides. Further investigation was conducted through IR spectrum, showing an intense stretching vibration at 1693 cm 21 indicating the presence of amide carbonyl groups and missing those characteristic stretching vibrations of the nitro functionality at 1553 and 1360 cm 21 as for psychrophilins A -D (Dalsgaard et al. 2004a(Dalsgaard et al. , 2004b2005). These findings suggested that 1 is a cyclic tripeptide resembling the psychrophilins except in the absence of the nitro group ( Figure 1).
Structural elucidation was continued through extensive 2D NMR spectral analyses including 1 H-1 H COSY and HSQC (see Supplementary materials, Figures S9 and S10) which confirmed the existence of an ortho-substituted benzene and indole ring systems similar to those in psychrophilins A -D representing an anthranilic acid (Abz) and tryptophan (Try) residues in addition to two aliphatic spin systems of the types X -CH(X 0 )-CH 2 -CH 2 -CH 2 -X 00 and -XNH -CH(X 0 ) -CH 2 -X 00 which were attributed to proline and tryptophan residues, respectively. The arrangement of amino acid residues was confirmed through the HMBC spectrum (see Supplementary materials, Figure S11) which revealed correlations between H-11 and C-12 (d C 166.2), 18-NH to C-19 (d C 167.4) and H-20 to C-1 (d C 172.4) together with the correlations of 2-NH, H-2 and a singlet methyl resonance at d H 2.04 (s, 3H) to C-24 (d C 170.1) which established the amino acid sequence of psychrophilin E (1) as cyclo-(N-acetyltryptophanprolyl-O-aminobenzoyl).
The absolute configuration of 1 was determined through implementing chiral-phase gas chromatographic (GC) analysis of methyl N-(trifluoroacetyl)proline derivative obtained from hydrolysis, using methanolic HCl followed by acetylation using trifluoroacetic acid anhydride (TFAA) compared with those of D-and L-proline standards (Stenerson & Lee 2011). Comparison of GC retention times of the derivatised proline with D-and L-proline standards unambiguously proved proline to possess the L-configuration assigning S configuration to C-20. The absolute configuration of C-2 cannot be directly established due to the destruction of N-acetyltryptophan during acid hydrolysis.
However, the ROESY spectrum (see Supplementary materials, Figures S14 and S15) of psychrophilin E (1) revealed key correlations which were used to determine the most likely configuration. Using the 3D models of the minimal energy conformation of (2R, 20S) and (2S, 20S) psychrophilin E (1) together with the ROESY correlations (see Supplementary material, Figure S15) between the a-proton in N-acetyltryptophan (H-2) and H-3b; H-2 and H-23a/b; and the correlations between H-3b and H-6; H-3a and H-11, the absolute stereochemistry of C-2 was assigned as R configuration and hence (2R, 20S) for psychrophilin E (1) unlike the absolute configuration reported for psychrophilins A -D (Dalsgaard et al. 2004a(Dalsgaard et al. , 2004b2005).

General experimental procedures
Optical rotation was recorded using a Perkin-Elmer-341 MC polarimeter (Perkin-Elmer w , Waltham, MA, USA) at 589 nm using a cylinder with 10 cm length at 208C. IR spectrum was recorded using Perkin-Elmer Spectrum One IR-Spectrometer (Perkin-Elmer w , Waltham, MA, USA). 1D and 2D NMR spectra were measured using Bruker ARX 500 and AVANCE DRX 600 NMR spectrometers (Bruker w , Billerica, MA, USA). MS (ESI) and HRMS (ESI) were determined with a ThermoFinnigan LCQ DECA (Thermo w , Waltham, MA, USA) and Micromass QTOF 2 (Waters w , Milford, MA, USA) mass spectrometers, respectively. Solvents were distilled prior to use, and spectral grade solvents were used for spectroscopic measurements.
For HPLC analysis, a Dionex P580 HPLC system coupled to a photodiode array detector (UVD340U) was used. Routine detection was conducted at 225, 250, 275 and 350 nm. The separation column (125 £ 4 mm ID) was pre-filled with C-18 Eurosphere, 5 mM (Knauer, Berlin, Germany). Separation was performed implementing a linear gradient from 10% MeOH to 100% MeOH over 40 min. For preparative HPLC separations, a separation column (250 £ 8 mm ID) pre-filled with C-18 Eurosphere, flow rate 3.0 mL/min and UV detection at 240 and 350 nm; the elution system comprised a linear gradient of CH 3 OH and nanopure H 2 O. TLC was performed using Polygram w , plastic sheets pre-coated with silica gel 60 F 254 (Macherey-Nagel, Düren, Germany). Chiral GC analysis was carried out on Shimadzu w GC-2010 Plus equipped with capillary CP-chirasil Dex CB column using helium as carrier gas and an isothermal program at 1208C for 15 min. Minimal energy conformations of psychrophilin E (1) was calculated using ChemDraw w Ultra 12.0 based on 3D models of the two conformers (2R, 20S) and (2S, 20S).

Isolation and cultivation of the microbial material
Two fungal strains of the genus Aspergillus (BM-05 and BM-05ML) were isolated from a brown algal species belonging to the genus Sargassum collected off Helgoland, North Sea, Germany in 2001. The two fungi were cultivated together as a co-culture under static conditions at 288C for four weeks in ten 1-L Erlenmeyer flasks containing the culture medium (500 mL/flask) composed of peptone from soya (4 g/L), maize starch (10 g/L), MgSO 4 (3.6 g/L), NaCl (20 g/L), yeast extract (2 g/L) and CaCO 3 (1.8 g/L) using demineralised water. The culture medium was sterilised in 1-L Erlenmeyer flasks at 1218C for 20 min. Agar plugs from plated culture were seeded into production medium (10 £ 500 mL). The fermentation time was reached when dark brown pigment of conidia was noticed.

Acid hydrolysis of psychrophilin E (1)
The compound (0.5 mg) was refluxed at 958C for 6 h with 6 N methanolic HCl. After cooling, the sample was evaporated under reduced pressure to dryness. Then, the remaining residue was dissolved in 1.0 mL of methylene chloride and 100 mL of TFAA and then it was refluxed at 608C for 20 min. The sample was then cooled and the remaining liquid was evaporated at room temperature. The residue obtained was dissolved in methylene chloride for chiral gas GC analysis. The same procedure was conducted for an unequal known mixture of D-and L-proline standards. The configuration of proline was determined by an isothermic GC analysis using CPchirasil Dex CB column at 1208C over 15 min. Retention times (in min) for the standards were proline, R, 7.12 min, and S, 7.24 min. Analysis of the derivative gave a retention time of 7.24 min, determining an S configuration for the proline residue.

Conclusions
Psychrophilins A -D were all reported from marine-derived psychrotolerant fungal species of the genus Penicillium, cultivated in the dark at 20 -258C, psychrophilin E (1) was obtained from a mixed culture of two marine alga-derived fungal strains of the genus Aspergillus which were cultivated at 288C. In addition, psychrophilin E (1) exhibited acetylation of a-amino group in tryptophan residue into an N-acetyl moiety, which was oxidised in psychrophilins A -D into a nitro group (Dalsgaard et al. 2004a(Dalsgaard et al. , 2004b2005). Furthermore, LC -MS screening of the fungal extract and purified fractions did not reveal the existence of other detectable psychrophilin derivatives which implies that this co-culture may have played a role in modifying the biosynthetic pathway to acetylation rather than oxidation of a-amino group in tryptophan residue.
Interestingly, the additional methoxy group in 5 potentiated its activity towards HCT116 and K562 cell lines (Table 1) as compared with sterigmatocystin (4) with IC 50 values of 4.4 and 13.4 mM with 5 and 10.3 and 57.0 mM with 4, respectively. These results may strengthen the significance of 5-methoxy group as a functional group important for anti-proliferative activity of compound (5).

Supplementary material
Supplementary material relating to this article is available online alongside Table S1 and Figures S1 -S15.